NORMAL GENOMIC

• Aguero-Chapin G et al.
• Non-linear models based on simple topological indices to identify RNaseIII protein members.
• J Theor Biol. 2011; 273: 167-78
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• Alignment-free classifiers are especially useful in the functionalclassification of protein classes with variable homology and differentdomain structures. Thus, the Topological Indices to BioPolymers (TI2BioP)methodology (Aguero-Chapin et al., 2010) inspired in both the TOPS-MODEand the MARCH-INSIDE methodologies allows the calculation of simpletopological indices (TIs) as alignment-free classifiers. These indiceswere derived from the clustering of the amino acids into four classes ofhydrophobicity and polarity revealing higher sequence-order informationbeyond the amino acid composition level. The predictability power of suchTIs was evaluated for the first time on the RNase III family, due to thehigh diversity of its members (primary sequence and domain organization).Three non-linear models were developed for RNase III class prediction:Decision Tree Model (DTM), Artificial Neural Networks (ANN)-model andHidden Markov Model (HMM). The first two are alignment-free approaches,using TIs as input predictors. Their performances were compared with anon-classical HMM, modified according to our amino acid clusteringstrategy. The alignment-free models showed similar performances on thetraining and the test sets reaching values above 90% in the overallclassification. The non-classical HMM showed the highest rate in theclassification with values above 95% in training and 100% in test.Although the higher accuracy of the HMM, the DTM showed simplicity for theRNase III classification with low computational cost. Such simplicity wasevaluated in respect to HMM and ANN models for the functional annotationof a new bacterial RNase III class member, isolated and annotated by ourgroup.

• Jain C
• Identification and characterization of growth suppressors of Escherichiacoli strains lacking phosphorolytic ribonucleases.
• J Bacteriol. 2009; 191: 5622-7
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• RNases are involved in critical aspects of RNA metabolism in allorganisms. Two classes of RNases that digest RNA from an end (exo-RNases)are known: RNases that use water as a nucleophile to catalyze RNAdegradation (hydrolytic RNases) and RNases that use inorganic phosphate(phosphorolytic RNases). It has been shown previously that the absence ofthe two known Escherichia coli phosphorolytic RNases, polynucleotidephosphorylase and RNase PH, leads to marked growth and ribosome assemblydefects. To investigate the basis for these defects, a screen for growthsuppressors was performed. The majority of suppressor mutations were foundto lie within nsrR, which encodes a nitric oxide (NO)-sensitivetranscriptional repressor. Further analysis showed that the suppressorsfunction not by inactivating nsrR but by causing overexpression of adownstream gene that encodes a hydrolytic RNase, RNase R. Additionalstudies revealed that overexpression of another hydrolytic RNase, RNaseII, similarly suppressed the growth defects. These results suggest thatthe requirement for phosphorolytic RNases for robust cellular growth andefficient ribosome assembly can be bypassed by increased expression ofhydrolytic RNases.

• Barbas A, Matos RG, Amblar M, Lopez-Vinas E, Gomez-Puertas P, Arraiano CM
• New insights into the mechanism of RNA degradation by ribonuclease II:identification of the residue responsible for setting the RNase II endproduct.
• J Biol Chem. 2008; 283: 13070-6
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• RNase II is a key exoribonuclease involved in the maturation, turnover,and quality control of RNA. RNase II homologues are components of theexosome, a complex of exoribonucleases. The structure of RNase IIunraveled crucial aspects of the mechanism of RNA degradation. Here weshow that mutations in highly conserved residues at the active site affectthe activity of the enzyme. Moreover, we have identified the residue thatis responsible for setting the end product of RNase II. In addition, wepresent for the first time the models of two members of the RNase IIfamily, RNase R from Escherichia coli and human Rrp44, also called Dis3.Our findings improve the present model for RNA degradation by the RNase IIfamily of enzymes.

