SMART MODE:

NORMAL GENOMIC

• Hett EC et al.
• Rational targeting of active-site tyrosine residues using sulfonyl fluoride probes.
• ACS Chem Biol. 2015; 10: 1094-8
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• This work describes the first rational targeting of tyrosine residues in a protein binding site by small-molecule covalent probes. Specific tyrosine residues in the active site of the mRNA-decapping scavenger enzyme DcpS were modified using reactive sulfonyl fluoride covalent inhibitors. Structure-based molecular design was used to create an alkyne-tagged probe bearing the sulfonyl fluoride warhead, thus enabling the efficient capture of the protein from a complex proteome. Use of the probe in competition experiments with a diaminoquinazoline DcpS inhibitor permitted the quantification of intracellular target occupancy. As a result, diaminoquinazoline upregulators of survival motor neuron protein that are used for the treatment of spinal muscular atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes designed to react with specific tyrosine residues of a protein and augments the chemical biology toolkit by these probes uses in target validation and molecular pharmacology.

• Hu G et al.
• A conserved mechanism of TOR-dependent RCK-mediated mRNA degradation regulates autophagy.
• Nat Cell Biol. 2015; 17: 930-42
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• Autophagy is an essential eukaryotic pathway requiring tight regulation to maintain homeostasis and preclude disease. Using yeast and mammalian cells, we report a conserved mechanism of autophagy regulation by RNA helicase RCK family members in association with the decapping enzyme Dcp2. Under nutrient-replete conditions, Dcp2 undergoes TOR-dependent phosphorylation and associates with RCK members to form a complex with autophagy-related (ATG) mRNA transcripts, leading to decapping, degradation and autophagy suppression. Simultaneous with the induction of ATG mRNA synthesis, starvation reverses the process, facilitating ATG mRNA accumulation and autophagy induction. This conserved post-transcriptional mechanism modulates fungal virulence and the mammalian inflammasome, the latter providing mechanistic insight into autoimmunity reported in a patient with a PIK3CD/p110delta gain-of-function mutation. We propose a dynamic model wherein RCK family members, in conjunction with Dcp2, function in controlling ATG mRNA stability to govern autophagy, which in turn modulates vital cellular processes affecting inflammation and microbial pathogenesis.

• Kelly SP, Bedwell DM
• Both the autophagy and proteasomal pathways facilitate the Ubp3p-dependent depletion of a subset of translation and RNA turnover factors during nitrogen starvation in Saccharomyces cerevisiae.
• RNA. 2015; 21: 898-910
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• Protein turnover is an important regulatory mechanism that facilitates cellular adaptation to changing environmental conditions. Previous studies have shown that ribosome abundance is reduced during nitrogen starvation by a selective autophagy mechanism termed ribophagy, which is dependent upon the deubiquitinase Ubp3p. In this study, we asked whether the abundance of various translation and RNA turnover factors are reduced following the onset of nitrogen starvation in Saccharomyces cerevisiae. We found distinct differences in the abundance of the proteins tested following nitrogen starvation: (1) The level of some did not change; (2) others were reduced with kinetics similar to ribophagy, and (3) a few proteins were rapidly depleted. Furthermore, different pathways differentially degraded the various proteins upon nitrogen starvation. The translation factors eRF3 and eIF4GI, and the decapping enhancer Pat1p, required an intact autophagy pathway for their depletion. In contrast, the deadenylase subunit Pop2p and the decapping enzyme Dcp2p were rapidly depleted by a proteasome-dependent mechanism. The proteasome-dependent depletion of Dcp2p and Pop2p was also induced by rapamycin, suggesting that the TOR1 pathway influences this pathway. Like ribophagy, depletion of eIF4GI, eRF3, Dcp2p, and Pop2p was dependent upon Ubp3p to varying extents. Together, our results suggest that the autophagy and proteasomal pathways degrade distinct translation and RNA turnover factors in a Ubp3p-dependent manner during nitrogen starvation. While ribophagy is thought to mediate the reutilization of scarce resources during nutrient limitation, our results suggest that the selective degradation of specific proteins could also facilitate a broader reprogramming of the post-transcriptional control of gene expression.

• Motomura K et al.
• Diffuse decapping enzyme DCP2 accumulates in DCP1 foci under heat stress in Arabidopsis thaliana.
• Plant Cell Physiol. 2015; 56: 107-15
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• The decapping enzymes DCP1 and DCP2 are components of a decapping complex that degrades mRNAs. DCP2 is the catalytic core and DCP1 is an auxiliary subunit. It has been assumed that DCP1 and DCP2 are consistently co-localized in cytoplasmic RNA granules called processing bodies (P-bodies). However, it has not been confirmed whether DCP1 and DCP2 co-localize in Arabidopsis thaliana. In this study, we generated DCP1-green fluorescent protein (GFP) and DCP2-GFP transgenic plants that complemented dcp1 and dcp2 mutants, respectively, to see whether localization of DCP2 is identical to that of DCP1. DCP2 was present throughout the cytoplasm, whereas DCP1 formed P-body-like foci. Use of DCP1-GFP/DCP2-red fluorescent protein (RFP) or DCP1-RFP/DCP2-GFP plants showed that heat treatment induced DCP2 assembly into DCP1 foci. In contrast, cold treatment did not induce DCP2 assembly, while the number of DCP1 foci increased. These changes in DCP1 and DCP2 localization during heat and cold treatments occurred without changes in DCP1 and DCP2 protein abundance. Our results show that DCP1 and DCP2 respond differently to environmental changes, indicating that P-bodies have diverse DCP1 and DCP2 proportions depending on environmental conditions. The localization changes of DCP1 and DCP2 may explain how specific mRNAs are degraded during changes in environmental conditions.

• Erickson SL et al.
• Competition between Decapping Complex Formation and Ubiquitin-Mediated Proteasomal Degradation Controls Human Dcp2 Decapping Activity.
• Mol Cell Biol. 2015; 35: 2144-53
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• mRNA decapping is a central step in eukaryotic mRNA decay that simultaneously shuts down translation initiation and activates mRNA degradation. A major complex responsible for decapping consists of the decapping enzyme Dcp2 in association with decapping enhancers. An important question is how the activity and accumulation of Dcp2 are regulated at the cellular level to ensure the specificity and fidelity of the Dcp2 decapping complex. Here, we show that human Dcp2 levels and activity are controlled by a competition between decapping complex assembly and Dcp2 degradation. This is mediated by a regulatory domain in the Dcp2 C terminus, which, on the one hand, promotes Dcp2 activation via decapping complex formation mediated by the decapping enhancer Hedls and, on the other hand, targets Dcp2 for ubiquitin-mediated proteasomal degradation in the absence of Hedls association. This competition between Dcp2 activation and degradation restricts the accumulation and activity of uncomplexed Dcp2, which may be important for preventing uncontrolled decapping or for regulating Dcp2 levels and activity according to cellular needs.

• Antic S, Wolfinger MT, Skucha A, Hosiner S, Dorner S
• General and MicroRNA-Mediated mRNA Degradation Occurs on Ribosome Complexes in Drosophila Cells.
• Mol Cell Biol. 2015; 35: 2309-20
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• The translation and degradation of mRNAs are two key steps in gene expression that are highly regulated and targeted by many factors, including microRNAs (miRNAs). While it is well established that translation and mRNA degradation are tightly coupled, it is still not entirely clear where in the cell mRNA degradation takes place. In this study, we investigated the possibility of mRNA degradation on the ribosome in Drosophila cells. Using polysome profiles and ribosome affinity purification, we could demonstrate the copurification of various deadenylation and decapping factors with ribosome complexes. Also, AGO1 and GW182, two key factors in the miRNA-mediated mRNA degradation pathway, were associated with ribosome complexes. Their copurification was dependent on intact mRNAs, suggesting the association of these factors with the mRNA rather than the ribosome itself. Furthermore, we isolated decapped mRNA degradation intermediates from ribosome complexes and performed high-throughput sequencing analysis. Interestingly, 93% of the decapped mRNA fragments (approximately 12,000) could be detected at the same relative abundance on ribosome complexes and in cell lysates. In summary, our findings strongly indicate the association of the majority of bulk mRNAs as well as mRNAs targeted by miRNAs with the ribosome during their degradation.

• Steffens A, Brautigam A, Jakoby M, Hulskamp M
• The BEACH Domain Protein SPIRRIG Is Essential for Arabidopsis Salt Stress Tolerance and Functions as a Regulator of Transcript Stabilization and Localization.
• PLoS Biol. 2015; 13: 1002188-1002188
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• Members of the highly conserved class of BEACH domain containing proteins (BDCPs) have been established as broad facilitators of protein-protein interactions and membrane dynamics in the context of human diseases like albinism, bleeding diathesis, impaired cellular immunity, cancer predisposition, and neurological dysfunctions. Also, the Arabidopsis thaliana BDCP SPIRRIG (SPI) is important for membrane integrity, as spi mutants exhibit split vacuoles. In this work, we report a novel molecular function of the BDCP SPI in ribonucleoprotein particle formation. We show that SPI interacts with the P-body core component DECAPPING PROTEIN 1 (DCP1), associates to mRNA processing bodies (P-bodies), and regulates their assembly upon salt stress. The finding that spi mutants exhibit salt hypersensitivity suggests that the local function of SPI at P-bodies is of biological relevance. Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi. We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs. Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN (Factor Associated with Neutral sphingomyelinase activation) interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies.

• Lee KT, Kwon H, Lee D, Bahn YS
• A Nudix Hydrolase Protein, Ysa1, Regulates Oxidative Stress Response and Antifungal Drug Susceptibility in Cryptococcus neoformans.
• Mycobiology. 2014; 42: 52-8
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• A nucleoside diphosphate-linked moiety X (Nudix) hydrolase-like gene, YSA1, has been identified as one of the gromwell plant extract-responsive genes in Cryptococcus neoformans. Ysa1 is known to control intracellular concentrations of ADP-ribose or O-acetyl-ADP-ribose, and has diverse biological functions, including the response to oxidative stress in the ascomycete yeast, Saccharomyces cerevisiae. In this study, we characterized the role of YSA1 in the stress response and adaptation of the basidiomycete yeast, C. neoformans. We constructed three independent deletion mutants for YSA1, and analyzed their mutant phenotypes. We found that ysa1 mutants did not show increased sensitivity to reactive oxygen species-producing oxidative damage agents, such as hydrogen peroxide and menadione, but exhibited increased sensitivity to diamide, which is a thiol-specific oxidant. Ysa1 was dispensable for the response to most environmental stresses, such as genotoxic, osmotic, and endoplasmic reticulum stress. In conclusion, modulation of YSA1 may regulate the cellular response and adaptation of C. neoformans to certain oxidative stresses and contribute to the evolution of antifungal drug resistance.

• Shrestha RK et al.
• Insights into the mechanism of deubiquitination by JAMM deubiquitinases from cocrystal structures of the enzyme with the substrate and product.
• Biochemistry. 2014; 53: 3199-217
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• AMSH, a conserved zinc metallo deubiquitinase, controls downregulation and degradation of cell-surface receptors mediated by the endosomal sorting complexes required for transport (ESCRT) machinery. It displays high specificity toward the Lys63-linked polyubiquitin chain, which is used as a signal for ESCRT-mediated endosomal-lysosomal sorting of receptors. Herein, we report the crystal structures of the catalytic domain of AMSH orthologue Sst2 from fission yeast, its ubiquitin (product)-bound form, and its Lys63-linked diubiquitin (substrate)-bound form at 1.45, 1.7, and 2.3 A, respectively. The structures reveal that the P-side product fragment maintains nearly all the contacts with the enzyme as seen with the P portion (distal ubiquitin) of the Lys63-linked diubiquitin substrate, with additional coordination of the Gly76 carboxylate group of the product with the active-site Zn(2+). One of the product-bound structures described herein is the result of an attempt to cocrystallize the diubiquitin substrate bound to an active site mutant presumed to render the enzyme inactive, instead yielding a cocrystal structure of the enzyme bound to the P-side ubiquitin fragment of the substrate (distal ubiquitin). This fragment was generated in situ from the residual activity of the mutant enzyme. In this structure, the catalytic water is seen placed between the active-site Zn(2+) and the carboxylate group of Gly76 of ubiquitin, providing what appears to be a snapshot of the active site when the product is about to depart. Comparison of this structure with that of the substrate-bound form suggests the importance of dynamics of a flexible flap near the active site in catalysis. The crystal structure of the Thr319Ile mutant of the catalytic domain of Sst2 provides insight into structural basis of microcephaly capillary malformation syndrome. Isothermal titration calorimetry yields a dissociation constant (KD) of 10.2 +/- 0.6 muM for the binding of ubiquitin to the enzyme, a value comparable to the KM of the enzyme catalyzing hydrolysis of the Lys63-linked diubiquitin substrate (~20 muM). These results, together with the previously reported observation that the intracellular concentration of free ubiquitin (~20 muM) exceeds that of Lys63-linked polyubiquitin chains, imply that the free, cytosolic form of the enzyme remains inhibited by being tightly bound to free ubiquitin. We propose that when AMSH associates with endosomes, inhibition would be relieved because of ubiquitin binding domains present on its endosomal binding partners that would shift the balance toward better recognition of polyubiquitin chains via the avidity effect.

• Wang L et al.
• Regulation of the Rhp26ERCC6/CSB chromatin remodeler by a novel conserved leucine latch motif.
• Proc Natl Acad Sci U S A. 2014; 111: 18566-71
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• CSB/ERCC6 (Cockayne syndrome B protein/excision repair cross-complementation group 6), a member of a subfamily of SWI2/SNF2 (SWItch/sucrose nonfermentable)-related chromatin remodelers, plays crucial roles in gene expression and the maintenance of genome integrity. Here, we report the mechanism of the autoregulation of Rhp26, which is the homolog of CSB/ERCC6 in Schizosaccharomyces pombe. We identified a novel conserved protein motif, termed the "leucine latch," at the N terminus of Rhp26. The leucine latch motif mediates the autoinhibition of the ATPase and chromatin-remodeling activities of Rhp26 via its interaction with the core ATPase domain. Moreover, we found that the C terminus of the protein counteracts this autoinhibition and that both the N- and C-terminal regions of Rhp26 are needed for its proper function in DNA repair in vivo. The presence of the leucine latch motif in organisms ranging from yeast to humans suggests a conserved mechanism for the autoregulation of CSB/ERCC6 despite the otherwise highly divergent nature of the N- and C-terminal regions.

• Mota S et al.
• Role of the DHH1 gene in the regulation of monocarboxylic acids transporters expression in Saccharomyces cerevisiae.
• PLoS One. 2014; 9: 111589-111589
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• Previous experiments revealed that DHH1, a RNA helicase involved in the regulation of mRNA stability and translation, complemented the phenotype of a Saccharomyces cerevisiae mutant affected in the expression of genes coding for monocarboxylic-acids transporters, JEN1 and ADY2 (Paiva S, Althoff S, Casal M, Leao C. FEMS Microbiol Lett, 1999, 170:301-306). In wild type cells, JEN1 expression had been shown to be undetectable in the presence of glucose or formic acid, and induced in the presence of lactate. In this work, we show that JEN1 mRNA accumulates in a dhh1 mutant, when formic acid was used as sole carbon source. Dhh1 interacts with the decapping activator Dcp1 and with the deadenylase complex. This led to the hypothesis that JEN1 expression is post-transcriptionally regulated by Dhh1 in formic acid. Analyses of JEN1 mRNAs decay in wild-type and dhh1 mutant strains confirmed this hypothesis. In these conditions, the stabilized JEN1 mRNA was associated to polysomes but no Jen1 protein could be detected, either by measurable lactate carrier activity, Jen1-GFP fluorescence detection or western blots. These results revealed the complexity of the expression regulation of JEN1 in S. cerevisiae and evidenced the importance of DHH1 in this process. Additionally, microarray analyses of dhh1 mutant indicated that Dhh1 plays a large role in metabolic adaptation, suggesting that carbon source changes triggers a complex interplay between transcriptional and post-transcriptional effects.

• Wu D et al.
• Lsm2 and Lsm3 bridge the interaction of the Lsm1-7 complex with Pat1 for decapping activation.
• Cell Res. 2014; 24: 233-46
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• The evolutionarily conserved Lsm1-7-Pat1 complex is the most critical activator of mRNA decapping in eukaryotic cells and plays many roles in normal decay, AU-rich element-mediated decay, and miRNA silencing, yet how Pat1 interacts with the Lsm1-7 complex is unknown. Here, we show that Lsm2 and Lsm3 bridge the interaction between the C-terminus of Pat1 (Pat1C) and the Lsm1-7 complex. The Lsm2-3-Pat1C complex and the Lsm1-7-Pat1C complex stimulate decapping in vitro to a similar extent and exhibit similar RNA-binding preference. The crystal structure of the Lsm2-3-Pat1C complex shows that Pat1C binds to Lsm2-3 to form an asymmetric complex with three Pat1C molecules surrounding a heptameric ring formed by Lsm2-3. Structure-based mutagenesis revealed the importance of Lsm2-3-Pat1C interactions in decapping activation in vivo. Based on the structure of Lsm2-3-Pat1C, a model of Lsm1-7-Pat1 complex is constructed and how RNA binds to this complex is discussed.

• Huch S, Nissan T
• Interrelations between translation and general mRNA degradation in yeast.
• Wiley Interdiscip Rev RNA. 2014; 5: 747-63
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• Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system.

• Steffens A, Jaegle B, Tresch A, Hulskamp M, Jakoby M
• Processing-body movement in Arabidopsis depends on an interaction between myosins and DECAPPING PROTEIN1.
• Plant Physiol. 2014; 164: 1879-92
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• Processing (P)-bodies are cytoplasmic RNA protein aggregates responsible for the storage, degradation, and quality control of translationally repressed messenger RNAs in eukaryotic cells. In mammals, P-body-related RNA and protein exchanges are actomyosin dependent, whereas P-body movement requires intact microtubules. In contrast, in plants, P-body motility is actin based. In this study, we show the direct interaction of the P-body core component DECAPPING PROTEIN1 (DCP1) with the tails of different unconventional myosins in Arabidopsis (Arabidopsis thaliana). By performing coexpression studies with AtDCP1, dominant-negative myosin fragments, as well as functional full-length myosin XI-K, the association of P-bodies and myosins was analyzed in detail. Finally, the combination of mutant analyses and characterization of P-body movement patterns showed that myosin XI-K is essential for fast and directed P-body transport. Together, our data indicate that P-body movement in plants is governed by myosin XI members through direct binding to AtDCP1 rather than through an adapter protein, as known for membrane-coated organelles. Interspecies and intraspecies interaction approaches with mammalian and yeast protein homologs suggest that this mechanism is evolutionarily conserved among eukaryotes.

• Weidner J, Wang C, Prescianotto-Baschong C, Estrada AF, Spang A
• The polysome-associated proteins Scp160 and Bfr1 prevent P body formation under normal growth conditions.
• J Cell Sci. 2014; 127: 1992-2004
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• Numerous mRNAs are degraded in processing bodies (P bodies) in Saccharomyces cerevisiae. In logarithmically growing cells, only 0-1 P bodies per cell are detectable. However, the number and appearance of P bodies change once the cell encounters stress. Here, we show that the polysome-associated mRNA-binding protein Scp160 interacts with P body components, such as the decapping protein Dcp2 and the scaffold protein Pat1, presumably, on polysomes. Loss of either Scp160 or its interaction partner Bfr1 caused the formation of Dcp2-positive structures. These Dcp2-positive foci contained mRNA, because their formation was inhibited by the presence of cycloheximide. In addition, Scp160 was required for proper P body formation because only a subset of bona fide P body components could assemble into the Dcp2-positive foci in Deltascp160 cells. In either Deltabfr1 or Deltascp160 cells, P body formation was uncoupled from translational attenuation as the polysome profile remained unchanged. Collectively, our data suggest that Bfr1 and Scp160 prevent P body formation under normal growth conditions.

• Livesay SB, Collier SE, Bitton DA, Bahler J, Ohi MD
• Structural and functional characterization of the N terminus of Schizosaccharomyces pombe Cwf10.
• Eukaryot Cell. 2013; 12: 1472-89
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• The spliceosome is a dynamic macromolecular machine that catalyzes the removal of introns from pre-mRNA, yielding mature message. Schizosaccharomyces pombe Cwf10 (homolog of Saccharomyces cerevisiae Snu114 and human U5-116K), an integral member of the U5 snRNP, is a GTPase that has multiple roles within the splicing cycle. Cwf10/Snu114 family members are highly homologous to eukaryotic translation elongation factor EF2, and they contain a conserved N-terminal extension (NTE) to the EF2-like portion, predicted to be an intrinsically unfolded domain. Using S. pombe as a model system, we show that the NTE is not essential, but cells lacking this domain are defective in pre-mRNA splicing. Genetic interactions between cwf10-DeltaNTE and other pre-mRNA splicing mutants are consistent with a role for the NTE in spliceosome activation and second-step catalysis. Characterization of Cwf10-NTE by various biophysical techniques shows that in solution the NTE contains regions of both structure and disorder. The first 23 highly conserved amino acids of the NTE are essential for its role in splicing but when overexpressed are not sufficient to restore pre-mRNA splicing to wild-type levels in cwf10-DeltaNTE cells. When the entire NTE is overexpressed in the cwf10-DeltaNTE background, it can complement the truncated Cwf10 protein in trans, and it immunoprecipitates a complex similar in composition to the late-stage U5.U2/U6 spliceosome. These data show that the structurally flexible NTE is capable of independently incorporating into the spliceosome and improving splicing function, possibly indicating a role for the NTE in stabilizing conformational rearrangements during a splice cycle.

• Liszczak G, Goldberg JM, Foyn H, Petersson EJ, Arnesen T, Marmorstein R
• Molecular basis for N-terminal acetylation by the heterodimeric NatA complex.
• Nat Struct Mol Biol. 2013; 20: 1098-105
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• N-terminal acetylation is ubiquitous among eukaryotic proteins and controls a myriad of biological processes. Of the N-terminal acetyltransferases (NATs) that facilitate this cotranslational modification, the heterodimeric NatA complex has the most diversity for substrate selection and modifies the majority of all N-terminally acetylated proteins. Here, we report the X-ray crystal structure of the 100-kDa holo-NatA complex from Schizosaccharomyces pombe, in the absence and presence of a bisubstrate peptide-CoA-conjugate inhibitor, as well as the structure of the uncomplexed Naa10p catalytic subunit. The NatA-Naa15p auxiliary subunit contains 13 tetratricopeptide motifs and adopts a ring-like topology that wraps around the NatA-Naa10p subunit, an interaction that alters the Naa10p active site for substrate-specific acetylation. These studies have implications for understanding the mechanistic details of other NAT complexes and how regulatory subunits modulate the activity of the broader family of protein acetyltransferases.

• Ullah A et al.
• Structural and functional analysis of transmembrane segment IV of the salt tolerance protein Sod2.
• J Biol Chem. 2013; 288: 24609-24
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• Sod2 is the plasma membrane Na(+)/H(+) exchanger of the fission yeast Schizosaccharomyces pombe. It provides salt tolerance by removing excess intracellular sodium (or lithium) in exchange for protons. We examined the role of amino acid residues of transmembrane segment IV (TM IV) ((126)FPQINFLGSLLIAGCITSTDPVLSALI(152)) in activity by using alanine scanning mutagenesis and examining salt tolerance in sod2-deficient S. pombe. Two amino acids were critical for function. Mutations T144A and V147A resulted in defective proteins that did not confer salt tolerance when reintroduced into S. pombe. Sod2 protein with other alanine mutations in TM IV had little or no effect. T144D and T144K mutant proteins were inactive; however, a T144S protein was functional and provided lithium, but not sodium, tolerance and transport. Analysis of sensitivity to trypsin indicated that the mutations caused a conformational change in the Sod2 protein. We expressed and purified TM IV (amino acids 125-154). NMR analysis yielded a model with two helical regions (amino acids 128-142 and 147-154) separated by an unwound region (amino acids 143-146). Molecular modeling of the entire Sod2 protein suggested that TM IV has a structure similar to that deduced by NMR analysis and an overall structure similar to that of Escherichia coli NhaA. TM IV of Sod2 has similarities to TM V of the Zygosaccharomyces rouxii Na(+)/H(+) exchanger and TM VI of isoform 1 of mammalian Na(+)/H(+) exchanger. TM IV of Sod2 is critical to transport and may be involved in cation binding or conformational changes of the protein.

• Hopkins K, Cherry S
• Bunyaviral cap-snatching vs. decapping: recycling cell cycle mRNAs.
• Cell Cycle. 2013; 12: 3711-2

• Goring ME et al.
• The ability of an arginine to tryptophan substitution in Saccharomyces cerevisiae tRNA nucleotidyltransferase to alleviate a temperature-sensitive phenotype suggests a role for motif C in active site organization.
• Biochim Biophys Acta. 2013; 1834: 2097-106
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• We report that the temperature-sensitive (ts) phenotype in Saccharomyces cerevisiae associated with a variant tRNA nucleotidyltransferase containing an amino acid substitution at position 189 results from a reduced ability to incorporate AMP and CMP into tRNAs. We show that this defect can be compensated for by a second-site suppressor converting residue arginine 64 to tryptophan. The R64W substitution does not alter the structure or thermal stability of the enzyme dramatically but restores catalytic activity in vitro and suppresses the ts phenotype in vivo. R64 is found in motif A known to be involved in catalysis and nucleotide triphosphate binding while E189 lies within motif C previously thought only to connect the head and neck domains of the protein. Although mutagenesis experiments indicate that residues R64 and E189 do not interact directly, our data suggest a critical role for residue E189 in enzyme structure and function. Both R64 and E189 may contribute to the organization of the catalytic domain of the enzyme. These results, along with overexpression and deletion analyses, show that the ts phenotype of cca1-E189F does not arise from thermal instability of the variant tRNA nucleotidyltransferase but instead from the inability of a partially active enzyme to support growth only at higher temperatures.

• Song MG, Bail S, Kiledjian M
• Multiple Nudix family proteins possess mRNA decapping activity.
• RNA. 2013; 19: 390-9
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• RNA decapping is an important contributor to gene expression and is a critical determinant of mRNA decay. The recent demonstration that mammalian cells harbor at least two distinct decapping enzymes that preferentially modulate a subset of mRNAs raises the intriguing possibility of whether additional decapping enzymes exist. Because both known decapping proteins, Dcp2 and Nudt16, are members of the Nudix hydrolase family, we set out to determine whether other members of this family of proteins also contain intrinsic RNA decapping activity. Here we demonstrate that six additional mouse Nudix proteins--Nudt2, Nudt3, Nudt12, Nudt15, Nudt17, and Nudt19--have varying degrees of decapping activity in vitro on both monomethylated and unmethylated capped RNAs. The decapping products from Nudt17 and Nudt19 were analogous to Dcp2 and predominantly generated m(7)GDP, while cleavage by Nudt2, Nudt3, Nudt12, and Nudt15 was more pleiotropic and generated both m(7)GMP and m(7)GDP. Interestingly, all six Nudix proteins as well as both Dcp2 and Nudt16 could hydrolyze the cap of an unmethylated capped RNA, indicating that decapping enzymes may be less constrained for the presence of the methyl moiety. Investigation of Saccharomyces cerevisiae Nudix proteins revealed that the yeast homolog of Nudt3, Ddp1p, also possesses decapping activity in vitro. Moreover, the bacterial Nudix pyrophosphohydrolase RppH displayed RNA decapping activity and released m(7)GDP product comparable to Dcp2, indicating that decapping is an evolutionarily conserved activity that preceded mammalian cap formation. These findings demonstrate that multiple Nudix family hydrolases may function in mRNA decapping and mRNA stability.

• He C et al.
• Structural analysis of Stc1 provides insights into the coupling of RNAi and chromatin modification.
• Proc Natl Acad Sci U S A. 2013; 110: 187988-187988
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• Noncoding RNAs can modulate gene expression by directing modifications to histones that alter chromatin structure. In fission yeast, siRNAs produced via the RNAi pathway direct modifications associated with heterochromatin formation. siRNAs associate with the RNAi effector protein Argonaute 1 (Ago1), targeting the Ago1-containing RNA-induced transcriptional silencing (RITS) complex to homologous nascent transcripts. This promotes recruitment of the Clr4 complex (CLRC), which mediates methylation of histone H3 on lysine 9 (H3K9me) in cognate chromatin. A key question is how the RNAi and chromatin modification machineries are connected. Stc1 is a small protein recently shown to associate with both Ago1 and CLRC and to play a pivotal role in mediating the RNAi-dependent recruitment of CLRC to chromatin. To understand its mode of action, we have performed a detailed structural and functional analysis of the Stc1 protein. Our analyses reveal that the conserved N-terminal region of Stc1 represents an unusual tandem zinc finger domain, with similarities to common LIM domains but distinguished by a lack of preferred relative orientation of the two zinc fingers. We demonstrate that this tandem zinc finger domain is involved in binding Ago1, whereas the nonconserved C-terminal region mediates association with CLRC. These findings elucidate the molecular basis for the coupling of RNAi to chromatin modification in fission yeast.

• Huang HT, Maruyama J, Kitamoto K
• Aspergillus oryzae AoSO is a novel component of stress granules upon heat stress in filamentous fungi.
• PLoS One. 2013; 8: 72209-72209
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• Stress granules are a type of cytoplasmic messenger ribonucleoprotein (mRNP) granule formed in response to the inhibition of translation initiation, which typically occurs when cells are exposed to stress. Stress granules are conserved in eukaryotes; however, in filamentous fungi, including Aspergillus oryzae, stress granules have not yet been defined. For this reason, here we investigated the formation and localization of stress granules in A. oryzae cells exposed to various stresses using an EGFP fusion protein of AoPab1, a homolog of Saccharomyces cerevisiae Pab1p, as a stress granule marker. Localization analysis showed that AoPab1 was evenly distributed throughout the cytoplasm under normal growth conditions, and accumulated as cytoplasmic foci mainly at the hyphal tip in response to stress. AoSO, a homolog of Neurospora crassa SO, which is necessary for hyphal fusion, colocalized with stress granules in cells exposed to heat stress. The formation of cytoplasmic foci of AoSO was blocked by treatment with cycloheximide, a known inhibitor of stress granule formation. Deletion of the Aoso gene had effects on the formation and localization of stress granules in response to heat stress. Our results suggest that AoSO is a novel component of stress granules specific to filamentous fungi. The authors would specially like to thank Hiroyuki Nakano and Kei Saeki for generously providing experimental and insightful opinions.

• Ziemniak M et al.
• Synthesis and evaluation of fluorescent cap analogues for mRNA labelling.
• RSC Adv. 2013; 3: 0-0
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• We describe the synthesis and properties of five dinucleotide fluorescent cap analogues labelled at the ribose of the 7-methylguanosine moiety with either anthraniloyl (Ant) or N-methylanthraniloyl (Mant), which have been designed for the preparation of fluorescent mRNAs via transcription in vitro. Two of the analogues bear a methylene modification in the triphosphate bridge, providing resistance against either the Dcp2 or DcpS decapping enzymes. All these compounds were prepared by ZnCl2-mediated coupling of a nucleotide P-imidazolide with a fluorescently labelled mononucleotide. To evaluate the utility of these compounds for studying interactions with cap-binding proteins and cap-related cellular processes, both biological and spectroscopic features of those compounds were determined. The results indicate acceptable quantum yields of fluorescence, pH independence, environmental sensitivity, and photostability. The cap analogues are incorporated by RNA polymerase into mRNA transcripts that are efficiently translated in vitro. Transcripts containing fluorescent caps but unmodified in the triphosphate chain are hydrolysed by Dcp2 whereas those containing a alpha-beta methylene modification are resistant. Model studies exploiting sensitivity of Mant to changes of local environment demonstrated utility of the synthesized compounds for studying cap-related proteins.

• Hanazono Y et al.
• Nonequivalence observed for the 16-meric structure of a small heat shock protein, SpHsp16.0, from Schizosaccharomyces pombe.
• Structure. 2013; 21: 220-8
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• Small heat shock proteins (sHsps) play a role in preventing the fatal aggregation of denatured proteins in the presence of stresses. The sHsps exist as monodisperse oligomers in their resting state. Because the hydrophobic N-terminal regions of sHsps are possible interaction sites for denatured proteins, the manner of assembly of the oligomer is critical for the activation and inactivation mechanisms. Here, we report the oligomer architecture of SpHsp16.0 from Schizosaccharomyces pombe determined with X-ray crystallography and small angle X-ray scattering. Both results indicate that eight dimers of SpHsp16.0 form an elongated sphere with 422 symmetry. The monomers show nonequivalence in the interaction with neighboring monomers and conformations of the N- and C-terminal regions. Variants for the N-terminal phenylalanine residues indicate that the oligomer formation ability is highly correlated with chaperone activity. Structural and biophysical results are discussed in terms of their possible relevance to the activation mechanism of SpHsp16.0.

• Sun W et al.
• Crystal structure of the yeast TSC1 core domain and implications for tuberous sclerosis pathological mutations.
• Nat Commun. 2013; 4: 2135-2135
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• Tuberous sclerosis complex is a disease caused by mutations in two tumor-suppressor genes, TSC1 and TSC2. The TSC1 protein, also known as hamartin, has a critical role in controlling mTOR signalling. TSC1 does not bear apparent sequence homology with other proteins. Here we show that the N-terminal half of yeast TSC1 forms a protease-resistant domain, which is evolutionarily conserved. The crystal structure of this yeast TSC1 core domain shows that it contains a pseudo-HEAT repeat fold with its C-terminal end capped by a helical subdomain. This allows us to model the three-dimensional structure of the human TSC1 N-terminal domain (TSC1-NTD), which anchors essentially all pathogenic TSC1 missense mutations found in tuberous sclerosis patients. Interestingly, most pathogenic mutations map inside of the folded TSC1-NTD structure, whereas most non-pathogenic variants are on the structural surface. This indicates that the disruption of the TSC1-NTD globular structure is a major cause of tuberous sclerosis.

• Molero C et al.
• The Schizosaccharomyces pombe fusion gene hal3 encodes three distinct activities.
• Mol Microbiol. 2013; 90: 367-82
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• Saccharomyces cerevisiae Hal3 and Vhs3 are moonlighting proteins, forming an atypical heterotrimeric decarboxylase (PPCDC) required for CoA biosynthesis, and regulating cation homeostasis by inhibition of the Ppz1 phosphatase. The Schizosaccharomyces pombe ORF SPAC15E1.04 (renamed as Sp hal3) encodes a protein whose amino-terminal half is similar to Sc Hal3 whereas its carboxyl-terminal half is related to thymidylate synthase (TS). We show that Sp Hal3 and/or its N-terminal domain retain the ability to bind to and modestly inhibit in vitro S. cerevisiae Ppz1 as well as its S. pombe homolog Pzh1, and also exhibit PPCDC activity in vitro and provide PPCDC function in vivo, indicating that Sp Hal3 is a monogenic PPCDC in fission yeast. Whereas the Sp Hal3 N-terminal domain partially mimics Sc Hal3 functions, the entire protein and its carboxyl-terminal domain rescue the S. cerevisiae cdc21 mutant, thus proving TS function. Additionally, we show that the 70 kDa Sp Hal3 protein is not proteolytically processed under diverse forms of stress and that, as predicted, Sp hal3 is an essential gene. Therefore, Sp hal3 represents a fusion event that joined three different functional activities in the same gene. The possible advantage derived from this surprising combination of essential proteins is discussed.

