Surveillance pathways rescuing eukaryotic ribosomes lost in translation

Translation is a multistep process leading to polypeptide synthesis from mRNA templates. It is carried out accurately and efficiently by the ribosome (a macromolecular complex composed of proteins and rRNAs) tRNAs and translation factors. Translation proceeds in four steps: initiation, elongation, termina-tion and recycling. In eukaryotes, trans lation initiation results in the recruitment of the 40S ribosomal subunit (SSU), initiation factor s and the initiator Met-tRNAi

Met to the start codon, followed by joining of the 60S ribosomal subunit to form the 80S ribosome. Elongation is the iterative cycle of delivery of correct aminoacyl-tRNAs to the ribosomal A-site, followed by peptide bond forma-tion and translocation to the next codon to produce a complete polypeptide. Translation terminates upon entry of a stop codon in the ribosomal A-site. These stop codons are not recognized by tRNAs but by eukaryotic release factor 1 (eRF1), which, together with the translational GTPase (trGTPase) eRF3, triggers release of newly synthesized proteins. Finally, ribosome subunits are dissociated and recycled for new rounds oftranslation.

Eukaryotic cells have evolved specific quality control mechanisms aimed at detect-ing and degrading faulty RNAs to preserve the production of functional proteins (for reviews, see REFS13). Thebest known

qualit y control process is the nonsense-mediate d decay (NMD) pathway, which clears cells from mRNAs containing in-frame premature termination codons4,5. Mechanisms involved in NMD (and in the related Staufen-mediated RNA decay pathway) and their medical implications have been extensively analysed and are reviewedelsewhere47.

Three translation-dependent surveil-lance pathways that specialize in the rapid degradation of RNA molecules trapped in stalled translation complexes have been described more recently: the non-stop decay (NSD) pathway degrades mRNAs that lack stop codons both in bacteria (BOX1) and in eukary otes810; the no-go decay (NGD) path-way degrades eukaryotic mRNAs that cause ribosomes to stall during elongation11; and the eukaryotic non-functional 18S-rRNA decay (18S-NRD) pathway degrades in active or immature 40S ribosomal subunits1214. These mechanisms are undoubtedly crucial, as in their absence stalling of one ribosome

would block all translation complexes located upstream on the same mRNA in a polysome, with deleterious consequences forcells.

Analyses of the mechanisms underlying the detection and release of paused trans-lational complexes and the degradation of associated faulty mRNAs have allowed the identification of factors that are involved in these quality control pathways and have offered a better understanding of eukaryotic translation termination. In this Opinion article, we summarize the current knowledge of these surveillance pathways and the recent results regarding the mechanisms involved in the detection, release and degradation of stalled translation complexes. Moreover, on the basis of these data, we suggest a molecu-lar model of function for the NSD, NGD and 18S-NRD pathways and draw parallels between these surveillance processes and the mechanism of eukaryotic translation termination. Finally, we outline questions that need to be addressed to ensure a more detailed understanding of ribosome stalling recovery.

Stalled ribosomes need to be releasedProblems at the mRNA or ribosome levels may cause eukaryotic ribosomes to stall dur-ing elongation. As stalled ribosomes cannot proceed to normal translation termination, several pathways that recognize and release them have evolved. These processes, which are undoubtedly responsible for the low occurrence of peptides derived from aber-rant mRNAs that induce stalling, contribute to the quality of gene expression.

Translation termination ensures ribosome recycling. In-frame stop codons are crucial for correct translation termination and ribo-some recycling. In eukaryotes, these events involve the concerted action of the transla-tion termination factors eRF1 and eRF3 for peptide release1517, together with the ATPase Rli1 (RNAse L inhibitor 1; also known as ABCE1 in mammals) for ribosome dissocia-tion1820 (FIG.1a). Translation termination occurs when one of the three stop codons (UAA, UGA and UAG) enters into the ribo-somal A-site. These codons are recognized by the class I release factor eRF1 (REF.16), a protein consisting of three domains

O P I N I O N

Surveillance pathways rescuing eukaryotic ribosomes lost in translationMarc Graille and Bertrand Sraphin

