In eukaryotic cells, the 3′ poly(A) tails found on mRNA influence their stability and translation. The discovery of a second nuclear poly(A) polymerase complex has fueled a series of reports defining a new and unexpected role for 3′ end poly(A) tails in the nuclear surveillance and turnover of noncoding RNAs and intergenic mRNAs of unknown function

Anderson, James T.

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RNA Turnover: Unexpected Consequences of Being Tailed 

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Marquette Universitye-Publications@MarquetteBiological Sciences Faculty Research andPublications Biological Sciences, Department of8-23-2005RNA Turnover: Unexpected Consequences ofBeing TailedJames T. AndersonMarquette University, james.anderson@marquette.eduAccepted version. Current Biology, Vol. 15, No. 16 (August 23, 2005): R635-R638. DOI. © 2005Elsevier B.V. Used with permission.                                                                                     Marquette Universitye-Publications@MarquetteBiology Faculty Research and Publications/College of Arts andSciencesThis paper is NOT THE PUBLISHED VERSION; but the author’s final, peer-reviewed manuscript.The published version may be accessed by following the link in the citation below.Curent Biology, Vol. 15, No. 16 (August, 2006) : R635-R638. DOI. This article is © Elsevier (CellPress) and permission has been granted for this version to appear in e-Publications@Marquette. Elsevier (Cell Press) does not grant permission for this article to befurther copied/distributed or hosted elsewhere without the express permission from Elsevier(Cell Press).RNA Turnover: Unexpected Consequences of Being TailedJames T. AndersonDepartment of Biological Sciences, Marquette University, Milwaukee, WIIn eukaryotic cells, the 3′ poly(A) tails found on mRNA influence their stability and translation. The discovery of a second nuclear poly(A) polymerase complex has fueled a series of reports defining a newand unexpected role for 3′ end poly(A) tails in the nuclear surveillance and turnover of noncoding RNAsand intergenic mRNAs of unknown function.The life cycle of RNA begins with transcription and ends upon degradation; in between are several processing steps that are required for maturation and function. One aspect of RNA maturation is 3′ endprocessing, which for most RNA occurs through endonucleolytic cleavage, and/or exonucleolyticdigestion, and subsequent addition of polyadenylate (poly(A)) tails to mRNA and CCA to tRNA, or noaddition in the case of rRNA, snRNA and snoRNAs. The synthesis of mRNA 3′ poly(A) tails is carried outby a conventional nuclear localized poly(A) polymerase, and the functions of poly(A) tails are to stabilize,facilitate localization and enhance translation of mRNAs.                                                                                                                                                    Recent studies have confirmed the identity of a second nuclear poly(A) polymerase in yeast that isevolutionarily conserved throughout eukaryotes. Genetic and biochemical characterization of thispoly(A) polymerase, Trf4p, and its associated factors1,2 establishes a new function for poly(A) tails intargeting the degradation of aberrant non-coding RNAs — tRNA, rRNA and snRNA — and intergenicmRNAs of unknown function.3 The first poly(A) polymerase enzymatic activity, Pap1, was identified in Escherichia coli, but theidentification of poly(A)+ RNAs in bacteria lagged behind this discovery. As it turns out, a small percentage of bacterial mRNA is polyadenylated by bacterial Pap1, and in some cases polyadenylation isassociated with increased mRNA turnover. Noncoding RNAs can also be polyadenylated in E. coli, andthe presence of the poly(A) tail on noncoding RNAs has been shown to control the expression of aregulatory RNA and target a mutant tRNA for degradation (reviewed in4,5).Interestingly, Piper et al.6 identified 3′ end poly(A) tails on yeast 5S rRNA in a mutant strain lacking a putative endonuclease required for proper 5S rRNA 3′end processing. More recently, polyadenylation ofsnRNA, snoRNA and 5S rRNA by the conventional poly(A) polymerase was observed in mutant strainslacking one or more subunit of the exosome,7,8,9 a multisubunit protein complex involved in manyaspects of RNA metabolism.10 While these observations were suggestive, their significance remained enigmatic until recently, when anumber of laboratories began investigating Trf4p, a novel yeast poly(A) polymerase, and the effects of Trf4p-derived poly(A) tails on mRNA and noncoding RNA expression. Trf4p belongs to the polymerase β-type nucleotidyltransferase superfamily;11 other members are DNA polymerase Xs, tRNA CCA addingenzymes, terminal deoxynucleotidyltransferases, Gld2 and Trf5p, a functionally redundant protein inyeast which exhibits 48% amino acid identity with Trf4p (my unpublished observation).While conducting a genetic screen in Schizosaccharoymces pombe designed to uncover genes involvedin replicational response, Saitoh et al.12 identified Cid13p, a cytoplasmic poly(A) polymerase relatedto Saccharomyces cerevisiaeTrf4/5p. During this work they showed that affinity