• Wang CY, Chen WL, Wang SW
• Pdc1 functions in the assembly of P bodies in Schizosaccharomyces pombe.
• Mol Cell Biol. 2013; 33: 1244-53
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• P bodies are cytoplasmic RNA granules containing the Dcp1-Dcp2 decapping enzymes where mRNA decay can occur. Here, we describe the characterization of P bodies in the fission yeast Schizosaccharomyces pombe. Most information on the property and function of P bodies stems from studies in the distantly related budding yeast Saccharomyces cerevisiae, and Edc3 was identified as a scaffold protein required for P-body assembly. However, we found that, unlike in S. cerevisiae, fission yeast Edc3 was dispensable for P-body formation. Pdc1, a novel partner of the fission yeast decapping enzyme, with a limited similarity to plant Edc4/Varicose that is required for the assembly of P bodies, was identified (tandem affinity purification-matrix-assisted laser desorption ionization tandem mass spectrometry [TAP-MALDI MS/MS]). Pdc1 interacts with Dcp2 through its C terminus and contains a coiled-coil region for self-interaction to mediate P-body formation. In line with the model that Pdc1 cross-bridges different proteins, additional interactions can be demonstrated with components such as Edc3 and Ste13. Although Pdc1 is not required for the interaction between Dcp1 and Dcp2, our data suggest that Pdc1 acts as a functional homologue of Edc4, a third component of the decapping enzymes that is thought to be absent from fungi. Together, these results highlight the diverse P-body protein compositions between different species and might help to provide insight into their evolutionary paths.

• Drazkowska K, Tomecki R, Stodus K, Kowalska K, Czarnocki-Cieciura M, Dziembowski A
• The RNA exosome complex central channel controls both exonuclease and endonuclease Dis3 activities in vivo and in vitro.
• Nucleic Acids Res. 2013; 41: 3845-58
• Display abstract

• The RNA exosome is an essential ribonuclease complex involved in RNA processing and decay. It consists of a 9-subunit catalytically inert ring composed of six RNase PH-like proteins forming a central channel and three cap subunits with KH/S1 domains located at the top. The yeast exosome catalytic activity is supplied by the Dis3 (also known as Rrp44) protein, which has both endo- and exoribonucleolytic activities and the nucleus-specific exonuclease Rrp6. In vitro studies suggest that substrates reach the Dis3 exonucleolytic active site following passage through the ring channel, but in vivo support is lacking. Here, we constructed an Rrp41 ring subunit mutant with a partially blocked channel that led to thermosensitivity and synthetic lethality with Rrp6 deletion. Rrp41 mutation caused accumulation of nuclear and cytoplasmic exosome substrates including the non-stop decay reporter, for which degradation is dependent on either endonucleolytic or exonucleolytic Dis3 activities. This suggests that the central channel also controls endonucleolytic activity. In vitro experiments performed using Chaetomium thermophilum exosomes reconstituted from recombinant subunits confirmed this notion. Finally, we analysed the impact of a lethal mutation of conserved basic residues in Rrp4 cap subunit and found that it inhibits digestion of single-stranded and structured RNA substrates.

• Boehringer J et al.
• Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation.
• Biochem J. 2012; 448: 55-65
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• The ubiquitin-proteasome system targets selected proteins for degradation by the 26S proteasome. Rpn12 is an essential component of the 19S regulatory particle and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. In the present paper we report the crystal structure of Rpn12, a proteasomal PCI-domain-containing protein. The structure helps to define a core structural motif for the PCI domain and identifies potential sites through which Rpn12 might form protein-protein interactions. We demonstrate that mutating residues at one of these sites impairs Rpn12 binding to Rpn10 in vitro and reduces Rpn10 incorporation into proteasomes in vivo.

• Liu X, Mitchell JM, Wozniak RW, Blobel G, Fan J
• Structural evolution of the membrane-coating module of the nuclear pore complex.
• Proc Natl Acad Sci U S A. 2012; 109: 16498-503
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• The coatomer module of the nuclear pore complex borders the cylinder-like nuclear pore-membrane domain of the nuclear envelope. In evolution, a single coatomer module increases in size from hetero-heptamer (Saccharomyces cerevisiae) to hetero-octamer (Schizosaccharomyces pombe) to hetero-nonamer (Metazoa). Notably, the heptamer-octamer transition proceeds through the acquisition of the nucleoporin Nup37. How Nup37 contacts the heptamer remained unknown. Using recombinant nucleoporins, we show that Sp-Nup37 specifically binds the Sp-Nup120 member of the hetero-heptamer but does not bind an Sc-Nup120 homolog. To elucidate the Nup37-Nup120 interaction at the atomic level, we carried out crystallographic analyses of Sp-Nup37 alone and in a complex with an N-terminal, ~110-kDa fragment of Sp-Nup120 comprising residues 1-950. Corroborating structural predictions, we determined that Nup37 folds into a seven-bladed beta-propeller. Several disordered surface regions of the Nup37 beta-propeller assume structure when bound to Sp-Nup120. The N-terminal domain of Sp-Nup120(1-950) also folds into a seven-bladed propeller with a markedly protruding 6D-7A insert and is followed by a contorted helical domain. Conspicuously, this 6D-7A insert contains an extension of 50 residues which also is highly conserved in Metazoa but is absent in Sc-Nup120. Strikingly, numerous contacts with the Nup37 beta-propeller are located on this extension of the 6D-7A insert. Another contact region is situated toward the end of the helical region of Sp-Nup120(1-950). Our findings provide information about the evolution and the assembly of the coatomer module of the nuclear pore complex.

• Parker R
• RNA degradation in Saccharomyces cerevisae.
• Genetics. 2012; 191: 671-702
• Display abstract

• All RNA species in yeast cells are subject to turnover. Work over the past 20 years has defined degradation mechanisms for messenger RNAs, transfer RNAs, ribosomal RNAs, and noncoding RNAs. In addition, numerous quality control mechanisms that target aberrant RNAs have been identified. Generally, each decay mechanism contains factors that funnel RNA substrates to abundant exo- and/or endonucleases. Key issues for future work include determining the mechanisms that control the specificity of RNA degradation and how RNA degradation processes interact with translation, RNA transport, and other cellular processes.

• Chao WC, Kulkarni K, Zhang Z, Kong EH, Barford D
• Structure of the mitotic checkpoint complex.
• Nature. 2012; 484: 208-13
• Display abstract

• In mitosis, the spindle assembly checkpoint (SAC) ensures genome stability by delaying chromosome segregation until all sister chromatids have achieved bipolar attachment to the mitotic spindle. The SAC is imposed by the mitotic checkpoint complex (MCC), whose assembly is catalysed by unattached chromosomes and which binds and inhibits the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome segregation. Here, using the crystal structure of Schizosaccharomyces pombe MCC (a complex of mitotic spindle assembly checkpoint proteins Mad2, Mad3 and APC/C co-activator protein Cdc20), we reveal the molecular basis of MCC-mediated APC/C inhibition and the regulation of MCC assembly. The MCC inhibits the APC/C by obstructing degron recognition sites on Cdc20 (the substrate recruitment subunit of the APC/C) and displacing Cdc20 to disrupt formation of a bipartite D-box receptor with the APC/C subunit Apc10. Mad2, in the closed conformation (C-Mad2), stabilizes the complex by optimally positioning the Mad3 KEN-box degron to bind Cdc20. Mad3 and p31(comet) (also known as MAD2L1-binding protein) compete for the same C-Mad2 interface, which explains how p31(comet) disrupts MCC assembly to antagonize the SAC. This study shows how APC/C inhibition is coupled to degron recognition by co-activators.

• Geisler S, Lojek L, Khalil AM, Baker KE, Coller J
• Decapping of long noncoding RNAs regulates inducible genes.
• Mol Cell. 2012; 45: 279-91
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• Decapping represents a critical control point in regulating expression of protein coding genes. Here, we demonstrate that decapping also modulates expression of long noncoding RNAs (lncRNAs). Specifically, levels of >100 lncRNAs in yeast are controlled by decapping and are degraded by a pathway that occurs independent of decapping regulators. We find many lncRNAs degraded by DCP2 are expressed proximal to inducible genes. Of these, we show several genes required for galactose utilization are associated with lncRNAs that have expression patterns inversely correlated with their mRNA counterpart. Moreover, decapping of these lncRNAs is critical for rapid and robust induction of GAL gene expression. Failure to destabilize a lncRNA known to exert repressive histone modifications results in perpetuation of a repressive chromatin state that contributes to reduced plasticity of gene activation. We propose that decapping and lncRNA degradation serve a vital role in transcriptional regulation specifically at inducible genes.

• Webb CJ, Zakian VA
• Schizosaccharomyces pombe Ccq1 and TER1 bind the 14-3-3-like domain of Est1, which promotes and stabilizes telomerase-telomere association.
• Genes Dev. 2012; 26: 82-91
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• The telomerase protein Est1 exists in multiple organisms, including Schizosaccharomyces pombe, humans, and Saccharomyces cerevisiae, but its function has only been closely examined in S. cerevisiae, where it is a recruiter/activator of telomerase. Here, we demonstrate that S. pombe Est1 was required for the telomere association of the telomerase holoenzyme, suggesting that it too has a recruitment role. Its association with telomeres was dependent on Trt1, the catalytic subunit, and Ccq1, a telomeric protein. Surprisingly, Est1 telomere binding was only partially dependent on TER1, the telomerase RNA, even though Est1 bound nucleotides 415-507 of TER1. A ter1-Delta415-507 strain had short telomeres and very low Est1 and Trt1 telomere association in late S phase but did not senesce. An unbiased search for mutations that reduced Est1-TER1 interaction identified mutations only in the Est1 14-3-3-like domain, a phosphoserine-binding motif, the first example of a 14-3-3-like domain with RNA-binding activity. These mutations also reduced Est1-Ccq1 binding. One such mutant prevented Est1 telomere association and caused telomere loss and slow senescence, similar to ccq1Delta. We propose that the Est1-Ccq1 interaction is critical for telomerase recruitment, while the Est1-TER1 interaction acts downstream from Ccq1-mediated recruitment to stabilize the holoenzyme at the telomere.

• Decker CJ, Parker R
• P-bodies and stress granules: possible roles in the control of translation and mRNA degradation.
• Cold Spring Harb Perspect Biol. 2012; 4: 12286-12286
• Display abstract

• The control of translation and mRNA degradation is important in the regulation of eukaryotic gene expression. In general, translation and steps in the major pathway of mRNA decay are in competition with each other. mRNAs that are not engaged in translation can aggregate into cytoplasmic mRNP granules referred to as processing bodies (P-bodies) and stress granules, which are related to mRNP particles that control translation in early development and neurons. Analyses of P-bodies and stress granules suggest a dynamic process, referred to as the mRNA Cycle, wherein mRNPs can move between polysomes, P-bodies and stress granules although the functional roles of mRNP assembly into higher order structures remain poorly understood. In this article, we review what is known about the coupling of translation and mRNA degradation, the properties of P-bodies and stress granules, and how assembly of mRNPs into larger structures might influence cellular function.

• Perea-Resa C, Hernandez-Verdeja T, Lopez-Cobollo R, del Mar Castellano M, Salinas J
• LSM proteins provide accurate splicing and decay of selected transcripts to ensure normal Arabidopsis development.
• Plant Cell. 2012; 24: 4930-47
• Display abstract

• In yeast and animals, SM-like (LSM) proteins typically exist as heptameric complexes and are involved in different aspects of RNA metabolism. Eight LSM proteins, LSM1 to 8, are highly conserved and form two distinct heteroheptameric complexes, LSM1-7 and LSM2-8,that function in mRNA decay and splicing, respectively. A search of the Arabidopsis thaliana genome identifies 11 genes encoding proteins related to the eight conserved LSMs, the genes encoding the putative LSM1, LSM3, and LSM6 proteins being duplicated. Here, we report the molecular and functional characterization of the Arabidopsis LSM gene family. Our results show that the 11 LSM genes are active and encode proteins that are also organized in two different heptameric complexes. The LSM1-7 complex is cytoplasmic and is involved in P-body formation and mRNA decay by promoting decapping. The LSM2-8 complex is nuclear and is required for precursor mRNA splicing through U6 small nuclear RNA stabilization. More importantly, our results also reveal that these complexes are essential for the correct turnover and splicing of selected development-related mRNAs and for the normal development of Arabidopsis. We propose that LSMs play a critical role in Arabidopsis development by ensuring the appropriate development-related gene expression through the regulation of mRNA splicing and decay.

• Lariviere L, Plaschka C, Seizl M, Wenzeck L, Kurth F, Cramer P
• Structure of the Mediator head module.
• Nature. 2012; 492: 448-51
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• Gene transcription by RNA polymerase (Pol) II requires the coactivator complex Mediator. Mediator connects transcriptional regulators and Pol II, and is linked to human disease. Mediator from the yeast Saccharomyces cerevisiae has a molecular mass of 1.4 megadaltons and comprises 25 subunits that form the head, middle, tail and kinase modules. The head module constitutes one-half of the essential Mediator core, and comprises the conserved subunits Med6, Med8, Med11, Med17, Med18, Med20 and Med22. Recent X-ray analysis of the S. cerevisiae head module at 4.3 A resolution led to a partial architectural model with three submodules called neck, fixed jaw and moveable jaw. Here we determine de novo the crystal structure of the head module from the fission yeast Schizosaccharomyces pombe at 3.4 A resolution. Structure solution was enabled by new structures of Med6 and the fixed jaw, and previous structures of the moveable jaw and part of the neck, and required deletion of Med20. The S. pombe head module resembles the head of a crocodile with eight distinct elements, of which at least four are mobile. The fixed jaw comprises tooth and nose domains, whereas the neck submodule contains a helical spine and one limb, with shoulder, arm and finger elements. The arm and the essential shoulder contact other parts of Mediator. The jaws and a central joint are implicated in interactions with Pol II and its carboxy-terminal domain, and the joint is required for transcription in vitro. The S. pombe head module structure leads to a revised model of the S. cerevisiae module, reveals a high conservation and flexibility, explains known mutations, and provides the basis for unravelling a central mechanism of gene regulation.

• Yates LA et al.
• Structural basis for the activity of a cytoplasmic RNA terminal uridylyl transferase.
• Nat Struct Mol Biol. 2012; 19: 782-7
• Display abstract

• Cytoplasmic terminal uridylyl transferases comprise a conserved family of enzymes that negatively regulate the stability or biological activity of a variety of eukaryotic RNAs, including mRNAs and tumor-suppressor let-7 microRNAs. Here we describe crystal structures of the Schizosaccharomyces pombe cytoplasmic terminal uridylyl transferase Cid1 in two apo conformers and bound to UTP. We demonstrate that a single histidine residue, conserved in mammalian Cid1 orthologs, is responsible for discrimination between UTP and ATP. We also describe a new high-affinity RNA substrate-binding mechanism of Cid1, which is essential for enzymatic activity and is mediated by three basic patches across the surface of the enzyme. Overall, our structures provide a basis for understanding the activity of Cid1 and a mechanism of UTP selectivity conserved in its human orthologs, suggesting potential implications for anticancer drug design.

• Miller JE, Reese JC
• Ccr4-Not complex: the control freak of eukaryotic cells.
• Crit Rev Biochem Mol Biol. 2012; 47: 315-33
• Display abstract

• The purpose of this review is to provide an analysis of the latest developments on the functions of the carbon catabolite-repression 4-Not (Ccr4-Not) complex in regulating eukaryotic gene expression. Ccr4-Not is a nine-subunit protein complex that is conserved in sequence and function throughout the eukaryotic kingdom. Although Ccr4-Not has been studied since the 1980s, our understanding of what it does is constantly evolving. Once thought to solely regulate transcription, it is now clear that it has much broader roles in gene regulation, such as in mRNA decay and quality control, RNA export, translational repression and protein ubiquitylation. The mechanism of actions for each of its functions is still being debated. Some of the difficulty in drawing a clear picture is that it has been implicated in so many processes that regulate mRNAs and proteins in both the cytoplasm and the nucleus. We will describe what is known about the Ccr4-Not complex in yeast and other eukaryotes in an effort to synthesize a unified model for how this complex coordinates multiple steps in gene regulation and provide insights into what questions will be most exciting to answer in the future.

• Ren J et al.
• Down-regulation of Decapping Protein 2 mediates chronic nicotine exposure-induced locomotor hyperactivity in Drosophila.
• PLoS One. 2012; 7: 52521-52521
• Display abstract

• Long-term tobacco use causes nicotine dependence via the regulation of a wide range of genes and is accompanied by various health problems. Studies in mammalian systems have revealed some key factors involved in the effects of nicotine, including nicotinic acetylcholine receptors (nAChRs), dopamine and other neurotransmitters. Nevertheless, the signaling pathways that link nicotine-induced molecular and behavioral modifications remain elusive. Utilizing a chronic nicotine administration paradigm, we found that adult male fruit flies exhibited locomotor hyperactivity after three consecutive days of nicotine exposure, while nicotine-naive flies did not. Strikingly, this chronic nicotine-induced locomotor hyperactivity (cNILH) was abolished in Decapping Protein 2 or 1 (Dcp2 or Dcp1) -deficient flies, while only Dcp2-deficient flies exhibited higher basal levels of locomotor activity than controls. These results indicate that Dcp2 plays a critical role in the response to chronic nicotine exposure. Moreover, the messenger RNA (mRNA) level of Dcp2 in the fly head was suppressed by chronic nicotine treatment, and up-regulation of Dcp2 expression in the nervous system blocked cNILH. These results indicate that down-regulation of Dcp2 mediates chronic nicotine-exposure-induced locomotor hyperactivity in Drosophila. The decapping proteins play a major role in mRNA degradation; however, their function in the nervous system has rarely been investigated. Our findings reveal a significant role for the mRNA decapping pathway in developing locomotor hyperactivity in response to chronic nicotine exposure and identify Dcp2 as a potential candidate for future research on nicotine dependence.

• Lee IJ, Wu JQ
• Characterization of Mid1 domains for targeting and scaffolding in fission yeast cytokinesis.
• J Cell Sci. 2012; 125: 2973-85
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• Division-site selection and contractile-ring assembly are two crucial steps in cytokinesis. In fission yeast, the anillin-like Mid1 protein specifies the division site at the cell equator by assembling cortical nodes, the precursors of the contractile ring. Thus, Mid1 is essential for linking the positional cues for the cleavage site to contractile-ring formation. However, how Mid1 domains cooperate to regulate cytokinesis is poorly understood. Here we unravel the functions of different Mid1 domains (motifs) by a series of truncations. We report that the conserved PH domain stabilizes Mid1 in nodes by binding to lipids and is required for Mid1 cortical localization during interphase in the absence of Cdr2 kinase. Mid1 lacking an internal region that is approximately one third of the full-length protein has higher nuclear and cortical concentration and suppresses the division-site positioning defects in cells with a deletion of the dual-specificity tyrosine-regulated kinase Pom1. The N-terminus of Mid1 physically interacts with cytokinesis node proteins. When fused to cortical node protein Cdr2, Mid1(1-100) is sufficient to assemble cytokinesis nodes and the contractile ring. Collectively, our study recognizes domains regulating Mid1 cortical localization and reveals domains sufficient for contractile-ring assembly.

• Li Y, Dai J, Song M, Fitzgerald-Bocarsly P, Kiledjian M
• Dcp2 decapping protein modulates mRNA stability of the critical interferon regulatory factor (IRF) IRF-7.
• Mol Cell Biol. 2012; 32: 1164-72
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• The mammalian Dcp2 mRNA-decapping protein functions primarily on a subset of mRNAs in a transcript-specific manner. Here we show that Dcp2 is an important modulator of genes involved in the type I interferon (IFN) response, which is the initial line of antiviral innate immune response elicited by a viral challenge. Mouse embryonic fibroblast cells with reduced Dcp2 levels (Dcp2(beta/beta)) contained significantly elevated levels of mRNAs encoding proteins involved in the type I IFN response. In particular, analysis of a key type I IFN transcription factor, IFN regulatory factor 7 (IRF-7), revealed an increase in both IRF-7 mRNA and protein in Dcp2(beta/beta) cells. Importantly, the increase in IRF-7 mRNA within the background of reduced Dcp2 levels was attributed to a stabilization of the IRF-7 mRNA, suggesting that Dcp2 normally modulates IRF-7 mRNA stability. Moreover, Dcp2 expression was also induced upon viral infection, consistent with a role in attenuating the antiviral response by promoting IRF-7 mRNA degradation. The induction of Dcp2 levels following a viral challenge and the specificity of Dcp2 in targeting the decay of IRF-7 mRNA suggest that Dcp2 may negatively contribute to the innate immune response in a negative feedback mechanism to restore normal homeostasis following viral infection.

• Xu J, Chua NH
• Dehydration stress activates Arabidopsis MPK6 to signal DCP1 phosphorylation.
• EMBO J. 2012; 31: 1975-84
• Display abstract

• Eukaryotic mRNA decapping proteins are essential for normal turnover of mRNA. Yet, the mechanism of bulk mRNA turnover during stress responses and its importance to stress tolerance are poorly understood. Here, we showed that dehydration stress activated MPK6 to phosphorylate serine 237 of Arabidopsis DCP1 and phospho-DCP1 preferentially associated with DCP5 to promote mRNA decapping in vivo. This process was essential for stress adaption as dcp5-1 and DCP1-S237A plants were hypersensitive to stress compared with wild-type (WT) plants. Microarray analysis revealed that dehydration-induced expression of many stress responsive genes was compromised in dcp5-1, whereas a subset of transcripts was over-represented in this mutant. Further analysis revealed that this subset of transcripts was likely the direct targets of stress-triggered mRNA decapping in WT. Our results suggest that mRNA decapping through MPK6-DCP1-DCP5 pathway serves as a rapid response to dehydration stress in Arabidopsis.

• Lai T et al.
• Structural basis of the PNRC2-mediated link between mrna surveillance and decapping.
• Structure. 2012; 20: 2025-37
• Display abstract

• Nonsense-mediated mRNA decay (NMD) is an important mRNA surveillance system, and human PNRC2 protein mediates the link between mRNA surveillance and decapping. However, the mechanism by which PNRC2 interacts with the mRNA surveillance machinery and stimulates NMD is unknown. Here, we present the crystal structure of Dcp1a in complex with PNRC2. The proline-rich region of PNRC2 is bound to the EVH1 domain of Dcp1a, while its NR-box mediates the interaction with the hyperphosphorylated Upf1. The mode of PNRC2 interaction with Dcp1a is distinct from those observed in other EVH1/proline-rich ligands interactions. Disruption of the interaction of PNRC2 with Dcp1a abolishes its P-body localization and ability to promote mRNA degradation when tethered to mRNAs. PNRC2 acts in synergy with Dcp1a to stimulate the decapping activity of Dcp2 by bridging the interaction between Dcp1a and Dcp2, suggesting that PNRC2 is a decapping coactivator in addition to its adaptor role in NMD.

• Thran M, Link K, Sonnewald U
• The Arabidopsis DCP2 gene is required for proper mRNA turnover and prevents transgene silencing in Arabidopsis.
• Plant J. 2012; 72: 368-77
• Display abstract

• Post-transcriptional gene silencing often limits the over-expression of transgenes in transgenic plants. It involves RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), which recognizes aberrant transcripts, such as inaccurately processed or uncapped mRNA, and triggers silencing of target transcripts. Here, we describe the isolation and characterization of an Arabidopsis mutant displaying increased transgene silencing (its1). Reduced accumulation of transgene mRNA in the its1 mutant background was accompanied by accumulation of transgene-specific siRNAs and was overcome by potyvirus infection. We therefore speculated that ITS1 is a suppressor of post-transcriptional gene silencing. Map-based cloning and subsequent complementation revealed that ITS1 encodes DECAPPING 2 (DCP2), which is crucial for decapping, a prerequisite for mRNA degradation. In agreement with the proposed function of DCP2, we found a reduced accumulation of uncapped mRNA in the its1 mutant. Furthermore, silencing in the its1 mutant was dependent on RDR6 function, suggesting that reduced decapping leads to accumulation of aberrant capped mRNA. Hence, we provide evidence for a class of aberrant mRNA that accumulates upon impaired mRNA decapping and triggers post-transcriptional gene silencing in Arabidopsis. As DCP2 knockouts cause post-embryo lethality, we isolated a hypomorphic dcp2 allele, providing insights into mRNA degradation and its interplay with post-transcriptional gene silencing.

• Bulfer SL, Hendershot JM, Trievel RC
• Crystal structure of homoisocitrate dehydrogenase from Schizosaccharomyces pombe.
• Proteins. 2012; 80: 661-6
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• Homoisocitrate dehydrogenase (HICDH) catalyzes the conversion of homoisocitrate to 2-oxoadipate, the third enzymatic step in the alpha-aminoadipate pathway by which lysine is synthesized in fungi and certain archaebacteria. This enzyme represents a potential target for anti-fungal drug design. Here, we describe the first crystal structures of a fungal HICDH, including structures of an apoenzyme and a binary complex with a glycine tri-peptide. The structures illustrate the homology of HICDH with other beta-hydroxyacid oxidative decarboxylases and reveal key differences with the active site of Thermus thermophilus HICDH that provide insights into the differences in substrate specificity of these enzymes.

• Medina B et al.
• The ubiquitin-associated (UBA) 1 domain of Schizosaccharomyces pombe Rhp23 is essential for the recognition of ubiquitin-proteasome system substrates both in vitro and in vivo.
• J Biol Chem. 2012; 287: 42344-51
• Display abstract

• The ubiquitin-proteasome system is essential for maintaining a functional cell. Not only does it remove incorrectly folded proteins, it also regulates protein levels to ensure their appropriate spatial and temporal distribution. Proteins marked for degradation by the addition of Lys(48)-linked ubiquitin (Ub) chains are recognized by shuttle factors and transported to the 26 S proteasome. One of these shuttle factors, Schizosaccharomyces pombe Rhp23, has an unusual domain architecture. It comprises an N-terminal ubiquitin-like domain that can recognize the proteasome followed by two ubiquitin-associated (UBA) domains, termed UBA1 and UBA2, which can bind Ub. This architecture is conserved up to humans, suggesting that both domains are important for Rhp23 function. Such an extent of conservation raises the question as to why, in contrast to all other shuttle proteins, does Rhp23 require two UBA domains? We performed in vitro Ub binding assays using domain swap chimeric proteins and mutated domains in isolation as well as in the context of the full-length protein to reveal that the Ub binding properties of the UBA domains are context-dependent. In vivo, the internal Rhp23 UBA1 domain provides sufficient Ub recognition for the protein to function without UBA2.

• Brook M, Gray NK
• The role of mammalian poly(A)-binding proteins in co-ordinating mRNA turnover.
• Biochem Soc Trans. 2012; 40: 856-64
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• The function of cytoplasmic PABPs [poly(A)-binding proteins] in promoting mRNA translation has been intensively studied. However, PABPs also have less clearly defined functions in mRNA turnover including roles in default deadenylation, a major rate-limiting step in mRNA decay, as well as roles in the regulation of mRNA turnover by cis-acting control elements and in the detection of aberrant mRNA transcripts. In the present paper, we review our current understanding of the complex roles of PABP1 in mRNA turnover, focusing on recent progress in mammals and highlighting some of the major questions that remain to be addressed.

• Beaudoin J, Thiele DJ, Labbe S, Puig S
• Dissection of the relative contribution of the Schizosaccharomyces pombe Ctr4 and Ctr5 proteins to the copper transport and cell surface delivery functions.
• Microbiology. 2011; 157: 1021-31
• Display abstract

• The Ctr1 family of proteins mediates high-affinity copper (Cu) acquisition in eukaryotic organisms. In the fission yeast Schizosaccharomyces pombe, Cu uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Unlike human and Saccharomyces cerevisiae Ctr1 proteins, Ctr4 and Ctr5 are unable to function independently in Cu acquisition. Instead, both proteins physically interact with each other to form a Ctr4-Ctr5 heteromeric complex, and are interdependent for secretion to the plasma membrane and Cu transport activity. In this study, we used S. cerevisiae mutants that are defective in high-affinity Cu uptake to dissect the relative contribution of Ctr4 and Ctr5 to the Cu transport function. Functional complementation and localization assays show that the conserved Met-X(3)-Met motif in transmembrane domain 2 of the Ctr5 protein is dispensable for the functionality of the Ctr4-Ctr5 complex, whereas the Met-X(3)-Met motif in the Ctr4 protein is essential for function and for localization of the hetero-complex to the plasma membrane. Moreover, Ctr4/Ctr5 chimeric proteins reveal unique properties found either in Ctr4 or in Ctr5, and are sufficient for Cu uptake on the cell surface of Sch. pombe cells. Functional chimeras contain the Ctr4 central and Ctr5 carboxyl-terminal domains (CTDs). We propose that the Ctr4 central domain mediates Cu transport in this hetero-complex, whereas the Ctr5 CTD functions in the regulation of trafficking of the Cu transport complex to the cell surface.

• Santiveri CM, Mirassou Y, Rico-Lastres P, Martinez-Lumbreras S, Perez-Canadillas JM
• Pub1p C-terminal RRM domain interacts with Tif4631p through a conserved region neighbouring the Pab1p binding site.
• PLoS One. 2011; 6: 24481-24481
• Display abstract

• Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM). Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to beta-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1-402) of yeast eIF4G1 (Tif4631p), very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role.

• Wu D, Raafat M, Pak E, Hammond S, Murashov AK
• MicroRNA machinery responds to peripheral nerve lesion in an injury-regulated pattern.
• Neuroscience. 2011; 190: 386-97
• Display abstract

• Recently, functional and potent RNA interference (RNAi) has been reported in peripheral nerve axons transfected with short-interfering RNA (siRNA). In addition, components of RNA-induced silencing complex (RISC) have been identified in axotomized sciatic nerve fibers as well as in regenerating dorsal root ganglia (DRG) neurons in vitro. Based on these observations, and on the fact that siRNA and microRNA (miRNA) share the same effector enzymes, we hypothesized that the endogenous miRNA biosynthetic pathway would respond to peripheral nerve injury. To answer this question, we investigated changes in the expression of miRNA biosynthetic enzymes following peripheral nerve crush injury in mice. Here, we show that several pivotal miRNA biosynthetic enzymes are expressed in an injury-regulated pattern in sciatic nerve in vivo, and in DRG axons in vitro. Moreover, the sciatic nerve lesion induced expression of mRNA-processing bodies (P-bodies), which are the local foci of mRNA degradation in DRG axons. In addition, a group of injury-regulated miRNAs was identified by miRNA microarray and validated by real-time quantitative PCR (qPCR) and in situ hybridization analyses. Taken together, our data support the hypothesis that the peripheral nerve regeneration processes may be regulated by miRNA pathway.

• van Hoof A, Wagner EJ
• A brief survey of mRNA surveillance.
• Trends Biochem Sci. 2011; 36: 585-92
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• Defective mRNAs are degraded more rapidly than normal mRNAs in a process called mRNA surveillance. Eukaryotic cells use a variety of mechanisms to detect aberrations in mRNAs and a variety of enzymes to preferentially degrade them. Recent advances in the field of RNA surveillance have provided new information regarding how cells determine which mRNA species should be subject to destruction and novel mechanisms by which a cell tags an mRNA once such a decision has been reached. In this review, we highlight recent progress in our understanding of these processes.

• Dutta A, Zheng S, Jain D, Cameron CE, Reese JC
• Intermolecular interactions within the abundant DEAD-box protein Dhh1 regulate its activity in vivo.
• J Biol Chem. 2011; 286: 27454-70
• Display abstract

• Dhh1 is a highly conserved DEAD-box protein that has been implicated in many processes involved in mRNA regulation. At least some functions of Dhh1 may be carried out in cytoplasmic foci called processing bodies (P-bodies). Dhh1 was identified initially as a putative RNA helicase based solely on the presence of conserved helicase motifs found in the superfamily 2 (Sf2) of DEXD/H-box proteins. Although initial mutagenesis studies revealed that the signature DEAD-box motif is required for Dhh1 function in vivo, enzymatic (ATPase or helicase) or ATP binding activities of Dhh1 or those of any its many higher eukaryotic orthologues have not been described. Here we provide the first characterization of the biochemical activities of Dhh1. Dhh1 has weaker RNA-dependent ATPase activity than other well characterized DEAD-box helicases. We provide evidence that intermolecular interactions between the N- and C-terminal RecA-like helicase domains restrict its ATPase activity; mutation of residues mediating these interactions enhanced ATP hydrolysis. Interestingly, the interdomain interaction mutant displayed enhanced mRNA turnover, RNA binding, and recruitment into cytoplasmic foci in vivo compared with wild type Dhh1. Also, we demonstrate that the ATPase activity of Dhh1 is not required for it to be recruited into cytoplasmic foci, but it regulates its association with RNA in vivo. We hypothesize that the activity of Dhh1 is restricted by interdomain interactions, which can be regulated by cellular factors to impart stringent control over this very abundant RNA helicase.

• Muller JJ et al.
• Structural and thermodynamic characterization of the adrenodoxin-like domain of the electron-transfer protein Etp1 from Schizosaccharomyces pombe.
• J Inorg Biochem. 2011; 105: 957-65
• Display abstract

• The protein Etp1 of Schizosaccharomyces pombe consists of an amino-terminal COX15-like domain and a carboxy-terminal ferredoxin-like domain, Etp1(fd), which is cleaved off after mitochondrial import. The physiological function of Etp1(fd) is supposed to lie in the participation in the assembly of iron-sulfur clusters and the synthesis of heme A. In addition, the protein was shown to be the first microbial ferredoxin being able to support electron transfer in mitochondrial steroid hydroxylating cytochrome P450 systems in vivo and in vitro, replacing thereby the native redox partner, adrenodoxin. Despite a sequence similarity of 39% and the fact that fission yeast is a mesophilic organism, thermodynamic studies revealed that Etp1(fd) has a melting temperature more than 20 degrees C higher than adrenodoxin. The three-dimensional structure of Etp1(fd) has been determined by crystallography. To the best of our knowledge it represents the first three-dimensional structure of a yeast ferredoxin. The structure-based sequence alignment of Etp1(fd) with adrenodoxin yields a rational explanation for their observed mutual exchangeability in the cytochrome P450 system. Analysis of the electron exchange with the S. pombe redox partner Arh1 revealed differences between Etp1(fd) and adrenodoxin, which might be linked to their different physiological functions in the mitochondria of mammals and yeast.

• Rzeczkowski K et al.
• c-Jun N-terminal kinase phosphorylates DCP1a to control formation of P bodies.
• J Cell Biol. 2011; 194: 581-96
• Display abstract

• Cytokines and stress-inducing stimuli signal through c-Jun N-terminal kinase (JNK) using a diverse and only partially defined set of downstream effectors. In this paper, the decapping complex subunit DCP1a was identified as a novel JNK target. JNK phosphorylated DCP1a at residue S315 in vivo and in vitro and coimmunoprecipitated and colocalized with DCP1a in processing bodies (P bodies). Sustained JNK activation by several different inducers led to DCP1a dispersion from P bodies, whereas IL-1 treatment transiently increased P body number. Inhibition of TAK1-JNK signaling also affected the number and size of P bodies and the localization of DCP1a, Xrn1, and Edc4. Transcriptome analysis further identified a central role for DCP1a in IL-1-induced messenger ribonucleic acid (mRNA) expression. Phosphomimetic mutation of S315 stabilized IL-8 but not IkappaBalpha mRNA, whereas overexpressed DCP1a blocked IL-8 transcription and suppressed p65 NF-kappaB nuclear activity. Collectively, these data reveal DCP1a as a multifunctional regulator of mRNA expression and suggest a novel mechanism controlling the subcellular localization of DCP1a in response to stress or inflammatory stimuli.

