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Turnover of mRNA in prokaryotes and lower eukaryotes
Turnover of mRNA in prokaryotes and lower eukaryotes Christopher F. Higgins, Stuart W. Peltz and Allan Jacobson University of O x f o r d , Oxford, UK a n d University of M a s s a c h u s e t t s Medical School, W o r c e s t e r , M a s s a c h u s e t t s , USA The turnover of mRNA plays an important role in the regulation of gene expression. The two best understood model systems are those of the prokaryote Escherichia coil and the lower eukaryote Saccharomyces cerevisiae. Considerable progress in recent years has helped define the general pathways by which mRNA is degraded in E coil Much less is known about the pathways of decay, or the enzymes involved, in eukawotic cells. However, both c/s-acting sequences and trans-acting factors have recently been characterized in S. cerevisiae and an indispensable role for translation has been identified. A comparison of these model species highlights both similarities and differences in mRNA turnover between prokaryotic and eukaryotic systems. Current Opinion in Genetics and Development 1992, 2:739-747
Introdudion The amount of a given protein synthesized in the cell depends, to a large degree, upon the abundance of its cognate mRNA. The fact that the steady-state level of mRNA depends on its rate of degradation, as well as its rate of synthesis is frequently forgotten. Nevertheless, it is now clear that cells possess a sophisticated apparatus for the degradation of mRNA, and that differences in the rate of mRNA degradation can play an important role in determining the level of gene expression. The vast majority of studies on mRNA turnover have concentrated on a small number of model species and a few specific messages in these species. This article is divided into two halves, first addressing recent studies on E. coli as a model prokaryore and then S. cerevisiae as a model eukaryote.
Turnover of mRNA in prokaryotes The pathways by which mRNA turnover in E. coli is mediated have been delineated in broad ternls, although many problems still remain to be answered. It is now clear that there is no single pathway but that different mRNA species are often degraded by different routes. These pathways operate in parallel such that, if the normal degradative pathway for an mRNA molecule is compromised (e.g. by mutation), an alternative pathway comes into operation. This default pathway may not be
much slower than the primary pathway and so elimination of the primary pathway does not necessarily have a major effect on stability. Indeed, it seems likely that for some messages two alternative pathways of degradation may operate at similar rates enabling different molecules within the population to be degraded by different routes. These overlapping and alternative pathways of degradation explain, at least in part, why an understanding of the factors that influence mRNA turnover has lagged behind our understanding of the control of mRNA synthesis.
Exoribonucleases The degradation of mRNA is mediated by a combination of endonucleotylic and exonucleolytic events (Fig. 1). In the simplest scenario, degradation of an mRNA molecule is mediated by two 3'-5' exonucleases, RNaselI and polynucleotide phosphorylase (PNPase) (Fig. la). These two enzymes attack the 3' end of a message, processively removing ribonucleotides as they proceed towards the 5' end. In the absence of any factor impeding their progress, the end-product of degradation by these enzymes (other than the recycled monoribonucleotides) are small oligoribonucleotides of 10-20 bp. An enzyme that may mediate the final degradation of these small oligoribonucleotides to mononucleotides has recently been identified and designated as RNaseI* [1..]. This enzyme is closely related to, but apparently distinct from, the periplasmic ribonuclease RNaseI.
Abbreviations PAB--poly(A)-binding protein; PAN--poly(A) nuclease;PNPase~polynucleotidephosphorylase; rRNA--ribosomal RNA; IRNA--transfer RNA; ts--temperature-sensitive; UTR--untranslated region.
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Fig. 1. Schematic illustration of the general,pathways for mRNA degradation in prokaryotes. Messenger RNA with an unblocked 3' end is degraded principally by two processive 3 -5 exonucleases, polynucleotide phosphorylase (PNPase)and RNasell (a,i). For many mRNAs the processive activities after interaction of these enzymes are blocked by a stem-loop structure, possibly after interaction with RNAbinding proteins that recognize such structures (a,ii). In many, and probably most, cases the block to exonu(slease activity is sufficiently long that decay is initiated by upstream endonuclease cleavage (b). This may be achieved by an endonuclease removing or destroying the terminal stem loop (b,i), or endonuclease cleavage at the 5' end and subsequently elsewhere in the mRNA, producing many small fragments (b,ii). RNaseE is the only endonuclease known to play a rather general role in these processes. RNaseK acts on the 5 end of at least some mRNAs, but it remains to be established whether or not RNaseK is entirely distinct from RNaseE. For a few specific mRNA molecules, RNaselll also plays a role. Of course, other enzymes that play specific or general roles might remain to be identified. Finally, the products of endonuclease cleavage are degraded to mononucleotides and small oligonucleotides by the two exonucleases, RNasell and PNPase, and the small oligonucleotides may be scavenged by RNaseP.
Most mRNAmolecules are not degraded by this simple route but include sequences that can potentially form secondary (stem-loop) structures in the message and impede the processive activities of these two exonucle-
ases (Fig. lb). Over the past few years, considerable evidence that stem-loop structures can stabilize upstream mRNA in vivo, presumably by impeding the activities of PNPase and RNaseII, has accrued (reviewed in [2]).
Turnover of mRNA in prokaryotes and lower eukaryotes Higgins, Peltz, Jacobson 741 These concepts have recendy been tested more direcdy in vitro [3"']. Stem-loop structures that stabilize upstream mRNA in vivo have been shown to impede the processive activities of both PNPase and RNaselI in vitro [3"']. Perhaps surprisingly, the period of time for which these exonucleases were impeded was relatively short compared with the increased half-life these sequences confer on mRNA in vivo. This implies that an additional factor, for example a stem-loop-binding protein, may play a role in the mRNA stabilization by such structures in vivo. evidence for such a protein has now been obtained (HC Causton and CF Higgins, unpublished data). The majority of messages have a stem-loop structure at the 3' end (rho-independent transcription terminators consist of a stem-loop structure). For these messages, degradation can potentially proceed by the exonucleases overcoming the barrier presented by the terminal structure. However, the weight of evidence now indicates that for most messages the terminal stem-loop structure provides an efficient block to exonucleolytic decay such that decay is initiated by upstream endonucleolytic cleavage (Fig. lb). It should be noted that there is, as yet, no evidence for a 5'-3' exoribonucleolytic activity in E. coli. Indeed, for the majority of message an exoribonuclease that attacks the free 5' end would be incompatible with the available data.