Abstract | Living cells require the continuous production of proteins by the ribosomes. Any problem enforcing these protein factories to stall during mRNA translation may then have deleterious cellular effects. To minimize these defects, eukaryotic cells have evolved dedicated surveillance pathways: non-stop decay (NSD), no-go decay (NGD) and non-functional 18S-rRNA decay (18S-NRD). Recent studies support a general molecular framework for these surveillance pathways, the mechanisms of which are intimately related to translation termination.

specific quality control mechanisms aimed at detecting and degrading faulty RNAs

PERSPECTIVES

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 13 | NOVEMBER 2012 | 727

2012 Macmillan Publishers Limited. All rights reserved

Nature Reviews | Molecular Cell Biology

E P A

50S

30S

AUG

tRNANascent peptide

SmpB

E P AAUG

tmRNA

mRNA

EF-Tu

E P A

AUG

RNases

mRNArelease anddegradation

E P A

Transpeptidationand translationof tmRNApeptide tag

Ribosomedissociation

Degradation oftagged protein

RF1/RF2

50S30S

+

arranged to mimic a tRNA molecule, with the amino-termina l and central domains corresponding to the tRNA anti codon stemloop and the aminoacyl acceptor arm, respectively21. The central domain harbours a universally conserved Gly-Gly-Gln motif, which binds to the ribosomal peptidyl trans-ferase centre and catalyses hydrolysis of the P-site tRNAnascent peptide bond, thereby releasing the newly synthesized protein. eRF1 interacts with the class II release factor eRF3 and enhances GTP (but not GDP) bind-ing to eRF317. The eRF3 GTPase activity is stimulate d by both eRF1 and the ribosome22.

Recruitment of the eRF1eRF3GTP complex to the A-site induces a rearrange-ment of the termination complex, leading to subsequent GTP hydrolysis followed by rapid hydrolysis of the peptidyl-tRNA ester bond and release of the nascent pro-tein(FIG.1a). After eRF3 dissociation, Rli1 binds to the ribosome and eRF1, promoting sub unit dissociation and eRF1 release after ATPhydrolysis23.

Non-stop decay. The absence of an in-frame stop codon within an mRNA (hereafter called non-stop mRNA) may result from

erroneous transcription termination or accidental mRNA severing. Although the formation of non-stop mRNAs may be triggered by mutations, natural examples of genes producing significant levels of such mRNAs are known2426. Ribosomes translatin g non-stop mRNAs stall at their 3 end, with the newly synthesized pro-tein covalently bound to the P-site tRNA. However, mRNAs lacking in-frame stop codons (non-stop mRNA) are rapidly degraded in yeast and mammals by the translation-dependent NSD pathway8,10. In yeast, NSD requires either the exo nucleolytic or endonucleolytic activities of the exosome subunit chromosome disjunction3 (Dis3; also known as Rrp44) and of the SKI com-plex and its associated factor super killer7 (Ski7; which is a yeast-specific member of the eukaryotic elongation factor1A (eEF1A) trGTPasefamily)10,2731(FIG.1b).

Natural NSD substrates are rarely expected to arise as a consequence of mutations in normal stop codons, as in-frame stop codons are likely to be present in the 3untranslated region (UTR). However, mutations of the stop codon into sense codons combined with the absence of in-frame stop codons are responsible for at least two human diseases: 2,8-dihydroxyadenin e urolithiasis and hypo-gonadotropic hypogonadism32,33. In both cases, the levels of non-stop mRNAs and the resulting proteins are significantly reduced, suggesting that these non-stop mRNAs are eliminated byNSD.

