NORMAL GENOMIC

• Grzechnik P, Tan-Wong SM, Proudfoot NJ
• Terminate and make a loop: regulation of transcriptional directionality.
• Trends Biochem Sci. 2014; 39: 319-27
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• Bidirectional promoters are a common feature of many eukaryotic organisms from yeast to humans. RNA Polymerase II that is recruited to this type of promoter can start transcribing in either direction using alternative DNA strands as the template. Such promiscuous transcription can lead to the synthesis of unwanted transcripts that may have negative effects on gene expression. Recent studies have identified transcription termination and gene looping as critical players in the enforcement of promoter directionality. Interestingly, both mechanisms share key components. Here, we focus on recent findings relating to the transcriptional output of bidirectional promoters.

• Mir-Montazeri B, Ammelburg M, Forouzan D, Lupas AN, Hartmann MD
• Crystal structure of a dimeric archaeal cleavage and polyadenylation specificity factor.
• J Struct Biol. 2011; 173: 191-5
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• Proteins of the metallo-beta-lactamase (MbetaL) fold form a large superfamily of metallo-hydrolase/oxidoreductases. Members of this family are found in all three domains of life and are involved in a variety of biological functions related to hydrolysis, redox processes, DNA repair and uptake, and RNA processing. We classified the archaeal homologs of this superfamily based on sequence similarity and characterized a subfamily of the Cleavage and Polyadenylation Specificity Factor (CPSF) with an uncommon domain composition: in addition to an extended MbetaL domain, which accommodates the active site for RNA cleavage, this group has two N-terminal KH domains. Here, we present the crystal structure of a member of this group from Methanosarcina mazei. It reveals a dimerization mode of the MbetaL domain that has not been observed before and suggests that RNA is bound across the dimer interface, recognized by the KH domains of one monomer, and cleaved at the active site of the other.

• Yang D, Shcheynikov N, Muallem S
• IRBIT: it is everywhere.
• Neurochem Res. 2011; 36: 1166-74
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• IRBIT (IP(3)Rs binding protein released with IP(3)) is a protein originally identified by the Mikoshiba group as an inhibitor of IP(3) receptors function. Subsequently it was found to have multiple functions and regulate the activity of diverse proteins, including regulation of HCO(3)(-) transporters to coordinate epithelial HCO(3)(-) secretion and to determine localization of the Fip1 subunit of the CPSF complex to regulate mRNA processing. This review highlights the remarkably divers functions of IRBIT that are likely only a fraction of all the potential functions of this protein.

• Reguera J, Weber F, Cusack S
• Bunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.
• PLoS Pathog. 2010; 6: 1001101-1001101
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• Bunyaviruses are a large family of segmented RNA viruses which, like influenza virus, use a cap-snatching mechanism for transcription whereby short capped primers derived by endonucleolytic cleavage of host mRNAs are used by the viral RNA-dependent RNA polymerase (L-protein) to transcribe viral mRNAs. It was recently shown that the cap-snatching endonuclease of influenza virus resides in a discrete N-terminal domain of the PA polymerase subunit. Here we structurally and functionally characterize a similar endonuclease in La Crosse orthobunyavirus (LACV) L-protein. We expressed N-terminal fragments of the LACV L-protein and found that residues 1-180 have metal binding and divalent cation dependent nuclease activity analogous to that of influenza virus endonuclease. The 2.2 A resolution X-ray crystal structure of the domain confirms that LACV and influenza endonucleases have similar overall folds and identical two metal binding active sites. The in vitro activity of the LACV endonuclease could be abolished by point mutations in the active site or by binding 2,4-dioxo-4-phenylbutanoic acid (DPBA), a known influenza virus endonuclease inhibitor. A crystal structure with bound DPBA shows the inhibitor chelating two active site manganese ions. The essential role of this endonuclease in cap-dependent transcription was demonstrated by the loss of transcriptional activity in a RNP reconstitution system in cells upon making the same point mutations in the context of the full-length LACV L-protein. Using structure based sequence alignments we show that a similar endonuclease almost certainly exists at the N-terminus of L-proteins or PA polymerase subunits of essentially all known negative strand and cap-snatching segmented RNA viruses including arenaviruses (2 segments), bunyaviruses (3 segments), tenuiviruses (4-6 segments), and orthomyxoviruses (6-8 segments). This correspondence, together with the well-known mapping of the conserved polymerase motifs to the central regions of the L-protein and influenza PB1 subunit, suggests that L-proteins might be architecturally, and functionally equivalent to a concatemer of the three orthomyxovirus polymerase subunits in the order PA-PB1-PB2. Furthermore, our structure of a known influenza endonuclease inhibitor bound to LACV endonuclease suggests that compounds targeting a potentially broad spectrum of segmented RNA viruses, several of which are serious or emerging human, animal and plant pathogens, could be developed using structure-based optimisation.