Endonucleases The most important advances in the last couple of years have been in the identification of endonucleases and characterization of their cleavage sites that, for many messages, provide the rate-determining step in degradation. These nucleases bypass the barrier to 3'-5' exonucleases, either by removing the terminal stem-loop structure (Fig. lb,i) or by cleavage at the 5' end of the message, which initiates subsequent decay (Fig. lb,ii). The products of endonuclease cleavage are then degraded to mononucleotides by the 3'-5' exonucleases PNPase and RNaseII. For many years the only endonuclease known to play a role in mRNA degradation has been RNaselII. RNaseIII recognizes large stem-loop structures and, as most messages lack such sites, cannot play a role in the degradation of more than a small subset of messages. Recently, RNaseE has been shown to play a central role in the degradation of a large number of messages. RNaseE was identified many years ago as a consequence of its role in processing the 9S ribosomal RNA (rRNA) precursor [4]. Despite earlier reports that this enzyme plays litde or no role in mRNA turnover [5] it is now clear that this is incorrect: mutants in the rne gene, defective in RNAaseE activity, result in the general stabilization of E. coli and bacteriophage mRNA and several individual mRNA's are stabilized in these mutants [6,7"',8"']. Several other studies have also identified defects in specific mRNA-processing events in rnemutants, including mRNA for p a p pili [9"], ompA [10.], dicB [11], ribosomal proteins S15 [12.] and $20 [13-], phage fl [14], and the antisense RNA1 that plays a role in determining ColE1
plasmid replication [15"]. Finally, the ares mutation identified several years ago as conferring altered mRNA stability, has been shown to be allelic with me. Two sequences for the rne (arm) gene have been published, one predicting a protein of 62kD [16], the other a protein of 91 kD [17"]. Both sequences may contain frameshifts and predict a prematurely terminated polypeptide since the relative mobility of the RNaseE protein indicates a molecular weight of 180 kD (EA Mudd and CF Higgins, unpublished data; IB Holland, personal communication). The cleavage specificity of RNaseE is still unclear. Following identification of the first few cleavage sites a consensus appeared to be emerging [6]. However, as more sites have been identified the existence of a consensus sequence is less convincing. Nevertheless, mutational studies at or near the site of cleavage show that RNaseE recognizes both sequence and structural elements [15"',18.',19"]: cleavage occurs upstream of a stem-loop structure and there is a preference for an AU dinucleotide 3' to the site of cleavage. Although RNaseE plays an important role in the degradation of many mRNA's, it is certainly not the only endonuclease involved. In rne mutants, even those mRNA molecules whose stability is increased eventually turn over: another endonuclease must initiate this alternative pathway. For some messages, an enzyme other than RNaseE may play an initiating role in degradation. The decay of b/a and ompA mRNA's is initiated by endonucleolytic cleavage events at the 5' end [20]. An endonuclease, designated RNaseK, has been purified that cleaves these mRNA's in vitro at sites similar to those observed in vivo [21..]. Whether this enzyme also cleaves other mRNA's is not yet known. Some of the sites in ompA mRNA that are cleaved by RNaseK in vitro are affected by rne mutations in vivo. This can be interpreted in one of two ways: RNaseE may regulate the expression/activity of RNaseK or, alternatively, RNaseK may not be a distinct enzyme but a fragment of RNaseE. The molecular weight of RNaseK is about 60kD [21-.] - - it is known that small proteolytic fragments of RNaseE can retain enzymic activity (EA Mudd and CF Higgins, unpublished data). This possibility needs to be rigorously excluded before we can be sure that RNaseK and RNaseE are two distinct enzymes. Finally, two broad-specificity endonucleases have recently been characterized, RNaseM and RNaseR [22.]; any general role they may play in mRNA turnover is, as yet, unclear. If we are to be able to predict from its sequence the stability of a message, it is important to identify the sites responsible for determining the initial endonucleolytic cleavage events that provide the rate-determining steps in degradation. At least for some messages this initial cleavage, and therefore mRNA turnover, appears to be regulated. For example, cleavage of ompA mRNA appears to be regulated by the growth rate [23], and cleavage of p u f m R N A is influenced by oxygen availability [24..]. For some messages, such as ompA secondary structures at the 5' end appear to play a central role in determining the rate-determining endonucleolytic cleavage events [25,26]. It seems that the sequence of the secondary structure is of less importance than the location of the secondary
742
Prokaryotes and lower eukaryotes structure with respect to the 5' end of the message [27-]. If the structure is more than 2 - 4 b p from the 5' end, its stabilizing role is lost [27..]. One of the maior challenges is to understand the events following an initial cleavage event at the 5' end. It is possible that, following initial cleavage near the 5' end, a 5'-3' exonuclease processively degrades the mRNA from the point of cleavage. However, there is no evidence for such an enzyme and the presence of many degradation intermediates would tend to argue against this mechm-tism. Alternatively, secondaw endonucleolytic cleavages may be promoted following recognition or cleavage at the 5' end of the message: these secondary cleavages could be mediated by the original enzTme or by a second enzyme [27.,]. Fragments generated by these secondary endonucleolytic cleavages would then be degraded by the 3'-5' exonucleases RNaselI and PNPase (Fig. lb,ii). This latter model does, however, pose a problem: why do the secondary cleavages not occur until an initial event at the 5' end has occurred? Finally, although the 5' end is clearly crucial for the decay of some messages, this is not necessarily generally true: for certain messages, multiple defined sites are cleaved and all must be removed if a message is to be stabilized [28,29].
Translation The role of translation in mRNA degradation has been controversial. Some early reports suggested that translation stimulates mRNA degradation, others that translation stabilizes mRNA. Many of these studies have been difficult to interpret, particularly those making use of translational inhibitors. Recent data show that, at lea.st for the ompA message, degradation that is initiated by an endonucleolytic cleavage at the 5' end is independent of translation rates [25]. For other messages, translation does affect deca), [30]. However, there is no evidence that translation is normally essential for degradation, and enzymes like RNaseE are not associated vdth ribosomes [31]. Most probably the effects of translation on mRNA turnover are indirect, influencing the accessibility of endonuclease cleavage sites.
Yeast as an experimental system in which to study mRNA turnover Work on mRNA decay in the yeast S. cerevisiae began more than two decades ago [32,33], but only recently has the subject attracted the efforts of a sufficiently large number of laboratories necessary for a concerted attack on the underlying regulatory mechanisms. Research has now reached a point where a simple and reliable procedure for the determination of decay rates has been established [34,35]: stable, unstable, and regulated mRNAs have been identified [34,36",37], chimeric mRNAs have been utilized to delineate cis-acting instability determinants (B Heaton et al., unpublished data) [38,39,40"], nucleases have been purified and begun to be characterized [41-44,45-,46,47], and an in vitro decay system
has been used to localize cleavage sites [48..]. Of particular significance to an understanding of the interplay between the turnover machinery and the translational apparatus, yeast mutants defective in mRNA turnover have now been isolated and are beginning to shed light on important trans-acting factors (SW Peltz et aL, unpublished data) [47,49"',50,51"].
C/s-acting sequences as determinants of mRNA instability In strains harboring the temperature-sensitive (ts) ppbl- I allele of RNA polymerase II, a shift to 36°C leads to the rapid and selective cessation of mRNA synthesis and to a reduction in the steady-state levels of pre-existing mRNAs [52]. This observation led to the development of a routine procedure for the measurement of mRNA-deca}, rates in which cells growing at 24°C were abruptly shifted to 36°C and changes in the relative anlounts of individual mRNAs were quantified by RNA-blotting procedures [34,35,52]. The half-lives of the different mRNAs measured by this procedure range from 1~50 min, a result consistent with the complex decay kinetics observed for the total mRNA population [34]. In an approach analogous to those used for other systems (see [53], for a review), cis-acting instability sequences have been identified in yeast that promote rapid mRNA decay when transferred to mRNAs that are normally stable (B Heaton et aL, unpublished data) [34,38]. In general, chimeric genes encoding portions of a stable and an unstable mRNA are constructed (the reading franle across the hybrid junction is maintained), introduced into cells (~12bl-1 mutants) on centromere vectors, and the decay rates of the resulting hybrid transcripts and their 'parental' mRNAs mea*sured using RNA isolated from temperature-shiftecl cells. The simplest expectation from this type of experiment is that instability elements will be modular; that is, hybrid mRNAs which contain sequences sufficient to promote rapid turnover will themselves exhibit rapid decw kinetics. Hybrid mRNAs that lack such sequences should be stable. These expectations have been borne out by the results of experiments in which chimeras have been constructed between the stable mRNAs encoded by the ACT] or PGK1 genes and the unstable mRNAs encoded by the MATotl, STE3, STE2, HIS3 and CDC4 genes (B Heaton el aL, unpublished data) [38,39,40"]. For example, a chimeric mRNA comprised of the stable PGK1 mRNA fused, in frmne, to the unstable k,D
Turnover of mRNA in prokaryotes and lower eukaryotes Higgins, Peltz, Jacobson 743 Experiments analogous to those described for MATkl have also been carried out with the unstable HIS3, STEP, STE3 and CDC4 mRNAs (B Heaton et al., unpublished data) [38,39,40°,]. Sequences essential for rapid decay of chimeric mRNAs have been found in the coding regions of all four mRNAs. Evidence for additional complexity of instability determinants was obtained in studies of the STE3 mRNA. Whereas deletion of STE3 sequences spanning codons 13-179 stabilizes a STE3-ACT1 chimeric mRNA approximately threefold, deletion of the same sequences from bona fide STE3 mRNA fails to stabilize that mRNA (B Heaton el al., unpublished data). This suggests tilat other regions of the STE3 mRNA can stimulate mRNA decay independently of the coding-region sequences. Further analysis demonstrated that the STE3 3' untranslated region (UTR) can act as the independent instability element (B Heaton et aL, unpublished data). Results suggesting tile presence of 3'-UTR instability elements have also been obtained for the STk~, HTB1 and MFA2 mRNAs (D Herrick and A Jacobson, unpublished data; A Sachs; R Parker, personal communications) [37]. For the MFA2 mRNA, analysis of poly(A) shortening both in z,it,o ancl hi vitro indicates that the 3'UTR controls tile rate and extent of poly(A) shortening. mRNAs containing the MFA2 3'-UTR are apparently destabilized as a consequence of rapid and complete poly(A) removal (A Sachs; R Parker, personal communications). For the transcript of the HTB1 gene, sequences in tile 3'UTR and in the adjacent coding region are responsible, in part, for tile periodic changes in histone mRNA abundance during the cell cycle [37]. Some c&acting sequences may act as mRNA stabilizing elements. Vreken el aL [54] have shown tilat insertion of poly(G)18 , but not otiler ribopolymers, into tile 3'UTR of the PGK1 mRNA causes a ~,ofolcl stabilization of this mRNA. Interestingly, tills insertion caused the accumulation of a stable 3' degradation intermediate extending front the poly(G) to the transcription termination site [55"]. The s~mle decay intermediate could also be detected in wild-type PGK1 mRNA, st,ggesting that poly(G) insertion may somehow reduce the rate at which primary cleavage products are subsequendy scavenged. This procedure may be generally useful as an approach to trapping decay intemlediates.