These are, however, exceptional cases, and the evolutionary pressure that has resulted in the maintenance of NSD in yeast and mammals stems most likely from the production of a non-negligible number of physiological NSD substrates. Some of these substrates are likely to be mRNAs with a 3poly(A) tail as these were initially used to describe NSD. In such cases, the poly(A) tail is translated into a carboxy-terminal poly-Lys tract. Natural transcripts of this type, arising from premature cleavage and polyadenylation, have, for example, been detected for yeast CBP1 (which encodes centromere-binding protein 1), chicken growth hormone receptor genes, Xenopus laevis tpm1 (which encodes -tropomyosin) and for genes encoding the metazoan poly-adenylation factor CSTF77 (cleavage stimu-lation factor 77 kDa subunit), and some of these mRNAs were shown to be NSD substrates8,2426,34. This situation is unlikely to be anecdotal because many eukaryotic genes contain putative 3 end processing signals within their coding regions8,35,36,

Box 1 | Degradation of bacterial mRNAs forcing ribosome stalling during elongation

Bacterial mRNAs lacking inframe stop codons are rapidly degraded by a nonstop decay (NSD) pathway, which is also used to quickly adapt to changing environmental conditions94. Indeed, under conditions of nutrient starvation, uncharged tRNAs bind to the ribosomal Asite and inhibit translational elongation. RelA binds to stalled ribosomes and synthesizes the (p) ppGpp signal nucleotide, which induces the recruitment of the RelE endonuclease to theribosomal Asite, where it specifically cleaves mRNAs and thereby eliminates downstream stop codons9598.

An mRNA that lacks an inframe stop codon (nonstop mRNA) and the associated nascent protein are degraded by the transtranslation mechanism (reviewed in REFS94,99). Fulllength mRNAs that contain a cluster of rare codons or a weak stop codon are also degraded by this mechanism. Transtranslation relies on two main factors: small stable 10S RNA (ssrA), an aminoacylated transfermessenger RNA (tmRNA) with properties both of a tRNA and an mRNA; and SmpB (small protein B). The trimeric complex SmpBtmRNAEFTu binds to the Asite of stalled ribosomes (see the figure). ThetmRNA tRNA domain is located close to the ribosome peptidyl transferase centre, and SmpB contacts the mRNA100. The nascent polypeptide is transferred from the Psite tRNA to the Asite tmRNA. The nonstop mRNA is consequently released and rapidly degraded by RNases (RNaseR in Escherichiacoli). Next, the mRNA moiety of tmRNA drives the synthesis of a peptide tag linked to the carboxyl terminus ofthe truncated nascent peptide. This tag triggers the rapid degradation of the protein fusion by cellular proteases99. Translation termination on the tmRNA stop codon and ribosome recycling are catalysed by the standard bacterial termination factors (peptide chain release factor1 (RF1) or RF2, RF3 and ribosomal recycling factor (RRF)). Interestingly, a growing body of evidence suggests that transtranslation also participates in other physiological quality control functions, such as monitoring of protein folding or correct protein secretion101.

P E R S P E C T I V E S

728 | NOVEMBER 2012 | VOLUME 13 www.nature.com/reviews/molcellbio



E P A

60S

40S

AUG (A)n

E P AAUG (A)n

E P A

60S

40S

AUG (A)n AAA AAA AAA

E P AAUG (A)n AAA AAA AAA

AUG

60S

60S

60S

60S

40S40S

40S

Ribosome dissociation,40S degradation

AUG (A)n AAA AAA A

Exosome

STOP

STOP

Ub

a Normal termination

c No-go decay d 18S-rRNA decay

b Non-stop decay

tRNA

eRF1eRF3

STOP

Ski7

E P A

60S

AUG (A)nSTOP

E P AAUG (A)nSTOP

(A)nSTOP

Dom34Hbs1

E P A

60S

AUG (A)nSTOP

E P AAUG (A)nSTOP

AUG (A)nSTOP

AUG

Xrn1

Stalling site

Nascent peptide

Peptide release,ribosome dissociation

mRNA endonucleolyticcleavage, degradation,ribosome dissociation

Protein ubiquitylation,ribosome dissociation,mRNA degradation

Ub

Dom34Hbs1

Dom34Hbs1

40S

40S40S Ski7

and accidental premature polyadenylation may occur at cryptic intronic sites, even if usage of such sites is normally suppressed inmetazoans37.

In addition, NSD substrates may arise follow ing endonucleolytic cleavage (as occurs during NMD in some species38,39) as a result of the NGD process (see below), following chemical damage (for example, alkaline hydrolysis) and from natural or accidental nuclease cleavage40, although these NSD substrates will lack a poly(A) tail. Moreover, in recent years, many short aberrant transcripts have been identified in eukaryotes41, and although many are preponderantly nuclear, some reach the cytoplasm, where a fraction is recognized as NSD substrates42. These aberrant transcripts, together with truncated mRNAs, probably form most of the natural NSD substrates.