purified tagged-Trf4ppossesses an intrinsic poly(A) polymerase activity in vitro.The role of Trf4p as a poly(A) polymerase in vivo was first implied from a genetic suppressor analysisused to identify genes required for the instability of a tRNAiMet lacking one modified nucleotide, 1-methyladenosine 58 in the TΨC loop. Along with TRF4, RRP44 andMTR4 were also found to be requiredfor degradation of the hypomethylated tRNAiMet .13 Rrp44p is a subunit of the exosome which functionsin the nucleus and cytoplasm10 and Mtr4p is a putative ATP-dependent RNA helicase that influences the processing and degradation of many RNAs by the nuclear exosome.14 In a sophisticated series of experiments, Kadaba and Krueger13 demonstrated that the hypomethylatedpre-tRNAiMet is polyadenylated, and that the length and abundance of the poly(A) tail is stronglyinfluenced by the presence of excess Trf4p. These results established a firm relationship between the polyadenylation of the hypomethylated tRNAiMet by Trf4p and degradation of the polyadenylated tRNAby the exosome.In accordance with the genetic relationship between TRF4 andMTR4,13 Trf4p, Mtr4p and a putative RNAbinding protein, Air2p,15 form a stable complex in vivo.16 Air2p shows 41% amino acid identity withAir1p, curiously reminiscent of the structural similarity between Trf4p and Trf5p.                                                                                                T, 5Clip'lito 7n,Gj:p..~ pr&-IIIVeoo "'8 TRAMP ----AAAAAAMA PlllYMl + Rt<IA.6 C=:J mRNA. E:L025c Curwnt 9iologv The Trf4p/Air2p/Mtr4p polyadenylation complex (TRAMP) has been the focus of recent studies whichhave demonstrated that TRAMP purified from yeast whole cell extracts has poly(A) polymeraseactivity1,2 that can polyadenylate small RNAs, including tRNA in vitro (Figure 1), which in turn stimulates degradation of the adenylated RNA by the exosome.Figure 1. TRAMP polyadenylates RNAs to trigger their degradation.After transcription, pre-5S rRNA, sn/snoRNAs, pre-tRNA and mRNAs that do not pass a quality controlpoint, because of a perturbation of structure or error in processing, are recognized and polyadenylatedby TRAMP, a complex of Trf4p (poly(A) polymerase), Air2p (RNA binding) and Mtr4p (putative ATPdependent RNA helicase). It is not known what role the highly related proteins Trf5p and Air1p play inthis pathway so they have been shown on the side highlighted by a question mark.LaCava et al.1 and Vanacova et al.2 independently identified the components of TRAMP by affinitypurification of Trf4p and mass spectrometry analysis of the copurifying proteins.1,2 They both found thatTrf4p, Mtr4p and Air2p form stable and stoichiometric complexes. Purified TRAMP exhibits significantpoly(A) polymerase activity when presented with a small RNA substrate and ATP, and this activity is ablated if mutations are introduced that replace two key glycine amino acids in thenucleotidyltransferase catalytic core of Trf4p with alanine.1,2 The presence of Trf5p and Air1p, in addition to Trf4p and Air2p, when Mtr4p-TAP is used for purificationof TRAMP has made it hard to discern the role each of these proteins play in TRAMP function. It hasbrought to light some important unanswered questions about the role of Trf4p/Trf5p and Air1p/Air2p inthe formation of TRAMP. Purification of Trf5p-TAP resulted in the copurification of Mtr4p, but no Air1por Air2p were detectable and no polyadenylation activity was detected,1,2 leaving the question what isthe role of Trf5p in TRAMP function?When Air1p-TAP was affinity purified, Mtr4p and, to a lesser extent, Trf4p copurified with Air1p and thiscomplex had a low-level of polyadenylation activity.2 Recombinant Trf4p failed to polyadenylate an RNAsubstrate unless it was incubated together with recombinant Air1p or Air2p and the RNA,2 indicatingthat Air1p or Air2p provide the RNA-binding activity of TRAMP complexes.                                                                                                                         Biology As Trf5p and Air1p are marginally required for adenylation and degradation of a tRNA substrate in vitro and for the adenylation and degradation of hypomethylated tRNAiMetin vivo (my and S. Kadaba’sunpublished data), do Trf5p, Air1p and Mtr4p form a distinct complex from Trf4p, Air2p and Mtr4p thatfunctions similarly to polyadenylate an unidentified substrate, or do they have a completely differentfunction? Consistent with the idea that their function may be to modify different substrates, Trf5p-GFPwas predominantly localized to the nucleolus, while Trf4p-GFP was distributed throughout the nucleus.17 LaCava et al.1 and Vanacova et al.2 showed that providing the exosome with a prepolyadenylated tRNAor purified TRAMP lacking Mtr4p was insufficient to convey degradation of the RNA substrate beyonddeadenylation. This is striking in the face of the observation that TRAMP does not require ATP tostimulate exosome degradation of a nonadenylated pre-tRNA substrate.1 What is the function of apoly(A) tail on the degradation of RNA substrates by the exosome? On the one hand it appears that the tRNA substrate requires adenylation for degradation by the exosome to proceed efficiently. This