Trans-acting factors
involved in m R N A decay
A combination of genetics and biochemistry has identified a number of tran.s:acting factors tllat appear to be involved, eitiler directly or indirectly, in wast mRNA decay. These include CCA1, XRN1, PAN1, RNA14 and RNA15, and UPF1 and UPF3.
ts352 has a defect in the CCA1 gene, which encodes transfer RNA (tRNA) nucleotidyltransferase, the enzyme that adds 3'CCA-termini to tRNAs [56]. Once shifted to the non-permissive temperature, ts352 cells rapidly cease protein synthesis, reduce the rates of degradation of all tile mRNAs tested by three- to fivefold, and increase bodl die relative number of ribosomes associated with mRNAs and the overall size of polysomes (SW Peltz el al., unpublished data). These results are analogous to those observed in wcloheximicle-treated cells (SW Peltz et al., unpublished data) and are generally consistent with models that invoke a role R)r translational elongation in the process of mRNA turnover.
XRN
Tile best characterized yeast ribonuclease is a 160kD 5'-3' exoribonuclease encoded by XRN1 [41-43]. This nuclease degrades RNAs with 5' monophosphates, while capped RNAs are resistant to degradation [41]. X/aVl has been cloned and sequenced [44] and has been shown to be identical to several independently isolated genes, namely, DST2, SEP1 and KEM1 [57~50]. Surprisingly, the latter genes were thought to encode factors involved in nuclear fuskm or in DNA strand exchange during genetic recombination. Cells that are deleted for XRN1 are viable, but grow slowly, have altered rRNA processing [44,45"], and appear to stabilize the decay of several mRNAs (A Stevens, personal communication). Whether such stabilization is a direct consequence of tile deletion of XRN1 on mRNA-decw pathways or a consequence of the slowgrowth phenotype or even an alteration of protein synthesis (attributable to altered rR~NA biogenesis) remains to be determined.
PAN Recognizing tile potential importance of" the poly(A)shortening reaction to an understanding of tile turnover of specifc mRNAs, A Sachs and colleagues (personal communication) exploited a novel property of poly(A)binding protein (PAB) mutations in order to purify and characterize the poly(A) nuclease (PAN). Capitalizing on earlier work that had shown that PAB-deficient strains fail to shorten poly(A) tails [60], they were able to identify PAN activity by supplementing extracts from PAB1- cells with purified PAB. PAN was subsequently purified and shown to be a 135kD protein that requires the presence of PAB in order to degrade poly(A) in vitro. The PAN-PAB complex nomlally shortens poly(A) tracts in vitro to a length of 15-25 bp. For some nlRNAs, sequence elements in the 3'-UTR allow complete deadenylation and dec W of the transcript in vitro (A Sachs, personal communication).
CCA1
RNA14 and RNA15
Screening a collection of ts mutants identified ts352, a mutant that accumulated moderately stable and unstable mRNAs after a shift from 23°C to 37°C [56]. The mutant
Two other genes that appear to affect poly(A) metabolism have been investigated by Minvielle-Sebastia et al. [51"]. Mutants ill RNA14 and R N A 1 5 w e r e isolated
744
Prokaryotesand lower eukaryotes on the basis of their simultaneous cordycepin sensitivity and temperature-sensitivity [61]. Compared with wildtype cells, mutants in either of these two genes show rapid reductions in poly(A)-tail lengths and reduced stability of the ACT1 mRNA at the non-permissive temperature [51",61]. This rapid poly(A) shortening occurs at the same rate as the poly(A) shortening observed in cells where transcription has been inhibited, but r n a l 4 and rna15 mutants show no significant reductions in transcriptional activity. As the rapid poly(A)-shortening phenotype is observed with both old and newly synthesized transcripts, it is unlikely that the rna14 and rna15 mutations simply affect nuclear polyadenylation. These phenotypes are compatible with defects that either increase poly(A)-shortening rates or decrease the rate of cytoplasmic poly(A) addition. Both RNA14 and RNA15 are single-copy essential genes. The primary sequence of RNA15 is similar to known RNA- and DNA-binding proteins in that it contains an anlino-terminal segment with RNP1 and RNP2 consensus sequences followed by glutamine- and asparagine-rich regions. Over-expression of either RNA14 or RNA15 leads to suppression of both the r n a l 4 and r n a l 5 mutations, indicating that the respective gene products probably interact [51"].
UPF1 and UPF3
In both prokaryotes and eukaryotes, nonsense mutations in a gene can enhance the decay rate of the mRNA transcribed from that gene, a phenomenon described as nonsense-mediated mRNA decay (see [40.,] for a review). Early work on nonsense mutations in the yeast URA3 gene showed that mRNA destabilization is linked to premature translational termination, because the nonsensecontaining mRNA is stabilized in a strain containing an amber suppressor-tRNA. In addition to tRNA mutants, nonsense suppressors in yeast can also be mutants in non-tRNA genes, which enhance the expression of nonsense-containing alleles by other mechanisms. The latter mutants include the allosuppressors, frameshift suppressors, and omnipotent suppressors [63,64]. Mutations in two genes that were were isolated as allosuppressors, UPF1 and UPF3, lead to the selective stabilization of mRNAs containing earl), nonsense mutations without affecting the decay rates of most other mRNAs [40°°,49-,50]. The UPF1 gene has been cloned and sequenced and shown to be non-essential for viability, capable of encoding a 109 kD protein with both zinc finger and nucleotide (GTP)-binding-site motifs, and partially homologous to the yeast SEN1 gene [50]. The latter encodes a non-catalytic subunit of the tRNA-splicing endonuclease complex [65], suggesting that U p f l p may also be part of a nuclease complex targeted specifically to nonsense-containing mRNAs.
Translation and turnover are intimately linked in yeast Experiments described above indicate that: (a) instability elements involved in the rapid decay of five mRNAs have been localized to the coding regions of the respective mRNAs; (b) inhibition of translational elongation can reduce mRNA-decay rates; (c) metabolism of the poly(A) tail, a structure recently shown to be involved in translational initiation [66], is an integral step in the decay of several mRNAs; and (d) premature translational termination can enhance mRNA decay rates. These observations indicate that the processes of mRNA translation and mRNA turnover must be intimately linked. Further evidence to support this conclusion comes from experiments in yeast that show that ribosome translocation up to, or through, the a ~ 7 ~ Z instability element is required for rapid decay of PGK1-MATotl and ACT1-MATotl chimeric mRNAs [38]. A requirement for specific sequences and translation could be accommodated by the occurrence of an), of the following events in the initiation of mRNA decay. First, the ribosome could pause at a specific sequence - - pausing may be a consequence of a mRNA-rRNA interaction [40..], clustered rare codons [38], or interactions promoted by the nascent polypeptide [67]. Second, a paused ribosome could activate a nuclease and/or expose a downstream cleavage site. Third, cleavage could be mediated by a metabolically unstable nuclease. The requirement for translation may not be limited to a single step in the turnover process, but, as in the first two points, may reflect a need for ongoing protein .wnthesis at several mutually dependent events.
Conclusion Our understanding of mRNA turnover in bacteria is somewhat more advanced than for eukaryotic systems. Many of tile concepts developed from bacterial studies are guiding studies and their interpretation in eukaryotes. It is perhaps relevant to consider how similar or dissimilar the pathways of turnover in prokaryotes and eukaryotes are. There is one important operational difference: bacterial mRNA is generally considered to be unstable unless stabilizing sequences (e.g. 3' terminal stem-loops) are present. In contrast, eukaryotic messages are generally considered stable unless instability signals are introduced. This difference is probably because eukaryotic messages must remain intact as they are transported from the nucleus to the cytoplasm: in contrast, prokaryotic mRNA is normally translated as it is being synthesized and long-term stability is less necessary. The intrinsic stability of eukaryotic messages is presumably, at least in part, achieved by the modified 3' (polyA) and 5' (capped) ends. A second difference, probably for reasons related to the above, is the role of translation.
Turnover of mRNA in prokaryotes and lower eukaryotes Higgins, Peltz, Jacobson Translation appears to play a more 'direct' role in mRNA decay in eukaryotic systems. This is understandable when considering that in eukaryotes mRNA is not translated immediately after it is synthesized but must first be transported to the cytoplasm. It would be inappropriate if a message were degraded before ribosomes had loaded. Nevertheless, despite these differences, the general pathways of turnover in prokaryotes and eukaryotes may be quite similar, involving a combination of endo- and exonucleolytic activities and specific sequences that serve as recognition sites for these enzymes and so determine stability. Most of the concepts and techniques are now in place for investigating mRNA turnover and we can look forward to considerable progress during the next few years.