• Rispal D, Henri J, van Tilbeurgh H, Graille M, Seraphin B
• Structural and functional analysis of Nro1/Ett1: a protein involved in translation termination in S. cerevisiae and in O2-mediated gene control in S. pombe.
• RNA. 2011; 17: 1213-24
• Display abstract

• In Saccharomyces cerevisiae, the putative 2-OG-Fe(II) dioxygenase Tpa1 and its partner Ett1 have been shown to impact mRNA decay and translation. Hence, inactivation of these factors was shown to influence stop codon read-though. In addition, Tpa1 represses, by an unknown mechanism, genes regulated by Hap1, a transcription factor involved in the response to levels of heme and O(2). The Schizosaccharomyces pombe orthologs of Tpa1 and Ett1, Ofd1, and its partner Nro1, respectively, have been shown to regulate the stability of the Sre1 transcription factor in response to oxygen levels. To gain insight into the function of Nro1/Ett1, we have solved the crystal structure of the S. pombe Nro1 protein deleted of its 54 N-terminal residues. Nro1 unexpectedly adopts a Tetratrico Peptide Repeat (TPR) fold, a motif often responsible for protein or peptide binding. Two ligands, a sulfate ion and an unknown molecule, interact with a cluster of highly conserved amino acids on the protein surface. Mutation of these residues demonstrates that these ligand binding sites are essential for Ett1 function in S. cerevisiae, as investigated by assaying for efficient translation termination.

• Lu G et al.
• hNUDT16: a universal decapping enzyme for small nucleolar RNA and cytoplasmic mRNA.
• Protein Cell. 2011; 2: 64-73
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• Human NUDT16 (hNUDT16) is a decapping enzyme initially identified as the human homolog to the Xenopus laevis X29. As a metalloenzyme, hNUDT16 relies on divalent cations for its cap-hydrolysis activity to remove m(7)GDP and m(2)(2)(7)GDP from RNAs. Metal also determines substrate specificity of the enzyme. So far, only U8 small nucleolar RNA (snoRNA) has been identified as the substrate of hNUDT16 in the presence of Mg(2)(+). Here we demonstrate that besides U8, hNUDT16 can also actively cleave the m(7)GDP cap from mRNAs in the presence of Mg(2)(+) or Mn(2)(+). We further show that hNUDT16 does not preferentially recognize U8 or mRNA substrates by our cross-inhibition and quantitative decapping assays. In addition, our mutagenesis analysis identifies several key residues involved in hydrolysis and confirms the key role of the REXXEE motif in catalysis. Finally an investigation into the subcellular localization of hNUDT16 revealed its abundance in both cytoplasm and nucleus. These findings extend the substrate spectrum of hNUDT16 beyond snoRNAs to also include mRNA, demonstrating the pleiotropic decapping activity of hNUDT16.

• Takeda K et al.
• Implications for proteasome nuclear localization revealed by the structure of the nuclear proteasome tether protein Cut8.
• Proc Natl Acad Sci U S A. 2011; 108: 16950-5
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• Degradation of nuclear proteins by the 26S proteasome is essential for cell viability. In yeast, the nuclear envelope protein Cut8 mediates nuclear proteasomal sequestration by an uncharacterized mechanism. Here we describe structures of Schizosaccharomyces pombe Cut8, which shows that it contains a unique, modular fold composed of an extended N-terminal, lysine-rich segment that when ubiquitinated binds the proteasome, a dimer domain followed by a six-helix bundle connected to a flexible C tail. The Cut8 six-helix bundle shows structural similarity to 14-3-3 phosphoprotein-binding domains, and binding assays show that this domain is necessary and sufficient for liposome and cholesterol binding. Moreover, specific mutations in the 14-3-3 regions corresponding to putative cholesterol recognition/interaction amino acid consensus motifs abrogate cholesterol binding. In vivo studies confirmed that the 14-3-3 region is necessary for Cut8 membrane localization and that dimerization is critical for its function. Thus, the data reveal the Cut8 organization at the nuclear envelope. Reconstruction of Cut8 evolution suggests that it was present in the last common ancestor of extant eukaryotes and accordingly that nuclear proteasomal sequestration is an ancestral eukaryotic feature. The importance of Cut8 for cell viability and its absence in humans suggests it as a possible target for the development of specific chemotherapeutics against invasive fungal infections.

• Borja MS, Piotukh K, Freund C, Gross JD
• Dcp1 links coactivators of mRNA decapping to Dcp2 by proline recognition.
• RNA. 2011; 17: 278-90
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• Cap hydrolysis is a critical step in several eukaryotic mRNA decay pathways and is carried out by the evolutionarily conserved decapping complex containing Dcp2 at the catalytic core. In yeast, Dcp1 is an essential activator of decapping and coactivators such as Edc1 and Edc2 are thought to enhance activity, though their mechanism remains elusive. Using kinetic analysis we show that a crucial function of Dcp1 is to couple the binding of coactivators of decapping to activation of Dcp2. Edc1 and Edc2 bind Dcp1 via its EVH1 proline recognition site and stimulate decapping by 1000-fold, affecting both the K(M) for mRNA and rate of the catalytic step. The C-terminus of Edc1 is necessary and sufficient to enhance the catalytic step, while the remainder of the protein likely increases mRNA binding to the decapping complex. Lesions in the Dcp1 EVH1 domain or the Edc1 proline-rich sequence are sufficient to block stimulation. These results identify a new role of Dcp1, which is to link the binding of coactivators to substrate recognition and activation of Dcp2.

• Li Y, Song M, Kiledjian M
• Differential utilization of decapping enzymes in mammalian mRNA decay pathways.
• RNA. 2011; 17: 419-28
• Display abstract

• mRNA decapping is a crucial step in the regulation of mRNA stability and gene expression. Dcp2 is an mRNA decapping enzyme that has been widely studied. We recently reported the presence of a second mammalian cytoplasmic decapping enzyme, Nudt16. Here we address the differential utilization of the two decapping enzymes in specified mRNA decay processes. Using mouse embryonic fibroblast (MEF) cell lines derived from a hypomorphic knockout of the Dcp2 gene with undetectable levels of Dcp2 or MEF cell lines harboring a Nudt16-directed shRNA to generate reduced levels of Nudt16, we demonstrate the distinct roles for Dcp2 and Nudt16 in nonsense-mediated mRNA decay (NMD), decay of ARE-containing mRNA and miRNA-mediated silencing. Our results indicated that NMD preferentially utilizes Dcp2 rather than Nudt16; Dcp2 and Nudt16 are redundant in miRNA-mediated silencing; and Dcp2 and Nudt16 are differentially utilized for ARE-mRNA decay. These data demonstrate that the two distinct decapping enzymes can uniquely function in specific mRNA decay processes in mammalian cells.

• Zhang Z, Kulkarni K, Hanrahan SJ, Thompson AJ, Barford D
• The APC/C subunit Cdc16/Cut9 is a contiguous tetratricopeptide repeat superhelix with a homo-dimer interface similar to Cdc27.
• EMBO J. 2010; 29: 3733-44
• Display abstract

• The anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase responsible for controlling cell cycle transitions, is a multisubunit complex assembled from 13 different proteins. Numerous APC/C subunits incorporate multiple copies of the tetratricopeptide repeat (TPR). Here, we report the crystal structure of Schizosaccharomyces pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26), showing that Cdc16/Cut9 is a contiguous TPR superhelix of 14 TPR units. A C-terminal block of TPR motifs interacts with Hcn1, whereas an N-terminal TPR block mediates Cdc16/Cut9 self-association through a homotypic interface. This dimer interface is structurally related to the N-terminal dimerization domain of Cdc27, demonstrating that both Cdc16/Cut9 and Cdc27 form homo-dimers through a conserved mechanism. The acetylated N-terminal Met residue of Hcn1 is enclosed within a chamber created from the Cut9 TPR superhelix. Thus, in complex with Cdc16/Cut9, the N-acetyl-Met residue of Hcn1, a putative degron for the Doa10 E3 ubiquitin ligase, is inaccessible for Doa10 recognition, protecting Hcn1/Cdc26 from ubiquitin-dependent degradation. This finding may provide a structural explanation for a mechanism to control the stoichiometry of proteins participating in multisubunit complexes.

• Geisler S, Coller J
• Alternate endings: a new story for mRNA decapping.
• Mol Cell. 2010; 40: 349-50
• Display abstract

• With most of the important players identified, the process of decapping is thought, for the most part, to be well understood. In this issue of Molecular Cell, Song et al. (2010) challenge this notion with the identification of a previously uncharacterized mRNA decapping enzyme.

• Haas G, Braun JE, Igreja C, Tritschler F, Nishihara T, Izaurralde E
• HPat provides a link between deadenylation and decapping in metazoa.
• J Cell Biol. 2010; 189: 289-302
• Display abstract

• Decapping of eukaryotic messenger RNAs (mRNAs) occurs after they have undergone deadenylation, but how these processes are coordinated is poorly understood. In this study, we report that Drosophila melanogaster HPat (homologue of Pat1), a conserved decapping activator, interacts with additional decapping factors (e.g., Me31B, the LSm1-7 complex, and the decapping enzyme DCP2) and with components of the CCR4-NOT deadenylase complex. Accordingly, HPat triggers deadenylation and decapping when artificially tethered to an mRNA reporter. These activities reside, unexpectedly, in a proline-rich region. However, this region alone cannot restore decapping in cells depleted of endogenous HPat but also requires the middle (Mid) and the very C-terminal domains of HPat. We further show that the Mid and C-terminal domains mediate HPat recruitment to target mRNAs. Our results reveal an unprecedented role for the proline-rich region and the C-terminal domain of metazoan HPat in mRNA decapping and suggest that HPat is a component of the cellular mechanism that couples decapping to deadenylation in vivo.

• Nakamura T et al.
• Structural and dynamic features of the MutT protein in the recognition of nucleotides with the mutagenic 8-oxoguanine base.
• J Biol Chem. 2010; 285: 444-52
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• Escherichia coli MutT hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, an event that can prevent the misincorporation of 8-oxoguanine opposite adenine in DNA. Of the several enzymes that recognize 8-oxoguanine, MutT exhibits high substrate specificity for 8-oxoguanine nucleotides; however, the structural basis for this specificity is unknown. The crystal structures of MutT in the apo and holo forms and in the binary and ternary forms complexed with the product 8-oxo-dGMP and 8-oxo-dGMP plus Mn(2+), respectively, were determined. MutT strictly recognizes the overall conformation of 8-oxo-dGMP through a number of hydrogen bonds. This recognition mode revealed that 8-oxoguanine nucleotides are discriminated from guanine nucleotides by not only the hydrogen bond between the N7-H and Odelta (N119) atoms but also by the syn glycosidic conformation that 8-oxoguanine nucleotides prefer. Nevertheless, these discrimination factors cannot by themselves explain the roughly 34,000-fold difference between the affinity of MutT for 8-oxo-dGMP and dGMP. When the binary complex of MutT with 8-oxo-dGMP is compared with the ligand-free form, ordering and considerable movement of the flexible loops surrounding 8-oxo-dGMP in the binary complex are observed. These results indicate that MutT specifically recognizes 8-oxoguanine nucleotides by the ligand-induced conformational change.

• Kilchert C, Weidner J, Prescianotto-Baschong C, Spang A
• Defects in the secretory pathway and high Ca2+ induce multiple P-bodies.
• Mol Biol Cell. 2010; 21: 2624-38
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• mRNA is sequestered and turned over in cytoplasmic processing bodies (PBs), which are induced by various cellular stresses. Unexpectedly, in Saccharomyces cerevisiae, mutants of the small GTPase Arf1 and various secretory pathway mutants induced a significant increase in PB number, compared with PB induction by starvation or oxidative stress. Exposure of wild-type cells to osmotic stress or high extracellular Ca(2+) mimicked this increase in PB number. Conversely, intracellular Ca(2+)-depletion strongly reduced PB formation in the secretory mutants. In contrast to PB induction through starvation or osmotic stress, PB formation in secretory mutants and by Ca(2+) required the PB components Pat1 and Scd6, and calmodulin, indicating that different stressors act through distinct pathways. Consistent with this hypothesis, when stresses were combined, PB number did not correlate with the strength of the translational block, but rather with the type of stress encountered. Interestingly, independent of the stressor, PBs appear as spheres of approximately 40-100 nm connected to the endoplasmic reticulum (ER), consistent with the idea that translation and silencing/degradation occur in a spatially coordinated manner at the ER. We propose that PB assembly in response to stress occurs at the ER and depends on intracellular signals that regulate PB number.

• Belasco JG
• All things must pass: contrasts and commonalities in eukaryotic and bacterial mRNA decay.
• Nat Rev Mol Cell Biol. 2010; 11: 467-78
• Display abstract

• Despite its universal importance for controlling gene expression, mRNA degradation was initially thought to occur by disparate mechanisms in eukaryotes and bacteria. This conclusion was based on differences in the structures used by these organisms to protect mRNA termini and in the RNases and modifying enzymes originally implicated in mRNA decay. Subsequent discoveries have identified several striking parallels between the cellular factors and molecular events that govern mRNA degradation in these two kingdoms of life. Nevertheless, some key distinctions remain, the most fundamental of which may be related to the different mechanisms by which eukaryotes and bacteria control translation initiation.

• Yoon JH, Choi EJ, Parker R
• Dcp2 phosphorylation by Ste20 modulates stress granule assembly and mRNA decay in Saccharomyces cerevisiae.
• J Cell Biol. 2010; 189: 813-27
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• Translation and messenger RNA (mRNA) degradation are important sites of gene regulation, particularly during stress where translation and mRNA degradation are reprogrammed to stabilize bulk mRNAs and to preferentially translate mRNAs required for the stress response. During stress, untranslating mRNAs accumulate both in processing bodies (P-bodies), which contain some translation repressors and the mRNA degradation machinery, and in stress granules, which contain mRNAs stalled in translation initiation. How signal transduction pathways impinge on proteins modulating P-body and stress granule formation and function is unknown. We show that during stress in Saccharomyces cerevisiae, Dcp2 is phosphorylated on serine 137 by the Ste20 kinase. Phosphorylation of Dcp2 affects the decay of some mRNAs and is required for Dcp2 accumulation in P-bodies and specific protein interactions of Dcp2 and for efficient formation of stress granules. These results demonstrate that Ste20 has an unexpected role in the modulation of mRNA decay and translation and that phosphorylation of Dcp2 is an important control point for mRNA decapping.

• Harigaya Y, Jones BN, Muhlrad D, Gross JD, Parker R
• Identification and analysis of the interaction between Edc3 and Dcp2 in Saccharomyces cerevisiae.
• Mol Cell Biol. 2010; 30: 1446-56
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• Cap hydrolysis is a critical control point in the life of eukaryotic mRNAs and is catalyzed by the evolutionarily conserved Dcp1-Dcp2 complex. In Saccharomyces cerevisiae, decapping is modulated by several factors, including the Lsm family protein Edc3, which directly binds to Dcp2. We show that Edc3 binding to Dcp2 is mediated by a short peptide sequence located C terminal to the catalytic domain of Dcp2. This sequence is required for Edc3 to stimulate decapping activity of Dcp2 in vitro, for Dcp2 to efficiently accumulate in P-bodies, and for efficient degradation of the RPS28B mRNA, whose decay is enhanced by Edc3. In contrast, degradation of YRA1 pre-mRNA, another Edc3-regulated transcript, occurs independently from this region, suggesting that the effect of Edc3 on YRA1 is independent of its interaction with Dcp2. Deletion of the sequence also results in a subtle but significant defect in turnover of the MFA2pG reporter transcript, which is not affected by deletion of EDC3, suggesting that the region affects some other aspect of Dcp2 function in addition to binding Edc3. These results raise a model for Dcp2 recruitment to specific mRNAs where regions outside the catalytic core promote the formation of different complexes involved in mRNA decapping.

• Nissan T, Rajyaguru P, She M, Song H, Parker R
• Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms.
• Mol Cell. 2010; 39: 773-83
• Display abstract

• Eukaryotic mRNA degradation often occurs in a process whereby translation initiation is inhibited and the mRNA is targeted for decapping. In yeast cells, Pat1, Scd6, Edc3, and Dhh1 all function to promote decapping by an unknown mechanism(s). We demonstrate that purified Scd6 and a region of Pat1 directly repress translation in vitro by limiting the formation of a stable 48S preinitiation complex. Moreover, while Pat1, Edc3, Dhh1, and Scd6 all bind the decapping enzyme, only Pat1 and Edc3 enhance its activity. We also identify numerous direct interactions between Pat1, Dcp1, Dcp2, Dhh1, Scd6, Edc3, Xrn1, and the Lsm1-7 complex. These observations identify three classes of decapping activators that function to directly repress translation initiation and/or stimulate Dcp1/2. Moreover, Pat1 is identified as critical in mRNA decay by first inhibiting translation initiation, then serving as a scaffold to recruit components of the decapping complex, and finally activating Dcp2.

• Braun JE et al.
• The C-terminal alpha-alpha superhelix of Pat is required for mRNA decapping in metazoa.
• EMBO J. 2010; 29: 2368-80
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• Pat proteins regulate the transition of mRNAs from a state that is translationally active to one that is repressed, committing targeted mRNAs to degradation. Pat proteins contain a conserved N-terminal sequence, a proline-rich region, a Mid domain and a C-terminal domain (Pat-C). We show that Pat-C is essential for the interaction with mRNA decapping factors (i.e. DCP2, EDC4 and LSm1-7), whereas the P-rich region and Mid domain have distinct functions in modulating these interactions. DCP2 and EDC4 binding is enhanced by the P-rich region and does not require LSm1-7. LSm1-7 binding is assisted by the Mid domain and is reduced by the P-rich region. Structural analysis revealed that Pat-C folds into an alpha-alpha superhelix, exposing conserved and basic residues on one side of the domain. This conserved and basic surface is required for RNA, DCP2, EDC4 and LSm1-7 binding. The multiplicity of interactions mediated by Pat-C suggests that certain of these interactions are mutually exclusive and, therefore, that Pat proteins switch decapping partners allowing transitions between sequential steps in the mRNA decapping pathway.

• Morozov IY et al.
• Distinct roles for Caf1, Ccr4, Edc3 and CutA in the co-ordination of transcript deadenylation, decapping and P-body formation in Aspergillus nidulans.
• Mol Microbiol. 2010; 76: 503-16
• Display abstract

• Transcript degradation is a key step in gene regulation. In eukaryotes, mRNA decay is generally initiated by removal of the poly(A) tail mediated by the Ccr4-Caf1-Not complex. Deadenylated transcripts are then rapidly degraded, primarily via the decapping-dependent pathway. Components of this pathway can be localized into highly dynamic cytoplasmic foci, the mRNA processing (P)-bodies. We have undertaken confocal fluorescence microscopy to monitor P-bodies in Aspergillus nidulans. As in other organisms a dynamic shift in P-body formation occurs in response to diverse physiological signals. Significantly, both this cellular response and the signalled degradation of specific transcripts are dependent on the nuclease activity of Caf1 but not Ccr4. P-body formation is disrupted in A. nidulans strains deleted for Edc3, an enhancer of decapping, or CutA, which encodes a nucleotidyltransferase that triggers mRNA decapping by the addition of a CUCU tag to the poly(A) tail. As with DeltacutA, Deltaedc3 led to reduced rates of transcript degradation. These data link P-bodies to both the optimization and regulation of transcript degradation.

• Fan JS et al.
• Solution and crystal structures of mRNA exporter Dbp5p and its interaction with nucleotides.
• J Mol Biol. 2009; 388: 1-10
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• DEAD-box protein 5 (Dbp5p) plays very important roles in RNA metabolism from transcription, to translation, to RNA decay. It is an RNA helicase and functions as an essential RNA export factor from nucleus. Here, we report the solution NMR structures of the N- and C-terminal domains (NTD and CTD, respectively) of Dbp5p from Saccharomyces cerevisiae (ScDbp5p) and X-ray crystal structure of Dbp5p from Schizosaccharomyces pombe (SpDbp5p) in the absence of nucleotides and RNA. The crystal structure clearly shows that SpDbp5p comprises two RecA-like domains that do not interact with each other. NMR results show that the N-terminal flanking region of ScDpbp5 (M1-E70) is intrinsically unstructured and the region Y71-R121 including the Q motif is highly dynamic on millisecond-microsecond timescales in solution. The C-terminal flanking region of ScDbp5p forms a short beta-strand and a long helix. This helix is unique for ScDbp5p and has not been observed in other DEAD-box proteins. Compared with other DEAD-box proteins, Dbp5p has an extra insert with six residues in the CTD. NMR structure reveals that the insert is located in a solvent-exposed loop capable of interacting with other proteins. ATP and ADP titration experiments show that both ADP and ATP bind to the consensus binding site in the NTD of ScDbp5p but do not interact with the CTD at all. Binding of ATP or ADP to NTD induces significant conformational rearrangement too.

• Parrish S, Hurchalla M, Liu SW, Moss B
• The African swine fever virus g5R protein possesses mRNA decapping activity.
• Virology. 2009; 393: 177-82
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• The African Swine Fever Virus (ASFV) encodes a single Nudix enzyme in its genome, termed the g5R protein (g5Rp). Nudix phosphohydrolases cleave a variety of substrates, such as nucleotides and diphosphoinositol polyphosphates. Previously, ASFV g5Rp was shown to hydrolyze diphosphoinositol polyphosphates and GTP, but was unable to cleave methylated mRNA cap analogues. In vaccinia virus (VACV), a distant relative of ASFV, the D9 and D10 Nudix enzymes were shown to cleave the mRNA cap, but only when the cap was attached to an RNA body. Here, we show that recombinant ASFV g5Rp hydrolyzes the mRNA cap when tethered to an RNA moiety, liberating m(7)GDP as a product. Mutations in the Nudix motif abolished mRNA decapping activity, confirming that g5Rp was responsible for cap cleavage. The decapping activity of g5Rp was potently inhibited by excess uncapped RNA but not by methylated cap analogues, suggesting that substrate recognition occurs by RNA binding.

• Sarmah B, Wente SR
• Dual functions for the Schizosaccharomyces pombe inositol kinase Ipk1 in nuclear mRNA export and polarized cell growth.
• Eukaryot Cell. 2009; 8: 134-46
• Display abstract

• The inositol 1,3,4,5,6-pentakisphosphate (IP(5)) 2-kinase (Ipk1) catalyzes the production of inositol hexakisphosphate (IP(6)) in eukaryotic cells. Previous studies have shown that IP(6) is required for efficient nuclear mRNA export in the budding yeast Saccharomyces cerevisiae. Here, we report the first functional analysis of ipk1(+) in Schizosaccharomyces pombe. S. pombe Ipk1 (SpIpk1) is unique among Ipk1 orthologues in that it harbors a novel amino (N)-terminal domain with coiled-coil structural motifs similar to those of BAR (Bin-amphiphysin-Rvs) domain proteins. Mutants with ipk1(+) deleted (ipk1Delta) had mRNA export defects as well as pleiotropic defects in polarized growth, cell morphology, endocytosis, and cell separation. The SpIpk1 catalytic carboxy-terminal domain was required to rescue these defects, and the mRNA export block was genetically linked to SpDbp5 function and, likely, IP(6) production. However, the overexpression of the N-terminal domain alone also inhibited these functions in wild-type cells. This revealed a distinct noncatalytic function for the N-terminal domain. To test for connections with other inositol polyphosphates, we also analyzed whether the loss of asp1(+) function, encoding an IP(6) kinase downstream of Ipk1, had an effect on ipk1Delta cells. The asp1Delta mutant alone did not block mRNA export, and its cell morphology, polarized growth, and endocytosis defects were less severe than those of ipk1Delta cells. Moreover, ipk1Delta asp1Delta double mutants had altered inositol polyphosphate levels distinct from those of the ipk1Delta mutant. This suggested novel roles for asp1(+) upstream of ipk1(+). We propose that IP(6) production is a key signaling linchpin for regulating multiple essential cellular processes.

• Gelinas AD et al.
• Telomere capping proteins are structurally related to RPA with an additional telomere-specific domain.
• Proc Natl Acad Sci U S A. 2009; 106: 19298-303
• Display abstract

• Telomeres must be capped to preserve chromosomal stability. The conserved Stn1 and Ten1 proteins are required for proper capping of the telomere, although the mechanistic details of how they contribute to telomere maintenance are unclear. Here, we report the crystal structures of the C-terminal domain of the Saccharomyces cerevisiae Stn1 and the Schizosaccharomyces pombe Ten1 proteins. These structures reveal striking similarities to corresponding subunits in the replication protein A complex, further supporting an evolutionary link between telomere maintenance proteins and DNA repair complexes. Our structural and in vivo data of Stn1 identify a new domain that has evolved to support a telomere-specific role in chromosome maintenance. These findings endorse a model of an evolutionarily conserved mechanism of DNA maintenance that has developed as a result of increased chromosomal structural complexity.

• Brooks MA, Ravelli RB, McCarthy AA, Strub K, Cusack S
• Structure of SRP14 from the Schizosaccharomyces pombe signal recognition particle.
• Acta Crystallogr D Biol Crystallogr. 2009; 65: 421-33
• Display abstract

• The signal recognition particle (SRP) Alu domain has been implicated in translation elongation arrest in yeasts and mammals. Fission yeast SRP RNA is similar to that of mammals, but has a minimal Alu-domain RNA lacking two stem-loops. The mammalian Alu-domain proteins SRP9 and SRP14 bind their cognate Alu RNA as a heterodimer. However, in yeasts, notably Saccharomyces cerevisiae, SRP14 is thought to bind Alu RNA as a homodimer, the SRP9 protein being replaced by SRP21, the function of which is not yet clear. Structural characterization of the Schizosaccharomyces pombe Alu domain may thus help to identify the critical features required for elongation arrest. Here, the crystal structure of the SRP14 subunit of S. pombe SRP (SpSRP14) which crystallizes as a homodimer, is presented. Comparison of the SpSRP14 homodimer with the known structure of human SRP9/14 in complex with Alu RNA suggests that many of the protein-RNA contacts centred on the conserved U-turn motif are likely to be conserved in fission yeast. Initial attempts to solve the structure using traditional selenomethionine SAD labelling failed. However, two As atoms originating from the cacodylate buffer were found to make cysteine adducts and strongly contributed to the anomalous substructure. These adducts were highly radiation-sensitive and this property was exploited using the RIP (radiation-damage-induced phasing) method. The combination of SAD and RIP phases yielded an interpretable electron-density map. This example will be of general interest to crystallographers attempting de novo phasing from crystals grown in cacodylate buffer.

• Chen CY, Zheng D, Xia Z, Shyu AB
• Ago-TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps.
• Nat Struct Mol Biol. 2009; 16: 1160-6
• Display abstract

• MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain largely unclear. Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.

• Lloyd J et al.
• A supramodular FHA/BRCT-repeat architecture mediates Nbs1 adaptor function in response to DNA damage.
• Cell. 2009; 139: 100-11
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• The Mre11/Rad50/Nbs1 protein complex plays central enzymatic and signaling roles in the DNA-damage response. Nuclease (Mre11) and scaffolding (Rad50) components of MRN have been extensively characterized, but the molecular basis of Nbs1 function has remained elusive. Here, we present a 2.3A crystal structure of the N-terminal region of fission yeast Nbs1, revealing an unusual but conserved architecture in which the FHA- and BRCT-repeat domains structurally coalesce. We demonstrate that diphosphorylated pSer-Asp-pThr-Asp motifs, recently identified as multicopy docking sites within Mdc1, are evolutionarily conserved Nbs1 binding targets. Furthermore, we show that similar phosphomotifs within Ctp1, the fission yeast ortholog of human CtIP, promote interactions with the Nbs1 FHA domain that are necessary for Ctp1-dependent resistance to DNA damage. Finally, we establish that human Nbs1 interactions with Mdc1 occur through both its FHA- and BRCT-repeat domains, suggesting how their structural and functional interdependence underpins Nbs1 adaptor functions in the DNA-damage response.

• Xu J, Chua NH
• Arabidopsis decapping 5 is required for mRNA decapping, P-body formation, and translational repression during postembryonic development.
• Plant Cell. 2009; 21: 3270-9
• Display abstract

• Eukaryotic processing bodies (P-bodies) are implicated in mRNA storage and mRNA decapping. We previously found that a decapping complex comprising Decapping 1 (DCP1), DCP2, and Varicose in Arabidopsis thaliana is essential for postembryonic development, but the underlying mechanism is poorly understood. Here, we characterized Arabidopsis DCP5, a homolog of human RNA-associated protein 55, as an additional P-body constituent. DCP5 associates with DCP1 and DCP2 and is required for mRNA decapping in vivo. In spite of its association with DCP2, DCP5 has no effect on DCP2 decapping activity in vitro, suggesting that the effect on decapping in vivo is indirect. In knockdown mutant dcp5-1, not only is mRNA decapping compromised, but the size of P-bodies is also significantly decreased. These results indicate that DCP5 is required for P-body formation, which likely facilitates efficient decapping. During wild-type seed germination, mRNAs encoding seed storage proteins (SSPs) are translationally repressed and degraded. By contrast, in dcp5-1, SSP mRNAs are translated, leading to accumulation of their products in germinated seedlings. In vitro experiments using wheat germ extracts confirmed that DCP5 is a translational repressor. Our results showed that DCP5 is required for translational repression and P-body formation and plays an indirect role in mRNA decapping.

• Jbel M, Mercier A, Pelletier B, Beaudoin J, Labbe S
• Iron activates in vivo DNA binding of Schizosaccharomyces pombe transcription factor Fep1 through its amino-terminal region.
• Eukaryot Cell. 2009; 8: 649-64
• Display abstract

• In Schizosaccharomyces pombe, the iron sensor Fep1 mediates the transcriptional repression of iron transport genes in response to high concentrations of iron. On the other hand, fep1(+) expression is downregulated under conditions of iron starvation by the CCAAT-binding factor Php4. In this study, we created a fep1Delta php4Delta double mutant strain where expression of fep1(+) was disengaged from its iron limitation-dependent repression by Php4 to examine the effects of iron on constitutively expressed functional fep1(+)-GFP and TAP-fep1(+) alleles and their gene products. In these cells, Fep1-green fluorescent protein was invariably localized in the nucleus under both iron-limiting and iron-replete conditions. Using chromatin immunoprecipitation assays, we found that Fep1 is associated with iron-responsive promoters in vivo. Chromatin binding was iron dependent, with a loss of binding observed in the presence of low iron. Functional dissection of the protein revealed that the N-terminal 241-residue segment that includes two consensus Cys(2)/Cys(2)-type zinc finger motifs and a Cys-rich region is required for optimal promoter occupancy by Fep1. Within this segment, a minimal module encompassing amino acids 60 to 241 is sufficient for iron-dependent chromatin binding. Using yeast one-hybrid analysis, we showed that the replacement of the repression domain of Fep1 by fusing the activation domain of VP16 to the chromatin-binding fragment of amino acids 1 to 241 of Fep1 converts the protein from an iron-dependent repressor into an iron-dependent transcriptional activator. Thus, the repression function of Fep1 can be replaced with that of a transcriptional activation function without the loss of its iron-dependent DNA-binding activity.

• Mariller C, Hardiville S, Hoedt E, Benaissa M, Mazurier J, Pierce A
• Proteomic approach to the identification of novel delta-lactoferrin target genes: Characterization of DcpS, an mRNA scavenger decapping enzyme.
• Biochimie. 2009; 91: 109-22
• Display abstract

• The expression of the transcription factor DeltaLf is deregulated in cancer cells. Its overexpression provokes cell cycle arrest along with antiproliferative effects and we recently showed that the Skp1 gene promoter was a target of DeltaLf. Skp1 belongs to the Skp1/Cullin-1/F-box ubiquitin ligase complex responsible for the ubiquitination and the proteosomal degradation of numerous cellular regulators. The transcriptional activity of DeltaLf is highly controlled and negatively regulated by O-GlcNAc, a dynamic post-translational modification known to regulate the functions of many intracellular proteins. We, therefore, constructed a DeltaLf-M4 mutant corresponding to a constitutively active DeltaLf isoform in which all the glycosylation sites were mutated. In order to discover novel targets of DeltaLf transcriptional activity and to investigate the impact of the O-GlcNAc regulation on this activity in situ we compared the proteome profiles of DeltaLf- and DeltaLf-M4-expressing HEK293 cells versus null plasmid transfected cells. A total of 14 differentially expressed proteins were visualized by 2D electrophoresis and silver staining and eight proteins were identified by mass spectrometry analyses (MALDI-TOF; LC-MS/MS), all of which were upregulated. The identified proteins are involved in several processes such as mRNA maturation and stability, cell viability, proteasomal degradation, protein and mRNA quality control. Among these proteins, only DcpS and TCPB were also upregulated at the mRNA level. Analysis of their respective promoters led to the detection of a cis-regulating element in the DcpS promoter. The S1(DcpS) is 80% identical to the S1 sequence previously described by He and Furmanski [Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA, Nature 373 (1995) 721-724]. Reporter gene analyses and ChIP assays demonstrated that DeltaLf interacts specifically with the DcpS promoter in vivo. These data established that DcpS, a key enzyme in mRNA decay, is a new target of DeltaLf transcriptional activity.

• Bulfer SL, Scott EM, Couture JF, Pillus L, Trievel RC
• Crystal structure and functional analysis of homocitrate synthase, an essential enzyme in lysine biosynthesis.
• J Biol Chem. 2009; 284: 35769-80
• Display abstract

• Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis in many fungi and certain Archaea and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is composed of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed alpha/beta topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCS-selective inhibitors.

• Cole SE, LaRiviere FJ, Merrikh CN, Moore MJ
• A convergence of rRNA and mRNA quality control pathways revealed by mechanistic analysis of nonfunctional rRNA decay.
• Mol Cell. 2009; 34: 440-50
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• Eukaryotes possess numerous quality control systems that monitor both the synthesis of RNA and the integrity of the finished products. We previously demonstrated that Saccharomyces cerevisiae possesses a quality control mechanism, nonfunctional rRNA decay (NRD), capable of detecting and eliminating translationally defective rRNAs. Here we show that NRD can be divided into two mechanistically distinct pathways: one that eliminates rRNAs with deleterious mutations in the decoding site (18S NRD) and one that eliminates rRNAs containing deleterious mutations in the peptidyl transferase center (25S NRD). 18S NRD is dependent on translation elongation and utilizes the same proteins as those participating in no-go mRNA decay (NGD). In cells that accumulate 18S NRD and NGD decay intermediates, both RNA types can be seen in P-bodies. We propose that 18S NRD and NGD are different observable outcomes of the same initiating event: a ribosome stalled inappropriately at a sense codon during translation elongation.

• Gao J, Davidson MK, Wahls WP
• Distinct regions of ATF/CREB proteins Atf1 and Pcr1 control recombination hotspot ade6-M26 and the osmotic stress response.
• Nucleic Acids Res. 2008; 36: 2838-51
• Display abstract

• The Atf1 protein of Schizosaccharomyces pombe contains a bZIP (DNA-binding/protein dimerization) domain characteristic of ATF/CREB proteins, but no other functional domains or clear homologs have been reported. Atf1-containing, bZIP protein dimers bind to CRE-like DNA sites, regulate numerous stress responses, and activate meiotic recombination at hotspots like ade6-M26. We defined systematically the organization of Atf1 and its heterodimer partner Pcr1, which is required for a subset of Atf1-dependent functions. Surprisingly, only the bZIP domain of Pcr1 is required for hotspot activity and tethering of Atf1 to ade6 promotes recombination in the absence of its bZIP domain and the Pcr1 protein. Therefore the recombination-activation domain of Atf1-Pcr1 heterodimer resides exclusively in Atf1, and Pcr1 confers DNA-binding site specificity in vivo. Atf1 has a modular organization in which distinct regions affect differentially the osmotic stress response (OSA) and meiotic recombination (HRA, HRR). The HRA and HRR regions are necessary and sufficient to activate and repress recombination, respectively. Moreover, Atf1 defines a family of conserved proteins with discrete sequence motifs in the functional domains (OSA, HRA, HRR, bZIP). These findings reveal the functional organization of Atf1 and Pcr1, and illustrate several mechanisms by which bZIP proteins can regulate multiple, seemingly disparate activities.