• Zhang J et al.
• A polyadenylation factor subunit implicated in regulating oxidative signaling in Arabidopsis thaliana.
• PLoS One. 2008; 3: 2410-2410
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• BACKGROUND: Plants respond to many unfavorable environmental conditions via signaling mediated by altered levels of various reactive oxygen species (ROS). To gain additional insight into oxidative signaling responses, Arabidopsis mutants that exhibited tolerance to oxidative stress were isolated. We describe herein the isolation and characterization of one such mutant, oxt6. METHODOLOGY/PRINCIPAL FINDINGS: The oxt6 mutation is due to the disruption of a complex gene (At1g30460) that encodes the Arabidopsis ortholog of the 30-kD subunit of the cleavage and polyadenylation specificity factor (CPSF30) as well as a larger, related 65-kD protein. Expression of mRNAs encoding Arabidopsis CPSF30 alone was able to restore wild-type growth and stress susceptibility to the oxt6 mutant. Transcriptional profiling and single gene expression studies show elevated constitutive expression of a subset of genes that encode proteins containing thioredoxin- and glutaredoxin-related domains in the oxt6 mutant, suggesting that stress can be ameliorated by these gene classes. Bulk poly(A) tail length was not seemingly affected in the oxt6 mutant, but poly(A) site selection was different, indicating a subtle effect on polyadenylation in the mutant. CONCLUSIONS/SIGNIFICANCE: These results implicate the Arabidopsis CPSF30 protein in the posttranscriptional control of the responses of plants to stress, and in particular to the expression of a set of genes that suffices to confer tolerance to oxidative stress.

• Dickson KS, Thompson SR, Gray NK, Wickens M
• Poly(A) polymerase and the regulation of cytoplasmic polyadenylation.
• J Biol Chem. 2001; 276: 41810-6
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• Translational activation in oocytes and embryos is often regulated via increases in poly(A) length. Cleavage and polyadenylation specificity factor (CPSF), cytoplasmic polyadenylation element binding protein (CPEB), and poly(A) polymerase (PAP) have each been implicated in cytoplasmic polyadenylation in Xenopus laevis oocytes. Cytoplasmic polyadenylation activity first appears in vertebrate oocytes during meiotic maturation. Data presented here shows that complexes containing both CPSF and CPEB are present in extracts of X. laevis oocytes prepared before or after meiotic maturation. Assessment of a variety of RNA sequences as polyadenylation substrates indicates that the sequence specificity of polyadenylation in egg extracts is comparable to that observed with highly purified mammalian CPSF and recombinant PAP. The two in vitro systems exhibit a sequence specificity that is similar, but not identical, to that observed in vivo, as assessed by injection of the same RNAs into the oocyte. These findings imply that CPSFs intrinsic RNA sequence preferences are sufficient to account for the specificity of cytoplasmic polyadenylation of some mRNAs. We discuss the hypothesis that CPSF is required for all polyadenylation reactions, but that the polyadenylation of some mRNAs may require additional factors such as CPEB. To test the consequences of PAP binding to mRNAs in vivo, PAP was tethered to a reporter mRNA in resting oocytes using MS2 coat protein. Tethered PAP catalyzed polyadenylation and stimulated translation approximately 40-fold; stimulation was exclusively cis-acting, but was independent of a CPE and AAUAAA. Both polyadenylation and translational stimulation required PAPs catalytic core, but did not require the putative CPSF interaction domain of PAP. These results demonstrate that premature recruitment of PAP can cause precocious polyadenylation and translational stimulation in the resting oocyte, and can be interpreted to suggest that the role of other factors is to deliver PAP to the mRNA.

• Zarudnaia MI, Govorun DN
• [Is it reality that the endonuclease that cleaves pre-mRNA on polyadenylation has not been discovered?].
• Ukr Biokhim Zh (1999). 2001; 73: 128-34
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• Specific cleavage of transcript by a complex of multisubunit proteins is the first stage of polyadenylation of eukaryotic pre-mRNAs. The main participant of this reaction--endonuclease--is not discovered yet. However it is known that proteins CPSF-30 (mammalian) and Yth 1p (yeast) are homologues of the drosofila protein clipper (CLP), which displays endoribonucleolytic activity. In the N-terminal region all three proteins contain five copies of CCCH zinc finger motif that are associated with nucleolytic activity in the case of CLP. Literature data on the three above-mentioned proteins has been analysed. The results of these works do not contradict the hypothesis that exactly CPSF-30 and its homologues are the actual nucleases that cleave pre-mRNA in the process of polyadenylation.