Structural Gene of Escherichia coll. Proc Nail Acad Sci USA 1991, 88:1-5. This reference, together with [7**], demonstrates that RNaseE has a general role in the degradation of mRNA in E coll. This is the first endonuclease shown to play a role in the turnover of man), different messages. Also shows that the rne and ares mutations are allelic. 9.
NILe;SONP, UHLIN BE: Differential Decay of a Polycistronic coli Transcript is Initiated by RNaseE-dep e n d e n t Endonucleolytic Processing. Mol Microbiol 1991, 5:1791-1799. Shows that rne mutations affect RNaseE activity and thus the processing of pap mRNA. •
10. .
MEI.EFORSO, VON GABAIN A: Genetic Studies of Cleavageinitiated mRNA Decay and Processing of Ribosomal 9S RNA Show that the Escherichia coli ares and r n e Loci are the Same. Mol Microbiol 1991, 5:857-864. Shows that ~vle mutants, defective in RNaseE activity, ,affect processing of ompA message in vivo. 11.
Acknowledgments This work was supported by a National Institutes of Health grant (GM27757) to A Jacobson, a postdoctoral fellowship to SW Peltz from the American Cancer Society, and by support to CF l-liggins from the Imperial Research Fund.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest CANNtSTRARO VJ, KENNEtL D: RNasel', a Form of RNase I, and mRNA Degradation in Escherichia coll. J Bacteriol 1991, 173:4653-4659. Characterization of RNasel*, a novel enzyme that probably has a role degrading residual short oligoribonucleotides to monoribonucleotides during the final stages of mRNA turnover.
BEI2VSCO J, HIGGINS CF: Mechanisms of mRNA Decay in Bacteria: a Perspective. Gene 1988, 72:15-23.
3. ••
MCLARENP~, NEWBURYSF, DANCE GSC, CAUSTON HC, HIGGINS CF: mRNA Degradation by Processive 3 ' - 5 ' Exoribonucleases in Vitro and the Implication for Prokaryotic mRNA Decay in Vivo. J Mol Biol 1991, 220:81-95. In vitro study on the ability of stem-loop structures to impede the activity of processive endonucleases. Differences between data obtained in vivo and in vitro imply the existence of stem-loop mRNA-binding proteins that play a role in mRNA turnover.
FAtlBLADIERM, C&~I K, BOUCHE J-P: Escherichia coli Cell Division Inhibitor DicF.RNA of the dicB Operon. J Mol Biol 1990, 212:461-471.
REGNIERP:, I-bXJNSDORFE: Decay of mRNA Encoding Ribosoma] Protein S15 of Escherichia coil is Initiated by an RNaseE E-dependent Endonucleolytic Cleavage that Rem o v e s the 3' Stabilizing Stem and Loop Structure. J Mol Biol 1991, 217:283-292. Demonstrates that rne mutations affect processing and decay of a ribosomal protein RNA. 12. •
13. •
MACKIEGA: Specific Endonucleolytic Cleavage of the mRNA for Ribosomal Protein S20 of Escherichia coil Requires the Product of the a m s Gene in Vivo and in Vitro. J Bacteriol 1991, 173:2488-2497. Cleavage of the $20 message by RNaseE (the ares-encoded product) determines its rate of degradation. 14.
1. ••
2.
Escherichia
KOKOSKARJ, BLUMERKJ, STEEGE DA: Phage fl mRNA Processing in Escherichia coli: Search for the Upstream Products of Endonuclease Cleavage, Requirement for the Product of the Altered mRNA Stability ( a m s ) Locus. Biochimie 1990, 72:803-811.
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LtN-CHAOS, COHEN SN: The Rate of Processing and Degrada. tion of Antisense RNAI Regulates the Replication of ColE1type Plasmids in Vivo. Cell 1991, 65:1233-1242. Demonstration that cleavage of the RNA1 antisense species by RNaseE plays a role in regulating plasmid copy-number and replication. 16.
CHAUHANAK, MICZAKA, TARASE\qCIENE L, APIPdON D: Sequencing and Expression of the r n e Gene of Escherichia colt Nucleic AcicL~ Res 1990, 125-129.
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CLAVERIE-MARTINF, DtAZ-TORRES/VlR, YANCEY SD, KUSHNERSR: Analysis of the Altered mRNA Stability ( a m s ) Gene from Escherichia coll. J Biol Chem 1991, 266:2843-2851. Sequence of the gene encoding RNaseE (also see [16]). 18.
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MUDDEA, CARPOUSlSAJ, KRISCH HM: Eschertchia coli RNase E has a Role in the Decay of Bacteriophage T4 mRNA. Genes Dev 1990, 4:873-881.
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MUDD EA, KmSCH HM, HIGGINS CF: RNase E, an Endoribonuclease, has a General Role in the Chemical Decay of Eschertchia coli mRNA: Evidence that r n e and ares are t he Same Genetic Locus. Mol Microbiol 1990, 4:2127-2135.
••
see [8"]. 8. ••
BABrrZKX P, KUSHNER SP,: T h e Ams (Altered mRNA Stability) Protein and Ribonuclease E are E n c o d e d by t h e Same
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21. ••
LUNDBERGU, VON GABAIN A, MELEFORS O: Cleagaves in t h e 5' Region of the o m p A and bl a mRNA Control Stability: Studies with an E. coli Mutant Altering mRNA Stability and a Novel Endoribonuclease. EMBO J 1990, 9:2731-2741.
745
746
Prokaryotes and lower eukaryotes Describes the purification of RNaseK, an endonuclease that clem,es the 5' end of ompA and bla mRNA. 22. •
SRIVASTAVASK, CANNISTRAROVJ, KENNEL D: Broad Specificity Endoribonucleases and mRNA Degradation in Escherichia coli. J Bacteriol 1992, 174:56452. Characterization of two relatively non-specific endonucleases, RNaseM mid RNaseR. Thus far, their roles in mRNA turnover are undefined. 23.
NUSSONG, BEbVSCOJG, COHEN SN, VON G,mAIN A: GrowthRate Dependent Regulation of mRNA Stability Ion Escherichia coli. Nature 1984, 312:75-77.
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37.
Xu H, JOHNSON L, GRUNSTF.INM: Coding and Non-coding Sequences at the Y End of Yeast Histone H2B mRNA Confer Cell-Cycle Regulation. Mol Cell Biol 1990, 10:2687-2694.
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PARKERR, JACOBSON A: Translation and a Forty-Two Nucleotide Segment within the Coding Region of the mRNA Encoded by the M A T k l Gene are Involved in Promoting Rapid mRNA Decay in Yeast. Proc Natl Acad Sci USA 1990, 87:2780-2784.
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HERRICKD, JACOBSON A: A segment of the Coding Region is Necessary but not Sufficient for Rapid Decay of the HIS3 mRNA in Yeast. Gene 1992, 114:35-41.
PELT'ZS\Xz, JACOBSONA: mRNA Turnover in Saccharomyces cerevisiae. In Control oJ'ml~VA stabilio,. Edited by Brawerman G and Belasco J. New York: Academic Press; 1992. A comprehensive review of the current state of mRNA turnover research in the y~lst Saccbarom3,ces cerevisiae. 40. •.
25.
EMORYSA, BEIASCO JG: The o m p A 5' Untranslated RNA Segment Functions in Escherichia coil as a Growth-Rateregulated mRNA Stabilizer w h o s e Activity is Unrelated to Translational Efficiency..1 Bacteriol 1990, 172:4472-4481.
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CHEN L-H, EMORY SA, BRICKER AL, BOtrVET P. BEL.kSCO JG: Structure and Function of a Bacterial mRNA Stabilizer: Analysis of the 5' Untranslated Region of ompA mRNA. J Bacteriol 1991, 173:4578-4586.
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Sll~VI'NSA: Purification and Characterization of a Saccha. romyces cerevisiae Exoribonuclease which Yields 5'-Monophosphates by a 5'-3' mode of Hydrolysis. J Biol Chem 1980, 255:3080-3085.
EMORYSA, BOt"VET P, BEt.,VSCOJG: A 5'-Terminal Stem-Loop Structure can Stabilise mRNA in Escherichia coll. Genes Det, 1992, 6:135-148. A detailed analysis of secondary structures at the 5' end of ompA mRNA that provides insights into the requirements R)r initial ribonuclease cle~avage and the propagation of RNA turnover follox~Sng this cleavage.
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STEVENSA, MAUl'IN MK: A 5'-3' Exoribonuclease of Saccharomyces cerevisiae. Size and Novel Substrate Specificity. Arch Biocbem Bioph|,s 1987, 252:339-347.
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IARIMERFkXs, S1TVENSA: Disruption of the Gene XRNI, Coding for a 5'-3' Exoribonuclease, Restricts Yeast Cell Growth. Gene 1990, 95:85-90.