• Gunawardana D, Cheng HC, Gayler KR
• Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2).
• Nucleic Acids Res. 2008; 36: 203-16
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• The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn(2+)- or Mg(2+)-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B.

• Floor SN, Jones BN, Gross JD
• Control of mRNA decapping by Dcp2: An open and shut case?
• RNA Biol. 2008; 5: 189-92
• Display abstract

• mRNA decapping by Dcp2 is a critical step in several major eukaryotic mRNA decay pathways. Dcp2 forms the catalytic core of a mRNP that is configured for processing diverse substrates by pathway-specific activators. Here we elaborate a model of catalysis by Dcp2 which posits that activity is controlled by a conformational equilibrium between an open, inactive and closed, active form of the enzyme. Structural studies on yeast Dcp2 indicate that the general activator Dcp1 and substrate promote the closed form of the enzyme. Kinetic studies indicate the catalytic step of decapping is rate-limiting and accelerated by Dcp1. We propose that regulation of conformational transitions in Dcp2 during a rate-limiting step after assembly of the decapping mRNP provides a checkpoint for determining if an mRNA is degraded or recycled to translation.

• Martin-Cuadrado AB et al.
• The Schizosaccharomyces pombe endo-1,3-beta-glucanase Eng1 contains a novel carbohydrate binding module required for septum localization.
• Mol Microbiol. 2008; 69: 188-200
• Display abstract

• Cell separation in Schizosaccharomyces pombe is achieved through the concerted action of the Eng1 endo-beta-1,3-glucanase and the Agn1 endo-alpha-1,3-glucanase, which are transported to the septum and localize to a ring-like structure that surrounds the septum. Correct localization of these hydrolases requires the presence of both the septins and the exocyst. In this work, we show that the glucanase Eng1 contains a region at the C-terminus that acts as a carbohydrate-binding module (CBM) and that it is not present in other members of glycoside hydrolases family 81 (GH81). In vitro, the purified CBM has affinity for beta-1,3-glucan chains with a minimum degree of polymerization of 30 glucose units. Deletion of the CBM results in a protein that is largely defective in complementing the separation defect of eng1Delta mutants. This defect is due to a reduction in the catalytic activity against insoluble substrates and to a defect in targeting of Eng1 to the septum, as the truncated protein localizes to the lateral cell wall of the cell. Thus, the targeting of Eng1 to the primary septum requires not only trans-factors (septins and the exocyst complex) but also a cis-element localized to the C-terminus of the protein.

• Kilkenny ML et al.
• Structural and functional analysis of the Crb2-BRCT2 domain reveals distinct roles in checkpoint signaling and DNA damage repair.
• Genes Dev. 2008; 22: 2034-47
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• Schizosaccharomyces pombe Crb2 is a checkpoint mediator required for the cellular response to DNA damage. Like human 53BP1 and Saccharomyces cerevisiae Rad9 it contains Tudor(2) and BRCT(2) domains. Crb2-Tudor(2) domain interacts with methylated H4K20 and is required for recruitment to DNA dsDNA breaks. The BRCT(2) domain is required for dimerization, but its precise role in DNA damage repair and checkpoint signaling is unclear. The crystal structure of the Crb2-BRCT(2) domain, alone and in complex with a phosphorylated H2A.1 peptide, reveals the structural basis for dimerization and direct interaction with gamma-H2A.1 in ionizing radiation-induced foci (IRIF). Mutational analysis in vitro confirms the functional role of key residues and allows the generation of mutants in which dimerization and phosphopeptide binding are separately disrupted. Phenotypic analysis of these in vivo reveals distinct roles in the DNA damage response. Dimerization mutants are genotoxin sensitive and defective in checkpoint signaling, Chk1 phosphorylation, and Crb2 IRIF formation, while phosphopeptide-binding mutants are only slightly sensitive to IR, have extended checkpoint delays, phosphorylate Chk1, and form Crb2 IRIF. However, disrupting phosphopeptide binding slows formation of ssDNA-binding protein (Rpa1/Rad11) foci and reduces levels of Rad22(Rad52) recombination foci, indicating a DNA repair defect.

• Biswas S, Bastia D
• Mechanistic insights into replication termination as revealed by investigations of the Reb1-Ter3 complex of Schizosaccharomyces pombe.
• Mol Cell Biol. 2008; 28: 6844-57
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• Relatively little is known about the interaction of eukaryotic replication terminator proteins with the cognate termini and the replication termination mechanism. Here, we report a biochemical analysis of the interaction of the Reb1 terminator protein of Schizosaccharomyces pombe, which binds to the Ter3 site present in the nontranscribed spacers of ribosomal DNA, located in chromosome III. We show that Reb1 is a dimeric protein and that the N-terminal dimerization domain of the protein is dispensable for replication termination. Unlike its mammalian counterpart Ttf1, Reb1 did not need an accessory protein to bind to Ter3. The two myb/SANT domains and an adjacent, N-terminal 154-amino-acid-long segment (called the myb-associated domain) were both necessary and sufficient for optimal DNA binding in vitro and fork arrest in vivo. The protein and its binding site Ter3 were unable to arrest forks initiated in vivo from ars of Saccharomyces cerevisiae in the cell milieu of the latter despite the facts that the protein retained the proper affinity of binding, was located in vivo at the Ter site, and apparently was not displaced by the "sweepase" Rrm3. These observations suggest that replication fork arrest is not an intrinsic property of the Reb1-Ter3 complex.

• Sharifmoghadam MR, Valdivieso MH
• The Schizosaccharomyces pombe Map4 adhesin is a glycoprotein that can be extracted from the cell wall with alkali but not with beta-glucanases and requires the C-terminal DIPSY domain for function.
• Mol Microbiol. 2008; 69: 1476-90
• Display abstract

• SUMMARY: In fungi, cell adhesion is required for flocculation, mating and virulence, and it is mediated by covalently bound cell wall proteins termed adhesins. Map4, an adhesin required for mating in Schizosaccharomyces pombe, is N-glycosylated and O-glycosylated, and is an endogenous substrate for the mannosyl transferase Oma4p. Map4 has a modular structure with an N-terminal signal peptide, a serine and threonine (S/T)-rich domain that includes nine repeats of 36 amino acids (rich in serine and threonine residues, but lacking glutamines), and a C-terminal DIPSY domain with no glycosylphosphatidyl inositol (GPI)-anchor signal. Map4 can be extracted from cell walls with SDS/mercaptoethanol sample buffer or with mild alkali solutions. After extensive extraction with hot sample buffer, no more protein can be released by beta-glucanases or alkali. Additionally, none of the cysteine residues of the protein is required for its retention at the cell wall. These results show that Map4 is not directly bound to beta-glucans and point to the existence of alkali- and SDS/mercaptoethanol-sensitive linkages between cell wall proteins. The N-terminal S/T-rich regions are required for cell wall attachment, but the C-terminal DIPSY domain is required for agglutination and mating in liquid and solid media.

• Stuwe T et al.
• The FACT Spt16 "peptidase" domain is a histone H3-H4 binding module.
• Proc Natl Acad Sci U S A. 2008; 105: 8884-9
• Display abstract

• The FACT complex is a conserved cofactor for RNA polymerase II elongation through nucleosomes. FACT bears histone chaperone activity and contributes to chromatin integrity. However, the molecular mechanisms behind FACT function remain elusive. Here we report biochemical, structural, and mutational analyses that identify the peptidase homology domain of the Schizosaccharomyces pombe FACT large subunit Spt16 (Spt16-N) as a binding module for histones H3 and H4. The 2.1-A crystal structure of Spt16-N reveals an aminopeptidase P fold whose enzymatic activity has been lost. Instead, the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails. Mutations within a conserved surface pocket in Spt16-N or posttranslational modification of the histone H4 tail reduce interaction in vitro, whereas the globular domains of H3-H4 and the H3 tail bind distinct Spt16-N surfaces. Our analysis suggests that the N-terminal domain of Spt16 may add to the known H2A-H2B chaperone activity of FACT by including a H3-H4 tail and H3-H4 core binding function mediated by the N terminus of Spt16. We suggest that these interactions may aid FACT-mediated nucleosome reorganization events.

• Shen V, Liu H, Liu SW, Jiao X, Kiledjian M
• DcpS scavenger decapping enzyme can modulate pre-mRNA splicing.
• RNA. 2008; 14: 1132-42
• Display abstract

• The human scavenger decapping enzyme, DcpS, functions to hydrolyze the resulting cap structure following cytoplasmic mRNA decay yet is, surprisingly, a nuclear protein by immunofluorescence. Here, we show that DcpS is a nucleocytoplasmic shuttling protein that contains separable nuclear import and Crm-1-dependent export signals. We postulated that the presence of DcpS in both cellular compartments and its ability to hydrolyze cap structure may impact other cellular events dependent on cap-binding proteins. An shRNA-engineered cell line with markedly diminished DcpS levels led to a corresponding reduction in cap-proximal intron splicing of a reporter minigene and endogenous genes. The impaired cap catabolism and resultant imbalanced cap concentrations were postulated to sequester the cap-binding complex (CBC) from its normal splicing function. In support of this explanation, DcpS efficiently displaced the nuclear cap-binding protein Cbp20 from cap structure, and complementation with Cbp20 reversed the reduced splicing, indicating that modulation of splicing by DcpS is mediated through Cbp20. Our studies demonstrate that the significance of DcpS extends beyond its well-characterized role in mRNA decay and involves a broader range of functions in RNA processing including nuclear pre-mRNA splicing.

• Pitt CW, Valente LP, Rhodes D, Simonsson T
• Identification and characterization of an essential telomeric repeat binding factor in fission yeast.
• J Biol Chem. 2008; 283: 2693-701
• Display abstract

• Whereas mammalian cells harbor two double strand telomeric repeat binding factors, TRF1 and TRF2, the fission yeast Schizosaccharomyces pombe has been thought to harbor solely the TRF1/TRF2 ortholog Taz1p to perform comparable functions. Here we report the identification of telomeric repeat binding factor 1 (Tbf1), a second TRF1/TRF2 ortholog in S. pombe. Like the Taz1p, the identified Tbf1p shares amino acid sequence similarity, as well as structural and functional characteristics, with the mammalian TRF1 and TRF2 proteins. This family of proteins shares a common architecture with two separate structural domains. An N-terminal domain is necessary and sufficient for the formation of homodimers, and a C-terminal MYB/homeodomain mediates sequence specific recognition of double-stranded telomeric DNA. The identified Tbf1p binds S. pombe telomeric DNA with high sequence specificity in vitro. Targeted deletion of the tbf1 gene reveals that it is essential for survival, and overexpression of the tbf1 gene leads to telomere elongation in vivo, which is dependent upon the MYB domain. These data suggest that fission yeast, like mammals, have two factors that bind double-stranded telomeric DNA and perform distinct roles in telomere length regulation.

• Passos DO, Parker R
• Analysis of cytoplasmic mRNA decay in Saccharomyces cerevisiae.
• Methods Enzymol. 2008; 448: 409-27
• Display abstract

• The yeast, Saccharomyces cerevisiae, is a model system for the study of eukaryotic mRNA degradation. In this organism, a variety of methods have been developed to measure mRNA decay rates, trap intermediates in the mRNA degradation process, and establish precursor-product relationships. In addition, the use of mutant strains lacking specific enzymes involved in mRNA destruction, or key regulatory proteins, allows one to determine the mechanisms by which individual mRNAs are degraded. In this chapter, we discuss methods for analyzing mRNA degradation in S. cerevisiae.

• Ebina H, Chatterjee AG, Judson RL, Levin HL
• The GP(Y/F) domain of TF1 integrase multimerizes when present in a fragment, and substitutions in this domain reduce enzymatic activity of the full-length protein.
• J Biol Chem. 2008; 283: 15965-74
• Display abstract

• Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the gamma-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining.

• Franks TM, Lykke-Andersen J
• The control of mRNA decapping and P-body formation.
• Mol Cell. 2008; 32: 605-15
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• mRNA decapping is a critical step in eukaryotic cytoplasmic mRNA turnover. Cytoplasmic mRNA decapping is catalyzed by Dcp2 in conjunction with its coactivator Dcp1 and is stimulated by decapping enhancer proteins. mRNAs associated with the decapping machinery can assemble into cytoplasmic mRNP granules called processing bodies (PBs). Evidence suggests that PB-associated mRNPs are translationally repressed and can be degraded or stored for subsequent translation. However, whether mRNP assembly into a PB is important for translational repression, decapping, or decay has remained controversial. Here, we discuss the regulation of decapping machinery recruitment to specific mRNPs and how their assembly into PBs is governed by the relative rates of translational repression, mRNP multimerization, and mRNA decay.

• Zhang J, Gao F, Zhang Q, Chen Q, Qi J, Yan J
• Crystallization and crystallographic analysis of human NUDT16.
• Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008; 64: 639-40
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• Human NUDT16, a decapping enzyme belonging to the Nudix superfamily, plays a pivotal role in U8 snoRNA stability. Recombinant NUDT16 expressed in Escherichia coli was crystallized using the hanging-drop vapour-diffusion method. The crystals, which diffracted to 2.10 A resolution, belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 44.47, b = 79.32, c = 97.20 A. The Matthews coefficient and the solvent content were calculated to be 1.92 A(3) Da(-1) and 35.84%, respectively, for two molecules per asymmetric unit.

• Liu SW, Rajagopal V, Patel SS, Kiledjian M
• Mechanistic and kinetic analysis of the DcpS scavenger decapping enzyme.
• J Biol Chem. 2008; 283: 16427-36
• Display abstract

• Decapping is an important process in the control of eukaryotic mRNA degradation. The scavenger decapping enzyme DcpS functions to clear the cell of cap structure following decay of the RNA body by catalyzing the hydrolysis of m(7)GpppN to m(7)Gp and ppN. Structural analysis has revealed that DcpS is a dimeric protein with a domain-swapped amino terminus. The protein dimer contains two cap binding/hydrolysis sites and displays a symmetric structure with both binding sites in the open conformation in the ligand-free state and an asymmetric conformation with one site open and one site closed in the ligand-bound state. The structural data are suggestive of a dynamic decapping mechanism where each monomer could alternate between an open and closed state. Using transient state kinetic studies, we show that both the rate-limiting step and rate of decapping are regulated by cap substrate. A regulatory mechanism is established by the intrinsic domain-swapped structure of the DcpS dimer such that the decapping reaction is very efficient at low cap substrate concentrations yet regulated with excess cap substrate. These data provide biochemical evidence to verify experimentally a dynamic and mutually exclusive cap hydrolysis activity of the two cap binding sites of DcpS and provide key insights into its regulation.

• Steyert SR, Messing SA, Amzel LM, Gabelli SB, Pineiro SA
• Identification of Bdellovibrio bacteriovorus HD100 Bd0714 as a Nudix dGTPase.
• J Bacteriol. 2008; 190: 8215-9
• Display abstract

• Bdellovibrio bacteriovorus bacteria are predatory organisms that attack other gram-negative bacteria. Here, we report that Bd0714 is a Nudix dGTPase from B. bacteriovorus HD100 with a substrate specificity similar to that of Escherichia coli MutT and complements an E. coli mutT-deficient strain. We observed different transcription levels of the gene throughout the predator life cycle.

• Yamochi T, Ohnuma K, Hosono O, Tanaka H, Kanai Y, Morimoto C
• SSA/Ro52 autoantigen interacts with Dcp2 to enhance its decapping activity.
• Biochem Biophys Res Commun. 2008; 370: 195-9
• Display abstract

• We identified human decapping enzyme 2 (hDCP2) as a binding protein with Ro52, being colocalized in processing bodies (p-bodies). We also showed that the N-terminus and C-terminus of Ro52 bound to hDCP2. Moreover, Ro52 enhanced decapping activity of hDCP2 in a dose-dependent manner. Our data support the novel notion of the association between Ro52 with hDCP2 protein in cytoplasmic p-bodies, playing a role in mRNA metabolism in response to cellular stimulation.

• Pinar M, Coll PM, Rincon SA, Perez P
• Schizosaccharomyces pombe Pxl1 is a paxillin homologue that modulates Rho1 activity and participates in cytokinesis.
• Mol Biol Cell. 2008; 19: 1727-38
• Display abstract

• Schizosaccharomyces pombe Rho GTPases regulate actin cytoskeleton organization and cell integrity. We studied the fission yeast gene SPBC4F6.12 based on its ability to suppress the thermosensitivity of cdc42-1625 mutant strain. This gene, named pxl1(+), encodes a protein with three LIM domains that is similar to paxillin. Pxl1 does not interact with Cdc42 but it interacts with Rho1, and it negatively regulates this GTPase. Fission yeast Pxl1 forms a contractile ring in the cell division region and deletion of pxl1(+) causes a delay in cell-cell separation, suggesting that it has a function in cytokinesis. Pxl1 N-terminal region is required and sufficient for its localization to the medial ring, whereas the LIM domains are necessary for its function. Pxl1 localization requires actin polymerization and the actomyosin ring, but it is independent of the septation initiation network (SIN) function. Moreover, Pxl1 colocalizes and interacts with Myo2, and Cdc15, suggesting that it is part of the actomyosin ring. Here, we show that in cells lacking Pxl1, the myosin ring is not correctly assembled and that actomyosin ring contraction is delayed. Together, these data suggest that Pxl1 modulates Rho1 GTPase signaling and plays a role in the formation and contraction of the actomyosin ring during cytokinesis.

• Beckham C, Hilliker A, Cziko AM, Noueiry A, Ramaswami M, Parker R
• The DEAD-box RNA helicase Ded1p affects and accumulates in Saccharomyces cerevisiae P-bodies.
• Mol Biol Cell. 2008; 19: 984-93
• Display abstract

• Recent results suggest that cytoplasmic mRNAs can form translationally repressed messenger ribonucleoprotein particles (mRNPs) capable of decapping and degradation, or accumulation into cytoplasmic processing bodies (P-bodies), which can function as sites of mRNA storage. The proteins that function in transitions between the translationally repressed mRNPs that accumulate in P-bodies and mRNPs engaged in translation are largely unknown. Herein, we demonstrate that the yeast translation initiation factor Ded1p can localize to P-bodies. Moreover, depletion of Ded1p leads to defects in P-body formation. Overexpression of Ded1p results in increased size and number of P-bodies and inhibition of growth in a manner partially suppressed by loss of Pat1p, Dhh1p, or Lsm1p. Mutations that inactivate the ATPase activity of Ded1p increase the overexpression growth inhibition of Ded1p and prevent Ded1p from localizing in P-bodies. Combined with earlier work showing Ded1p can have a positive effect on translation, these results suggest that Ded1p is a bifunctional protein that can affect both translation initiation and P-body formation.

• Pilkington GR, Parker R
• Pat1 contains distinct functional domains that promote P-body assembly and activation of decapping.
• Mol Cell Biol. 2008; 28: 1298-312
• Display abstract

• The control of mRNA degradation and translation are important aspects of gene regulation. Recent results suggest that translation repression and mRNA decapping can be intertwined and involve the formation of a quiescent mRNP, which can accumulate in cytoplasmic foci referred to as P bodies. The Pat1 protein is a key component of this complex and an important activator of decapping, yet little is known about its function. In this work, we analyze Pat1 in Saccharomyces cerevisiae function by deletion and functional analyses. Our results identify two primary functional domains in Pat1: one promoting translation repression and P-body assembly and a second domain promoting mRNA decapping after assembly of the mRNA into a P-body mRNP. In addition, we provide evidence that Pat1 binds RNA and has numerous domain-specific interactions with mRNA decapping factors. These results indicate that Pat1 is an RNA binding protein and a multidomain protein that functions at multiple stages in the process of translation repression and mRNA decapping.

• Wilson MA, Meaux S, van Hoof A
• Diverse aberrancies target yeast mRNAs to cytoplasmic mRNA surveillance pathways.
• Biochim Biophys Acta. 2008; 1779: 550-7
• Display abstract

• Eukaryotic gene expression is a complex, multistep process that needs to be executed with high fidelity and two general methods help achieve the overall accuracy of this process. Maximizing accuracy in each step in gene expression increases the fraction of correct mRNAs made. Fidelity is further improved by mRNA surveillance mechanisms that degrade incorrect or aberrant mRNAs that are made when a step is not perfectly executed. Here, we review how cytoplasmic mRNA surveillance mechanisms selectively recognize and degrade a surprisingly wide variety of aberrant mRNAs that are exported from the nucleus into the cytoplasm.

• Linder T et al.
• Two conserved modules of Schizosaccharomyces pombe Mediator regulate distinct cellular pathways.
• Nucleic Acids Res. 2008; 36: 2489-504
• Display abstract

• Mediator is an evolutionary conserved coregulator complex required for transcription of almost all RNA polymerase II-dependent genes. The Schizosaccharomyces pombe Mediator consists of two dissociable components-a core complex organized into a head and middle domain as well as the Cdk8 regulatory subcomplex. In this work we describe a functional characterization of the S. pombe Mediator. We report the identification of the S. pombe Med20 head subunit and the isolation of ts alleles of the core head subunit encoding med17+. Biochemical analysis of med8(ts), med17(ts), Deltamed18, Deltamed20 and Deltamed27 alleles revealed a stepwise head domain molecular architecture. Phenotypical analysis of Cdk8 and head module alleles including expression profiling classified the Mediator mutant alleles into one of two groups. Cdk8 module mutants flocculate due to overexpression of adhesive cell-surface proteins. Head domain-associated mutants display a hyphal growth phenotype due to defective expression of factors required for cell separation regulated by transcription factor Ace2. Comparison with Saccharomyces cerevisiae Mediator expression data reveals that these functionally distinct modules are conserved between S. pombe and S. cerevisiae.

• Nolen BJ, Pollard TD
• Structure and biochemical properties of fission yeast Arp2/3 complex lacking the Arp2 subunit.
• J Biol Chem. 2008; 283: 26490-8
• Display abstract

• Arp2/3 (actin-related protein 2/3) complex is a seven-subunit complex that nucleates branched actin filaments in response to cellular signals. Nucleation-promoting factors such as WASp/Scar family proteins activate the complex by facilitating the activating conformational change and recruiting the first actin monomer for the daughter branch. Here we address the role of the Arp2 subunit in the function of Arp2/3 complex by isolating a version of the complex lacking Arp2 (Arp2Delta Arp2/3 complex) from fission yeast. An x-ray crystal structure of the DeltaArp2 Arp2/3 complex showed that the rest of the complex is unperturbed by the loss of Arp2. However, the Arp2Delta Arp2/3 complex was inactive in actin nucleation assays, indicating that Arp2 is essential to form a branch. A fluorescence anisotropy assay showed that Arp2 does not contribute to the affinity of the complex for Wsp1-VCA, a Schizosaccharomyces pombe nucleation-promoting factor protein. Fluorescence resonance energy transfer experiments showed that the loss of Arp2 does not prevent VCA from recruiting an actin monomer to the complex. Truncation of the N terminus of ARPC5, the smallest subunit in the complex, increased the yield of Arp2Delta Arp2/3 complex during purification but did not compromise nucleation activity of the full Arp2/3 complex.

• Decker CJ, Teixeira D, Parker R
• Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae.
• J Cell Biol. 2007; 179: 437-49
• Display abstract

• Processing bodies (P-bodies) are cytoplasmic RNA granules that contain translationally repressed messenger ribonucleoproteins (mRNPs) and messenger RNA (mRNA) decay factors. The physical interactions that form the individual mRNPs within P-bodies and how those mRNPs assemble into larger P-bodies are unresolved. We identify direct protein interactions that could contribute to the formation of an mRNP complex that consists of core P-body components. Additionally, we demonstrate that the formation of P-bodies that are visible by light microscopy occurs either through Edc3p, which acts as a scaffold and cross-bridging protein, or via the "prionlike" domain in Lsm4p. Analysis of cells defective in P-body formation indicates that the concentration of translationally repressed mRNPs and decay factors into microscopically visible P-bodies is not necessary for basal control of translation repression and mRNA decay. These results suggest a stepwise model for P-body assembly with the initial formation of a core mRNA-protein complex that then aggregates through multiple specific mechanisms.

• Teixeira D, Parker R
• Analysis of P-body assembly in Saccharomyces cerevisiae.
• Mol Biol Cell. 2007; 18: 2274-87
• Display abstract

• Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), that contain untranslating mRNAs in conjunction with proteins involved in translation repression and mRNA decapping and degradation. However, the order of protein assembly into P-bodies and the interactions that promote P-body assembly are unknown. To gain insight into how yeast P-bodies assemble, we examined the P-body accumulation of Dcp1p, Dcp2p, Edc3p, Dhh1p, Pat1p, Lsm1p, Xrn1p, Ccr4p, and Pop2p in deletion mutants lacking one or more P-body component. These experiments revealed that Dcp2p and Pat1p are required for recruitment of Dcp1p and of the Lsm1-7p complex to P-bodies, respectively. We also demonstrate that P-body assembly is redundant and no single known component of P-bodies is required for P-body assembly, although both Dcp2p and Pat1p contribute to P-body assembly. In addition, our results indicate that Pat1p can be a nuclear-cytoplasmic shuttling protein and acts early in P-body assembly. In contrast, the Lsm1-7p complex appears to primarily function in a rate limiting step after P-body assembly in triggering decapping. Taken together, these results provide insight both into the function of individual proteins involved in mRNA degradation and the mechanisms by which yeast P-bodies assemble.

• Deshmukh MV, Oku Y, Gross JD
• Backbone and sidechain methyl Ile (delta1), Leu and Val resonance assignments of the catalytic domain of the yeast mRNA decapping enzyme, Dcp2.
• Biomol NMR Assign. 2007; 1: 17-8
• Display abstract

• Eukaryotic mRNA decapping by Dcp2 is the penultimate step in several mRNA decay pathways. To understand regulation of Dcp2 by ligand interactions, we have assigned the backbone and sidechain methyl Ile (delta1), Leu and Val chemical shifts of the catalytic domain of the S. Cerevisiae enzyme.

• Chen CY et al.
• Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells.
• RNA. 2007; 13: 1775-86
• Display abstract

• Development of transcriptional pulsing approaches using the c-fos and Tet-off promoter systems greatly facilitated studies of mRNA turnover in mammalian cells. However, optimal protocols for these approaches vary for different cell types and/or physiological conditions, limiting their widespread application. In this study, we have further optimized transcriptional pulsing systems for different cell lines and developed new protocols to facilitate investigation of various aspects of mRNA turnover. We apply the Tet-off transcriptional pulsing strategy to investigate ARE-mediated mRNA decay in human erythroleukemic K562 cells arrested at various phases of the cell cycle by pharmacological inhibitors. This application facilitates studies of the role of mRNA stability in control of cell-cycle dependent gene expression. To advance the investigation of factors involved in mRNA turnover and its regulation, we have also incorporated recently developed transfection and siRNA reagents into the transcriptional pulsing approach. Using these protocols, siRNA and DNA plasmids can be effectively cotransfected into mouse NIH3T3 cells to obtain high knockdown efficiency. Moreover, we have established a tTA-harboring stable line using human bronchial epithelial BEAS-2B cells and applied the transcriptional pulsing approach to monitor mRNA deadenylation and decay kinetics in this cell system. This broadens the application of the transcriptional pulsing system to investigate the regulation of mRNA turnover related to allergic inflammation. Critical factors that need to be considered when employing these approaches are characterized and discussed.

• Brengues M, Parker R
• Accumulation of polyadenylated mRNA, Pab1p, eIF4E, and eIF4G with P-bodies in Saccharomyces cerevisiae.
• Mol Biol Cell. 2007; 18: 2592-602
• Display abstract

• Recent experiments have shown that mRNAs can move between polysomes and P-bodies, which are aggregates of nontranslating mRNAs associated with translational repressors and the mRNA decapping machinery. The transitions between polysomes and P-bodies and how the poly(A) tail and the associated poly(A) binding protein 1 (Pab1p) may affect this process are unknown. Herein, we provide evidence that poly(A)(+) mRNAs can enter P-bodies in yeast. First, we show that both poly(A)(-) and poly(A)(+) mRNA become translationally repressed during glucose deprivation, where mRNAs accumulate in P-bodies. In addition, both poly(A)(+) transcripts and/or Pab1p can be detected in P-bodies during glucose deprivation and in stationary phase. Cells lacking Pab1p have enlarged P-bodies, suggesting that Pab1p plays a direct or indirect role in shifting the equilibrium of mRNAs away from P-bodies and into translation, perhaps by aiding in the assembly of a type of mRNP within P-bodies that is poised to reenter translation. Consistent with this latter possibility, we observed the translation initiation factors (eIF)4E and eIF4G in P-bodies at a low level during glucose deprivation and at high levels in stationary phase. Moreover, Pab1p exited P-bodies much faster than Dcp2p when stationary phase cells were given fresh nutrients. Together, these results suggest that polyadenylated mRNAs can enter P-bodies, and an mRNP complex including poly(A)(+) mRNA, Pab1p, eIF4E, and eIF4G2 may represent a transition state during the process of mRNAs exchanging between P-bodies and translation.

• Siddiqui N, Mangus DA, Chang TC, Palermino JM, Shyu AB, Gehring K
• Poly(A) nuclease interacts with the C-terminal domain of polyadenylate-binding protein domain from poly(A)-binding protein.
• J Biol Chem. 2007; 282: 25067-75
• Display abstract

• The poly(A)-binding protein (PABP) is an essential protein found in all eukaryotes and is involved in an extensive range of cellular functions, including translation, mRNA metabolism, and mRNA export. Its C-terminal region contains a peptide-interacting PABC domain that recruits proteins containing a highly specific PAM-2 sequence motif to the messenger ribonucleoprotein complex. In humans, these proteins, including Paip1, Paip2, eRF3 (eukaryotic release factor 3), Ataxin-2, and Tob2, are all found to regulate translation through varying mechanisms. The following reports poly(A) nuclease (PAN) as a PABC-interacting partner in both yeast and humans. Their interaction is mediated by a PAM-2 motif identified within the PAN3 subunit. This site was identified in various fungal and animal species suggesting that the interaction is conserved throughout evolution. Our results indicate that PABP is directly involved in recruiting a deadenylase to the messenger ribonucleoprotein complex. This demonstrates a novel role for the PABC domain in mRNA metabolic processes and gives further insight into the function of PABP in mRNA maturation, export, and turnover.

• Hausmann S, Ramirez A, Schneider S, Schwer B, Shuman S
• Biochemical and genetic analysis of RNA cap guanine-N2 methyltransferases from Giardia lamblia and Schizosaccharomyces pombe.
• Nucleic Acids Res. 2007; 35: 1411-20
• Display abstract

• RNA cap guanine-N2 methyltransferases such as Schizosaccharomyces pombe Tgs1 and Giardia lamblia Tgs2 catalyze methylation of the exocyclic N2 amine of 7-methylguanosine. Here we performed a mutational analysis of Giardia Tgs2, entailing an alanine scan of 17 residues within the minimal active domain. Alanine substitutions at Phe18, Thr40, Asp76, Asn103 and Asp140 reduced methyltransferase specific activity to <3% of wild-type Tgs2, thereby defining these residues as essential. Alanines at Pro142, Tyr148 and Pro185 reduced activity to 7-12% of wild-type. Structure-activity relationships at Phe18, Thr40, Asp76, Asn103, Asp140 and Tyr148, and at three other essential residues defined previously (Asp68, Glu91 and Trp143) were gleaned by testing the effects of 18 conservative substitutions. Our results engender a provisional map of the Tgs2 active site, which we discuss in light of crystal structures of related methyltransferases. A genetic analysis of S. pombe Tgs1 showed that it is nonessential. An S. pombe tgs1Delta strain grows normally, notwithstanding the absence of 2,2,7-trimethylguanosine caps on its U1, U2, U4 and U5 snRNAs. However, we find that S. pombe requires cap guanine-N7 methylation catalyzed by the enzyme Pcm1. Deletion of the pcm1(+) gene was lethal, as were missense mutations in the Pcm1 active site. Thus, whereas m(7)G caps are essential in both S. pombe and S. cerevisiae, m(2,2,7)G caps are not.

• Jung J, Ahn YJ, Kang LW
• Overexpression, crystallization and preliminary X-ray crystallographic analysis of Nudix hydrolase Orf141 from Escherichia coli K-1.
• Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007; 63: 812-5
• Display abstract

• Nudix hydrolases are a family of proteins that contain the characteristic amino-acid sequence GX(5)EX(7)REUXEEXGU (where U is usually I, L or V), the Nudix signature sequence. They catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives such as nucleoside triphosphates, nucleotide sugars, ADP-ribose, dinucleotide coenzymes, diadenosine oligophosphates and capped RNAs. Recently, three new Nudix hydrolases have been found from Escherichia coli; one of them is Orf141, which cleaves pyrimidine deoxynucleoside triphosphates. Orf141 was cloned directly from E. coli K1 strain and was overexpressed in E. coli without any extra residues. Orf141 crystals were successfully obtained using polyethylene glycol 1500 as a precipitant at 285 K. X-ray diffraction data were collected to 3.1 A resolution using synchrotron radiation. The crystal is a member of the rhombohedral space group H32, with unit-cell parameters a = b = 182.2, c = 62.3 A, alpha = 90, beta = 90, gamma = 120 degrees (hexagonal setting). Two or three monomers are likely to be present in the asymmetric unit, with corresponding V(M) values of 2.92 and 1.95 A3 Da(-1) and solvent contents of 57.9 and 36.9%, respectively.

• Sha Z, Yen HC, Scheel H, Suo J, Hofmann K, Chang EC
• Isolation of the Schizosaccharomyces pombe proteasome subunit Rpn7 and a structure-function study of the proteasome-COP9-initiation factor domain.
• J Biol Chem. 2007; 282: 32414-23
• Display abstract

• Proper assembly of the 26 S proteasome is required to efficiently degrade polyubiquitinated proteins. Many proteasome subunits contain the proteasome-COP9-initiation factor (PCI) domain, thus raising the possibility that the PCI domain may play a role in mediating proteasome assembly. We have previously characterized the PCI protein Yin6, a fission yeast ortholog of the mammalian Int6 that has been implicated in breast oncogenesis, and demonstrated that it binds and regulates the assembly of the proteasome. In this study, we isolated another PCI proteasome subunit, Rpn7, as a high copy suppressor that rescued the proteasome defects in yin6 null cells. To better define the function of the PCI domain, we aligned protein sequences to identify a conserved leucine residue that is present in nearly all known PCI domains. Replacing it with aspartate in yeast Rpn7, Yin6, and Rpn5 inactivated these proteins, and mutant human Int6 mislocalized in HeLa cells. Rpn7 and Rpn5 bind Rpn9 with high affinity, but their mutant versions do not. Our data suggest that this leucine may interact with several hydrophobic amino acid residues to influence the spatial arrangement either within the N-terminal tandem alpha-helical repeats or between these repeats and the more C-terminal winged helix subdomain. Disruption of such an arrangement in the PCI domain may substantially inactivate many PCI proteins and block their binding to other proteins.