• Wilusz CJ, Wormington M, Peltz SW
• The cap-to-tail guide to mRNA turnover.
• Nat Rev Mol Cell Biol. 2001; 2: 237-46
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• The levels of cellular messenger RNA transcripts can be regulated by controlling the rate at which the mRNA decays. Because decay rates affect the expression of specific genes, they provide a cell with flexibility in effecting rapid change. Moreover, many clinically relevant mRNAs--including several encoding cytokines, growth factors and proto-oncogenes--are regulated by differential RNA stability. But what are the sequence elements and factors that control the half-lives of mRNAs?

• Dickson KS, Bilger A, Ballantyne S, Wickens MP
• The cleavage and polyadenylation specificity factor in Xenopus laevis oocytes is a cytoplasmic factor involved in regulated polyadenylation.
• Mol Cell Biol. 1999; 19: 5707-17
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• During early development, specific mRNAs receive poly(A) in the cytoplasm. This cytoplasmic polyadenylation reaction correlates with, and in some cases causes, translational stimulation. Previously, it was suggested that a factor similar to the multisubunit nuclear cleavage and polyadenylation specificity factor (CPSF) played a role in cytoplasmic polyadenylation. A cDNA encoding a cytoplasmic form of the 100-kDa subunit of Xenopus laevis CPSF has now been isolated. The protein product is 91% identical at the amino acid sequence level to nuclear CPSF isolated from Bos taurus thymus. This report provides three lines of evidence that implicate the X. laevis homologue of the 100-kDa subunit of CPSF in the cytoplasmic polyadenylation reaction. First, the protein is predominantly localized to the cytoplasm of X. laevis oocytes. Second, the 100-kDa subunit of X. laevis CPSF forms a specific complex with RNAs that contain both a cytoplasmic polyadenylation element (CPE) and the polyadenylation element AAUAAA. Third, immunodepletion of the 100-kDa subunit of X. laevis CPSF reduces CPE-specific polyadenylation in vitro. Further support for a cytoplasmic form of CPSF comes from evidence that a putative homologue of the 30-kDa subunit of nuclear CPSF is also localized to the cytoplasm of X. laevis oocytes. Overexpression of influenza virus NS1 protein, which inhibits nuclear polyadenylation through an interaction with the 30-kDa subunit of nuclear CPSF, prevents cytoplasmic polyadenylation, suggesting that the cytoplasmic X. laevis form of the 30-kDa subunit of CPSF is involved in this reaction. Together, these results indicate that a distinct, cytoplasmic form of CPSF is an integral component of the cytoplasmic polyadenylation machinery.

• Bilger A, Fox CA, Wahle E, Wickens M
• Nuclear polyadenylation factors recognize cytoplasmic polyadenylation elements.
• Genes Dev. 1994; 8: 1106-16
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• In the cytoplasm of oocytes and early embryos, addition of poly(A) to mRNAs can activate their translation. We demonstrate that despite many differences between poly(A) addition in the cytoplasm and nucleus, these two forms of polyadenylation may involve identical trans-acting factors. Nuclear polyadenylation requires the sequence AAUAAA, the AAUAAA-binding cleavage and polyadenylation specificity factor (CPSF), and a poly(A) polymerase (PAP). We show that CPSF and PAP, purified from calf thymus, exhibit the same sequence specificity observed in the cytoplasm during frog oocyte maturation, requiring both AAUAAA and a proximal U-rich sequence. The enhanced polyadenylation of RNAs containing U-rich sequences is caused by their increased affinity for CPSF. Frog nuclear polyadenylation factors display cytoplasmic sequence specificity when dilute, suggesting that a difference in their concentrations in the nucleus and cytoplasm underlies the different sequence specificities in the two compartments. Because polyadenylation in extracts prepared from oocytes before maturation is stimulated by addition of CPSF, the onset of polyadenylation during early development may be attributable to the activation or synthesis of a CPSF-like factor. We suggest that sequences upstream of AAUAAA that are required for cleavage and polyadenylation of certain pre-mRNAs in the nucleus may be functionally equivalent to the upstream, U-rich sequences that function in the cytoplasm, enhancing CPSF binding. We propose that CPSF and PAP comprise a core polyadenylation apparatus in the cytoplasm of oocytes and early embryos.