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CHEN C-YA, Bl't,vsco JG: Degradation of pufIa}lX mRNA in Rhodobacter capsulatus is Initiated by Nonrandom Endonucleolytic Cleavage. J Bacteriol 1990, 172:4578-4586. KI.UGG, COHEN SN: Combined Actions of Multiple Hairpin Loop Structures and Sites of Rate-limiting Endonucleolytic Cleavage Determine Differential Degradation Rates of Individual Segments within Polycistronic p u f O p e r o n mRNA..I Bacteriol 1990, 172:5140-5146. KLUG G, COHEN SN: Effects of Translation on Degradation of mRNA Segments Transcribed from the Polycistronic p u f Operon of R h o d o b a c t e r capsulatus. J 13acteriol 1991, 173:1478-1484.
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MIC;'.AKA, SRIX',XSTAVARAK, APIRION D: Location of the RNAprocessing Enzymes RNase III, RNase E and RNase P in the Escherichia coli Cell. Mol Microbiol 1991, 5:1801-1810.
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HUTCHIS, ON HT, 1-1AR'D,VEH.LH, McLMJGHHN CS: Temperaturesensitive Yeast Mutant Defective in Ribonucleic Acid Production. J Bacteriol 1969, 99:807-814.
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KUO SC, CANO FR, L~XlI'EN JO: Lomofungin, an Inhibitor of Ribonucleic Acid Synthesis in Yeast Protoplasts: its Effect on Enzyme Formation. Antimicrob Ag Chemother 1973, 3:716-722.
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HERmCKD, PARKER R, J^COBSON A: Identification and Comparison of Stable and Unstable mRNAs in the Yeast Saccharomyces cerevisiae. Mol Cell Biol 1990, 10:2269-2284.
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36. •
PARKERR, HERRICKD, PELTZSW, JACOBSONA: Measurement of mRNA Decay Rates in S a c c h a r o m y c e s cerevisiae. In Metly otis in Enzymology: Molecular Biology of Saccharomyces cer@ visiae. Edited by Guthrie C, Fink G. New York: Academic Press; 1991:415-423.
PRESLrI'nC, CLAFRE S.-& BOZZONt 1: The Ribosomal Protein L2 in S. cerevisiae Controls the Level of Accumulation of its O w n mRNA. ElVlBO J 1991, 10:2215-2221. An example of an autogenously regulated yeast mRNA. This study reports that over-expression of ribosomal protein L2 diminishes the relative abundance of the L2 mRNA.
45. •
Sla:.VI'NSA, l-lstt CL, ISHAMKR, L.XRIMI¢Rl"XXr:Fragments of the Internal Transcribed Spacer 1 of pre-rRNA Accumulate in Saccharomyces cerevisiae Lacking 5'-3' Exoribonuclease I. J Bacteriol 1991, 173:7024-7028. This paper describes the initial characterization of the mutant deficient in 5'-3' endonuclease. 46.
Sll"VENS A: I~,rimidine.specific Cleavage by an Endoribonuclease of Saccharomyces cerevisiae. J Bacleriol 1985, 164:57-62.
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VREKEN P, BUDDE'I2.1EIJER N, RAUE HA: A Cell-free Extract from Yeast Cells for Studying mRNA Turnover. Nucleic ,,Icid~ Res 1992, 20:2503-2510. The first ex~unple of an in vitro mRNA-decay system for 3,east. 49. ..
LEEDSP, PELT'/. SW, JACOBSON A, CUmEa'rSON MR: The Product of the Yeast UPFI Gene is Required for Rapid Turnover of mRNAs Containing a Premature Translational Termination Codon. Genes Det, 1991, 5:2303-2314. This paper demonstrates that nonsense-mediated mRNA decay, but not the decay of inherently unstable mRNAs, is dependent on the product of the UPF1 gene. 50.
LEEDS P, WOOD JM, LEE B.-S~ CUI.BERTSON MR: Gene Products that Promote mRNA Turnover in Saccharomyces cerevisiae. Mol Cell Biol 1992, 12:2165-2177.
51. °°
MINVIF.I.LE-SEBASTIAL, WINSOR B, BONNEAUD N, LACROUTE F: Mutations in the Yeast RNA14 and R N A I 5 Genes Result in an Abnormal mRNA Decay Rate; Sequence Analysis Reveals an RNA-binding Domain in the R N A I 5 Protein. Mol Cell Biol 1991, 11:3075-3087. Describes the isolation and characterization of two genes that affect both poly(A) metabolism and mRNA stability. 52.
NONETM, SCAFE C, SEXTON J, YOUNG R: Eucaryotic RNA Polymerase Conditional Mutant that Rapidly Ceases mRNA Synthesis. Mol Cell Biol 1987, 7:1602-1611.
Turnover of mRNA in prokaryotes and lower eukaryotes Higgins, Peltz, Jacobson 53.
PELTZSW, BREwEr G, BERNSTEIN P, ROSS J: Regulation of mRNA Turnover in Eukaryotic Cells. Crit Rev Euk Gene F_.xp 1991, 1:99-126.
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BLOCH JC, PERRIN F, LACROUTEF: Yeast T e m p e r a t u r e Sensitive Mutants Impaired in Processing of poly(A)-containing RNAs. Mol Gen Genet 1978, 165:123-127.
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VREKENP, VAN DER VEEN P, DE REG'FVCHF, DE MAATAL, PLANTA RJ, RAUE HA: Turnover Rate of Yeast PGKI mRNA can be Changed by Specific Alterations in its Trailer Structure. BiodMmie 1991, 73:729-737.
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LOSSON R, LACROUTEF: Interference of Nonsense Mutations with Eukaryotic Messenger RNA Stability. Proc Nail Acad Sci USA 1979, 76:5134-5137.
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HINNEBUSCH AG, LIEBbIANSW: Protein Synthesis and Translational Control in Saccharomyces cerevisiae. In The MolecMar a n d Celhdar Biologl, of the Yeast Saccharono,ces: Volu m e L Genome Dynamics, Protein Synthesis, a n d Energetics. Edited by Broach JR, Pringle JR, Jones EW. Cold Spring Harbor: Cold Spring Harbor ktboratory Press; 1991:627-735.
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WINEY M, CULBERTSON MR: Mutations Affecting the tRNAsplicing Endonuclease Activity of Saccharomyces cerevisiae. Genetics 1988, 118:607-617.
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MUNROE D, JACOBSONA: Tales of p o l y ( A ) - - a Review. Gene 1990, 91:151-158.
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CLEVELANDDW: Autoregulated Instability of Tubulin mRNAs: a Novel Eukaryotic Regulatory Mechanism. Trends Biochem Sci 1988, 13:339-343.
55. •
VREKENP, RAtJE HA: The Rate-limiting Step in Yeast PGK1 mRNA Degradation is an Endonucleolytic Cleavage in the 3'-Terminal Part of the Coding Region. Mol Cell Biol 1992, 12:2986-2996. The first example in yeast of specific decay intermediates.
56
57.
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59.
60.
AEBI M, KIRSCHNERG, CHENJ.-Y, VIJAYRAGHAVANU, JACOBSONA, MARTIN NC, ABEI£ONJ: Isolation of a T e m p e r a t u r e Sensitive Mutant with Altered tRNA Nucleotidyl Transferase Activities and Cloning of the Gene Encoding tRNA Nucleotidyltransferase in the Yeast Saccharomyces cerevisiae. J Biol Chem 1990, 265:16216-16220. DYKSTRACC, HAM&TAKERK, SUGINO A: DNA Strand Transfer Protein from Yeast Mitotic Cells Differs from Strand Transfer Protein at from Meiotic Cells. J Biol Chem 1990, 265:10968-10973. D'CKSTRACC, KITADAK, CLARKAB, HAblATAKERK, SUGINO A: Cloning and Characterization of DST2, the G e n e for DNA Strand Transfer Protein from S a c c h a r o m y c e s cerevisiae. Mol Cell Biol 1991, 11:2583-2592. KIM J, LJUNGDAHLPO, FINK GR: k e m Mutations Affect Nuclear Fusion in Saccharomyces cerevisiae. Genetics 1990, 126:799-812.
CF Higgins, ICRF Laboratories, Institute of Molecular Medicine, Universit), of Oxford, John Radcliffe Hospital Oxford OX3'9DU, UK.
SACHSAB, DAVtS RW: The poly(A)-binding Protein is Required for poly(A) Shortening and 60S Ribosomal Subunit Dependent Translation Initiation. Cell 1989, 58:857-867.
SW Peltz, A Jacobson, Department of Molecular Genetics and lVlicrobiology, University of lVlassachusetts Medical School, Worcester, Massachusetts 01655, USA.