• Tritschler F et al.
• A divergent Sm fold in EDC3 proteins mediates DCP1 binding and P-body targeting.
• Mol Cell Biol. 2007; 27: 8600-11
• Display abstract

• Members of the (L)Sm (Sm and Sm-like) protein family are found across all kingdoms of life and play crucial roles in RNA metabolism. The P-body component EDC3 (enhancer of decapping 3) is a divergent member of this family that functions in mRNA decapping. EDC3 is composed of a N-terminal LSm domain, a central FDF domain, and a C-terminal YjeF-N domain. We show that this modular architecture enables EDC3 to interact with multiple components of the decapping machinery, including DCP1, DCP2, and Me31B. The LSm domain mediates DCP1 binding and P-body localization. We determined the three-dimensional structures of the LSm domains of Drosophila melanogaster and human EDC3 and show that the domain adopts a divergent Sm fold that lacks the characteristic N-terminal alpha-helix and has a disrupted beta4-strand. This domain remains monomeric in solution and lacks several features that canonical (L)Sm domains require for binding RNA. The structures also revealed a conserved patch of surface residues that are required for the interaction with DCP1 but not for P-body localization. The conservation of surface and of critical structural residues indicates that LSm domains in EDC3 proteins adopt a similar fold that has separable novel functions that are absent in canonical (L)Sm proteins.

• Stribinskis V, Ramos KS
• Rpm2p, a protein subunit of mitochondrial RNase P, physically and genetically interacts with cytoplasmic processing bodies.
• Nucleic Acids Res. 2007; 35: 1301-11
• Display abstract

• The RPM2 gene of Saccharomyces cerevisiae codes for a protein subunit of mitochondrial RNase P and has another unknown essential function. We previously demonstrated that Rpm2p localizes to the nucleus and acts as a transcriptional activator. Rpm2p influences the level of mRNAs that encode components of the mitochondrial import apparatus and essential mitochondrial chaperones. Evidence is presented here that Rpm2p interacts with Dcp2p, a subunit of mRNA decapping enzyme in the two-hybrid assay, and is enriched in cytoplasmic P bodies, the sites of mRNA degradation and storage in yeast and mammalian cells. When overexpressed, GFP-Rpm2p does not impact the number and size of P bodies; however, it prevents their disappearance when translation elongation is inhibited by cycloheximide. Proteasome mutants, ump1-2 and pre4-2, that bypass essential Rpm2p function, also stabilize P bodies. The stabilization of P bodies by Rpm2p may occur through reduced protein degradation since GFP-Rpm2p expressing cells have lower levels of ubiquitin. Genetic analysis revealed that overexpression of Dhh1p (a DEAD box helicase localized to P bodies) suppresses temperature-sensitive growth of the rpm2-100 mutant. Overexpression of Pab1p (a poly (A)-binding protein) also suppresses rpm2-100, suggesting that Rpm2p functions in at least two aspects of mRNA metabolism. The results presented here, and the transcriptional activation function demonstrated earlier, implicate Rpm2p as a coordinator of transcription and mRNA storage/decay in P bodies.

• Tan H, Janiak-Spens F, West AH
• Functional characterization of the phosphorelay protein Mpr1p from Schizosaccharomyces pombe.
• FEMS Yeast Res. 2007; 7: 912-21
• Display abstract

• Histidine-containing phosphotransfer (HPt) proteins play an essential role in multistep histidine-aspartate phosphorelay signal transduction systems in prokaryotes and eukaryotes. The putative HPt protein in Schizosaccharomyces pombe, Mpr1p (also known as Spy1p), is a 295 amino acid protein that appears to be composed of more than one functional domain. The amino acid sequence of the N-terminal region of Mpr1p lacks homology to other known proteins, whereas the C-terminal domain is predicted to have structural similarity to the Ypd1p HPt protein from Saccharomyces cerevisiae. This study provides both in vitro and in vivo evidence that the C-terminal domain of Mpr1p indeed functions as an HPt protein in shuttling phosphoryl groups from one response regulator domain to another. Furthermore, we find that various deletions of the N-terminal region diminish both the phosphotransfer activity of Mpr1p and its affinity for response regulator domains, suggesting a possible role for the N-terminal domain in HPt-response regulator domain interactions.

• Simon E, Camier S, Seraphin B
• New insights into the control of mRNA decapping.
• Trends Biochem Sci. 2006; 31: 241-3
• Display abstract

• mRNA decapping irreversibly targets mRNAs for fast decay. Cap removal is catalyzed by decapping protein Dcp2 but also requires Dcp1. Recently, two groups have provided a first glimpse of the regulation mechanism of this crucial step in gene expression. Resolution of the yeast Dcp2 structure has enabled identification of the residues that are important for its interaction with Dcp1. However, the human decapping machinery seems to be more complex because a third component, Hedls, is required for a functional Dcp1-Dcp2 interaction.

• Peng K, Radivojac P, Vucetic S, Dunker AK, Obradovic Z
• Length-dependent prediction of protein intrinsic disorder.
• BMC Bioinformatics. 2006; 7: 208-208
• Display abstract

• BACKGROUND: Due to the functional importance of intrinsically disordered proteins or protein regions, prediction of intrinsic protein disorder from amino acid sequence has become an area of active research as witnessed in the 6th experiment on Critical Assessment of Techniques for Protein Structure Prediction (CASP6). Since the initial work by Romero et al. (Identifying disordered regions in proteins from amino acid sequences, IEEE Int. Conf. Neural Netw., 1997), our group has developed several predictors optimized for long disordered regions (>30 residues) with prediction accuracy exceeding 85%. However, these predictors are less successful on short disordered regions (< or =30 residues). A probable cause is a length-dependent amino acid compositions and sequence properties of disordered regions. RESULTS: We proposed two new predictor models, VSL2-M1 and VSL2-M2, to address this length-dependency problem in prediction of intrinsic protein disorder. These two predictors are similar to the original VSL1 predictor used in the CASP6 experiment. In both models, two specialized predictors were first built and optimized for short (< or = 30 residues) and long disordered regions (>30 residues), respectively. A meta predictor was then trained to integrate the specialized predictors into the final predictor model. As the 10-fold cross-validation results showed, the VSL2 predictors achieved well-balanced prediction accuracies of 81% on both short and long disordered regions. Comparisons over the VSL2 training dataset via 10-fold cross-validation and a blind-test set of unrelated recent PDB chains indicated that VSL2 predictors were significantly more accurate than several existing predictors of intrinsic protein disorder. CONCLUSION: The VSL2 predictors are applicable to disordered regions of any length and can accurately identify the short disordered regions that are often misclassified by our previous disorder predictors. The success of the VSL2 predictors further confirmed the previously observed differences in amino acid compositions and sequence properties between short and long disordered regions, and justified our approaches for modelling short and long disordered regions separately. The VSL2 predictors are freely accessible for non-commercial use at http://www.ist.temple.edu/disprot/predictorVSL2.php.

• Hosoda N, Lejeune F, Maquat LE
• Evidence that poly(A) binding protein C1 binds nuclear pre-mRNA poly(A) tails.
• Mol Cell Biol. 2006; 26: 3085-97
• Display abstract

• In mammalian cells, poly(A) binding protein C1 (PABP C1) has well-known roles in mRNA translation and decay in the cytoplasm. However, PABPC1 also shuttles in and out of the nucleus, and its nuclear function is unknown. Here, we show that PABPC1, like the major nuclear poly(A) binding protein PABPN1, associates with nuclear pre-mRNAs that are polyadenylated and intron containing. PABPC1 does not bind nonpolyadenylated histone mRNA, indicating that the interaction of PABPC1 with pre-mRNA requires a poly(A) tail. Consistent with this conclusion, UV cross-linking results obtained using intact cells reveal that PABPC1 binds directly to pre-mRNA poly(A) tails in vivo. We also show that PABPC1 immunopurifies with poly(A) polymerase, suggesting that PABPC1 is acquired by polyadenylated transcripts during poly(A) tail synthesis. Our findings demonstrate that PABPC1 associates with polyadenylated transcripts earlier in mammalian mRNA biogenesis than previously thought and offer insights into the mechanism by which PABPC1 is recruited to newly synthesized poly(A). Our results are discussed in the context of pre-mRNA processing and stability and mRNA trafficking and the pioneer round of translation.

• Gupta M, Brewer G
• MicroRNAs: new players in an old game.
• Proc Natl Acad Sci U S A. 2006; 103: 3951-2

• Jiao X, Wang Z, Kiledjian M
• Identification of an mRNA-decapping regulator implicated in X-linked mental retardation.
• Mol Cell. 2006; 24: 713-22
• Display abstract

• Two major decapping enzymes are involved in the decay of eukaryotic mRNA, Dcp2 and DcpS. Despite the detection of robust DcpS decapping activity in cell extract, minimal to no decapping is detected from human Dcp2 (hDcp2) in extract. We now demonstrate that one reason for the lack of detectable hDcp2 activity in extract is due to the presence of inhibitory trans factor(s). Furthermore, we demonstrate that a previously identified testis-specific protein of unknown function implicated in nonspecific X-linked mental retardation, VCX-A, can function as an inhibitor of hDcp2 decapping in vitro and in cells. VCX-A is a noncanonical cap-binding protein that binds to capped RNA but not cap structure lacking an RNA. Its cap association is enhanced by hDcp2 to further augment the ability of VCX-A to inhibit decapping. Our data demonstrate that VCX-A can regulate mRNA stability and that it is an example of a tissue-specific decapping regulator.

• Shen V, Kiledjian M
• Decapper comes into focus.
• Structure. 2006; 14: 171-2

• Bail S, Kiledjian M
• More than 1 + 2 in mRNA decapping.
• Nat Struct Mol Biol. 2006; 13: 7-9
• Display abstract

• Decapping of messenger RNA was thought to involve a complex of only Dcp1 and Dcp2, but new data suggest that a larger multisubunit decapping complex exists in mammals. The larger complex includes a protein that facilitates the association of the two Dcp proteins and can be recruited by specific factors that promote mRNA decay.

• Elmlund H et al.
• The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II.
• Proc Natl Acad Sci U S A. 2006; 103: 15788-93
• Display abstract

• CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II.

• Parrish S, Moss B
• Characterization of a vaccinia virus mutant with a deletion of the D10R gene encoding a putative negative regulator of gene expression.
• J Virol. 2006; 80: 553-61
• Display abstract

• The D9 and D10 proteins of vaccinia virus are 25% identical to each other, contain a mutT motif characteristic of nudix hydrolases, and are conserved in all sequenced poxviruses. Previous studies indicated that overexpression of D10 and, to a lesser extent, D9 decreased the levels of capped mRNAs and their translation products. Here, we further characterized the D10 protein and showed that only trace amounts are associated with purified virions and that it is expressed exclusively at late times after vaccinia virus infection. A viable deletion mutant (vdeltaD10) produced smaller plaques and lower virus yields than either wild-type virus or a D9R deletion mutant (vdeltaD9). Purified vdeltaD10 virions appeared normal by microscopic examination and biochemical analysis but produced 6- to 10-fold-fewer plaques at the same concentration as wild-type or vdeltaD9 virions. When 4 PFU per cell of wild-type or vdeltaD9 virions or equal numbers of vdeltaD10 virions were used for inoculation, nearly all cells were infected in each case, but viral early and late transcription was initiated more slowly in vdeltaD10-infected cells than in the others. However, viral early transcripts accumulated to higher levels in vdeltaD10-infected cells than in cells infected with the wild type or vdeltaD9. In addition, viral early and late mRNAs and cellular actin mRNA persisted longer in vdeltaD10-infected cells than in others. Furthermore, analysis of pulse-labeled proteins indicated prolonged synthesis of cellular and viral early proteins. These results are consistent with a role for D10 in regulating RNA levels in poxvirus-infected cells.

• Ford AS, Guan Q, Neeno-Eckwall E, Culbertson MR
• Ebs1p, a negative regulator of gene expression controlled by the Upf proteins in the yeast Saccharomyces cerevisiae.
• Eukaryot Cell. 2006; 5: 301-12
• Display abstract

• Mutations in EBS1 were identified in Saccharomyces cerevisiae that cosuppress missense, frameshift, and nonsense mutations. Evidence from studies of loss of function and overexpression of EBS1 suggests that Ebs1p affects gene expression by inhibiting translation and that a loss of EBS1 function causes suppression by increasing the rate of translation. Changes in EBS1 expression levels alter the expression of wild-type genes, but, in general, no changes in mRNA abundance were associated with a loss of function or overexpression of EBS1. Translation of a lacZ reporter was increased in strains carrying an ebs1-Delta mutant gene, whereas translation was decreased when EBS1 was overexpressed. The cap binding protein eIF-4E copurifies with Ebs1p in the absence of RNA, suggesting that the two proteins interact in vivo. Although physical and genetic interactions were detected between Ebs1p and Dcp1p, copurification was RNase sensitive, and changes in the expression of Ebs1p had little to no effect on decapping of the MFA2 transcript. The combined results suggest that Ebs1p inhibits translation, most likely through effects on eIF-4E rather than on decapping. Finally, EBS1 transcript levels are under the control of nonsense-mediated mRNA decay (NMD), providing the first example of an NMD-sensitive transcript whose protein product influences a step in gene expression required for NMD.

• Fenger-Gron M, Fillman C, Norrild B, Lykke-Andersen J
• Multiple processing body factors and the ARE binding protein TTP activate mRNA decapping.
• Mol Cell. 2005; 20: 905-15
• Display abstract

• Decapping is a key step in mRNA turnover. However, the composition and regulation of the human decapping complex is poorly understood. Here, we identify three proteins that exist in complex with the decapping enzyme subunits hDcp2 and hDcp1: hEdc3, Rck/p54, and a protein in decapping we name Hedls. Hedls is important in decapping because it enhances the activity of the catalytic hDcp2 subunit and promotes complex formation between hDcp2 and hDcp1. Specific decapping factors interact with the mRNA decay activators hUpf1 and TTP, and TTP enhances decapping of a target AU-rich element (ARE) RNA in vitro. Each decapping protein localizes in cytoplasmic processing bodies (PBs), and overexpression of Hedls produces aberrant PBs and concomitant accumulation of a deadenylated ARE-mediated mRNA decay intermediate. These observations suggest that multiple proteins involved in human decapping are important subunits of PBs and are activated on ARE-mRNAs by the protein TTP.

• Dennes A et al.
• The novel Drosophila lysosomal enzyme receptor protein mediates lysosomal sorting in mammalian cells and binds mammalian and Drosophila GGA adaptors.
• J Biol Chem. 2005; 280: 12849-57
• Display abstract

• Biogenesis of lysosomes depends in mammalian cells on the specific recognition and targeting of mannose 6-phosphate-containing lysosomal enzymes by two mannose 6-phosphate receptors (MPR46, MPR300), key components of the extensively studied receptor-mediated lysosomal sorting system in complex metazoans. In contrast, the biogenesis of lysosomes is poorly investigated in the less complex metazoan Drosophila melanogaster. We identified the novel type I transmembrane protein lysosomal enzyme receptor protein (LERP) with partial homology to the mammalian MPR300 encoded by Drosophila gene CG31072. LERP contains 5 lumenal repeats that share homology to the 15 lumenal repeats found in all identified MPR300. Four of the repeats display the P-lectin type pattern of conserved cysteine residues. However, the arginine residues identified to be essential for mannose 6-phosphate binding are not conserved. The recombinant LERP protein was expressed in mammalian cells and displayed an intracellular localization pattern similar to the mammalian MPR300. The LERP cytoplasmic domain shows highly conserved interactions with Drosophila and mammalian GGA adaptors known to mediate Golgi-endosome traffic of MPRs and other transmembrane cargo. Moreover, LERP rescues missorting of soluble lysosomal enzymes in MPR-deficient cells, giving strong evidence for a function that is equivalent to the mammalian counterpart. However, unlike the mammalian MPRs, LERP did not bind to the multimeric mannose 6-phosphate ligand phosphomannan. Thus ligand recognition by LERP does not depend on mannose 6-phosphate but may depend on a common feature present in mammalian lysosomal enzymes. Our data establish a potential important role for LERP in biogenesis of Drosophila lysosomes and suggest a GGA function also in the receptor-mediated lysosomal transport system in the fruit fly.

• Bianchin C, Mauxion F, Sentis S, Seraphin B, Corbo L
• Conservation of the deadenylase activity of proteins of the Caf1 family in human.
• RNA. 2005; 11: 487-94
• Display abstract

• The yeast Pop2 protein, belonging to the eukaryotic Caf1 family, is required for mRNA deadenylation in vivo. It also catalyzes poly(A) degradation in vitro, even though this property has been questioned. Caf1 proteins are related to RNase D, a feature supported by the recently published structure of Pop2. Yeast Pop2 contains, however, a divergent active site while its human homologs harbor consensus catalytic residues. Given these differences, we tested whether its deadenylase activity is conserved in the human homologs Caf1 and Pop2. Our data demonstrate that both human factors degrade poly(A) tails indicating their involvement in mRNA metabolism.

• Kuai L, Das B, Sherman F
• A nuclear degradation pathway controls the abundance of normal mRNAs in Saccharomyces cerevisiae.
• Proc Natl Acad Sci U S A. 2005; 102: 13962-7
• Display abstract

• We previously demonstrated an increased degradation of mRNAs in mutants of Saccharomyces cerevisiae having blocks in nuclear export. The degradation activity, designated DRN (degradation of mRNA in the nucleus), requires Cbc1p, a nuclear cap-binding protein, and Rrp6p, a nuclear exosome component. Microarray procedures were used to determine the half-lives of mRNAs from normal and mutant strains, leading to the tentative identification of hundreds of normal mRNAs that were notably stabilized when either CBC1 or RRP6 were deleted. Northern blot analysis of representative mRNAs confirmed the diminished degradation. One representative of this group, SKS1 mRNA, was also shown by a cytological procedure to be preferentially retained in the nucleus compared with typical mRNAs. We suggest that all normal mRNAs are subjected to degradation by DRN, but the degree of degradation is determined by the degree of nuclear retention. Furthermore, these mRNAs particularly susceptible to DRN were also diminished by overproduction of Cbc1p, demonstrating a regulatory role for CBC1. This conclusion was corroborated by finding an inverse relationship of the CBC1 and SKS1 mRNA levels in normal strains grown under different conditions.

• Hoeppner S, Baumli S, Cramer P
• Structure of the mediator subunit cyclin C and its implications for CDK8 function.
• J Mol Biol. 2005; 350: 833-42
• Display abstract

• Cyclin C binds the cyclin-dependent kinases CDK8 and CDK3, which regulate mRNA transcription and the cell cycle, respectively. The crystal structure of cyclin C reveals two canonical five-helix repeats and a specific N-terminal helix. In contrast to other cyclins, the N-terminal helix is short, mobile, and in an exposed position that allows for interactions with proteins other than the CDKs. A model of the CDK8/cyclin C pair reveals two regions in the interface with apparently distinct roles. A conserved region explains promiscuous binding of cyclin C to CDK8 and CDK3, and a non-conserved region may be responsible for discrimination of CDK8 against other CDKs involved in transcription. A conserved and cyclin C-specific surface groove may recruit substrates near the CDK8 active site. Activation of CDKs generally involves phosphorylation of a loop at a threonine residue. In CDK8, this loop is longer and the threonine is absent, suggesting an alternative mechanism of activation that we discuss based on a CDK8-cyclin C model.

• Yamashita A et al.
• Concerted action of poly(A) nucleases and decapping enzyme in mammalian mRNA turnover.
• Nat Struct Mol Biol. 2005; 12: 1054-63
• Display abstract

• In mammalian cells, the enzymatic pathways involved in cytoplasmic mRNA decay are incompletely defined. In this study, we have used two approaches to disrupt activities of deadenylating and/or decapping enzymes to monitor effects on mRNA decay kinetics and trap decay intermediates. Our results show that deadenylation is the key first step that triggers decay of both wild-type stable and nonsense codon-containing unstable beta-globin mRNAs in mouse NIH3T3 fibroblasts. PAN2 and CCR4 are the major poly(A) nucleases active in cytoplasmic deadenylation that have biphasic kinetics, with PAN2 initiating deadenylation followed by CCR4-mediated poly(A) shortening. DCP2-mediated decapping takes place after deadenylation and may serve as a backup mechanism for triggering mRNA decay when initial deadenylation by PAN2 is compromised. Our findings reveal a functional link between deadenylation and decapping and help to define in vivo pathways for mammalian cytoplasmic mRNA decay.

• Mildvan AS et al.
• Structures and mechanisms of Nudix hydrolases.
• Arch Biochem Biophys. 2005; 433: 129-43
• Display abstract

• Nudix hydrolases catalyze the hydrolysis of nucleoside diphosphates linked to other moieties, X, and contain the sequence motif or Nudix box, GX(5)EX(7)REUXEEXGU. The mechanisms of Nudix hydrolases are highly diverse in the position on the substrate at which nucleophilic substitution occurs, and in the number of required divalent cations. While most proceed by associative nucleophilic substitutions by water at specific internal phosphorus atoms of a diphosphate or polyphosphate chain, members of the GDP-mannose hydrolase sub-family catalyze dissociative nucleophilic substitutions, by water, at carbon. The site of substitution is likely determined by the positions of the general base and the entering water. The rate accelerations or catalytic powers of Nudix hydrolases range from 10(9)- to 10(12)-fold. The reactions are accelerated 10(3)-10(5)-fold by general base catalysis by a glutamate residue within, or beyond the Nudix box, or by a histidine beyond the Nudix box. Lewis acid catalysis, which contributes 10(3)-10(5)-fold to the rate acceleration, is provided by one, two, or three divalent cations. One divalent cation is coordinated by two or three conserved residues of the Nudix box, the initial glycine and one or two glutamate residues, together with a remote glutamate or glutamine ligand from beyond the Nudix box. Some Nudix enzymes require one (MutT) or two additional divalent cations (Ap(4)AP), to neutralize the charge of the polyphosphate chain, to help orient the attacking hydroxide or oxide nucleophile, and/or to facilitate the departure of the anionic leaving group. Additional catalysis (10-10(3)-fold) is provided by the cationic side chains of lysine and arginine residues and by H-bond donation by tyrosine residues, to orient the general base, or to promote the departure of the leaving group. The overall rate accelerations can be explained by both independent and cooperative effects of these catalytic components.

• Trempe JF et al.
• Mechanism of Lys48-linked polyubiquitin chain recognition by the Mud1 UBA domain.
• EMBO J. 2005; 24: 3178-89
• Display abstract

• The ubiquitin-pathway associated (UBA) domain is a 40-residue polyubiquitin-binding motif. The Schizosaccharomyces pombe protein Mud1 is an ortholog of the Saccharomyces cerevisiae DNA-damage response protein Ddi1 and binds to K48-linked polyubiquitin through its UBA domain. We have solved the crystal structure of Mud1 UBA at 1.8 angstroms resolution, revealing a canonical three-helical UBA fold. We have probed the interactions of this domain using mutagenesis, surface plasmon resonance, NMR and analytical ultracentrifugation. We show that the ubiquitin-binding surface of Mud1 UBA extends beyond previously recognized motifs and can be functionally dissected into primary and secondary ubiquitin-binding sites. Mutation of Phe330 to alanine, a residue exposed between helices 2 and 3, significantly reduces the affinity of the Mud1 UBA domain for K48-linked polyubiquitin, despite leaving the primary binding surface functionally intact. Moreover, K48-linked diubiquitin binds a single Mud1 UBA domain even in the presence of excess UBA. We therefore propose a mechanism for the recognition of K48-linked polyubiquitin chains by Mud1 in which diubiquitin units are specifically recognized by a single UBA domain.

• Escobar-Cabrera E, Venkatesan M, Desautels M, Hemmingsen SM, McIntosh LP
• Dissecting the domain structure of Cdc4p, a myosin essential light chain involved in Schizosaccharomyces pombe cytokinesis.
• Biochemistry. 2005; 44: 12136-48
• Display abstract

• Cytokinesis is the process by which one cell divides into two. Key in the cytokinetic mechanism of Schizosaccharomyces pombe is the contractile ring myosin, which consists of two heavy chains (Myo2p), two essential light chains (Cdc4p), and two regulatory light chains (Rlc1p). Cdc4p is a dumbbell-shaped EF-hand protein composed of N- and C-terminal domains separated by a flexible linker. The properties of these two domains are of particular interest because each is hypothesized to have independent functions in binding different components of the cytokinesis machinery. To help define these properties, we used NMR spectroscopy to compare the structure, stability, and dynamics of the isolated N- and C-terminal domains with one another and with native Cdc4p. On the basis of invariant chemical shifts, the N-domain retains the same structure in isolation as in the context of the full-length Cdc4p, whereas the C-domain appears markedly perturbed. This perturbation results from intramolecular binding of the residual linker sequence at the N-terminus of the C-domain in a mode similar to that used by native Cdc4p to associate with target polypeptide sequences. NMR relaxation, thermal denaturation, and amide hydrogen exchange experiments also indicate that the C-domain is less stable and more dynamic than the N-domain, both in isolation and in the full-length protein. We hypothesize that these properties reflect a conformational plasticity of the C-domain, which may allow Cdc4p to interact with several regulatory or contractile ring proteins necessary for cytokinesis.

• Ahmad F, Stewart E
• The N-terminal region of the Schizosaccharomyces pombe RecQ helicase, Rqh1p, physically interacts with Topoisomerase III and is required for Rqh1p function.
• Mol Genet Genomics. 2005; 273: 102-14
• Display abstract

• The Schizosaccharomyces pombe rqh1+ gene encodes a member of the RecQ DNA helicase family. Members of this protein family are essential for the maintenance of genetic integrity. Thus, mutations in the genes encoding the human RecQ homologues Blm, Wrn and RecQ4 cause Bloom syndrome, Werner syndrome and Rothmund-Thomson syndrome, respectively-diseases which result from genome instability. S. pombe cells that lack a functional rqh1+ gene show reduced viability and display defective chromosome segregation, particularly after UV irradiation or S-phase arrest. In this study we used an rqh1+ deletion series to show that the N-terminal portion of Rqh1 is essential for Rqh1 function. Moreover, the conserved Helicase and RNaseD C-terminal (HRDC) domain of Rqh1 also plays a role in allowing cells to tolerate exposure to DNA damaging agents and the S-phase inhibitor hydroxyurea (HU). We also demonstrate that Topoisomerase III (Top3) binds to a site within the first 322 N-terminal amino acids of Rqh1 and that this binding correlates with Rqh1 function. Genetic analysis of rqh1- top3delta mutants reveals that, in the presence of functional or partially functional Rqh1 protein, Top3 is required to maintain genome integrity and cell viability.

• Liu J, Valencia-Sanchez MA, Hannon GJ, Parker R
• MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies.
• Nat Cell Biol. 2005; 7: 719-23
• Display abstract

• Small RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs) can silence target genes through several different effector mechanisms. Whereas siRNA-directed mRNA cleavage is increasingly understood, the mechanisms by which miRNAs repress protein synthesis are obscure. Recent studies have revealed the existence of specific cytoplasmic foci, referred to herein as processing bodies (P-bodies), which contain untranslated mRNAs and can serve as sites of mRNA degradation. Here we demonstrate that Argonaute proteins--the signature components of the RNA interference (RNAi) effector complex, RISC--localize to mammalian P-bodies. Moreover, reporter mRNAs that are targeted for translational repression by endogenous or exogenous miRNAs become concentrated in P-bodies in a miRNA-dependent manner. These results provide a link between miRNA function and mammalian P-bodies and suggest that translation repression by RISC delivers mRNAs to P-bodies, either as a cause or as a consequence of inhibiting protein synthesis.

• Baez MV, Boccaccio GL
• Mammalian Smaug is a translational repressor that forms cytoplasmic foci similar to stress granules.
• J Biol Chem. 2005; 280: 43131-40
• Display abstract

• Cytoplasmic events depending on RNA-binding proteins contribute to the fine-tuning of gene expression. Sterile alpha motif-containing RNA-binding proteins constitute a novel family of post-transcriptional regulators that recognize a specific RNA sequence motif known as Smaug recognition element (SRE). The Drosophila member of this family, dSmaug, triggers the translational repression and deadenylation of maternal mRNAs by independent mechanisms, and the yeast homologue Vts1 stimulates degradation of SRE-containing messengers. Two homologous genes are present in the mammalian genome. Here we showed that hSmaug 1, encoded in human chromosome 14, represses the translation of reporter transcripts carrying SRE motifs. When expressed in fibroblasts, hSmaug 1 forms cytoplasmic granules that contain polyadenylated mRNA and the RNA-binding proteins Staufen, TIAR, TIA-1, and HuR. Smaug 1 foci are distinct from degradation foci. The murine protein mSmaug 1 is expressed in the central nervous system and is abundant in post-synaptic densities, a subcellular region where translation is tightly regulated by synaptic stimulation. Biochemical analysis indicated that mSmaug 1 is present in synaptoneurosomal 20 S particles. These results suggest a role for mammalian Smaug 1 in RNA granule formation and translation regulation in neurons.

• Sen GL, Blau HM
• Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies.
• Nat Cell Biol. 2005; 7: 633-6
• Display abstract

• RNA interference (RNAi) is an important means of eliminating mRNAs, but the intracellular location of RNA-induced silencing complex (RISC) remains unknown. We show here that Argonaute 2, a key component of RISC, is not randomly distributed but concentrates in mRNA decay centres that are known as cytoplasmic bodies. The localization of Argonaute 2 in decay centres is not altered by the presence or absence of small interfering RNAs or their targeted mRNAs. However, RNA is required for the integrity of cytoplasmic bodies because RNase eliminates Argonaute 2 localization. In addition, Argonaute 1, another Argonaute family member, is concentrated in cytoplasmic bodies. These results provide new insight into the mechanism of RNAi function.

• Sun X, Zhang H, Wang D, Ma D, Shen Y, Shang Y
• DLP, a novel Dim1 family protein implicated in pre-mRNA splicing and cell cycle progression.
• J Biol Chem. 2004; 279: 32839-47
• Display abstract

• In eukaryotes, primary transcripts undergo a splicing process that removes intronic sequences by a macromolecular enzyme known as the spliceosome. Both genetic and biochemical studies have revealed that essential components of the spliceosome include five small RNAs, U1, U2, U4, U5, and U6, and as many as 300 distinct proteins. Here we report the molecular cloning and functional analysis of a novel cDNA encoding for a protein of 149 amino acids. This protein has 38% amino acid sequence identity with and is evolutionally related to yeast Dim1 protein. Hence we named this protein DLP for Dim1-like protein. We showed that DLP is required for S/G(2) transition. We also demonstrated that DLP functions in cell nucleus and interacts with the U5-102-kDa protein subunit of the spliceosome, and blocking DLP protein activity led to an insufficient pre-mRNA splicing, suggesting that DLP is yet another protein component involved in pre-mRNA splicing. Collectively, our experiments indicated that DLP is implicated in not only cell cycle progression but also in a more specific molecular process such as pre-mRNA splicing.

• Parker R, Song H
• The enzymes and control of eukaryotic mRNA turnover.
• Nat Struct Mol Biol. 2004; 11: 121-7
• Display abstract

• The degradation of eukaryotic mRNAs plays important roles in the modulation of gene expression, quality control of mRNA biogenesis and antiviral defenses. In the past five years, many of the enzymes involved in this process have been identified and mechanisms that modulate their activities have begun to be identified. In this review, we describe the enzymes of mRNA degradation and their properties. We highlight that there are a variety of enzymes with different specificities, suggesting that individual nucleases act on distinct subpopulations of transcripts within the cell. In several cases, translation factors that bind mRNA inhibit these nucleases. In addition, recent work has begun to identify distinct mRNP complexes that recruit the nucleases to transcripts through different mRNA-interacting proteins. These properties and complexes suggest multiple mechanisms by which mRNA degradation could be regulated.

• Weiser DC, Sikes S, Li S, Shenolikar S
• The inhibitor-1 C terminus facilitates hormonal regulation of cellular protein phosphatase-1: functional implications for inhibitor-1 isoforms.
• J Biol Chem. 2004; 279: 48904-14
• Display abstract

• Inhibitor-1 (I-1) is a selective inhibitor of protein phosphatase-1 (PP1) and regulates several PP1-dependent signaling pathways, including cardiac contractility and regulation of learning and memory. The human I-1 gene has been spliced to generate two alternative mRNAs, termed I-1alpha and I-1beta, encoding polypeptides that differ from I-1 in their C-terminal sequences. Reverse transcription-PCR established that I-1alpha and I-1beta mRNAs are expressed in a developmental and tissue-specific manner. Functional analysis of I-1 in a Saccharomyces cerevisiae strain dependent on human I-1 for viability established that a novel domain encompassing amino acids 77-110 is necessary for PP1 inhibition in yeast. Expression of human I-1 in S. cerevisiae with a partial loss-of-function eukaryotic initiation factor-2alpha (eIF2alpha) kinase (Gcn2p) mutation permitted growth during amino acid starvation, consistent with the inhibition of Glc7p/PP1, the yeast eIF2alpha phosphatase. In contrast, human I-1alpha, which lacks amino acids 83-134, and I-1 with C-terminal deletions were significantly less effective in promoting yeast growth under starvation conditions. These data suggest that C-terminal sequences of I-1 enhance regulation of the eukaryotic eIF2alpha phosphatase. In vitro studies established that C-terminal sequences, deleted in both I-1alpha and I-1beta, enhance PP1 binding and inhibition. Expression of full-length and C-terminally truncated I-1 in HEK293T cells established the importance of the I-1 C terminus in transducing cAMP signals that promote eIF2alpha phosphorylation. This study demonstrates that multiple domains in I-1 target cellular PP1 complexes and establishes I-1 as a cellular regulator of eIF2alpha phosphorylation.

• Hoja U et al.
• HFA1 encoding an organelle-specific acetyl-CoA carboxylase controls mitochondrial fatty acid synthesis in Saccharomyces cerevisiae.
• J Biol Chem. 2004; 279: 21779-86
• Display abstract

• The Saccharomyces cerevisiae gene, HFA1, encodes a >250-kDa protein, which is required for mitochondrial function. Hfa1p exhibits 72% overall sequence similarity (54% identity) to ACC1-encoded yeast cytoplasmic acetyl-CoA carboxylase. Nevertheless, HFA1 and ACC1 functions are not overlapping because mutants of the two genes have different phenotypes and do not complement each other. Whereas ACC1 is involved in cytoplasmic fatty acid synthesis, the phenotype of hfa1Delta disruptants resembles that of mitochondrial fatty-acid synthase mutants. They fail to grow on lactate or glycerol, and the mitochondrial cofactor, lipoic acid, is reduced to <10% of its normal cellular concentration. Other than Acc1p, the N-terminal sequence of Hfa1p comprises a canonical mitochondrial targeting signal together with a matrix protease cleavage site. Accordingly, the HFA1-encoded protein was specifically assigned by Western blotting of appropriate cell fractions to the mitochondrial compartment. Removal of the mitochondrial targeting sequence abolished the competence of HFA1 DNA to complement hfal null mutants. Conversely and in contrast to the intact HFA1 sequence, the signal sequence-free HFA1 gene complemented the mutational loss of cytoplasmic acetyl-CoA carboxylase. Expression of HFA1 under the control of the ACC1 promoter restored cellular ACC activity in ACC1-defective yeast mutants to wild type levels. From this finding, it is concluded that HFA1 encodes a specific mitochondrial acetyl-CoA carboxylase providing malonyl-CoA for intraorganellar fatty acid and, in particular, lipoic acid synthesis.