• Machwe A, Xiao L, Orren DK
• Length-dependent degradation of single-stranded 3' ends by the Wernersyndrome protein (WRN): implications for spatial orientation andcoordinated 3' to 5' movement of its ATPase/helicase and exonucleasedomains.
• BMC Mol Biol. 2006; 7: 6-6
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• BACKGROUND: The cancer-prone and accelerated aging disease Werner syndromeis caused by loss of function of the WRN gene product that possessesATPase, 3' to 5' helicase and 3' to 5' exonuclease activities. AlthoughWRN has been most prominently suggested to function in telomeremaintenance, resolution of replication blockage and/or recombinationalrepair, its exact role in DNA metabolism remains unclear. WRN is the onlyhuman RecQ family member to possess both helicase and exonucleaseactivity, but the mechanistic relationship between these activities isunknown. In this study, model single-stranded and 3' overhang DNAsubstrates of varying length and structure were used to examine potentialcoordination between the ATPase/helicase and exonuclease activities ofWRN. RESULT: Our results show that WRN can not only bind to but alsocatalyze the 3' to 5' degradation of single-stranded and 3' overhang DNAsubstrates, structures that were previously thought to be refractory toWRN exonuclease activity. The length of the single-stranded regions inthese structures is a critical parameter in determining both the bindingaffinity and the level of exonuclease activity of WRN. Most importantly,specific nucleotide cofactors dramatically stimulate WRN exonucleaseactivity on these substrates, with conditions that permit ATP hydrolysisnot only resulting in enhanced exonuclease activity but also altering itslength dependence on these structures. Parallel experiments show that adeletion mutant containing only the WRN exonuclease domain lacks both thisDNA length and nucleotide cofactor dependence, demonstrating that theinteraction of the ATPase/helicase domain of WRN with the DNA substratehas a profound influence on exonuclease activity. CONCLUSION: Our resultsindicate that, under conditions that permit ATP hydrolysis, there is adynamic and cooperative relationship between the distinct ATPase/helicaseand exonuclease domains of WRN with regard to their orientation on DNA.Based on these results, models are proposed for the coordinated,unidirectional 3' to 5' movement of the helicase and exonuclease domainsof WRN on DNA that should be informative for elucidating its function ingenome maintenance.

• Panigrahi AK et al.
• Identification of novel components of Trypanosoma brucei editosomes.
• RNA. 2003; 9: 484-92
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• The editosome is a multiprotein complex that catalyzes the insertion anddeletion of uridylates that occurs during RNA editing in trypanosomatids.We report the identification of nine novel editosome proteins inTrypanosoma brucei. They were identified by mass spectrometric analysis offunctional editosomes that were purified by serial ion exchange/gelpermeation chromatography, immunoaffinity chromatography specific to theTbMP63 editosome protein, or tandem affinity purification based on atagged RNA editing ligase. The newly identified proteins have ribonucleaseand/or RNA binding motifs suggesting nuclease function for at least someof these. Five of the proteins are interrelated, as are two others, andone is related to four previously identified editosome proteins. Theimplications of these findings are discussed.

• Daugeron MC, Mauxion F, Seraphin B
• The yeast POP2 gene encodes a nuclease involved in mRNA deadenylation.
• Nucleic Acids Res. 2001; 29: 2448-55
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• The major mRNA degradation pathway involves deadenylation of the targetmolecule followed by decapping and, finally, 5'-->3' exonuclease digestionof the mRNA body. While yeast factors involved in the decapping andexonuclease degradation steps have been identified, the nature of thefactor(s) involved in the deadenylation step remained elusive. Databasesearches for yeast proteins related to the mammalian deadenylase PARNidentified the Pop2 protein (Pop2p) as a potential deadenylase. WhilePop2p was previously identified as a factor affecting transcription, weidentified a non-canonical RNase D sequence signature in its sequence.Analysis of the fate of a reporter mRNA in a pop2 mutant demonstrates thatPop2p is required for efficient mRNA degradation in vivo. Characterisationof mRNA degradation intermediates accumulating in this mutant supports theinvolvement of Pop2p in mRNA deadenylation in vivo. Similar phenotypes areobserved in yeast strains lacking the Ccr4 protein, which is known to beassociated with Pop2p. A recombinant Pop2p fragment encompassing theputative catalytic domain degrades poly(A) in vitro demonstrating thatPop2p is a nuclease. We also demonstrate that poly(A) is a bettercompetitor than poly(G) or poly(C) of the Pop2p nuclease activity.Altogether, our study indicates that Pop2p is a nuclease subunit of theyeast deadenylase and suggests that Pop2p homologues in other species mayhave similar functions.

• Lai L, Yokota H, Hung LW, Kim R, Kim SH
• Crystal structure of archaeal RNase HII: a homologue of human major RNaseH.
• Structure. 2000; 8: 897-904
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• BACKGROUND: RNases H are present in all organisms and cleave RNAs inRNA/DNA hybrids. There are two major types of RNases H that have littlesimilarity in sequence, size and specificity. The structure of RNase HI,the smaller enzyme and most abundant in bacteria, has been extensivelystudied. However, no structural information is available for the largerRNase H, which is most abundant in eukaryotes and archaea. Mammalian RNaseH participates in DNA replication, removal of the Okazaki fragments andpossibly DNA repair. RESULTS: The crystal structure of RNase HII from thehypothermophile Methanococcus jannaschii, which is homologous to mammalianRNase H, was solved using a multiwavelength anomalous dispersion (MAD)phasing method at 2 A resolution. The structure contains two compactdomains. Despite the absence of sequence similarity, the large N-terminaldomain shares a similar fold with the RNase HI of bacteria. The activesite of RNase HII contains three aspartates: Asp7, Asp112 and Asp149. Thenucleotide-binding site is located in the cleft between the N-terminal andC-terminal domains. CONCLUSIONS: Despite a lack of any detectablesimilarity in primary structure, RNase HII shares a similar structuraldomain with RNase HI, suggesting that the two classes of RNases H have acommon catalytic mechanism and possibly a common evolutionary origin. Theinvolvement of the unique C-terminal domain in substrate recognitionexplains the different reaction specificity observed between the twoclasses of RNase H.