747
Introdudion The amount of a given protein synthesized in the cell depends, to a large degree, upon the abundance of its cognate mRNA. The fact that the steady-state level of mRNA depends on its rate of degradation, as well as its rate of synthesis is frequently forgotten. Nevertheless, it is now clear that cells possess a sophisticated apparatus for the degradation of mRNA, and that differences in the rate of mRNA degradation can play an important role in determining the level of gene expression. The vast majority of studies on mRNA turnover have concentrated on a small number of model species and a few specific messages in these species. This article is divided into two halves, first addressing recent studies on E. coli as a model prokaryore and then S. cerevisiae as a model eukaryote.
Turnover of mRNA in prokaryotes The pathways by which mRNA turnover in E. coli is mediated have been delineated in broad ternls, although many problems still remain to be answered. It is now clear that there is no single pathway but that different mRNA species are often degraded by different routes. These pathways operate in parallel such that, if the normal degradative pathway for an mRNA molecule is compromised (e.g. by mutation), an alternative pathway comes into operation. This default pathway may not be
much slower than the primary pathway and so elimination of the primary pathway does not necessarily have a major effect on stability. Indeed, it seems likely that for some messages two alternative pathways of degradation may operate at similar rates enabling different molecules within the population to be degraded by different routes. These overlapping and alternative pathways of degradation explain, at least in part, why an understanding of the factors that influence mRNA turnover has lagged behind our understanding of the control of mRNA synthesis.
Exoribonucleases The degradation of mRNA is mediated by a combination of endonucleotylic and exonucleolytic events (Fig. 1). In the simplest scenario, degradation of an mRNA molecule is mediated by two 3'-5' exonucleases, RNaselI and polynucleotide phosphorylase (PNPase) (Fig. la). These two enzymes attack the 3' end of a message, processively removing ribonucleotides as they proceed towards the 5' end. In the absence of any factor impeding their progress, the end-product of degradation by these enzymes (other than the recycled monoribonucleotides) are small oligoribonucleotides of 10-20 bp. An enzyme that may mediate the final degradation of these small oligoribonucleotides to mononucleotides has recently been identified and designated as RNaseI* [1..]. This enzyme is closely related to, but apparently distinct from, the periplasmic ribonuclease RNaseI.
Abbreviations PAB--poly(A)-binding protein; PAN--poly(A) nuclease;PNPase~polynucleotidephosphorylase; rRNA--ribosomal RNA; IRNA--transfer RNA; ts--temperature-sensitive; UTR--untranslated region.
(~) Current Biology Lid ISSN 0959-437X
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Fig. 1. Schematic illustration of the general,pathways for mRNA degradation in prokaryotes. Messenger RNA with an unblocked 3' end is degraded principally by two processive 3 -5 exonucleases, polynucleotide phosphorylase (PNPase)and RNasell (a,i). For many mRNAs the processive activities after interaction of these enzymes are blocked by a stem-loop structure, possibly after interaction with RNAbinding proteins that recognize such structures (a,ii). In many, and probably most, cases the block to exonu(slease activity is sufficiently long that decay is initiated by upstream endonuclease cleavage (b). This may be achieved by an endonuclease removing or destroying the terminal stem loop (b,i), or endonuclease cleavage at the 5' end and subsequently elsewhere in the mRNA, producing many small fragments (b,ii). RNaseE is the only endonuclease known to play a rather general role in these processes. RNaseK acts on the 5 end of at least some mRNAs, but it remains to be established whether or not RNaseK is entirely distinct from RNaseE. For a few specific mRNA molecules, RNaselll also plays a role. Of course, other enzymes that play specific or general roles might remain to be identified. Finally, the products of endonuclease cleavage are degraded to mononucleotides and small oligonucleotides by the two exonucleases, RNasell and PNPase, and the small oligonucleotides may be scavenged by RNaseP.
Most mRNAmolecules are not degraded by this simple route but include sequences that can potentially form secondary (stem-loop) structures in the message and impede the processive activities of these two exonucle-
ases (Fig. lb). Over the past few years, considerable evidence that stem-loop structures can stabilize upstream mRNA in vivo, presumably by impeding the activities of PNPase and RNaseII, has accrued (reviewed in [2]).
Turnover of mRNA in prokaryotes and lower eukaryotes Higgins, Peltz, Jacobson 741 These concepts have recendy been tested more direcdy in vitro [3"']. Stem-loop structures that stabilize upstream mRNA in vivo have been shown to impede the processive activities of both PNPase and RNaselI in vitro [3"']. Perhaps surprisingly, the period of time for which these exonucleases were impeded was relatively short compared with the increased half-life these sequences confer on mRNA in vivo. This implies that an additional factor, for example a stem-loop-binding protein, may play a role in the mRNA stabilization by such structures in vivo. evidence for such a protein has now been obtained (HC Causton and CF Higgins, unpublished data). The majority of messages have a stem-loop structure at the 3' end (rho-independent transcription terminators consist of a stem-loop structure). For these messages, degradation can potentially proceed by the exonucleases overcoming the barrier presented by the terminal structure. However, the weight of evidence now indicates that for most messages the terminal stem-loop structure provides an efficient block to exonucleolytic decay such that decay is initiated by upstream endonucleolytic cleavage (Fig. lb). It should be noted that there is, as yet, no evidence for a 5'-3' exoribonucleolytic activity in E. coli. Indeed, for the majority of message an exoribonuclease that attacks the free 5' end would be incompatible with the available data.
Endonucleases The most important advances in the last couple of years have been in the identification of endonucleases and characterization of their cleavage sites that, for many messages, provide the rate-determining step in degradation. These nucleases bypass the barrier to 3'-5' exonucleases, either by removing the terminal stem-loop structure (Fig. lb,i) or by cleavage at the 5' end of the message, which initiates subsequent decay (Fig. lb,ii). The products of endonuclease cleavage are then degraded to mononucleotides by the 3'-5' exonucleases PNPase and RNaseII. For many years the only endonuclease known to play a role in mRNA degradation has been RNaselII. RNaseIII recognizes large stem-loop structures and, as most messages lack such sites, cannot play a role in the degradation of more than a small subset of messages. Recently, RNaseE has been shown to play a central role in the degradation of a large number of messages. RNaseE was identified many years ago as a consequence of its role in processing the 9S ribosomal RNA (rRNA) precursor [4]. Despite earlier reports that this enzyme plays litde or no role in mRNA turnover [5] it is now clear that this is incorrect: mutants in the rne gene, defective in RNAaseE activity, result in the general stabilization of E. coli and bacteriophage mRNA and several individual mRNA's are stabilized in these mutants [6,7"',8"']. Several other studies have also identified defects in specific mRNA-processing events in rnemutants, including mRNA for p a p pili [9"], ompA [10.], dicB [11], ribosomal proteins S15 [12.] and $20 [13-], phage fl [14], and the antisense RNA1 that plays a role in determining ColE1
plasmid replication [15"]. Finally, the ares mutation identified several years ago as conferring altered mRNA stability, has been shown to be allelic with me. Two sequences for the rne (arm) gene have been published, one predicting a protein of 62kD [16], the other a protein of 91 kD [17"]. Both sequences may contain frameshifts and predict a prematurely terminated polypeptide since the relative mobility of the RNaseE protein indicates a molecular weight of 180 kD (EA Mudd and CF Higgins, unpublished data; IB Holland, personal communication). The cleavage specificity of RNaseE is still unclear. Following identification of the first few cleavage sites a consensus appeared to be emerging [6]. However, as more sites have been identified the existence of a consensus sequence is less convincing. Nevertheless, mutational studies at or near the site of cleavage show that RNaseE recognizes both sequence and structural elements [15"',18.',19"]: cleavage occurs upstream of a stem-loop structure and there is a preference for an AU dinucleotide 3' to the site of cleavage. Although RNaseE plays an important role in the degradation of many mRNA's, it is certainly not the only endonuclease involved. In rne mutants, even those mRNA molecules whose stability is increased eventually turn over: another endonuclease must initiate this alternative pathway. For some messages, an enzyme other than RNaseE may play an initiating role in degradation. The decay of b/a and ompA mRNA's is initiated by endonucleolytic cleavage events at the 5' end [20]. An endonuclease, designated RNaseK, has been purified that cleaves these mRNA's in vitro at sites similar to those observed in vivo [21..]. Whether this enzyme also cleaves other mRNA's is not yet known. Some of the sites in ompA mRNA that are cleaved by RNaseK in vitro are affected by rne mutations in vivo. This can be interpreted in one of two ways: RNaseE may regulate the expression/activity of RNaseK or, alternatively, RNaseK may not be a distinct enzyme but a fragment of RNaseE. The molecular weight of RNaseK is about 60kD [21-.] - - it is known that small proteolytic fragments of RNaseE can retain enzymic activity (EA Mudd and CF Higgins, unpublished data). This possibility needs to be rigorously excluded before we can be sure that RNaseK and RNaseE are two distinct enzymes. Finally, two broad-specificity endonucleases have recently been characterized, RNaseM and RNaseR [22.]; any general role they may play in mRNA turnover is, as yet, unclear. If we are to be able to predict from its sequence the stability of a message, it is important to identify the sites responsible for determining the initial endonucleolytic cleavage events that provide the rate-determining steps in degradation. At least for some messages this initial cleavage, and therefore mRNA turnover, appears to be regulated. For example, cleavage of ompA mRNA appears to be regulated by the growth rate [23], and cleavage of p u f m R N A is influenced by oxygen availability [24..]. For some messages, such as ompA secondary structures at the 5' end appear to play a central role in determining the rate-determining endonucleolytic cleavage events [25,26]. It seems that the sequence of the secondary structure is of less importance than the location of the secondary
742
Prokaryotes and lower eukaryotes structure with respect to the 5' end of the message [27-]. If the structure is more than 2 - 4 b p from the 5' end, its stabilizing role is lost [27..]. One of the maior challenges is to understand the events following an initial cleavage event at the 5' end. It is possible that, following initial cleavage near the 5' end, a 5'-3' exonuclease processively degrades the mRNA from the point of cleavage. However, there is no evidence for such an enzyme and the presence of many degradation intermediates would tend to argue against this mechm-tism. Alternatively, secondaw endonucleolytic cleavages may be promoted following recognition or cleavage at the 5' end of the message: these secondary cleavages could be mediated by the original enzTme or by a second enzyme [27.,]. Fragments generated by these secondary endonucleolytic cleavages would then be degraded by the 3'-5' exonucleases RNaselI and PNPase (Fig. lb,ii). This latter model does, however, pose a problem: why do the secondary cleavages not occur until an initial event at the 5' end has occurred? Finally, although the 5' end is clearly crucial for the decay of some messages, this is not necessarily generally true: for certain messages, multiple defined sites are cleaved and all must be removed if a message is to be stabilized [28,29].