• Ghosh T, Peterson B, Tomasevic N, Peculis BA
• Xenopus U8 snoRNA binding protein is a conserved nuclear decapping enzyme.
• Mol Cell. 2004; 13: 817-28
• Display abstract

• U8 snoRNP is required for accumulation of mature 5.8S and 28S rRNA in vertebrates. We are identifying proteins that bind U8 RNA with high specificity to understand how U8 functions in ribosome biogenesis. Here, we characterize a Xenopus 29 kDa protein (X29), which we previously showed binds U8 RNA with high affinity. X29 and putative homologs in other vertebrates contain a NUDIX domain found in MutT and other nucleotide diphosphatases. Recombinant X29 protein has diphosphatase activity that removes m(7)G and m(227)G caps from U8 and other RNAs in vitro; the putative 29 kDa human homolog also displays this decapping activity. X29 is primarily nucleolar in Xenopus tissue culture cells. We propose that X29 is a member of a conserved family of nuclear decapping proteins that function in regulating the level of U8 snoRNA and other nuclear RNAs with methylated caps.

• Bobe R et al.
• Identification, expression, function, and localization of a novel (sixth) isoform of the human sarco/endoplasmic reticulum Ca2+ATPase 3 gene.
• J Biol Chem. 2004; 279: 24297-306
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• Understanding of Ca(2+) signaling requires the knowledge of proteins involved in this process. Among these proteins are sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) that pump Ca(2+) into the endoplasmic reticulum (ER). Recently, the human SERCA3 gene was shown to give rise to five isoforms (SERCA3a-e (h3a-h3e)). Here we demonstrate the existence of an additional new member, termed SERCA3f (h3f). By reverse transcriptase-PCR using monocytic U937 cell RNA, h3f mRNA was found to exclude the antepenultimate exon 21. h3f mRNA expression appeared as a human-specific splice variant. It was not found in rats or mice. h3f mRNA gave rise to an h3f protein differing in its C terminus from h3a-h3e. Of particular interest, h3f diverged in the first amino acids after the first splice site but presented the same last 21 amino acids as h3b. Consequently, we further investigated the structure-function-location relationships of the h3b and h3f isoforms. Comparative functional study of h3b and h3f recombinant proteins in intact HEK-293 cells and in fractionated membranes showed the following distinct characteristics: (i) resting cytosolic Ca(2+) concentration ([Ca(2+)](c)) and (ii) ER Ca(2+) content ([Ca(2+)](er)); similar characteristics were shown for the following: (i) the effects of the SERCA inhibitor, thapsigargin, on Ca(2+) release ([Ca(2+)](Tg)) and subsequent Ca(2+) entry ([Ca(2+)](e)) and (ii) the low apparent Ca(2+) affinity and the enhanced rate of dephosphorylation of the E(2)P phosphoenzyme intermediate. Subcellular location of h3b and h3f by immunofluorescence and/or confocal microscopy using the h3b- and h3f-specific polyclonal and the pan-h3 monoclonal (PL/IM430) antibodies suggested overlapping but distinct ER location. The endogenous expression of h3f protein was also proved in U937 cells. Altogether these data suggest that the SERCA3 isoforms have a more widespread role in cellular Ca(2+) signaling than previously appreciated.

• Harger JW, Dinman JD
• Evidence against a direct role for the Upf proteins in frameshifting or nonsense codon readthrough.
• RNA. 2004; 10: 1721-9
• Display abstract

• The Upf proteins are essential for nonsense-mediated mRNA decay (NMD). They have also been implicated in the modulation of translational fidelity at viral frameshift signals and premature termination codons. How these factors function in both mRNA turnover and translational control remains unclear. In this study, mono- and bicistronic reporter systems were used in the yeast Saccharomyces cerevisae to differentiate between effects at the levels of mRNA turnover and those at the level of translation. We confirm that upfDelta mutants do not affect programmed frameshifting, and show that this is also true for mutant forms of eIF1/Sui1p. Further, bicistronic reporters did not detect defects in translational readthrough due to deletion of the UPF genes, suggesting that their function in termination is not as general a phenomenon as was previously believed. The demonstration that upf sui1 double mutants are synthetically lethal demonstrates an important functional interaction between the NMD and translation initiation pathway.

• Anantharaman V, Aravind L
• Novel conserved domains in proteins with predicted roles in eukaryotic cell-cycle regulation, decapping and RNA stability.
• BMC Genomics. 2004; 5: 45-45
• Display abstract

• BACKGROUND: The emergence of eukaryotes was characterized by the expansion and diversification of several ancient RNA-binding domains and the apparent de novo innovation of new RNA-binding domains. The identification of these RNA-binding domains may throw light on the emergence of eukaryote-specific systems of RNA metabolism. RESULTS: Using sensitive sequence profile searches, homology-based fold recognition and sequence-structure superpositions, we identified novel, divergent versions of the Sm domain in the Scd6p family of proteins. This family of Sm-related domains shares certain features of conventional Sm domains, which are required for binding RNA, in addition to possessing some unique conserved features. We also show that these proteins contain a second previously uncharacterized C-terminal domain, termed the FDF domain (after a conserved sequence motif in this domain). The FDF domain is also found in the fungal Dcp3p-like and the animal FLJ22128-like proteins, where it fused to a C-terminal domain of the YjeF-N domain family. In addition to the FDF domains, the FLJ22128-like proteins contain yet another divergent version of the Sm domain at their extreme N-terminus. We show that the YjeF-N domains represent a novel version of the Rossmann fold that has acquired a set of catalytic residues and structural features that distinguish them from the conventional dehydrogenases. CONCLUSIONS: Several lines of contextual information suggest that the Scd6p family and the Dcp3p-like proteins are conserved components of the eukaryotic RNA metabolism system. We propose that the novel domains reported here, namely the divergent versions of the Sm domain and the FDF domain may mediate specific RNA-protein and protein-protein interactions in cytoplasmic ribonucleoprotein complexes. More specifically, the protein complexes containing Sm-like domains of the Scd6p family are predicted to regulate the stability of mRNA encoding proteins involved in cell cycle progression and vesicular assembly. The Dcp3p and FLJ22128 proteins may localize to the cytoplasmic processing bodies and possibly catalyze a specific processing step in the decapping pathway. The explosive diversification of Sm domains appears to have played a role in the emergence of several uniquely eukaryotic ribonucleoprotein complexes, including those involved in decapping and mRNA stability.

• Robalino J, Joshi B, Fahrenkrug SC, Jagus R
• Two zebrafish eIF4E family members are differentially expressed and functionally divergent.
• J Biol Chem. 2004; 279: 10532-41
• Display abstract

• Eukaryotic translation initiation factor 4E (eIF4E) is an essential component of the translational machinery that binds m(7)GTP and mediates the recruitment of capped mRNAs by the small ribosomal subunit. Recently, a number of proteins with homology to eIF4E have been reported in plants, invertebrates, and mammals. Together with the prototypical translation factor, these constitute a new family of structurally related proteins. To distinguish the prototypical translation factor eIF4E from other family members, it has been termed eIF4E-1 (Keiper, B. D., Lamphear, B. J., Deshpande, A. M., Jankowska-Anyszka, M., Aamodt, E. J., Blumenthal, T., and Rhoads, R. E. (2000) J. Biol. Chem. 275, 10590-10596). We describe the characterization of two eIF4E family members in the zebrafish Danio rerio. Based on their relative identities with human eIF4E-1, these zebrafish proteins are termed eIF4E-1A (82%) and eIF4E-1B (66%). eIF4E-1B, originally termed eIF4E(L), has been reported previously as the zebrafish eIF4E-1 counterpart (Fahrenkrug, S. C., Dahlquist, M. O., Clark, K., and Hackett, P. B. (1999) Differentiation 65, 191-201; Fahrenkrug, S. C., Joshi, B., Hackett, P. B., and Jagus, R. (2000) Differentiation 66, 15-22). Sequence comparisons suggest that the two genes probably evolved from a duplication event that occurred during vertebrate evolution. eIF4E-1A is expressed ubiquitously in zebrafish, whereas expression of eIF4E-1B is restricted to early embryonic development and to gonads and muscle of the tissues investigated. The ability of these two zebrafish proteins to bind m(7)GTP, eIF4G, and 4E-BP, as well as to complement yeast conditionally deficient in functional eIF4E, show that eIF4E-1A is a functional equivalent of human eIF4E-1. Surprisingly, although eIF4E-1B possesses all known residues thought to be required for interaction with the cap structure, eIF4G, and 4E-BPs, it fails to interact with any of these components, suggesting that this protein serves a role other than that assigned to eIF4E.

• Maquat LE
• Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics.
• Nat Rev Mol Cell Biol. 2004; 5: 89-99
• Display abstract

• Studies of nonsense-mediated mRNA decay in mammalian cells have proffered unforeseen insights into changes in mRNA-protein interactions throughout the lifetime of an mRNA. Remarkably, mRNA acquires a complex of proteins at each exon-exon junction during pre-mRNA splicing that influences the subsequent steps of mRNA translation and nonsense-mediated mRNA decay. Complex-loaded mRNA is thought to undergo a pioneer round of translation when still bound by cap-binding proteins CBP80 and CBP20 and poly(A)-binding protein 2. The acquisition and loss of mRNA-associated proteins accompanies the transition from the pioneer round to subsequent rounds of translation, and from translational competence to substrate for nonsense-mediated mRNA decay.

• Gotting C et al.
• Analysis of the DXD motifs in human xylosyltransferase I required for enzyme activity.
• J Biol Chem. 2004; 279: 42566-73
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• Human xylosyltransferase I (XT-I) is the initial enzyme involved in the biosynthesis of the glycosaminoglycan linker region in proteoglycans. Here, we tested the importance of the DXD motifs at positions 314-316 and 745-747 for enzyme activity and the nucleotide binding capacity of human XT-I. Mutations of the 314DED316 motif did not have any effect on enzyme activity, whereas alterations of the 745DWD747 motif resulted in reduced XT-I activity. Loss of function was observed after exchange of the highly conserved aspartic acid at position 745 with glycine. However, mutation of Asp745 to glutamic acid retained full enzyme activity, indicating the importance of an acidic amino acid at this position. Reduced substrate affinity was observed for mutants D747G (Km=6.9 microm) and D747E (Km=4.4 microm) in comparison with the wild-type enzyme (Km=0.9 microm). Changing the central tryptophan to a neutral, basic, or acidic amino acid resulted in a 6-fold lower Vmax, with Km values comparable with those of the wild-type enzyme. Despite the major effect of the DWD motif on XT-I activity, nucleotide binding was not abolished in the D745G and D747G mutants, as revealed by UDP-bead binding assays. Ki values for inhibition by UDP were determined to be 1.9-24.6 microm for the XT-I mutants. The properties of binding of XT-I to heparin-beads, the Ki constants for noncompetitive inhibition by heparin, and the activation by protamine were not altered by the generated mutations.

• Wang CH, Balasubramanian MK, Dokland T
• Structure, crystal packing and molecular dynamics of the calponin-homology domain of Schizosaccharomyces pombe Rng2.
• Acta Crystallogr D Biol Crystallogr. 2004; 60: 1396-403
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• Schizosaccharomyces pombe Rng2 is an IQGAP protein that is essential for the assembly of an actomyosin ring during cytokinesis. Rng2 contains an amino-terminal calponin-homology (CH) domain, 11 IQ repeats and a RasGAP-homology domain. CH domains are known mainly for their ability to bind F-actin, although they have other ligands in vivo and there are only few examples of actin-binding single CH domains. The structures of several CH domains have already been reported, but this is only the third report of an actin-binding protein that contains a single CH domain (the structures of calponin and EB1 have been reported previously). The 2.21 A resolution crystal structure of the amino-terminal 190 residues of Rng2 from Br- and Hg-derivatives includes 40 residues (150-190) carboxyl-terminal to the CH domain that resemble neither the extended conformation seen in utrophin, nor the compact conformation seen in fimbrin, although residues 154-160 form an unstructured coil which adopts a substructure similar to dystrophin residues 240-246 in the carboxyl-terminal portion of the CH2 domain. This region wraps around the stretch of residues that would be equivalent to the proposed actin-binding site ABS1 and ABS2 from dystrophin. This distinctive feature is absent from previously published CH-domain structures. Another feature revealed by comparing the two derivatives is the presence of two loop conformations between Tyr92 and Arg99.

• Davoodi J, Lin L, Kelly J, Liston P, MacKenzie AE
• Neuronal apoptosis-inhibitory protein does not interact with Smac and requires ATP to bind caspase-9.
• J Biol Chem. 2004; 279: 40622-8
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• The neuronal apoptosis-inhibitory protein (NAIP) is the founding member of the mammalian family of inhibitor of apoptosis (IAP) proteins (also known as BIRC proteins) and has been shown to be antiapoptotic both in vivo and in vitro. The 160-kDa NAIP contains three distinct regions: an amino-terminal cluster of three baculoviral inhibitory repeat (BIR) domains, a central nucleotide binding oligomerization domain (NOD), and a carboxyl-terminal leucine-rich repeat (LRR) domain. The presence of the NOD and LRR domains renders NAIP unique among the IAPs and suggests that NAIP activity is regulated in a manner distinct from that of other members of the family. In this report, we examined the interaction of various regions of NAIP with caspase-9 and Smac. Recombinant NAIPs with truncations of the carboxyl-terminal LRR or NOD-LRR regions bound to caspase-9. In contrast, the full-length protein did not, suggesting some form of structural autoregulation. However, the association of the wild type full-length protein with caspase-9 was observed when interaction analysis was performed in the presence of ATP. Furthermore, mutation of the NAIP ATP binding pocket allowed full-length protein to interact with caspase-9. Thus, we conclude that NAIP binds to caspase-9 with a structural requirement for ATP and that in the absence of ATP the LRR domain negatively regulates the caspase-9-inhibiting activity of the BIR domains. Interestingly, and in contrast to the X-chromosome-linked inhibitor of apoptosis protein (XIAP), NAIP-mediated inhibition of caspase-9 was not countered by a peptide containing an amino-terminal IAP binding motif (IBM). Consistent with this observation was the failure of Smac protein to interact with the NAIP BIR domains. These results demonstrate that NAIP is distinct from the other IAPs, both in demonstrating a ligand-dependent caspase-9 interaction and in demonstrating a distinct mechanism of inhibition.

• Yang SW et al.
• Expression of the telomeric repeat binding factor gene NgTRF1 is closely coordinated with the cell division program in tobacco BY-2 suspension culture cells.
• J Biol Chem. 2003; 278: 21395-407
• Display abstract

• Telomeres are vital for preserving chromosome integrity during cell division. Several genes encoding potential telomere-binding proteins have recently been identified in higher plants, but nothing is known about their function or regulation during cell division. In this study, we have isolated and characterized a cDNA clone, pNgTRF1, encoding a putative double-stranded telomeric repeat binding factor of Nicotiana glutinosa, a diploid tobacco plant. The predicted protein sequence of NgTRF1 (Mr = 75,000) contains a single Myb-like domain with significant homology to a corresponding motif in human TRF1/Pin2 and TRF2. Gel retardation assays revealed that bacterially expressed full-length NgTRF1 was able to form a specific complex only with probes containing three or more contiguous telomeric TTTAGGG repeats. The Myb-like domain of NgTRF1 is essential, but not sufficient, to bind the telomeric repeat sequence. The glutamine-rich extreme C-terminal region, which does not exist in animal proteins, was additionally required to form a specific telomere-protein complex. The dissociation constant (Kd) of the Myb motif plus the glutamine-rich domain of NgTRF1 to the two-telomeric repeat sequence was evaluated to be 4.5 +/- 0.2 x 10-9 m, which is comparable to that of the Myb domain of human TRF1. Expression analysis showed that NgTRF1 gene activity was inversely correlated with the cell division capacity of tobacco root cells and during the 9-day culture period of BY-2 suspension cells, while telomerase activity was positively correlated with cell division. In synchronized BY-2 cells, NgTRF1 was selectively expressed in G1 phase, whereas telomerase activity peaked in S phase. These findings suggest that telomerase activity and NgTRF1 expression are differentially regulated in an opposing fashion during growth and cell division in tobacco plants. The possible physiological functions of NgTRF1 in tobacco cells are also discussed.

• Yamakami M, Yoshimori T, Yokosawa H
• Tom1, a VHS domain-containing protein, interacts with tollip, ubiquitin, and clathrin.
• J Biol Chem. 2003; 278: 52865-72
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• The gene for Tom1 was initially identified as a specific target of the oncogene v-myb. The Tom1 protein belongs to the VHS domain-containing protein family, and it has a GAT domain in a central part as well as an N-terminal VHS domain. VHS domain-containing proteins, including Hrs/Vps27, STAM, and GGA proteins, have been implicated in intracellular trafficking and sorting, but the role of Tom1 has not yet been elucidated. In this study, we found that Tom1 binds directly with ubiquitin chains and Tollip, which was initially isolated as a mediator of interleukin-1 signaling and has a capacity to bind ubiquitin chains. Gel filtration and subsequent Western blot analysis showed that endogenous Tom1 associates with Tollip to form a complex. In addition, Tom1 was found to be capable of binding to clathrin heavy chain through a typical clathrin-binding motif. Fluorescence microscopic analysis revealed that green fluorescent protein-Tom1 was localized predominantly in the cytoplasm, whereas its mutant with deletion of the clathrin-binding motif had a diffuse localization throughout the cell. Thus, we propose that a Tom1-Tollip complex functions as a factor that links polyubiquitinated proteins to clathrin.

• Slater LM, Allen MD, Bycroft M
• Structural variation in PWWP domains.
• J Mol Biol. 2003; 330: 571-6
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• The PWWP domain is a ubiquitous eukaryotic protein module characterised by a region of sequence similarity of approximately 80 amino acids containing a highly conserved PWWP motif. It is frequently found in proteins associated with chromatin. We have determined the structure of a PWWP domain from the S. pombe protein SPBC215.07c using NMR spectroscopy. The structure is composed of a five stranded beta barrel followed by two alpha helices. Comparison to the recently reported structure of a homologous domain from the mammalian DNA methyltransferase Dnmt3b reveals substantial differences both in the C-terminal helical region and in the PWWP motif.

• Cristillo AD, Nie L, Macri MJ, Bierer BE
• Cloning and characterization of N4WBP5A, an inducible, cyclosporine-sensitive, Nedd4-binding protein in human T lymphocytes.
• J Biol Chem. 2003; 278: 34587-97
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• We have cloned and characterized a human cDNA, designated N4WBP5A, that belongs to the family of Nedd4-binding proteins. We originally identified N4WBP5A as an unknown expressed sequence tag (AA770150) represented in a cDNA microarray analysis that was up-regulated upon activation of T cells and inhibited by cell treatment with the calcineurin phosphatase inhibitors, cyclosporine (CsA) and tacrolimus (FK506). The predicted N4WBP5A amino acid sequence of 242 amino acid residues reveals an open reading frame of 729 nucleotides with a corresponding molecular mass of 27.1 kDa. Detection of N4WBP5A mRNA by reverse transcription-PCR was consistent with the induction of N4WBP5A following mitogenic stimulation of T lymphocytes and inhibition by CsA. Immunoblot analysis revealed endogenous N4WBP5A protein to be up-regulated following T cell activation and inhibited by CsA. This regulation of N4WBP5A mRNA expression differed from that of its homologue (51% identical; 65% similar) N4WBP5. Like N4WBP5, however, expression of epitope-tagged N4WBP5A indicated that the protein is localized predominantly to the Golgi network. Here we show by co-precipitation experiments that N4WBP5A interacts with the WW domains of Nedd4, an E3 ubiquitin ligase. Taken together, our data suggest that N4WBP5A may play a regulatory role in modulating Nedd4 activity at the level of the Golgi apparatus in T lymphocytes.

• Beaudoin J, Mercier A, Langlois R, Labbe S
• The Schizosaccharomyces pombe Cuf1 is composed of functional modules from two distinct classes of copper metalloregulatory transcription factors.
• J Biol Chem. 2003; 278: 14565-77
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• In fission yeast, the genes encoding proteins that are components of the copper transporter family are controlled at the transcriptional level by the Cuf1 transcription factor. Under low copper availability, Cuf1 induces expression of the copper transporter genes. In contrast, sufficient levels of copper inactivate Cuf1 and expression of its target genes. Our study reveals that Cuf1 harbors a putative copper-binding motif, Cys-X-Cys-X(3)-Cys-X-Cys-X(2)-Cys-X(2)-His, within its carboxyl-terminal region to sense changing environmental copper levels. Binding studies reveal that the amino-terminal 174-residue segment of Cuf1 expressed as a fusion protein in Escherichia coli specifically interacts with the cis-acting copper transporter promoter element CuSE (copper-signaling element). Within this region, the first 61 amino acids of Cuf1 exhibit more overall homology to the Saccharomyces cerevisiae Ace1 copper-detoxifying factor (from residues 1 to 63) than to Mac1, its functional ortholog. Consistently, we demonstrate that a chimeric Cuf1 protein bearing the amino-terminal 63-residue segment of Ace1 complements cuf1 Delta null phenotypes. Furthermore, we show that Schizosaccharomyces pombe cuf1Delta mutant cells expressing the full-length S. cerevisiae Ace1 protein are hypersensitive to copper ions, with a concomitant up-regulation of CuSE-mediated gene expression in fission yeast. Taken together, these studies reveal that S. cerevisiae Ace1 1-63 is functionally exchangeable with S. pombe Cuf1 1-61, and the nature of the amino acids located downstream of this amino-terminal conserved region may be crucial in dictating the type of regulatory response required to establish and maintain copper homeostasis.

• Hausmann S, Pei Y, Shuman S
• Homodimeric quaternary structure is required for the in vivo function and thermal stability of Saccharomyces cerevisiae and Schizosaccharomyces pombe RNA triphosphatases.
• J Biol Chem. 2003; 278: 30487-96
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• Saccharomyces cerevisiae Cet1 and Schizosaccharomyces pombe Pct1 are the essential RNA triphosphatase components of the mRNA capping apparatus of budding and fission yeast, respectively. Cet1 and Pct1 share a baroque active site architecture and a homodimeric quaternary structure. The active site is located within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel) that rests on a globular core domain (the pedestal) composed of elements from both protomers of the homodimer. Earlier studies of the effects of alanine cluster mutations at the crystallographic dimer interface of Cet1 suggested that homodimerization is important for triphosphatase function in vivo, albeit not for catalysis. Here, we studied the effects of 14 single-alanine mutations on Cet1 activity and thereby pinpointed Asp280 as a critical side chain required for dimer formation. We find that disruption of the dimer interface is lethal in vivo and renders Cet1 activity thermolabile at physiological temperatures in vitro. In addition, we identify individual residues within the pedestal domain (Ile470, Leu519, Ile520, Phe523, Leu524, and Ile530) that stabilize Cet1 in vivo and in vitro. In the case of Pct1, we show that dimerization depends on the peptide segment 41VPKIEMNFLN50 located immediately prior to the start of the Pct1 catalytic domain. Deletion of this peptide converts Pct1 into a catalytically active monomer that is defective in vivo in S. pombe and hypersensitive to thermal inactivation in vitro. Our findings suggest an explanation for the conservation of quaternary structure in fungal RNA triphosphatases, whereby the delicate tunnel architecture of the active site is stabilized by the homodimeric pedestal domain.

• Piccirillo C, Khanna R, Kiledjian M
• Functional characterization of the mammalian mRNA decapping enzyme hDcp2.
• RNA. 2003; 9: 1138-47
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• Regulation of decapping is a critical determinant of mRNA stability. We recently identified hDcp2 as a human decapping enzyme with intrinsic decapping activity. This activity is specific to N(7)-methylated guanosine containing RNA. The hDcp2 enzyme does not function on the cap structure alone and is not sensitive to competition by cap analog, suggesting that hDcp2 requires the RNA for cap recognition. We now demonstrate that hDcp2 is an RNA-binding protein and its recognition and hydrolysis of the cap substrate is dependent on an initial interaction with the RNA moiety. A biochemical characterization of hDcp2 revealed that a 163 amino acid region containing two evolutionarily conserved regions, the Nudix fold hydrolase domain and the adjacent Box B region contained methyl-cap-specific hydrolysis activity. Maximum decapping activity for wild-type as well as truncation mutants of hDcp2 required Mn(2+) as a divalent cation. The demonstration that hDcp2 is an RNA-binding protein with an RNA-dependent decapping activity will now provide new approaches to identify specific mRNAs that are regulated by this decapping enzyme as well as provide novel avenues to control mRNA decapping and turnover by influencing the RNA-binding property of hDcp2.

• Abdelghany HM, Bailey S, Blackburn GM, Rafferty JB, McLennan AG
• Analysis of the catalytic and binding residues of the diadenosine tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans by site-directed mutagenesis.
• J Biol Chem. 2003; 278: 4435-9
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• The contributions to substrate binding and catalysis of 13 amino acid residues of the Caenorhabditis elegans diadenosine tetraphosphate pyrophosphohydrolase (Ap(4)A hydrolase) predicted from the crystal structure of an enzyme-inhibitor complex have been investigated by site-directed mutagenesis. Sixteen glutathione S-transferase-Ap(4)A hydrolase fusion proteins were expressed and their k(cat) and K(m) values determined after removal of the glutathione S-transferase domain. As expected for a Nudix hydrolase, the wild type k(cat) of 23 s(-1) was reduced by 10(5)-, 10(3)-, and 30-fold, respectively, by replacement of the conserved P(4)-phosphate-binding catalytic residues Glu(56), Glu(52), and Glu(103) by Gln. K(m) values were not affected, indicating a lack of importance for substrate binding. In contrast, mutating His(31) to Val or Ala and Lys(83) to Met produced 10- and 16-fold increases in K(m) compared with the wild type value of 8.8 microm. These residues stabilize the P(1)-phosphate. H31V and H31A had a normal k(cat) but K83M showed a 37-fold reduction in k(cat). Lys(36) also stabilizes the P(1)-phosphate and a K36M mutant had a 10-fold reduced k(cat) but a relatively normal K(m). Thus both Lys(36) and Lys(83) may play a role in catalysis. The previously suggested roles of Tyr(27), His(38), Lys(79), and Lys(81) in stabilizing the P(2) and P(3)-phosphates were not confirmed by mutagenesis, indicating the absence of phosphate-specific binding contacts in this region. Also, mutating both Tyr(76) and Tyr(121), which clamp one substrate adenosine moiety between them in the crystal structure, to Ala only increased K(m) 4-fold. It is concluded that interactions with the P(1)- and P(4)-phosphates are minimum and sufficient requirements for substrate binding by this class of enzyme, indicating that it may have a much wider substrate range then previously believed.

• Sanchez R, Franco A, Gacto M, Notario V, Cansado J
• Characterization of gdp1+ as encoding a GDPase in the fission yeast Schizosaccharomyces pombe.
• FEMS Microbiol Lett. 2003; 228: 33-8
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• We have isolated the gdp1+ gene from Schizosaccharomyces pombe coding for a membrane protein with guanosine diphosphatase (GDPase) activity, which is highly homologous to Golgi GDPases isolated from other yeast species. The gdp1+ product, Gdp1p, displays both GDPase and uridine diphosphatase (UDPase) activities in vitro, with a strong dependence for calcium and manganese cations. The observation of a defect in N-glycosylation of invertase in S. pombe Deltagdp1 cells together with the ability of gdp1+ to functionally complement the defective O-mannosylation of chitinase in Saccharomyces cerevisiae cells disrupted in the GDA1 gene (gdp1+ homolog), suggests a main role for Gdp1p in protein glycosylation in fission yeast.

• Bauer S, Kemter K, Bacher A, Huber R, Fischer M, Steinbacher S
• Crystal structure of Schizosaccharomyces pombe riboflavin kinase reveals a novel ATP and riboflavin-binding fold.
• J Mol Biol. 2003; 326: 1463-73
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• The essential redox cofactors riboflavin monophosphate (FMN) and flavin adenine dinucleotide (FAD) are synthesised from their precursor, riboflavin, in sequential reactions by the metal-dependent riboflavin kinase and FAD synthetase. Here, we describe the 1.6A crystal structure of the Schizosaccharomyces pombe riboflavin kinase. The enzyme represents a novel family of phosphoryl transferring enzymes. It is a monomer comprising a central beta-barrel clasped on one side by two C-terminal helices that display an L-like shape. The opposite side of the beta-barrel serves as a platform for substrate binding as demonstrated by complexes with ADP and FMN. Formation of the ATP-binding site requires significant rearrangements in a short alpha-helix as compared to the substrate free form. The diphosphate moiety of ADP is covered by the glycine-rich flap I formed from parts of this alpha-helix. In contrast, no significant changes are observed upon binding of riboflavin. The ribityl side-chain might be covered by a rather flexible flap II. The unusual metal-binding site involves, in addition to the ADP phosphates, only the strictly conserved Thr45. This may explain the preference for zinc observed in vitro.

• Eystathioy T, Jakymiw A, Chan EK, Seraphin B, Cougot N, Fritzler MJ
• The GW182 protein colocalizes with mRNA degradation associated proteins hDcp1 and hLSm4 in cytoplasmic GW bodies.
• RNA. 2003; 9: 1171-3
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• A novel cytoplasmic compartment referred to as GW bodies (GWBs) was initially identified using antibodies specific to a 182-kD protein termed GW182. GW182 was characterized by multiple glycine(G)-tryptophan(W) repeats and an RNA recognition motif (RRM) that bound a subset of HeLa cell messenger RNAs (mRNAs). The function of GWBs was not known; however, more recent evidence suggested similarities between GWBs and cytoplasmic structures that contain hLSm proteins and hDcp1, the human homolog to a yeast decapping enzyme subunit. In this study, we used antibodies to hLSm4 and hDcp1 to show that both of these markers of an mRNA degradation pathway colocalize to the same structures as GW182. Our studies demonstrate that GW182, hLSm4, and hDcp1 are found in the same cytoplasmic structures and suggest that GW182 is involved in the same mRNA processing pathway as hLSm4 and hDcp1.

• Ueno M et al.
• Molecular characterization of the Schizosaccharomyces pombe nbs1+ gene involved in DNA repair and telomere maintenance.
• Mol Cell Biol. 2003; 23: 6553-63
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• The human MRN complex is a multisubunit nuclease that is composed of Mre11, Rad50, and Nbs1 and is involved in homologous recombination and DNA damage checkpoints. Mutations of the MRN genes cause genetic disorders such as Nijmegen breakage syndrome. Here we identified a Schizosaccharomyces pombe nbs1(+) homologue by screening for mutants with mutations that caused methyl methanesulfonate (MMS) sensitivity and were synthetically lethal with the rad2Delta mutation. Nbs1 physically interacts with the C-terminal half of Rad32, the Schizosaccharomyces pombe Mre11 homologue, in a yeast two-hybrid assay. nbs1 mutants showed sensitivities to gamma-rays, UV, MMS, and hydroxyurea and displayed telomere shortening similar to the characteristics of rad32 and rad50 mutants. nbs1, rad32, and rad50 mutant cells were elongated and exhibited abnormal nuclear morphology. These findings indicate that S. pombe Nbs1 forms a complex with Rad32-Rad50 and is required for homologous recombination repair, telomere length regulation, and the maintenance of chromatin structure. Amino acid sequence features and some characteristics of the DNA repair function suggest that the S. pombe Rad32-Rad50-Nbs1 complex has functional similarity to the corresponding MRN complexes of higher eukaryotes. Therefore, S. pombe Nbs1 will provide an additional model system for studying the molecular function of the MRN complex associated with genetic diseases.

• Vartiainen MK, Sarkkinen EM, Matilainen T, Salminen M, Lappalainen P
• Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated.
• J Biol Chem. 2003; 278: 34347-55
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• Twinfilin is a highly conserved actin monomer-binding protein that regulates cytoskeletal dynamics in organisms from yeast to mammals. In addition to the previously characterized mammalian twinfilin-1, a second protein with approximately 65% sequence identity to twinfilin-1 exists in mouse and humans. However, previous studies failed to identify any actin binding activity in this protein (Rohwer, A., Kittstein, W., Marks, F., and Gschwendt, M. (1999) Eur. J. Biochem. 263, 518-525). Here we show that this protein, which we named twinfilin-2, is indeed an actin monomer-binding protein. Similar to twinfilin-1, mouse twinfilin-2 binds ADP-G-actin with a higher affinity (KD = 0.12 microM) than ATP-G-actin (KD = 1.96 microM) and efficiently inhibits actin filament assembly in vitro. Both mouse twinfilins inhibit the nucleotide exchange on actin monomers and directly interact with capping protein. Furthermore, the actin interactions of mouse twinfilin-1 and twinfilin-2 are inhibited by phosphatidylinositol (4,5)-bisphosphate. Although biochemically very similar, our Northern blots and in situ hybridizations show that these two proteins display distinct expression patterns. Twinfilin-1 is the major isoform in embryos and in most adult mouse non-muscle cell-types, whereas twinfilin-2 is the predominant isoform of adult heart and skeletal muscles. Studies with isoform-specific antibodies demonstrated that although the two proteins show similar localizations in unstimulated cells, they are regulated by different mechanisms. The small GTPases Rac1 and Cdc42 induce the redistribution of twinfilin-1 to membrane ruffles and cell-cell contacts, respectively, but do not affect the localization of twinfilin-2. Taken together, these data show that mammals have two twinfilin isoforms, which are differentially expressed and regulated through distinct cellular signaling pathways.

• Pei Y, Schwer B, Shuman S
• Interactions between fission yeast Cdk9, its cyclin partner Pch1, and mRNA capping enzyme Pct1 suggest an elongation checkpoint for mRNA quality control.
• J Biol Chem. 2003; 278: 7180-8
• Display abstract

• RNA polymerase II (pol II) is subject to an early elongation delay induced by negative factors Spt5/Spt4 and NELF, which is overcome by the positive factor P-TEFb (Cdk9/cyclin T), a protein kinase that phosphorylates the pol II C-terminal domain (CTD) and the transcription elongation factor Spt5. Although the rationale for this arrest and restart is unclear, recent studies suggest a connection to mRNA capping, which is coupled to transcription elongation via physical and functional interactions between the cap-forming enzymes, the CTD-PO(4), and Spt5. Here we identify a novel interaction between fission yeast RNA triphosphatase Pct1, the enzyme that initiates cap formation, and Schizosaccharomyces pombe Cdk9. The C-terminal segment of SpCdk9 comprises a Pct1-binding domain distinct from the N-terminal Cdk domain. We show that the Cdk domain interacts with S. pombe Pch1, a homolog of cyclin T, and that the purified recombinant SpCdk9/Pch1 heterodimer can phosphorylate both the pol II CTD and the C-terminal domain of S. pombe Spt5. We provide genetic evidence that SpCdk9 and Pch1 are functional orthologs of the Saccharomyces cerevisiae CTD kinase Bur1/Bur2, a putative yeast P-TEFb. Mutations of the kinase active site and the regulatory T-loop of SpCdk9 abolish its activity in vivo. Deleting the C-terminal domain of SpCdk9 causes a severe growth defect. We suggest a model whereby Spt5-induced arrest of early elongation ensures a temporal window for recruitment of the capping enzymes, which in turn attract Cdk9 to alleviate the arrest. This elongation checkpoint may avoid wasteful rounds of transcription of uncapped pre-mRNAs.

• Mazzoni C et al.
• A truncated form of KlLsm4p and the absence of factors involved in mRNA decapping trigger apoptosis in yeast.
• Mol Biol Cell. 2003; 14: 721-9
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• The LSM4 gene of Saccharomyces cerevisiae codes for an essential protein involved in pre-mRNA splicing and also in mRNA decapping, a crucial step for mRNA degradation. We previously demonstrated that the first 72 amino acids of the Kluyveromyces lactis Lsm4p (KlLsm4p), which contain the Sm-like domains, can restore cell viability in both K. lactis and S. cerevisiae cells not expressing the endogenous protein. However, the absence of the carboxy-terminal region resulted in a remarkable loss of viability in stationary phase cells (). Herein, we demonstrate that S. cerevisiae cells expressing the truncated LSM4 protein of K. lactis showed the phenotypic markers of yeast apoptosis such as chromatin condensation, DNA fragmentation, and accumulation of reactive oxygen species. The study of deletion mutants revealed that apoptotic markers were clearly evident also in strains lacking genes involved in mRNA decapping, such as LSM1, DCP1, and DCP2, whereas a slight effect was observed in strains lacking the genes DHH1 and PAT1. This is the first time that a connection between mRNA stability and apoptosis is reported in yeast, pointing to mRNA decapping as the crucial step responsible of the observed apoptotic phenotypes.