is inagreement with the in vivo data demonstrating that hypomethylated tRNAiMet is not polyadenylated orefficiently degraded in strains lacking a functional Trf4p (S. Kadaba’s and my unpublished data).It is not clear from the reports whether stimulation of tRNA degradation by TRAMP in the absence ofadded ATP results in the same processive degradation observed when the exosome, TRAMP and ATP areincubated together with the substrate, or if substrate degradation stalls and intermediate sizeddegradation products accumulate.It is possible that initial rounds of degradation occur slowly in the absence of poly(A) and cannot reachcompletion unless initiated by adenylation and multiple subsequent rounds of adenylation as has beenproposed (Figure 2).1,2,3 If this hypothesis is true, then the 3′ poly(A) might be functioning as anunstructured fuse to prime the more difficult task of degrading the highly structured parts of RNAtargets, but in its absence RNA degradation is slow and unprocessive. This is consistent with theobserved accumulation of degradation intermediates seen when RNA is incubated with the exosomealone.1,2 Figure 2. Proposed model for degradation of poly(A)+ tRNA by exosome.A defective tRNA is polyadenylated by TRAMP, which stimulates degradation by the exosome. This initial round of degradation may result in stalling by the exosome at highly structured regions of the tRNA,leaving an intact but shortened tRNA (tR). This shortened tRNA undergoes a secondary round ofadenylation by TRAMP, which stimulates the exosome to re-engage and continue degradation of thissubstrate RNA. This cycle continues until the tRNA is completely degraded.                                                                                                                                                                               The work of Wyers et al.3 expands our understanding of TRAMP and the exosome to include turnover ofintergenic mRNAs. Intergenic mRNAs were discovered in studies characterizing the transcriptional topography of whole genomes, which ultimately led to the birth of the ‘transcriptome’. The intergenicmRNAs identified in this work3 are products of RNA polymerase II, as they contain a 5′ cap identical tocoding mRNAs and a mutation that blocks RNA polymerase II function reduced their abundance. Oneintergenic mRNA, NEL025c, was found to accumulate in yeast as heterogeneous length poly(A)+species in a strain lacking Rrp6p, a subunit of the exosome that is restricted to the nucleus.Wyers et al.3 went on to show that the heterogeneity of these mRNAs was due to a difference in their3′ termini and that polyadenylation of these shortened mRNAs is dependent on TRAMP. Are thesemRNAs functional or do they represent transcriptional noise? Whether they are transcriptional noise or functional mRNAs, yeast have developed and retained a mechanism for regulating their abundance byoligoadenylation, which hastens their degradation.The role of polyadenylation to facilitate degradation of RNA now stretches from aberrantly structured or processed noncoding RNA to intergenic mRNAs. Is it possible this mechanism of RNA turnover is moregeneral and used to degrade aged RNAs? New evidence suggests that tRNAs undergo retrogrademovement from the cytoplasm back to the nucleus (18 and H. Shaheen and A. Hopper, personalcommunication). Could this return to the nucleus by tRNA represent a method of colocalizing tRNA witha highly efficient RNA degrading machine to enable tRNA turnover?One of the key molecular questions to be answered is that of how TRAMP distinguishes between asubstrate that is to be adenylated and degraded, such as tRNAiMet lacking m1A58, and one that shouldnot, such as tRNAiMet possessing m1A58. If recognition of a tRNA designated for degradation is conferredby the absence of m1A58, then all tRNAs that normally contain m1A58 having lost this modifiednucleotide should be degraded, but this is not seen (my unpublished data).Vanacova et al.1 showed that tRNAiMet isolated from a wild-type yeast strain cannot be polyadenylatedby TRAMP while the fully unmodified form of the same tRNA made by in vitro transcription can. Giventhis result, it is more likely that a loss of tRNA tertiary structure triggers adenylation of thehypomethylated tRNAiMetin vivo and not the lack of m1A58.13,19 This is consistent with the finding thatTRAMP efficiently adenylated a tRNAala containing two point mutations predicted to disrupt its structure while the wild-type tRNAala was inefficiently adenylated in vitro.2 This result also implies that all the components needed to recognize aberrantly structured RNAs are present within purified TRAMP.The same structural perturbations may be required for the adenylation of snRNA, snoRNA and 5s rRNAs,but since most of these RNAs have been characterized as full-length or their 5′ and 3′ ends remainuncharacterized this seems unlikely for this set of RNAs. These new findings promise to provide insightsinto how nuclear RNA surveillance plays an important role in regulating eukaryotic gene expression.References1 J. LaCava, J. Houseley, C. Saveanu, E. Petfalski, E. Thompson, A. Jacquier, D. 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