Translation The role of translation in mRNA degradation has been controversial. Some early reports suggested that translation stimulates mRNA degradation, others that translation stabilizes mRNA. Many of these studies have been difficult to interpret, particularly those making use of translational inhibitors. Recent data show that, at lea.st for the ompA message, degradation that is initiated by an endonucleolytic cleavage at the 5' end is independent of translation rates [25]. For other messages, translation does affect deca), [30]. However, there is no evidence that translation is normally essential for degradation, and enzymes like RNaseE are not associated vdth ribosomes [31]. Most probably the effects of translation on mRNA turnover are indirect, influencing the accessibility of endonuclease cleavage sites.
Yeast as an experimental system in which to study mRNA turnover Work on mRNA decay in the yeast S. cerevisiae began more than two decades ago [32,33], but only recently has the subject attracted the efforts of a sufficiently large number of laboratories necessary for a concerted attack on the underlying regulatory mechanisms. Research has now reached a point where a simple and reliable procedure for the determination of decay rates has been established [34,35]: stable, unstable, and regulated mRNAs have been identified [34,36",37], chimeric mRNAs have been utilized to delineate cis-acting instability determinants (B Heaton et al., unpublished data) [38,39,40"], nucleases have been purified and begun to be characterized [41-44,45-,46,47], and an in vitro decay system
has been used to localize cleavage sites [48..]. Of particular significance to an understanding of the interplay between the turnover machinery and the translational apparatus, yeast mutants defective in mRNA turnover have now been isolated and are beginning to shed light on important trans-acting factors (SW Peltz et aL, unpublished data) [47,49"',50,51"].
C/s-acting sequences as determinants of mRNA instability In strains harboring the temperature-sensitive (ts) ppbl- I allele of RNA polymerase II, a shift to 36°C leads to the rapid and selective cessation of mRNA synthesis and to a reduction in the steady-state levels of pre-existing mRNAs [52]. This observation led to the development of a routine procedure for the measurement of mRNA-deca}, rates in which cells growing at 24°C were abruptly shifted to 36°C and changes in the relative anlounts of individual mRNAs were quantified by RNA-blotting procedures [34,35,52]. The half-lives of the different mRNAs measured by this procedure range from 1~50 min, a result consistent with the complex decay kinetics observed for the total mRNA population [34]. In an approach analogous to those used for other systems (see [53], for a review), cis-acting instability sequences have been identified in yeast that promote rapid mRNA decay when transferred to mRNAs that are normally stable (B Heaton et aL, unpublished data) [34,38]. In general, chimeric genes encoding portions of a stable and an unstable mRNA are constructed (the reading franle across the hybrid junction is maintained), introduced into cells (~12bl-1 mutants) on centromere vectors, and the decay rates of the resulting hybrid transcripts and their 'parental' mRNAs mea*sured using RNA isolated from temperature-shiftecl cells. The simplest expectation from this type of experiment is that instability elements will be modular; that is, hybrid mRNAs which contain sequences sufficient to promote rapid turnover will themselves exhibit rapid decw kinetics. Hybrid mRNAs that lack such sequences should be stable. These expectations have been borne out by the results of experiments in which chimeras have been constructed between the stable mRNAs encoded by the ACT] or PGK1 genes and the unstable mRNAs encoded by the MATotl, STE3, STE2, HIS3 and CDC4 genes (B Heaton el aL, unpublished data) [38,39,40"]. For example, a chimeric mRNA comprised of the stable PGK1 mRNA fused, in frmne, to the unstable k,D
Turnover of mRNA in prokaryotes and lower eukaryotes Higgins, Peltz, Jacobson 743 Experiments analogous to those described for MATkl have also been carried out with the unstable HIS3, STEP, STE3 and CDC4 mRNAs (B Heaton et al., unpublished data) [38,39,40°,]. Sequences essential for rapid decay of chimeric mRNAs have been found in the coding regions of all four mRNAs. Evidence for additional complexity of instability determinants was obtained in studies of the STE3 mRNA. Whereas deletion of STE3 sequences spanning codons 13-179 stabilizes a STE3-ACT1 chimeric mRNA approximately threefold, deletion of the same sequences from bona fide STE3 mRNA fails to stabilize that mRNA (B Heaton el al., unpublished data). This suggests tilat other regions of the STE3 mRNA can stimulate mRNA decay independently of the coding-region sequences. Further analysis demonstrated that the STE3 3' untranslated region (UTR) can act as the independent instability element (B Heaton et aL, unpublished data). Results suggesting tile presence of 3'-UTR instability elements have also been obtained for the STk~, HTB1 and MFA2 mRNAs (D Herrick and A Jacobson, unpublished data; A Sachs; R Parker, personal communications) [37]. For the MFA2 mRNA, analysis of poly(A) shortening both in z,it,o ancl hi vitro indicates that the 3'UTR controls tile rate and extent of poly(A) shortening. mRNAs containing the MFA2 3'-UTR are apparently destabilized as a consequence of rapid and complete poly(A) removal (A Sachs; R Parker, personal communications). For the transcript of the HTB1 gene, sequences in tile 3'UTR and in the adjacent coding region are responsible, in part, for tile periodic changes in histone mRNA abundance during the cell cycle [37]. Some c&acting sequences may act as mRNA stabilizing elements. Vreken el aL [54] have shown tilat insertion of poly(G)18 , but not otiler ribopolymers, into tile 3'UTR of the PGK1 mRNA causes a ~,ofolcl stabilization of this mRNA. Interestingly, tills insertion caused the accumulation of a stable 3' degradation intermediate extending front the poly(G) to the transcription termination site [55"]. The s~mle decay intermediate could also be detected in wild-type PGK1 mRNA, st,ggesting that poly(G) insertion may somehow reduce the rate at which primary cleavage products are subsequendy scavenged. This procedure may be generally useful as an approach to trapping decay intemlediates.
Trans-acting factors
involved in m R N A decay
A combination of genetics and biochemistry has identified a number of tran.s:acting factors tllat appear to be involved, eitiler directly or indirectly, in wast mRNA decay. These include CCA1, XRN1, PAN1, RNA14 and RNA15, and UPF1 and UPF3.
ts352 has a defect in the CCA1 gene, which encodes transfer RNA (tRNA) nucleotidyltransferase, the enzyme that adds 3'CCA-termini to tRNAs [56]. Once shifted to the non-permissive temperature, ts352 cells rapidly cease protein synthesis, reduce the rates of degradation of all tile mRNAs tested by three- to fivefold, and increase bodl die relative number of ribosomes associated with mRNAs and the overall size of polysomes (SW Peltz el al., unpublished data). These results are analogous to those observed in wcloheximicle-treated cells (SW Peltz et al., unpublished data) and are generally consistent with models that invoke a role R)r translational elongation in the process of mRNA turnover.