• Bremer KA, Stevens A, Schoenberg DR
• An endonuclease activity similar to Xenopus PMR1 catalyzes the degradation of normal and nonsense-containing human beta-globin mRNA in erythroid cells.
• RNA. 2003; 9: 1157-67
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• beta-globin mRNA bearing a nonsense codon is degraded in the cytoplasm of erythroid cells by endonuclease cleavage, preferentially at UG dinucleotides. An endonuclease activity in polysomes of MEL cells cleaved beta-globin and albumin mRNA in vitro at many of the same sites as PMR1, an mRNA endonuclease purified from Xenopus liver. Stable transfection of MEL cells expressing normal human beta-globin mRNA with a plasmid vector expressing the catalytically active form of PMR1 reduced the half-life of beta-globin mRNA from 12 to 1-2 h without altering GAPDH mRNA decay. The reduced stability of beta-globin mRNA in these cells was accompanied by an increase in the production of mRNA decay products corresponding to those seen in the degradation of nonsense-containing beta-globin mRNA. Therefore, beta-globin mRNA is cleaved in vivo by an endonuclease with properties similar to PMR1. Inhibiting translation with cycloheximide stabilized nonsense-containing beta-globin mRNA, resulting in a fivefold increase in its steady-state level. Taken together, our results indicate that the surveillance of nonsense-containing beta-globin mRNA in erythroid cells is a cytoplasmic process that functions on translating mRNA, and endonucleolytic cleavage constitutes one step in the process of beta-globin mRNA decay.

• Kwasnicka DA, Krakowiak A, Thacker C, Brenner C, Vincent SR
• Coordinate expression of NADPH-dependent flavin reductase, Fre-1, and Hint-related 7meGMP-directed hydrolase, DCS-1.
• J Biol Chem. 2003; 278: 39051-8
• Display abstract

• A novel human cytosolic flavin reductase, Nr1, was recently described that contains FMN, FAD, and NADPH cofactors. Though the targets of the related NADPH-dependent flavoprotein reductases, cytochrome P450 reductase, methionine synthase reductase, and nitric oxide synthase, are known, the cellular function of Nr1 is not clear. To explore expression and regulation of Nr1, we cloned fre-1, the Caenorhabditis elegans ortholog of Nr1, and discovered that it is transcribed as a bicistronic pre-mRNA together with dcs-1, the ortholog of the recently described scavenger mRNA decapping enzyme. We used the novel substrate, 7meGpppBODIPY, to demonstrate that DCS-1 has low micromolar specificity for guanine ribonucleotides with the 7me modification, whereas trimethylated G substrates are poor competitors. Contrary to earlier classification, DCS-1 is not a pyrophosphatase but a distant member of the Hint branch of the histidine triad superfamily of nucleotide hydrolases and transferases. These observations are consistent with the hypothesis that DCS-1 homologs may function in the metabolism of capped oligonucleotides generated following exosome-dependent degradation of short-lived mRNA transcripts. We find that fre-1 and dcs-1 are coordinately expressed through worm development, are induced by heat shock, and have a nearly identical expression profile in human tissues. Furthermore, immunocytochemical analysis of the endogenous proteins in COS cells indicates that both are present in the nucleus and concentrated in a distinct perinuclear structure. Though no connection between these enzymes had been anticipated, our data and data from global expression and protein association studies suggest that the two enzymes jointly participate in responses to DNA damage, heat shock, and other stresses.

• Sciorra VA, Rudge SA, Wang J, McLaughlin S, Engebrecht J, Morris AJ
• Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes.
• J Cell Biol. 2002; 159: 1039-49
• Display abstract

• Phospholipase D (PLD) generates lipid signals that coordinate membrane trafficking with cellular signaling. PLD activity in vitro and in vivo is dependent on phosphoinositides with a vicinal 4,5-phosphate pair. Yeast and mammalian PLDs contain an NH2-terminal pleckstrin homology (PH) domain that has been speculated to specify both subcellular localization and regulation of PLD activity through interaction with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2). We report that mutation of the PH domains of yeast and mammalian PLD enzymes generates catalytically active PI(4,5)P2-regulated enzymes with impaired biological functions. Disruption of the PH domain of mammalian PLD2 results in relocalization of the protein from the PI(4,5)P2-containing plasma membrane to endosomes. As a result of this mislocalization, mutations within the PH domain render the protein unresponsive to activation in vivo. Furthermore, the integrity of the PH domain is vital for yeast PLD function in both meiosis and secretion. Binding of PLD2 to model membranes is enhanced by acidic phospholipids. Studies with PLD2-derived peptides suggest that this binding involves a previously identified polybasic motif that mediates activation of the enzyme by PI(4,5)P2. By comparison, the PLD2 PH domain binds PI(4,5)P2 with lower affinity but sufficient selectivity to function in concert with the polybasic motif to target the protein to PI(4,5)P2-rich membranes. Phosphoinositides therefore have a dual role in PLD regulation: membrane targeting mediated by the PH domain and stimulation of catalysis mediated by the polybasic motif.

• Rader SD, Guthrie C
• A conserved Lsm-interaction motif in Prp24 required for efficient U4/U6 di-snRNP formation.
• RNA. 2002; 8: 1378-92
• Display abstract

• The assembly of the U4 and U6 snRNPs into the U4/U6 di-snRNP is necessary for pre-mRNA splicing, and in Saccharomyces cerevisiae requires the splicing factor Prp24. We have identified a family of Prp24 homologs that includes the human protein SART3/p110nrb, which had been identified previously as a surface antigen in several cancers. Sequence conservation among the Prp24 homologs reveals the existence of a fourth previously unidentified RNA recognition motif (RRM) in Prp24, which we demonstrate is necessary for growth of budding yeast at 37 degrees C. The family is also characterized by a highly conserved 12-amino-acid motif at the extreme C terminus. Deletion of this motif in Prp24 causes a cold-sensitive growth phenotype and a decrease in base-paired U4/U6 levels in vivo. The mutant protein also has a reduced association with U6 snRNA in extract, and is unable to interact with the U6 Lsm proteins by two-hybrid assay. In vitro annealing assays demonstrate that deletion of the motif causes a defect in U4/U6 formation by reducing binding of Prp24 to its substrate. We conclude that the conserved C-terminal motif of Prp24 interacts with the Lsm proteins to promote U4/U6 formation.

• Ramirez CV, Vilela C, Berthelot K, McCarthy JE
• Modulation of eukaryotic mRNA stability via the cap-binding translation complex eIF4F.
• J Mol Biol. 2002; 318: 951-62
• Display abstract

• Decapping by Dcp1 in Saccharomyces cerevisiae is a key step in mRNA degradation. However, the cap also binds the eukaryotic initiation factor (eIF) complex 4F and its associated proteins. Characterisation of the relationship between decapping and interactions involving eIF4F is an essential step towards understanding polysome disassembly and mRNA decay. Three types of observation suggest how changes in the functional status of eIF4F modulate mRNA stability in vivo. First, partial disruption of the interaction between eIF4E and eIF4G, caused by mutations in eIF4E or the presence of the yeast 4E-binding protein p20, stabilised mRNAs. The interactions of eIF4G and p20 with eIF4E may therefore act to modulate the decapping process. Since we also show that the in vitro decapping rate is not directly affected by the nature of the body of the mRNA, this suggests that changes in eIF4F structure could play a role in triggering decapping during mRNA decay. Second, these effects were seen in the absence of extreme changes in global translation rates in the cell, and are therefore relevant to normal mRNA turnover. Third, a truncated form of eIF4E (Delta196) had a reduced capacity to inhibit Dcp1-mediated decapping in vitro, yet did not change cellular mRNA half-lives. Thus, the accessibility of the cap to Dcp1 in vivo is not simply controlled by competition with eIF4E, but is subject to switching between molecular states with different levels of access.

• Lykke-Andersen J
• Identification of a human decapping complex associated with hUpf proteins in nonsense-mediated decay.
• Mol Cell Biol. 2002; 22: 8114-21
• Display abstract

• Decapping is a key step in general and regulated mRNA decay. In Saccharomyces cerevisiae it constitutes a rate-limiting step in the nonsense-mediated decay pathway that rids cells of mRNAs containing premature termination codons. Here two human decapping enzymes are identified, hDcp1a and hDcp2, as well as a homolog of hDcp1a, termed hDcp1b. Transiently expressed hDcp1a and hDcp2 proteins localize primarily to the cytoplasm and form a complex in human cell extracts. hDcp1a and hDcp2 copurify with decapping activity, an activity sensitive to mutation of critical hDcp residues. Importantly, coimmunoprecipitation assays demonstrate that hDcp1a and hDcp2 interact with the nonsense-mediated decay factor hUpf1, both in the presence and in the absence of the other hUpf proteins, hUpf2, hUpf3a, and hUpf3b. These data suggest that a human decapping complex may be recruited to mRNAs containing premature termination codons by the hUpf proteins.

• Wang Z, Jiao X, Carr-Schmid A, Kiledjian M
• The hDcp2 protein is a mammalian mRNA decapping enzyme.
• Proc Natl Acad Sci U S A. 2002; 99: 12663-8
• Display abstract

• Decapping of mRNA is a critical step in eukaryotic mRNA turnover, yet the proteins involved in this activity remain elusive in mammals. We identified the human Dcp2 protein (hDcp2) as an enzyme containing intrinsic decapping activity. hDcp2 specifically hydrolyzed methylated capped RNA to release m(7)GDP; however, it did not function on the cap structure alone. hDcp2 is therefore functionally distinct from the recently identified mammalian scavenger decapping enzyme, DcpS. hDcp2-mediated decapping required a functional Nudix (nucleotide diphosphate linked to an X moiety) pyrophosphatase motif as mutations in conserved amino acids within this motif disrupted the decapping activity. hDcp2 is detected exclusively in the cytoplasm and predominantly cosediments with polysomes. Consistent with the localization of hDcp2, endogenous Dcp2-like decapping activity was detected in polysomal fractions prepared from mammalian cells. Similar to decapping in yeast, the presence of the poly(A) tail was inhibitory to the endogenous decapping activity, yet unlike yeast, competition of cap-binding proteins by cap analog did not influence the efficiency of decapping. Therefore the mammalian homologue of the yeast Dcp2 protein is an mRNA decapping enzyme demonstrated to contain intrinsic decapping activity.

• Decker CJ, Parker R
• mRNA decay enzymes: decappers conserved between yeast and mammals.
• Proc Natl Acad Sci U S A. 2002; 99: 12512-4

• Terwilliger TC
• Automated structure solution, density modification and model building.
• Acta Crystallogr D Biol Crystallogr. 2002; 58: 1937-40
• Display abstract

• The approaches that form the basis of automated structure solution in SOLVE and RESOLVE are described. The use of a scoring scheme to convert decision making in macromolecular structure solution to an optimization problem has proven very useful and in many cases a single clear heavy-atom solution can be obtained and used for phasing. Statistical density modification is well suited to an automated approach to structure solution because the method is relatively insensitive to choices of numbers of cycles and solvent content. The detection of non-crystallographic symmetry (NCS) in heavy-atom sites and checking of potential NCS operations against the electron-density map has proven to be a reliable method for identification of NCS in most cases. Automated model building beginning with an FFT-based search for helices and sheets has been successful in automated model building for maps with resolutions as low as 3 A. The entire process can be carried out in a fully automatic fashion in many cases.

• Bailey S et al.
• The crystal structure of diadenosine tetraphosphate hydrolase from Caenorhabditis elegans in free and binary complex forms.
• Structure. 2002; 10: 589-600
• Display abstract

• The crystal structure of C. elegans Ap(4)A hydrolase has been determined for the free enzyme and a binary complex at 2.0 A and 1.8 A, respectively. Ap(4)A hydrolase has a key role in regulating the intracellular Ap(4)A levels and hence potentially the cellular response to metabolic stress and/or differentiation and apoptosis via the Ap(3)A/Ap(4)A ratio. The structures reveal that the enzyme has the mixed alpha/beta fold of the Nudix family and also show how the enzyme binds and locates its substrate with respect to the catalytic machinery of the Nudix motif. These results suggest how the enzyme can catalyze the hydrolysis of a range of related dinucleoside tetraphosphate, but not triphosphate, compounds through precise orientation of key elements of the substrate.

• Sousa S et al.
• The ARO4 gene of Candida albicans encodes a tyrosine-sensitive DAHP synthase: evolution, functional conservation and phenotype of Aro3p-, Aro4p-deficient mutants.
• Microbiology. 2002; 148: 1291-303
• Display abstract

• The enzyme 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyses the first step in aromatic amino acid biosynthesis in prokaryotes, plants and fungi. Cells of Saccharomyces cerevisiae contain two catalytically redundant DAHP synthases, encoded by the genes ARO3 and ARO4, whose activities are feedback-inhibited by phenylalanine and tyrosine, respectively. ARO3/4 gene transcription is controlled by GCN4. The authors previously cloned an ARO3 gene orthologue from Candida albicans and found that: (1) it can complement an aro3 aro4 double mutation in S. cerevisiae, an effect inhibited by excess phenylalanine, and (2) a homozygous aro3-deletion mutant of C. albicans is phenotypically Aro(+), suggesting the existence of another isozyme(s). They now report the identification and functional characterization of the C. albicans orthologue of S. cerevisiae Aro4p. The two Aro4p enzymes share 68% amino acid identity. Phylogenetic analysis places the fungal DAHP synthases in a cluster separate from prokaryotic orthologues and suggests that ARO3 and ARO4 arose from a single gene via a gene duplication event early in fungal evolution. C. albicans ARO4 mRNA is elevated upon amino acid starvation, consistent with the presence of three putative Gcn4p-responsive elements (GCREs) in the gene promoter sequence. C. albicans ARO4 complements an aro3 aro4 double mutation in S. cerevisiae, an effect inhibited by excess tyrosine. The authors engineered Deltaaro3/Deltaaro3 Deltaaro4/MET3p::ARO4 cells of C. albicans (with one wild-type copy of ARO4 placed under control of the repressible MET3 promoter) and found that they fail to grow in the absence of aromatic amino acids when ARO4 expression is repressed, and that this growth defect can be partially rescued by aromatic amino acids and certain aromatic amino acid pathway intermediates. It is concluded that, like S. cerevisiae, C. albicans contains two DAHP synthases required for the first step in the aromatic amino acid biosynthetic pathway.

• Jiang L, Lee CM, Shen SH
• Functional characterization of the Candida albicans homologue of secretion-associated and Ras-related (Sar1) protein.
• Yeast. 2002; 19: 423-8
• Display abstract

• Secretion-associated and Ras-related protein (Sar1p) plays an essential role during the protein transport from the endoplasmic reticulum to the Golgi apparatus. The cDNA sequence of the Sar1 gene has been identified and characterized from the human yeast pathogen, Candida albicans. This cDNA encodes a protein of 190 amino acids, which shares a 78% sequence identity with Saccharomyces cerevisiae Sar1p and contains the conserved GTP-binding motifs of the small GTPase superfamily. Complementation studies confirmed that this cDNA encodes the functional homologue of ScSar1p. The recombinant C. albicans Sar1p exhibits GTP-binding activity in vitro that was abolished by deletion of one of the three GTP-binding motifs.

• Ingelfinger D, Arndt-Jovin DJ, Luhrmann R, Achsel T
• The human LSm1-7 proteins colocalize with the mRNA-degrading enzymes Dcp1/2 and Xrnl in distinct cytoplasmic foci.
• RNA. 2002; 8: 1489-501
• Display abstract

• Sm and Sm-like (LSm) proteins form heptameric complexes that are involved in various steps of RNA metabolism. In yeast, the Lsm1-7 complex functions in mRNA degradation and is associated with several enzymes of this pathway, while the complex LSm2-8, the composition of which largely overlaps with that of LSm1-7, has a role in pre-mRNA splicing. A human gene encoding an LSm1 homolog has been identified, but its role in mRNA degradation has yet to be elucidated. We performed subcellular localization studies and found hLSm1 predominantly in the cytoplasm. However, it is not distributed evenly; rather, it is highly enriched in small, discrete foci. The endogenous hLSm4 is similarly localized, as are the overexpressed proteins hLSm1-7, but not hLSm8. The foci also contain two key factors in mRNA degradation, namely the decapping enzyme hDcp1/2 and the exonuclease hXrn1. Moreover, coexpression of wild-type and mutant LSm proteins, as well as fluorescence resonance energy transfer (FRET) studies, indicate that the mammalian proteins hLSm1-7 form a complex similar to the one found in yeast, and that complex formation is required for enrichment of the proteins in the cytoplasmic foci. Therefore, the foci contain a partially or fully assembled machinery for the degradation of mRNA.

• Valasek L, Phan L, Schoenfeld LW, Valaskova V, Hinnebusch AG
• Related eIF3 subunits TIF32 and HCR1 interact with an RNA recognition motif in PRT1 required for eIF3 integrity and ribosome binding.
• EMBO J. 2001; 20: 891-904
• Display abstract

• eIF3 binds to 40S ribosomal subunits and stimulates recruitment of Met-tRNAiMet and mRNA to the pre-initiation complex. Saccharomyces cerevisiae contains an ortholog of human eIF3 subunit p35, HCR1, whose interactions with yeast eIF3 are not well defined. We found that HCR1 has a dual function in translation initiation: it binds to, and stabilizes, the eIF3-eIF5- eIF1-eIF2 multifactor complex and is required for the normal level of 40S ribosomes. The RNA recognition motif (RRM) of eIF3 subunit PRT1 interacted simultaneously with HCR1 and with an internal domain of eIF3 subunit TIF32 that has sequence and functional similarity to HCR1. PRT1, HCR1 and TIF32 were also functionally linked by genetic suppressor analysis. We propose that HCR1 stabilizes or modulates interaction between TIF32 and the PRT1 RRM. Removal of the PRT1 RRM resulted in dissociation of TIF32, NIP1, HCR1 and eIF5 from eIF3 in vivo, and destroyed 40S ribosome binding by the residual PRT1-TIF34-TIF35 subcomplex. Hence, the PRT1 RRM is crucial for the integrity and ribosome-binding activity of eIF3.

• Tucker M, Valencia-Sanchez MA, Staples RR, Chen J, Denis CL, Parker R
• The transcription factor associated Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae.
• Cell. 2001; 104: 377-86
• Display abstract

• The major pathways of mRNA turnover in eukaryotes initiate with shortening of the poly(A) tail. We demonstrate by several criteria that CCR4 and CAF1 encode critical components of the major cytoplasmic deadenylase in yeast. First, both Ccr4p and Caf1p are required for normal mRNA deadenylation in vivo. Second, both proteins localize to the cytoplasm. Third, purification of Caf1p copurifies with a Ccr4p-dependent poly(A)-specific exonuclease activity. We also provide evidence that the Pan2p/Pan3p nuclease complex encodes the predominant alternative deadenylase. These results, and previous work on Pan2p/Pan3p, define the mRNA deadenylases in yeast. The strong conservation of Ccr4p, Caf1p, Pan2p, and Pan3p indicates that they will function as deadenylases in other eukaryotes. Interestingly, because Ccr4p and Caf1p interact with transcription factors, these results suggest an unexpected link between mRNA synthesis and turnover.

• Dunckley T, Parker R
• Yeast mRNA decapping enzyme.
• Methods Enzymol. 2001; 342: 226-33

• Bond AT, Mangus DA, He F, Jacobson A
• Absence of Dbp2p alters both nonsense-mediated mRNA decay and rRNA processing.
• Mol Cell Biol. 2001; 21: 7366-79
• Display abstract

• Dbp2p, a member of the large family of DEAD-box proteins and a yeast homolog of human p68, was shown to interact with Upf1p, an essential component of the nonsense-mediated mRNA decay pathway. Dbp2p:Upf1p interaction occurs within a large conserved region in the middle of Upf1p that is largely distinct from its Nmd2p and Sup35/45p interaction domains. Deletion of DBP2, or point mutations within its highly conserved DEAD-box motifs, increased the abundance of nonsense-containing transcripts, leading us to conclude that Dbp2p also functions in the nonsense-mediated mRNA decay pathway. Dbp2p, like Upf1p, acts before or at decapping, is predominantly cytoplasmic, and associates with polyribosomes. Interestingly, Dbp2p also plays an important role in rRNA processing. In dbp2Delta cells, polyribosome profiles are deficient in free 60S subunits and the mature 25S rRNA is greatly reduced. The ribosome biogenesis phenotype, but not the mRNA decay function, of dbp2Delta cells can be complemented by the human p68 gene. We propose a unifying model in which Dbp2p affects both nonsense-mediated mRNA decay and rRNA processing by altering rRNA structure, allowing specific processing events in one instance and facilitating dissociation of the translation termination complex in the other.

• Gao M, Wilusz CJ, Peltz SW, Wilusz J
• A novel mRNA-decapping activity in HeLa cytoplasmic extracts is regulated by AU-rich elements.
• EMBO J. 2001; 20: 1134-43
• Display abstract

• While decapping plays a major role in mRNA turnover in yeast, biochemical evidence for a similar activity in mammalian cells has been elusive. We have now identified a decapping activity in HeLa cytoplasmic extracts that releases (7me)GDP from capped transcripts. Decapping is activated in extracts by the addition of (7me)GpppG, which specifically sequesters cap-binding proteins such as eIF4E and the deadenylase DAN/PARN. Similar to in vivo observations, the presence of a poly(A) tail represses decapping of RNAs in vitro in a poly(A)-binding protein-dependent fashion. AU-rich elements (AREs), which act as regulators of mRNA stability in vivo, are potent stimulators of decapping in vitro. The stimulation of decapping by AREs requires sequence-specific ARE-binding proteins. These data suggest that cap recognition and decapping play key roles in mediating mRNA turnover in mammalian cells.

• Sassoon J, Lilie H, Baumann U, Kohli J
• Biochemical characterization of the structure-specific DNA-binding protein Cmb1 from Schizosaccharomyces pombe.
• J Mol Biol. 2001; 309: 1101-15
• Display abstract

• Cmb1, a novel HMG box protein from Schizosaccharomyces pombe, has been characterized biochemically using glutaraldehyde cross-linking, gel-filtration and analytical ultracentrifugation. It was identified as a monomeric, non-spherical protein, with a tendency to aggregate in solution. Limited proteolysis with trypsin and chymotrypsin showed that the C-terminal HMG box was a compact, proteolytically stable domain and the N-terminal region of Cmb1 was relatively unstructured and more easily digested. As Cmb1 was previously identified as a potential mismatch-binding protein, the binding constants and stoichiometry for both homoduplex and heteroduplex DNA were determined using an IASys resonant mirror biosensor. Cmb1 indeed demonstrated a tighter association with mismatched DNA, especially with the C/Delta-mismatch. Expression constructs of Cmb1 were made to study the sections of the protein involved in DNA binding. Constructs with the N-terminal region absent revealed that the C-terminal HMG box was the primary DNA-binding region. The presence of the N-terminal region did, however, facilitate tighter binding to both homoduplex and heteroduplex DNA. The amino acid residues isoleucine 14 and leucine 39 were located as putative intercalating residues using structure guided homology modelling. The model templates were derived from two distinct HMG:DNA complexes: HMG-D bound to homoduplex DNA and HMG 1 bound to cisplatin DNA. Binding studies using the Cmb1 HMG box with point mutations in these residues showed that isoleucine 14 was important for the binding of Cmb1 to homoduplex DNA, but affected binding to mismatches to a lesser extent. In contrast, leucine 39 appeared to have a more significant function in binding to mismatched DNA.

• Albig AR, Decker CJ
• The target of rapamycin signaling pathway regulates mRNA turnover in the yeast Saccharomyces cerevisiae.
• Mol Biol Cell. 2001; 12: 3428-38
• Display abstract

• The target of rapamycin (TOR) signaling pathway is an important mechanism by which cell growth is regulated by nutrient availability in eukaryotes. We provide evidence that the TOR signaling pathway controls mRNA turnover in Saccharomyces cerevisiae. During nutrient limitation (diauxic shift) or after treatment with rapamycin (a specific inhibitor of TOR), multiple mRNAs were destabilized, whereas the decay of other mRNAs was unaffected. Our findings suggest that the regulation of mRNA decay by the TOR pathway may play a significant role in controlling gene expression in response to nutrient depletion. The inhibition of the TOR pathway accelerated the major mRNA decay mechanism in yeast, the deadenylation-dependent decapping pathway. Of the destabilized mRNAs, two different responses to rapamycin were observed. Some mRNAs were destabilized rapidly, while others were affected only after prolonged exposure. Our data suggest that the mRNAs that respond rapidly are destabilized because they have short poly(A) tails prematurely either as a result of rapid deadenylation or reduced polyadenylation. In contrast, the mRNAs that respond slowly are destabilized by rapid decapping. In summary, the control of mRNA turnover by the TOR pathway is complex in that it specifically regulates the decay of some mRNAs and not others and that it appears to control decay by multiple mechanisms.

• Martinez-Ruiz A et al.
• RNase U2 and alpha-sarcin: a study of relationships.
• Methods Enzymol. 2001; 341: 335-51

• Ptushkina M, Berthelot K, von der Haar T, Geffers L, Warwicker J, McCarthy JE
• A second eIF4E protein in Schizosaccharomyces pombe has distinct eIF4G-binding properties.
• Nucleic Acids Res. 2001; 29: 4561-9
• Display abstract

• The eukaryotic cap-binding proteins belonging to the eIF4E family are generally involved in mediating the recruitment of ribosomes to capped mRNA. We described previously a cap-binding protein (now called eIF4E1) in Schizosaccharomyces pombe that appears to have all of the usual structural and functional attributes of an eIF4E. We have now characterised a new type of cap-binding protein (eIF4E2) from this organism, which at the amino acid sequence level, is 52% identical and 59% similar to eIF4E1. eIF4E2 is not essential in S.pombe but has some novel properties that may be related to a special function in the cell. The ratio of eIF4E2:eIF4E1 in the cell shifts in favour of eIF4E2 at higher temperatures. Despite having all of the dorsal face amino acids that have so far been associated with eIF4G binding to eIF4E1, eIF4E2 binds the eIF4E-binding domain of S.pombe eIF4G >10(2)-times weaker than eIF4E1 in vitro. The eIF4E2 cap-binding affinity is in the typical micromolar range. The results suggest that eIF4E2 is not active on the main pathway of translation initiation in fission yeast but might play a role in the adaptation strategy of this organism under specific growth conditions. Moreover, they provide insight into the molecular characteristics required for tight binding to eIF4G.

• Tsutsui Y, Khasanov FK, Shinagawa H, Iwasaki H, Bashkirov VI
• Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe.
• Genetics. 2001; 159: 91-105
• Display abstract

• Schizosaccharomyces pombe Rhp55 and Rhp57 are RecA-like proteins involved in double-strand break (DSB) repair. Here we demonstrate that Rhp55 and Rhp57 proteins strongly interact in vivo, similar to Saccharomyces cerevisiae Rad55p and Rad57p. Mutations in the conserved ATP-binding/hydrolysis folds of both the Rhp55 and Rhp57 proteins impaired their function in DNA repair but not in cell proliferation. However, when combined, ATPase fold mutations in Rhp55p and Rhp57p resulted in severe defects of both functions, characteristic of the deletion mutants. Yeast two-hybrid analysis also revealed other multiple in vivo interactions among S. pombe proteins involved in recombinational DNA repair. Similar to S. cerevisiae Rad51p-Rad54p, S. pombe Rhp51p and Rhp54p were found to interact. Both putative Rad52 homologs in S. pombe, Rad22p and Rti1p, were found to interact with the C-terminal region of Rhp51 protein. Moreover, Rad22p and Rti1p exhibited mutual, as well as self-, interactions. In contrast to the S. cerevisiae interacting pair Rad51p-Rad55p, S. pombe Rhp51 protein strongly interacted with Rhp57 but not with Rhp55 protein. In addition, the Rti1 and Rad22 proteins were found to form a complex with the large subunit of S. pombe RPA. Our data provide compelling evidence that most, but not all, of the protein-protein interactions found in S. cerevisiae DSB repair are evolutionarily conserved.

• Serin G, Gersappe A, Black JD, Aronoff R, Maquat LE
• Identification and characterization of human orthologues to Saccharomyces cerevisiae Upf2 protein and Upf3 protein (Caenorhabditis elegans SMG-4).
• Mol Cell Biol. 2001; 21: 209-23
• Display abstract

• Nonsense-mediated mRNA decay (NMD), also called mRNA surveillance, is an important pathway used by all organisms that have been tested to degrade mRNAs that prematurely terminate translation and, as a consequence, eliminate the production of aberrant proteins that could be potentially harmful. In mammalian cells, NMD appears to involve splicing-dependent alterations to mRNA as well as ribosome-associated components of the translational apparatus. To date, human (h) Upf1 protein (p) (hUpf1p), a group 1 RNA helicase named after its Saccharomyces cerevisiae orthologue that functions in both translation termination and NMD, has been the only factor shown to be required for NMD in mammalian cells. Here, we describe human orthologues to S. cerevisiae Upf2p and S. cerevisiae Upf3p (Caenorhabditis elegans SMG-4) based on limited amino acid similarities. The existence of these orthologues provides evidence for a higher degree of evolutionary conservation of NMD than previously appreciated. Interestingly, human orthologues to S. cerevisiae Upf3p (C. elegans SMG-4) derive from two genes, one of which is X-linked and both of which generate multiple isoforms due to alternative pre-mRNA splicing. We demonstrate using immunoprecipitations of epitope-tagged proteins transiently produced in HeLa cells that hUpf2p interacts with hUpf1p, hUpf3p-X, and hUpf3p, and we define the domains required for the interactions. Furthermore, we find by using indirect immunofluorescence that hUpf1p is detected only in the cytoplasm, hUpf2p is detected primarily in the cytoplasm, and hUpf3p-X localizes primarily to nuclei. The finding that hUpf3p-X is a shuttling protein provides additional indication that NMD has both nuclear and cytoplasmic components.

• Wilusz CJ, Gao M, Jones CL, Wilusz J, Peltz SW
• Poly(A)-binding proteins regulate both mRNA deadenylation and decapping in yeast cytoplasmic extracts.
• RNA. 2001; 7: 1416-24
• Display abstract

• The pathway of mRNA degradation has been extensively studied in the yeast, Saccharomyces cerevisiae, and it is now clear that many mRNAs decay by a deadenylation-dependent mechanism. Although several of the factors required for mRNA decay have been identified, the regulation and precise roles of many of the proteins involved remains unclear. We have developed an in vitro system that recapitulates both the deadenylation and the decapping steps of mRNA decay. Furthermore, both deadenylation and decapping are inhibited by poly(A) binding proteins in our assay. Our system has allowed us to separate the decay process from translation and we have shown that the poly(A) tail is capable of inhibiting decapping in an eIF4E-independent manner. Our in vitro system should prove invaluable in dissecting the mechanisms of mRNA turnover.

• Gabelli SB, Bianchet MA, Bessman MJ, Amzel LM
• The structure of ADP-ribose pyrophosphatase reveals the structural basis for the versatility of the Nudix family.
• Nat Struct Biol. 2001; 8: 467-72
• Display abstract

• Regulation of cellular levels of ADP-ribose is important in preventing nonenzymatic ADP-ribosylation of proteins. The Escherichia coli ADP-ribose pyrophosphatase, a Nudix enzyme, catalyzes the hydrolysis of ADP-ribose to ribose-5-P and AMP, compounds that can be recycled as part of nucleotide metabolism. The structures of the apo enzyme, the active enzyme and the complex with ADP-ribose were determined to 1.9 A, 2.7 A and 2.3 A, respectively. The structures reveal a symmetric homodimer with two equivalent catalytic sites, each formed by residues of both monomers, requiring dimerization through domain swapping for substrate recognition and catalytic activity. The structures also suggest a role for the residues conserved in each Nudix subfamily. The Nudix motif residues, folded as a loop-helix-loop tailored for pyrophosphate hydrolysis, compose the catalytic center; residues conferring substrate specificity occur in regions of the sequence removed from the Nudix motif. This segregation of catalytic and recognition roles provides versatility to the Nudix family.

• Fromont-Racine M et al.
• Genome-wide protein interaction screens reveal functional networks involving Sm-like proteins.
• Yeast. 2000; 17: 95-110
• Display abstract

• A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein-protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale.

• Schwer B, Saha N, Mao X, Chen HW, Shuman S
• Structure-function analysis of yeast mRNA cap methyltransferase and high-copy suppression of conditional mutants by AdoMet synthase and the ubiquitin conjugating enzyme Cdc34p.
• Genetics. 2000; 155: 1561-76
• Display abstract

• Here we present a genetic analysis of the yeast cap-methylating enzyme Abd1p. To identify individual amino acids required for Abd1p function, we introduced alanine mutations at 35 positions of the 436-amino acid yeast protein. Two new recessive lethal mutations, F256A and Y330A, were identified. Alleles F256L and Y256L were viable, suggesting that hydrophobic residues at these positions sufficed for Abd1p function. Conservative mutations of Asp-178 established that an acidic moiety is essential at this position (i.e. , D178E was viable whereas D178N was not). Phe-256, Tyr-330, and Asp-178 are conserved in all known cellular cap methyltransferases. We isolated temperature-sensitive abd1 alleles and found that abd1-ts cells display a rapid shut-off of protein synthesis upon shift to the restrictive temperature, without wholesale reduction in steady-state mRNA levels. These in vivo results are consistent with classical biochemical studies showing a requirement for the cap methyl group in cap-dependent translation. We explored the issue of how cap methylation might be regulated in vivo by conducting a genetic screen for high-copy suppressors of the ts growth defect of abd1 mutants. The identification of the yeast genes SAM2 and SAM1, which encode AdoMet synthase, as abd1 suppressors suggests that Abd1p function can be modulated by changes in the concentration of its substrate AdoMet. We also identified the ubiquitin conjugating enzyme Cdc34p as a high-copy abd1 suppressor. We show that mutations of Cdc34p that affect its ubiquitin conjugation activity or its capacity to interact with the E3-SCF complex abrogate its abd1 suppressor function. Moreover, the growth defect of abd1 mutants is exacerbated by cdc34-2. These findings suggest a novel role for Cdc34p in gene expression and engender a model whereby cap methylation or cap utilization is negatively regulated by a factor that is degraded when Cdc34p is overexpressed.

• Bouveret E, Rigaut G, Shevchenko A, Wilm M, Seraphin B
• A Sm-like protein complex that participates in mRNA degradation.
• EMBO J. 2000; 19: 1661-71
• Display abstract

• In eukaryotes, seven Sm proteins bind to the U1, U2, U4 and U5 spliceosomal snRNAs while seven Smlike proteins (Lsm2p-Lsm8p) are associated with U6 snRNA. Another yeast Sm-like protein, Lsm1p, does not interact with U6 snRNA. Surprisingly, using the tandem affinity purification (TAP) method, we identified Lsm1p among the subunits associated with Lsm3p. Coprecipitation experiments demonstrated that Lsm1p, together with Lsm2p-Lsm7p, forms a new seven-subunit complex. We purified the two related Sm-like protein complexes and identified the proteins recovered in the purified preparations by mass spectrometry. This confirmed the association of the Lsm2p-Lsm8p complex with U6 snRNA. In contrast, the Lsm1p-Lsm7p complex is associated with Pat1p and Xrn1p exoribonuclease, suggesting a role in mRNA degradation. Deletions of LSM1, 6, 7 and PAT1 genes increased the half-life of reporter mRNAs. Interestingly, accumulating mRNAs were capped, suggesting a block in mRNA decay at the decapping step. These results indicate the involvement of a new conserved Sm-like protein complex and a new factor, Pat1p, in mRNA degradation and suggest a physical connection between decapping and exonuclease trimming.