• Barrett C, Hughey R, Karplus K
• Scoring hidden Markov models.
• Comput Appl Biosci. 1997; 13: 191-9
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• MOTIVATION: Statistical sequence comparison techniques, such as hiddenMarkov models and generalized profiles, calculate the probability that asequence was generated by a given model. Log-odds scoring is a means ofevaluating this probability by comparing it to a null hypothesis, usuallya simpler statistical model intended to represent the universe ofsequences as a whole, rather than the group of interest. Such scoringleads to two immediate questions: what should the null model be, and whatthreshold of log-odds score should be deemed a match to the model.RESULTS: This paper analyses these two issues experimentally. Within thecontext of the Sequence Alignment and Modeling software suite (SAM), weconsider a variety of null models and suitable thresholds. Additionally,we consider HMMer's log-odds scoring and SAM's original Z-scoring method.Among the null model choices, a simple looping null model that emitscharacters according to the geometric mean of the character probabilitiesin the columns modeled by the hidden Markov model (HMM) performs well orbest across all four discrimination experiments.

• Pepe CM, Maslesa-Galic S, Simons RW
• Decay of the IS10 antisense RNA by 3' exoribonucleases: evidence thatRNase II stabilizes RNA-OUT against PNPase attack.
• Mol Microbiol. 1994; 13: 1133-42
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• RNA-OUT, the 69-nucleotide antisense RNA that regulates Tn10/IS10transposition folds into a simple stem-loop structure. The unusually highmetabolic stability of RNA-OUT is dependent, in part, on the integrity ofits stem-domain: mutations that disrupt stem-domain structure (Class IImutations) render RNA-OUT unstable, and restoration of structure restoresstability. Indeed, there is a strong correlation between the thermodynamicand metabolic stabilities of RNA-OUT. We show here that stem-domainintegrity determines RNA-OUT's resistance to 3' exoribonucleolytic attack:Class II mutations are almost completely suppressed in Escherichia colicells lacking its principal 3' exoribonucleases, ribonuclease II (RNaseII) and polynucleotide phosphorylase (PNPase). RNase II and PNPase areindividually able to degrade various RNA-OUT species, albeit withdifferent efficiencies: RNA-OUT secondary structure provides greaterresistance to RNase II than to PNPase. Surprisingly, RNA-OUT is threefoldmore stable in wild-type cells than in cells deficient for RNase IIactivity, suggesting that RNase II somehow lessens PNPase attack onRNA-OUT. We discuss how this might occur. We also show that wild-typeRNA-OUT stability changes only two-fold across the normal range ofphysiological growth temperatures (30-44 degrees C) in wild-type cells,which has important implications for IS10 biology.

• Koonin EV, Deutscher MP
• RNase T shares conserved sequence motifs with DNA proofreadingexonucleases.
• Nucleic Acids Res. 1993; 21: 2521-2

• Katayanagi K, Okumura M, Morikawa K
• Crystal structure of Escherichia coli RNase HI in complex with Mg2+ at 2.8A resolution: proof for a single Mg(2+)-binding site.
• Proteins. 1993; 17: 337-46
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• To obtain more precise insight into the Mg(2+)-binding site essential forRNase HI catalytic activity, we have determined the crystal structure ofE. coli RNase HI in complex with Mg2+. The analyzed cocrystal, which isnot isomorphous with the Mg(2+)-free crystal previously refined at 1.48 Aresolution, was grown at a high MgSO4 concentration more than 100 mM sothat even weakly bound Mg2+ sites could be identified. The structure wassolved by the molecular replacement method, using the Mg(2+)-free crystalstructure as a search model, and was refined to give a final R-value of0.190 for intensity data from 10 to 2.8 A, using the XPLOR and PROLSQprograms. The backbone structures are in their entirety very similar toeach other between the Mg(2+)-bound and the metal-free crystals, exceptfor minor regions in the enzyme interface with the DNA/RNA hybrid. Theactive center clearly revealed a single Mg2+ atom located at a positionalmost identical to that previously found by the soaking method. Althoughthe two metal-ion mechanism had been suggested by another group (Yang, W.,Hendrickson, W.A., Crouch, R.J., Satow, Y. Science 249:1398-1405, 1990)and partially supported by the crystallographic study of inactive HIV-1 RTRNase H fragment (Davies, J.F., II, Hostomska, Z., Hostomsky, Z., Jordan,S.R., Matthews, D. Science 252:88-95, 1991), the present result excludesthe possibility that RNase HI requires two metal-binding sites foractivity. In contrast to the features in the metal-free enzyme, the sidechains of Asn-44 and Glu-48 are found to form coordinate bonds with Mg2+in the metal-bound crystal.