XRN
Tile best characterized yeast ribonuclease is a 160kD 5'-3' exoribonuclease encoded by XRN1 [41-43]. This nuclease degrades RNAs with 5' monophosphates, while capped RNAs are resistant to degradation [41]. X/aVl has been cloned and sequenced [44] and has been shown to be identical to several independently isolated genes, namely, DST2, SEP1 and KEM1 [57~50]. Surprisingly, the latter genes were thought to encode factors involved in nuclear fuskm or in DNA strand exchange during genetic recombination. Cells that are deleted for XRN1 are viable, but grow slowly, have altered rRNA processing [44,45"], and appear to stabilize the decay of several mRNAs (A Stevens, personal communication). Whether such stabilization is a direct consequence of tile deletion of XRN1 on mRNA-decw pathways or a consequence of the slowgrowth phenotype or even an alteration of protein synthesis (attributable to altered rR~NA biogenesis) remains to be determined.
PAN Recognizing tile potential importance of" the poly(A)shortening reaction to an understanding of tile turnover of specifc mRNAs, A Sachs and colleagues (personal communication) exploited a novel property of poly(A)binding protein (PAB) mutations in order to purify and characterize the poly(A) nuclease (PAN). Capitalizing on earlier work that had shown that PAB-deficient strains fail to shorten poly(A) tails [60], they were able to identify PAN activity by supplementing extracts from PAB1- cells with purified PAB. PAN was subsequently purified and shown to be a 135kD protein that requires the presence of PAB in order to degrade poly(A) in vitro. The PAN-PAB complex nomlally shortens poly(A) tracts in vitro to a length of 15-25 bp. For some nlRNAs, sequence elements in the 3'-UTR allow complete deadenylation and dec W of the transcript in vitro (A Sachs, personal communication).
CCA1
RNA14 and RNA15
Screening a collection of ts mutants identified ts352, a mutant that accumulated moderately stable and unstable mRNAs after a shift from 23°C to 37°C [56]. The mutant
Two other genes that appear to affect poly(A) metabolism have been investigated by Minvielle-Sebastia et al. [51"]. Mutants ill RNA14 and R N A 1 5 w e r e isolated
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Prokaryotesand lower eukaryotes on the basis of their simultaneous cordycepin sensitivity and temperature-sensitivity [61]. Compared with wildtype cells, mutants in either of these two genes show rapid reductions in poly(A)-tail lengths and reduced stability of the ACT1 mRNA at the non-permissive temperature [51",61]. This rapid poly(A) shortening occurs at the same rate as the poly(A) shortening observed in cells where transcription has been inhibited, but r n a l 4 and rna15 mutants show no significant reductions in transcriptional activity. As the rapid poly(A)-shortening phenotype is observed with both old and newly synthesized transcripts, it is unlikely that the rna14 and rna15 mutations simply affect nuclear polyadenylation. These phenotypes are compatible with defects that either increase poly(A)-shortening rates or decrease the rate of cytoplasmic poly(A) addition. Both RNA14 and RNA15 are single-copy essential genes. The primary sequence of RNA15 is similar to known RNA- and DNA-binding proteins in that it contains an anlino-terminal segment with RNP1 and RNP2 consensus sequences followed by glutamine- and asparagine-rich regions. Over-expression of either RNA14 or RNA15 leads to suppression of both the r n a l 4 and r n a l 5 mutations, indicating that the respective gene products probably interact [51"].
UPF1 and UPF3
In both prokaryotes and eukaryotes, nonsense mutations in a gene can enhance the decay rate of the mRNA transcribed from that gene, a phenomenon described as nonsense-mediated mRNA decay (see [40.,] for a review). Early work on nonsense mutations in the yeast URA3 gene showed that mRNA destabilization is linked to premature translational termination, because the nonsensecontaining mRNA is stabilized in a strain containing an amber suppressor-tRNA. In addition to tRNA mutants, nonsense suppressors in yeast can also be mutants in non-tRNA genes, which enhance the expression of nonsense-containing alleles by other mechanisms. The latter mutants include the allosuppressors, frameshift suppressors, and omnipotent suppressors [63,64]. Mutations in two genes that were were isolated as allosuppressors, UPF1 and UPF3, lead to the selective stabilization of mRNAs containing earl), nonsense mutations without affecting the decay rates of most other mRNAs [40°°,49-,50]. The UPF1 gene has been cloned and sequenced and shown to be non-essential for viability, capable of encoding a 109 kD protein with both zinc finger and nucleotide (GTP)-binding-site motifs, and partially homologous to the yeast SEN1 gene [50]. The latter encodes a non-catalytic subunit of the tRNA-splicing endonuclease complex [65], suggesting that U p f l p may also be part of a nuclease complex targeted specifically to nonsense-containing mRNAs.
Translation and turnover are intimately linked in yeast Experiments described above indicate that: (a) instability elements involved in the rapid decay of five mRNAs have been localized to the coding regions of the respective mRNAs; (b) inhibition of translational elongation can reduce mRNA-decay rates; (c) metabolism of the poly(A) tail, a structure recently shown to be involved in translational initiation [66], is an integral step in the decay of several mRNAs; and (d) premature translational termination can enhance mRNA decay rates. These observations indicate that the processes of mRNA translation and mRNA turnover must be intimately linked. Further evidence to support this conclusion comes from experiments in yeast that show that ribosome translocation up to, or through, the a ~ 7 ~ Z instability element is required for rapid decay of PGK1-MATotl and ACT1-MATotl chimeric mRNAs [38]. A requirement for specific sequences and translation could be accommodated by the occurrence of an), of the following events in the initiation of mRNA decay. First, the ribosome could pause at a specific sequence - - pausing may be a consequence of a mRNA-rRNA interaction [40..], clustered rare codons [38], or interactions promoted by the nascent polypeptide [67]. Second, a paused ribosome could activate a nuclease and/or expose a downstream cleavage site. Third, cleavage could be mediated by a metabolically unstable nuclease. The requirement for translation may not be limited to a single step in the turnover process, but, as in the first two points, may reflect a need for ongoing protein .wnthesis at several mutually dependent events.
Conclusion Our understanding of mRNA turnover in bacteria is somewhat more advanced than for eukaryotic systems. Many of tile concepts developed from bacterial studies are guiding studies and their interpretation in eukaryotes. It is perhaps relevant to consider how similar or dissimilar the pathways of turnover in prokaryotes and eukaryotes are. There is one important operational difference: bacterial mRNA is generally considered to be unstable unless stabilizing sequences (e.g. 3' terminal stem-loops) are present. In contrast, eukaryotic messages are generally considered stable unless instability signals are introduced. This difference is probably because eukaryotic messages must remain intact as they are transported from the nucleus to the cytoplasm: in contrast, prokaryotic mRNA is normally translated as it is being synthesized and long-term stability is less necessary. The intrinsic stability of eukaryotic messages is presumably, at least in part, achieved by the modified 3' (polyA) and 5' (capped) ends. A second difference, probably for reasons related to the above, is the role of translation.
Turnover of mRNA in prokaryotes and lower eukaryotes Higgins, Peltz, Jacobson Translation appears to play a more 'direct' role in mRNA decay in eukaryotic systems. This is understandable when considering that in eukaryotes mRNA is not translated immediately after it is synthesized but must first be transported to the cytoplasm. It would be inappropriate if a message were degraded before ribosomes had loaded. Nevertheless, despite these differences, the general pathways of turnover in prokaryotes and eukaryotes may be quite similar, involving a combination of endo- and exonucleolytic activities and specific sequences that serve as recognition sites for these enzymes and so determine stability. Most of the concepts and techniques are now in place for investigating mRNA turnover and we can look forward to considerable progress during the next few years.
Structural Gene of Escherichia coll. Proc Nail Acad Sci USA 1991, 88:1-5. This reference, together with [7**], demonstrates that RNaseE has a general role in the degradation of mRNA in E coll. This is the first endonuclease shown to play a role in the turnover of man), different messages. Also shows that the rne and ares mutations are allelic. 9.
NILe;SONP, UHLIN BE: Differential Decay of a Polycistronic coli Transcript is Initiated by RNaseE-dep e n d e n t Endonucleolytic Processing. Mol Microbiol 1991, 5:1791-1799. Shows that rne mutations affect RNaseE activity and thus the processing of pap mRNA. •
10. .
MEI.EFORSO, VON GABAIN A: Genetic Studies of Cleavageinitiated mRNA Decay and Processing of Ribosomal 9S RNA Show that the Escherichia coli ares and r n e Loci are the Same. Mol Microbiol 1991, 5:857-864. Shows that ~vle mutants, defective in RNaseE activity, ,affect processing of ompA message in vivo. 11.
Acknowledgments This work was supported by a National Institutes of Health grant (GM27757) to A Jacobson, a postdoctoral fellowship to SW Peltz from the American Cancer Society, and by support to CF l-liggins from the Imperial Research Fund.
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