• Ben-Yehuda S, Dix I, Russell CS, McGarvey M, Beggs JD, Kupiec M
• Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.
• Genetics. 2000; 156: 1503-17
• Display abstract

• The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.

• Cagney G, Uetz P, Fields S
• High-throughput screening for protein-protein interactions using two-hybrid assay.
• Methods Enzymol. 2000; 328: 3-14

• Bonnerot C, Boeck R, Lapeyre B
• The two proteins Pat1p (Mrt1p) and Spb8p interact in vivo, are required for mRNA decay, and are functionally linked to Pab1p.
• Mol Cell Biol. 2000; 20: 5939-46
• Display abstract

• We report here the characterization of a bypass suppressor of pab1Delta which leads to a fourfold stabilization of the unstable MFA2 mRNA. Cloning of the wild-type gene for that suppressor reveals that it is identical to PAT1 (YCR077c), a gene whose product was reported to interact with Top2p. PAT1 is not an essential gene, but its deletion leads to a thermosensitive phenotype. Further analysis has shown that PAT1 is allelic with mrt1-3, a mutation previously reported to affect decapping and to bypass suppress pab1Delta, as is also the case for dcp1, spb8, and mrt3. Coimmunoprecipitation experiments show that Pat1p is associated with Spb8p. On sucrose gradients, the two proteins cosediment with fractions containing the polysomes. In the absence of Pat1p, however, Spb8p no longer cofractionates with the polysomes, while the removal of Spb8p leads to a sharp decrease in the level of Pat1p. Our results suggest that some of the factors involved in mRNA degradation could be associated with the mRNA that is still being translated, awaiting a specific signal to commit the mRNA to the degradation pathway.

• Zhang S, Williams CJ, Wormington M, Stevens A, Peltz SW
• Monitoring mRNA decapping activity.
• Methods. 1999; 17: 46-51
• Display abstract

• mRNA decapping is a common step shared between two important mRNA decay pathways in yeast, Saccharomyces cerevisiae. To investigate how mRNAs are decapped, we have developed an assay that can be easily used to measure the decapping activity. This assay has been used to isolate yeast strains with altered decapping activities. The results demonstrated that decreased decapping activity in vitro corresponds well with the decapping-deficient phenotype in vivo. This assay has been applied to the purified yeast decapping enzyme Dcp1 protein as well as crude yeast extracts and Xenopus oocyte extracts.

• McLennan AG
• The MutT motif family of nucleotide phosphohydrolases in man and human pathogens (review).
• Int J Mol Med. 1999; 4: 79-89
• Display abstract

• Human cells express at least eight members of the MutT motif protein (or nudix hydrolase) family. These enzymes are believed to eliminate toxic nucleotide derivatives from the cell and regulate the levels of important signalling nucleotides and their metabolites. Six have been fully or partially characterized: i) hMTH1 is a nucleoside triphosphatase which restricts AT-->CG transversions by specifically degrading the oxidized nucleotide 8-oxo-dGTP; ii) hAPAH1 preferentially degrades the signalling dinucleotide Ap4A; iii) DIPP is unusual in hydrolysing two seemingly unrelated signalling substrate groups - the dinucleotides Ap6A and Ap5A, and the diphosphoinositol polyphosphates; iv) DIPP2 is closely related to DIPP; v) hYSAH1 is an NDP-sugar hydrolase which prefers ADP-ribose, and vi) hGFG is a protein of unknown function encoded by the antisense transcript of the basic fibroblast growth factor gene. Although not yet associated with known hereditary or acquired disorders, the functional loss of any one of these hydrolases would be expected to be detrimental to cellular function. Furthermore, the ialA invasion gene of Bartonella bacilliformis and other invasive pathogens encodes a MutT motif Ap4A hydrolase while poxviruses express two MutT motif proteins, at least one of which is essential for infectivity. This protein family, therefore, occupies a position of some importance in controlling human health and disease.

• Ford LP, Watson J, Keene JD, Wilusz J
• ELAV proteins stabilize deadenylated intermediates in a novel in vitro mRNA deadenylation/degradation system.
• Genes Dev. 1999; 13: 188-201
• Display abstract

• We have developed an in vitro mRNA stability system using HeLa cell cytoplasmic S100 extracts and exogenous polyadenylated RNA substrates that reproduces regulated aspects of mRNA decay. The addition of cold poly(A) competitor RNA activated both a sequence-specific deadenylase activity in the extracts as well as a potent, ATP-dependent ribonucleolytic activity. The rates of both deadenylation and degradation were up-regulated by the presence of a variety of AU-rich elements in the body of substrate RNAs. Competition analyses demonstrated that trans-acting factors were required for RNA destabilization by AU-rich elements. The approximately 30-kD ELAV protein HuR specifically bound to RNAs containing an AU-rich element derived from the TNF-alpha mRNA in the in vitro system. Interaction of HuR with AU-rich elements, however, was not associated with RNA destabilization. Interestingly, recombinant ELAV proteins specifically stabilized deadenylated intermediates generated from the turnover of AU-rich element-containing substrate RNAs. These data suggest that mammalian ELAV proteins play a role in regulating mRNA stability by influencing the access of degradative enzymes to RNA substrates.

• Katan-Khaykovich Y, Spiegel I, Shaul Y
• The dimerization/repression domain of RFX1 is related to a conserved region of its yeast homologues Crt1 and Sak1: a new function for an ancient motif.
• J Mol Biol. 1999; 294: 121-37
• Display abstract

• The RFX protein family includes members from yeast to humans, which function in various biological systems, and share a DNA-binding domain and a conserved C-terminal region. In the human transcription regulator RFX1, the conserved C terminus is an independent functional domain, which mediates dimerization and transcriptional repression. This dimerization domain has a unique ability to mediate the formation of two alternative homodimeric DNA-protein complexes, the upper of which has been linked to repression. Here, we localize the complex formation capacity to several different RFX1 C-terminal subregions, each of which can function independently to generate the upper complex and repress transcription, thus correlating complex formation with repression. To gain an evolutionary perspective, we have examined whether the different properties of the RFX1 C terminus exist in the two yeast RFX proteins, which are involved in signaling pathways. Replacement of the RFX1 C terminus with those of Sak1 and Crt1, its orthologues from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively, and analysis of fusions with the Gal4 DNA-binding domain, revealed that the ability to generate the two alternative complexes is conserved in the RFX family, from S. cerevisiae to man. While sharing this unique biochemical property, the three C termini differed from each other in their ability to mediate dimerization and transcriptional repression. In both functions, RFX1, Sak1, and Crt1 showed high capacity, moderate capacity, and no capacity, respectively. This comparative analysis of the RFX proteins, representing different evolutionary stages, suggests a gradual development of the conserved C terminus, from the appearance of the ancestral motif (Crt1), to the later acquisition of the dimerization/repression functions (Sak1), and finally to the enhancement of these functions to generate a domain mediating highly stable protein-protein interactions and potent transcriptional repression (RFX1).

• Czaplinski K, Ruiz-Echevarria MJ, Gonzalez CI, Peltz SW
• Should we kill the messenger? The role of the surveillance complex in translation termination and mRNA turnover.
• Bioessays. 1999; 21: 685-96
• Display abstract

• Eukaryotes have evolved conserved mechanisms to rid cells of faulty gene products that can interfere with cell function. mRNA surveillance is an example of a pathway that monitors the translation termination process and promotes degradation of transcripts harboring premature translation termination codons. Studies on the mechanism of mRNA surveillance in yeast and humans suggest a common mechanism where a "surveillance complex" monitors the translation process and determines whether translation termination has occurred at the correct position within the mRNA. A model will be presented that suggests that the surveillance complex assesses translation termination by monitoring the transition of an RNP as it is converted from a nuclear to a cytoplasmic form during the initial rounds of translation.

• Terwilliger TC, Berendzen J
• Automated MAD and MIR structure solution.
• Acta Crystallogr D Biol Crystallogr. 1999; 55: 849-61
• Display abstract

• Obtaining an electron-density map from X-ray diffraction data can be difficult and time-consuming even after the data have been collected, largely because MIR and MAD structure determinations currently require many subjective evaluations of the qualities of trial heavy-atom partial structures before a correct heavy-atom solution is obtained. A set of criteria for evaluating the quality of heavy-atom partial solutions in macromolecular crystallography have been developed. These have allowed the conversion of the crystal structure-solution process into an optimization problem and have allowed its automation. The SOLVE software has been used to solve MAD data sets with as many as 52 selenium sites in the asymmetric unit. The automated structure-solution process developed is a major step towards the fully automated structure-determination, model-building and refinement procedure which is needed for genomic scale structure determinations.

• Mao H, White SA, Williamson JR
• A novel loop-loop recognition motif in the yeast ribosomal protein L30 autoregulatory RNA complex.
• Nat Struct Biol. 1999; 6: 1139-47
• Display abstract

• The yeast Saccharomyces cerevisiae ribosomal protein L30 negatively autoregulates its production by binding to a helix-loop-helix structure formed in its pre-mRNA and its mRNA. A three-dimensional solution structure of the L30 protein in complex with its regulatory RNA has been solved using NMR spectroscopy. In the complex, the helix-loop-helix RNA adopts a sharply bent conformation at the internal loop region. Unusual RNA features include a purine stack, a reverse Hoogsteen base pair (G11anti-G56syn) and highly distorted backbones. The L30 protein is folded in a three-layer alpha/beta/alpha sandwich topology, and three loops at one end of the sandwich make base-specific contacts with the RNA internal loop. The protein-RNA binding interface is divided into two clusters, including hydrophobic and aromatic stacking interactions centering around G56, and base-specific hydrogen-bonding contacts to A57, G58 and G10-U60 wobble base pair. Both the protein and the RNA exhibit a partially induced fit for binding, where loops in the protein and the internal loop in the RNA become more ordered upon complex formation. The specific interactions formed between loops on L30 and the internal loop on the mRNA constitute a novel loop-loop recognition motif where an intimate RNA-protein interface is formed between regions on both molecules that lack regular secondary structure.

• Ebihara K, Nakamura Y
• C-terminal interaction of translational release factors eRF1 and eRF3 of fission yeast: G-domain uncoupled binding and the role of conserved amino acids.
• RNA. 1999; 5: 739-50
• Display abstract

• Translation termination in eukaryotes requires a stop codon-responsive (class-I) release factor, eRF1, and a guanine nucleotide-responsive (class-II) release factor, eRF3. Schizosaccharomyces pombe eRF3 has an N-terminal polypeptide similar in size to the prion-like domain of Saccharomyces cerevisiae eRF3 in addition to the EF-1alpha-like catalytic domain. By in vivo two-hybrid assay as well as by an in vitro pull-down analysis using purified proteins of S. pombe as well as of S. cerevisiae, eRF1 bound to the C-terminal one-third domain of eRF3, named eRF3C, but not to the N-terminal two-thirds, which was inconsistent with the previous report by Paushkin et al. (1997, Mol Cell Biol 17:2798-2805). The activity of S. pombe eRF3 in eRF1 binding was affected by Ala substitutions for the C-terminal residues conserved not only in eRF3s but also in elongation factors EF-Tu and EF-1alpha. These single mutational defects in the eRF1-eRF3 interaction became evident when either truncated protein eRF3C or C-terminally altered eRF1 proteins were used for the authentic protein, providing further support for the presence of a C-terminal interaction. Given that eRF3 is an EF-Tu/EF-1alpha homolog required for translation termination, the apparent dispensability of the N-terminal domain of eRF3 for binding to eRF1 is in contrast to importance, direct or indirect, in EF-Tu/EF-1alpha for binding to aminoacyl-tRNA, although both eRF3 and EF-Tu/EF-1alpha share some common amino acids for binding to eRF1 and aminoacyl-tRNA, respectively. These differences probably reflect the independence of eRF1 binding in relation to the G-domain function of eRF3 (i.e., probably uncoupled with GTP hydrolysis), whereas aminoacyl-tRNA binding depends on that of EF-Tu/EF-1alpha(i.e., coupled with GTP hydrolysis), which sheds some light on the mechanism of eRF3 function.

• Zuk D, Belk JP, Jacobson A
• Temperature-sensitive mutations in the Saccharomyces cerevisiae MRT4, GRC5, SLA2 and THS1 genes result in defects in mRNA turnover.
• Genetics. 1999; 153: 35-47
• Display abstract

• In a screen for factors involved in mRNA turnover, four temperature-sensitive yeast strains (ts1189, ts942, ts817, and ts1100) exhibited defects in the decay of several mRNAs. Complementation of the growth and mRNA decay defects, and genetic experiments, revealed that ts1189 is mutated in the previously unknown MRT4 gene, ts942 is mutated in GRC5 (encoding the L9 ribosomal protein), ts817 contains a mutation in SLA2 (encoding a membrane protein), and ts1100 contains a mutation in THS1 (encoding the threonyl-tRNA synthetase). Three of the four mutants (mrt4, grc5, and sla2) were not defective in protein synthesis, suggesting that these strains contain mutations in factors that may play a specific role in mRNA decay. The mRNA stabilization observed in the ths1 strain, however, could be due to the significant drop in translation observed in this mutant at 37 degrees. While the three interesting mutants appear to encode novel mRNA decay factors, at least one could be linked to a previously characterized mRNA decay pathway. The growth and mRNA decay defects of ts942 (grc5) cells were suppressed by overexpression of the NMD3 gene, encoding a protein shown to participate in a two-hybrid interaction with the nonsense-mediated decay protein Upf1p.

• Zhang S, Williams CJ, Hagan K, Peltz SW
• Mutations in VPS16 and MRT1 stabilize mRNAs by activating an inhibitor of the decapping enzyme.
• Mol Cell Biol. 1999; 19: 7568-76
• Display abstract

• Decapping is a rate-limiting step in the decay of many yeast mRNAs; the activity of the decapping enzyme therefore plays a significant role in determining RNA stability. Using an in vitro decapping assay, we have identified a factor, Vps16p, that regulates the activity of the yeast decapping enzyme, Dcp1p. Mutations in the VPS16 gene result in a reduction of decapping activity in vitro and in the stabilization of both wild-type and nonsense-codon-containing mRNAs in vivo. The mrt1-3 allele, previously shown to affect the turnover of wild-type mRNAs, results in a similar in vitro phenotype. Extracts from both vps16 and mrt1 mutant strains inhibit the activity of purified Flag-Dcp1p. We have identified a 70-kDa protein which copurifies with Flag-Dcp1p as the abundant Hsp70 family member Ssa1p/2p. Intriguingly, the interaction with Ssa1p/2p is enhanced in strains with mutations in vps16 or mrt1. We propose that Hsp70s may be involved in the regulation of mRNA decapping.

• Hilleren P, Parker R
• mRNA surveillance in eukaryotes: kinetic proofreading of proper translation termination as assessed by mRNP domain organization?
• RNA. 1999; 5: 711-9
• Display abstract

• In the last few years it has become clear that a conserved mRNA degradation system, referred to as mRNA surveillance, exists in eukaryotic cells to degrade aberrant mRNAs. This process plays an important role in checking that mRNAs have been properly synthesized and functions, at least in part, to increase the fidelity of gene expression by degrading aberrant mRNAs that, if translated, would produce truncated proteins. A critical issue is how normal and aberrant mRNAs are distinguished and how that distinction leads to differences in mRNA stability. Recent results suggest a model with three main points. First, mRNPs have a domain organization that is, in part, a reflection of the completion of nuclear pre-mRNA processing events. Second, the critical aspect of distinguishing a normal from an aberrant mRNA is the environment of the translation termination codon as determined by the organization of the mRNP domains. Third, the cell distinguishes proper from improper termination through an internal clock that is the rate of ATP hydrolysis by Upf1p. If termination is completed before ATP hydrolysis, the mRNA is protected from mRNA degradation. Conversely, if termination is slow, then ATP hydrolysis and a structural rearrangement occurs before termination is completed, which affects the fate of the terminating ribosome in a manner that fails to stabilize the mRNA. This proposed system of distinguishing normal from aberrant transcripts is similar to, but distinct from other systems of kinetic proofreading that affect the accuracy of other biogenic processes such as translation accuracy and spliceosome assembly.

• He W, Parker R
• Analysis of mRNA decay pathways in Saccharomyces cerevisiae.
• Methods. 1999; 17: 3-10
• Display abstract

• The analysis of mRNA turnover often requires a knowledge of the pathway by which a particular mRNA is being degraded. In this article we describe experimental procedures that can be used to determine the mechanism of degradation for yeast transcripts. These approaches include the insertion of strong secondary structures to block exonuclease cleavage and thereby trap decay intermediates. In addition, mRNA decay pathways can be analyzed by using regulatable promoters to perform transcriptional pulse-chase experiments, thereby allowing the determination of precursor-product relationships during the mRNA decay process. Finally, the mechanism of mRNA degradation can also now be determined by using trans-acting mutations specific for distinct mRNA turnover pathways. Most importantly, the combination of these three approaches can often clearly define the mechanism(s) by which a given transcript is degraded.

• Brunger AT et al.
• Crystallography & NMR system: A new software suite for macromolecular structure determination.
• Acta Crystallogr D Biol Crystallogr. 1998; 54: 905-21
• Display abstract

• A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.

• Sun X, Perlick HA, Dietz HC, Maquat LE
• A mutated human homologue to yeast Upf1 protein has a dominant-negative effect on the decay of nonsense-containing mRNAs in mammalian cells.
• Proc Natl Acad Sci U S A. 1998; 95: 10009-14
• Display abstract

• All eukaryotic cells analyzed have developed mechanisms to eliminate the production of mRNAs that prematurely terminate translation. The mechanisms are thought to exist to protect cells from the deleterious effects of in-frame nonsense codons that are generated by routine inefficiencies and inaccuracies in RNA metabolism such as pre-mRNA splicing. Depending on the particular mRNA and how it is produced, nonsense codons can mediate a reduction in mRNA abundance either (i) before its release from an association with nuclei into the cytoplasm, presumably but not certainly while the mRNA is being exported to the cytoplasm and translated by cytoplasmic ribosomes, or (ii) in the cytoplasm. Here, we provide evidence for a factor that functions to eliminate the production of nonsense-containing RNAs in mammalian cells. The factor, variously referred to as Rent1 (regulator of nonsense transcripts) or HUPF1 (human Upf1 protein), was identified by isolating cDNA for a human homologue to Saccharomyces cerevisiae Upf1p, which is a group I RNA helicase that functions in the nonsense-mediated decay of mRNA in yeast. Using monkey COS cells and human HeLa cells, we demonstrate that expression of human Upf1 protein harboring an arginine-to-cysteine mutation at residue 844 within the RNA helicase domain acts in a dominant-negative fashion to abrogate the decay of nonsense-containing mRNA that takes place (i) in association with nuclei or (ii) in the cytoplasm. These findings provide evidence that nonsense-mediated mRNA decay is related mechanistically in yeast and in mammalian cells, regardless of the cellular site of decay.

• Cartwright JL, McLennan AG
• The MutT motif-containing ORF163w protein from the yeast Saccharomyces cerevisiae encodes a (di)nucleoside polyphosphate hydrolase.
• Biochem Soc Trans. 1997; 25: 580-580

• Haberland J, Becker J, Gerke V
• The acidic C-terminal domain of rna1p is required for the binding of Ran.GTP and for RanGAP activity.
• J Biol Chem. 1997; 272: 24717-26
• Display abstract

• The small GTP binding protein Ran is an essential component of the nuclear protein import machinery whose GTPase cycle is regulated by the nuclear guanosine nucleotide exchange factor RCC1 and by the cytosolic GTPase activating protein RanGAP. In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae the RanGAP activity is encoded by the RNA1 genes which are essential for cell viability and nucleocytoplasmic transport in vivo. Although of limited sequence identity the two yeast proteins show a conserved structural organization characterized by an N-terminal domain of eight leucine-rich repeats, motifs implicated in protein-protein interactions, and a C-terminal domain rich in acidic amino acid residues. By analyzing the RanGAP activity of a series of recombinantly expressed rna1p mutant derivatives, we show that the highly acidic sequence in the C-terminal domain of both yeast proteins is indispensable for activating Ran-mediated GTP hydrolysis. Chemical cross-linking reveals that the same sequence in rna1p is required for rna1p.Ran complex formation indicating that the loss of GAP activity in the C-terminally truncated rna1p mutants results from an impaired interaction with Ran. The predominant species stabilized through the covalent cross-link is a rna1p.Ran heterodimer whose formation requires the GTP-bound conformation of Ran. As the acidic C-terminal domain of rna1p is required for establishing the interaction with Ran, the leucine-rich repeats domain in rna1p is potentially available for additional protein interactions perhaps required for directing a fraction of rna1p to the nuclear pore.

• Uesono Y, Toh-e A, Kikuchi Y
• Ssd1p of Saccharomyces cerevisiae associates with RNA.
• J Biol Chem. 1997; 272: 16103-9
• Display abstract

• The SSD1 gene has been isolated as a single copy suppressor of many mutants, such as sit4, slk1/bck1, pde2, and rpc31, in the yeast Saccharomyces cerevisiae. Ssd1p has domains showing weak but significant homology with RNase II-related proteins, Cyt4p, Dss1p, VacB, and RNase II, which are involved in the modification of RNA. We found that Ssd1p had the ability to bind RNA, preferably poly(rA), as well as single-stranded DNA. Interestingly, the most conserved domain among the RNase II-related proteins was not necessary for interaction with RNA. Indirect immunofluorescence staining with anti-Ssd1p antibody revealed that Ssd1p was detected mainly in the cytoplasm. Furthermore, sucrose gradient sedimentation analysis demonstrated that Ssd1p was not cofractionated with polyribosomes, suggesting that Ssd1p is not particularly bound to a translationally active subpopulation of mRNA in the cytoplasm.

• Caponigro G, Parker R
• Mechanisms and control of mRNA turnover in Saccharomyces cerevisiae.
• Microbiol Rev. 1996; 60: 233-49

• Ptushkina M et al.
• Schizosaccharomyces pombe has a novel eukaryotic initiation factor 4F complex containing a cap-binding protein with the human eIF4E C-terminal motif KSGST.
• J Biol Chem. 1996; 271: 32818-24
• Display abstract

• Genetic and biochemical analyses were performed on the cytoplasmic cap-binding complex (eukaryotic initiation factor (eIF) 4F) of Schizosaccharomyces pombe. Genomic and cDNA sequencing of the S. pombe gene (tif1) encoding the cap-binding component eIF4E revealed the presence of two introns in a reading frame of 219 codons. The encoded sequence of 218 amino acids shows a greater degree of identity to the mammalian eIF4E sequence than does its counterpart from Saccharomyces cerevisiae. In particular, unlike its S. cerevisiae counterpart, S.pombe eIF4E has a C-terminal Ser209 within the motif KSGST that is a site of phosphorylation in hamster and rabbit eIF4E. Of relevance to its potential regulatory role, eIF4E was found to be encoded by an mRNA with a six-nucleotide leader and to be of low abundance in vivo. Cross-linking experiments identified S. pombe eIF4E as the major cap-binding protein while a further protein, p36, also showed cap-dependent binding. eIF4A was not associated with the cap-binding complex. While S. pombe eIF4E was shown capable of binding S. cerevisiae p20, an equivalent protein was absent from the eIF4F complex isolated from S. pombe cells. S. pombe 4F therefore shows a remarkable combination of structural and functional properties, some of which it shares with its higher and its lower eukaryotic counterparts.

• James P, Halladay J, Craig EA
• Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.
• Genetics. 1996; 144: 1425-36
• Display abstract

• The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.

• Abeygunawardana C et al.
• Solution structure of the MutT enzyme, a nucleoside triphosphate pyrophosphohydrolase.
• Biochemistry. 1995; 34: 14997-5005
• Display abstract

• The MutT enzyme (129 residues) catalyzes the hydrolysis of normal and mutagenic nucleoside triphosphates, such as 8-oxo-dGTP, by substitution at the rarely attacked beta-P, to yield NMP and pyrophosphate. Previous heteronuclear NMR studies of MutT have shown the secondary structure to consist of a five-stranded mixed beta-sheet connected by the loop I-alpha-helix I--loop II motif, by two tight turns, and by loop III, and terminated by loop IV--alpha-helix II [Abeygunawardana et al. (1993) Biochemistry 32, 13071-13080; Weber et al. (1993) Biochemistry 32, 13081-13087). Complete side-chain assignments of 1H and 13C resonances have now been made by 3D C(CO)NH and HCCH-TOCSY experiments. A total of 1461 interproton proximities (11 per residue), obtained by 3D 15N-resolved NOESY-HSQC and 3D 13C-resolved NOESY-HSQC spectra, including 372 long-range NOEs, as well as 65 dihedral angle (phi) restraints and 34 backbone hydrogen bond restraints were used to determine the tertiary structure of MutT by distance geometry, simulated annealing, and energy minimization with the program X-PLOR. The structure is globular and compact with the parallel portion of the beta-sheet sandwiched between the two alpha-helices, forming an alpha+beta fold. The essential divalent cation has previously been shown to bind near residues Gly-37, Gly-38, Lys-39, and Glu-57, and nucleotides have been shown to bind near residues Leu-54 and Val-58 by NMR relaxation methods [Frick et al. (1995) Biochemistry 34, 5577-5586].(ABSTRACT TRUNCATED AT 250 WORDS)

• He F, Jacobson A
• Identification of a novel component of the nonsense-mediated mRNA decay pathway by use of an interacting protein screen.
• Genes Dev. 1995; 9: 437-54
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• Rapid turnover of nonsense-containing mRNAs in yeast in dependent on the product of the UPF1 gene (Upf1p). Mutations in UPF1 lead to the selective stabilization of mRNAs containing early nonsense mutations without affecting the decay rates of most other mRNAs. To identify other integral components of this decay pathway, we have employed a two-hybrid screen, seeking those cellular factors that specifically interact with Upf1p. Screening of yeast genomic libraries identified six genes encoding potential Upf1p-interacting proteins. These include four previously uncharacterized genes, NMD1-4 (nonsense-mediated mRNA decay), DBP2, a gene encoding a putative RNA helicase with homology to mammalian p68 RNA helicase, and SNP1, a gene encoding a U1 snRNP 70-kD protein homolog. In this paper we report the identification and characterization of NMD2, a yeast gene that encodes a specific Upf1p-interacting protein. Disruption of NMD2 yields a nonsense-mediated mRNA decay phenotype identical to that obtained in UPF1-deletion strains, indicating that the NMD2 gene product (Nmd2p) is a new factor in the nonsense-mediated mRNA decay pathway. Deletion analysis demonstrated that the acidic carboxyl terminus of Nmd2p constituted the Upf1p-interacting domain. High-level expression of a fragment of Nmd2p containing this domain had a dominant-negative effect on nonsense-mediated mRNA decay when the protein was localized the cytoplasm but not when it was localized to the nucleus, indicating that this decay pathway has a cytoplasmic component. The association of a dominant-negative phenotype with a gene fragment identified in a two-hybrid screen suggests a generalized approach to confirming the function of genes identified in such screens.

• Mejean V, Salles C, Bullions LC, Bessman MJ, Claverys JP
• Characterization of the mutX gene of Streptococcus pneumoniae as a homologue of Escherichia coli mutT, and tentative definition of a catalytic domain of the dGTP pyrophosphohydrolases.
• Mol Microbiol. 1994; 11: 323-30
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• We show that deletion of a gene of Streptococcus pneumoniae, which we call mutX, confers a mutator phenotype to resistance to streptomycin. Analysis of the DNA sequence changes that occurred in several streptomycin-resistant mutants showed that mutations are unidirectional AT to CG transversions. The mutX gene is located immediately downstream of the previously identified ung gene and genetic evidence suggests that the two genes are co-ordinately regulated. Nucleotide sequence determination reveals that the mutX gene encodes a 17,870 Da protein (154 residues) which exhibits significant homology with the MutT protein of Escherichia coli, a nucleoside triphosphatase (dGTP pyrophosphohydrolase). The mutX gene complements the E. coli mutT mutator phenotype when introduced on a plasmid. Site-directed mutagenesis and analysis of nitrosoguanidine-induced mutT mutants suggest that a small region of high homology between the two proteins (61% identity over 23 residues) is part of the catalytic site of the nucleoside triphosphatase. Computer searching for sequence homology to MutX uncovered a second E. coli protein, the product of orf17, a gene of unknown function located near the ruvC gene. The region of high homology between MutX and MutT is also conserved in this protein, which raises the interesting possibility that the orf17 gene plays some role in determining mutation rates in E. coli. Finally, a small set of proteins, including a family of virus-encoded proteins and two evolutionarily conserved proteins encoded by an antisense transcript from the Xenopus laevis and human bFGF genes, were also found to harbour significant homology to this highly conserved region.

• Burgess SM, Guthrie C
• A mechanism to enhance mRNA splicing fidelity: the RNA-dependent ATPase Prp16 governs usage of a discard pathway for aberrant lariat intermediates.
• Cell. 1993; 73: 1377-91
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• PRP16 encodes an RNA-dependent ATPase required for the second step of mRNA splicing in S. cerevisiae. We have isolated seven alleles of PRP16 that, like the original allele prp16-1, allow splicing of introns with a mutant branch site (UACUAAC to UACUACC), by forming lariat intermediates at the mutant C nucleotide. Every suppressor mutation maps to the region of PRP16 common to RNA-dependent ATPases. We purified three of the mutant proteins and found that all exhibit reduced ATPase activity, as does Prp16-1. An in vivo analysis of the steady-state levels of the splicing intermediates and products provides evidence for a pathway, under the genetic control of PRP16, to discard incorrectly branched substrates. We propose that decreasing the rate of ATP hydrolysis by Prp16 allows aberrantly formed lariat intermediates more time to proceed through the productive rather than the discard branch of this pathway.

• Koonin EV
• A highly conserved sequence motif defining the family of MutT-related proteins from eubacteria, eukaryotes and viruses.
• Nucleic Acids Res. 1993; 21: 4847-4847

• Seraphin B
• The HIT protein family: a new family of proteins present in prokaryotes, yeast and mammals.
• DNA Seq. 1992; 3: 177-9
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• By comparing the sequence of a putative translation product from a Saccharomyces cerevisiae split gene with several data-bases, I have uncovered a new protein family. Members of this family are found in prokaryotes as well as in lower and higher eukaryotes. The function of these proteins is unknown but they share a characteristic histidine triad that may be involved in zinc binding. This group of protein has been named the HIT protein family.

• Kawamukai M et al.
• Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe.
• Mol Biol Cell. 1992; 3: 167-80
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• We have identified, cloned, and studied a gene, cap, encoding a protein that is associated with adenylyl cyclase in the fission yeast Schizosaccharomyces pombe. This protein shares significant sequence homology with the adenylyl cyclase-associated CAP protein in the yeast Saccharomyces cerevisiae. CAP is a bifunctional protein; the N-terminal domain appears to be involved in cellular responsiveness to RAS, whereas loss of the C-terminal portion is associated with morphological and nutritional defects. S. pombe cap can suppress phenotypes associated with deletion of the C-terminal CAP domain in S. cerevisiae but does not suppress phenotypes associated with deletion of the N-terminal domain. Analysis of cap disruptants also mapped the function of cap to two domains. The functional loss of the C-terminal region of S. pombe cap results in abnormal cellular morphology, slow growth, and failure to grow at 37 degrees C. Increases in mating and sporulation were observed when the entire gene was disrupted. Overproduction of both cap and adenylyl cyclase results in highly elongated large cells that are sterile and have measurably higher levels of adenylyl cyclase activity. Our results indicate that cap is required for the proper function of S. pombe adenylyl cyclase but that the C-terminal domain of cap has other functions that are shared with the C-terminal domain of S. cerevisiae CAP.

• Olesen JT, Fikes JD, Guarente L
• The Schizosaccharomyces pombe homolog of Saccharomyces cerevisiae HAP2 reveals selective and stringent conservation of the small essential core protein domain.
• Mol Cell Biol. 1991; 11: 611-9
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• The fission yeast Schizosaccharomyces pombe is immensely diverged from budding yeast (Saccharomyces cerevisiae) on an evolutionary time scale. We have used a fission yeast library to clone a homolog of S. cerevisiae HAP2, which along with HAP3 and HAP4 forms a transcriptional activation complex that binds to the CCAAT box. The S. pombe homolog php2 (S. pombe HAP2) was obtained by functional complementation in an S. cerevisiae hap2 mutant and retains the ability to associate with HAP3 and HAP4. We have previously demonstrated that the HAP2 subunit of the CCAAT-binding transcriptional activation complex from S. cerevisiae contains a 65-amino-acid "essential core" structure that is divisible into subunit association and DNA recognition domains. Here we show that Php2 contains a 60-amino-acid block that is 82% identical to this core. The remainder of the 334-amino-acid protein is completely without homology to HAP2. The function of php2 in S. pombe was investigated by disrupting the gene. Strikingly, like HAP2 in S. cerevisiae, the S. pombe gene is specifically involved in mitochondrial function. This contrasts to the situation in mammals, in which the homologous CCAAT-binding complex is a global transcriptional activator.

• Leeds P, Peltz SW, Jacobson A, Culbertson MR
• The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon.
• Genes Dev. 1991; 5: 2303-14
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• mRNA decay rates often increase when translation is terminated prematurely due to a frameshift or nonsense mutation. We have identified a yeast gene, UPF1, that codes for a trans-acting factor whose function is necessary for enhanced turnover of mRNAs containing a premature stop codon. In the absence of UPF1 function, frameshift or nonsense mutations in the HIS4 or LEU2 genes that normally cause rapid mRNA decay fail to have this effect. Instead, the mRNAs decay at rates similar to the corresponding wild-type mRNAs. The stabilization of frameshift or nonsense mRNAs observed in upf1- strains does not appear to result from enhanced readthrough of the termination signal. Loss of UPF1 function has no effect on the accumulation or stability of HIS4+ or LEU2+ mRNA, suggesting that the UPF1 product functions only in response to a premature termination signal. When we examined the accumulation and stability of other wild-type mRNAs in the presence or absence of UPF1, including MAT alpha 1, STE3, ACT1, PGK1, PAB1, and URA3 mRNAs, only the URA3 transcript was affected. On the basis of these and other results, the UPF1 product appears to participate in a previously uncharacterized pathway leading to the degradation of a limited class of yeast transcripts.

• Stevens A
• An mRNA decapping enzyme from ribosomes of Saccharomyces cerevisiae.
• Biochem Biophys Res Commun. 1980; 96: 1150-5

• Bergmann IE, Brawerman G
• Loss of the polyadenylate segment from mammalian messenger RNA. Selective cleavage of this sequence from polyribosomes.
• J Mol Biol. 1980; 139: 439-54

• Dornan V, Cook EA, Carey NH
• The polyA regions of hen oviduct RNA.
• Nucleic Acids Res. 1974; 1: 1561-71
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• Total RNA from hen oviduct has been hydrolysed with a mixture of T(1) and pancreatic ribonucleases. Poly(A) tracts in the digestion product have been isolated by binding to oligo(dT) cellulose. Of the four major ribonucleotides, the product has been shown to contain only adenylic acid. When separated on polyacrylamide gels, the poly(A) gave two peaks corresponding to average apparent lengths of 270-280 and 540-550 nucleotides.

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