To mimic these events experimentally, artificial reporters were engineered encoding non-stop mRNA molecules that were endo-nucleolytically cleaved at their 3end through the action of a hammerhead ribozyme (an RNA sequence that catalyses its own cleavage), and thus lack a poly(A) tail. This revealed that poly(A)-less non-stop mRNAs are highly unstable and targeted by a process slightly different from the NSD pathway originally described for poly(A)-carryin g non-stop mRNAs. In particular, whereas degradation of poly(A)-carrying non-stop mRNAs requires both the C-terminal domain and the N-terminal domain of Ski7, decay of poly(A)-less non-stop mRNAs only requires the Ski7 N-terminal domain40.

Interestingly, proteins resulting from the translation of non-stop mRNAs (non-stop proteins) are present at reduced levels compared with non-stop mRNA levels in a cell. This results from two additional control levels beyond mRNA turnover: translational repression and non-stop pro-tein destabiliz ation4345. Evidence suggests that the low levels of non-stop proteins result from an inhibition of their transla-tion owing to defects in recycling-associated ribosomes44. It was also observed that non-stop protein levels, but not non-stop mRNA levels, are affected by the activities of proteasome-associate d factors, including the ribosom e-associated protein listerin 1 (Ltn1), which is an E3 ubiquitin ligase. This indicates that nascent non-stop mRNA-derived proteins are subject to increased degradation by the proteasome46,47.

Finally, the degradation of non-stop mRNAs lacking a poly(A) tail and of the cor-responding aberrant peptides is also strongly influenced by the presence of Dom34 and

Figure 1 | Releasing ribosomes at the termination codon or when stalled during translation elon-gation. a | During normal translation termination, a stop codon entering the ribosome A-site recruits eukaryotic release factor 1 (eRF1) and eRF3GTP. GTP hydrolysis by eRF3 triggers eRF1 rearrangements that induce nascent protein release and eRF3GDP dissociation. Recruitment of Rli1 (RNase L inhibi-tor1) ensues and triggers subunit dissociation after ATP hydrolysis. Note that some termination events are associated with activation of mRNA decay by nonsense-mediated decay. b | Release of a stalled ribosome at a mRNA 3 end is an example of non-stop decay (NSD) of a poly(A)-carrying mRNAs. After translation of the poly(A) tail, attachment of a poly-Lys extension to the nascent polypeptide and recruitment of the super killer 7 (Ski7)exosome complex lead to ribosome dissociation and exosome-mediated mRNA degradation. The Dom34Hbs1 complex was also recently shown to be involved in this process. The released protein is degraded by the proteasome after ubiquitylation by E3 ligases (not shown). c | Degradation of mRNAs that induce translational stalls by the no-go decay (NGD) pathway. Ribosome stalling can result from faulty mRNAs that carry a very stable stemloop near the stalling site, an in-frame poly(A) stretch (at least 18 adenine residues) or a damaged base within the ribosomal A-site. In yeast, such mRNAs are endonucleolytically cleaved by Dom34 and Hbs1 and degraded by the 5 to 3 exoribonuclease Xrn1. The nascent peptide is degraded by Ltn1Not4-mediated proteasomal degradation (not shown). d | In the 18S-rRNA decay (18S-NRD) pathway, ribosomes unable to elongate owing to a defective 40S subunit recruit Dom34, Hbs1 and Ski7, leading to 18S rRNA degradation. Details of this process, including the fate of the mRNA and of the nascent peptide, are unclear.




Hbs1, two proteins related to eukaryotic translation termination factors that have a dominant role in quality control pathways (see below). Interestingly, these factors were also recently reported to dissociate stalled ribosomes that are present at the 3 end of poly(A)-carrying mRNA NSD substrates, thus stimulating their decay48.

No-go decay. Different faulty mRNAs that induce ribosome stalling during elongation are degraded by the NGD pathway. NGD relies on the endonucleolytic cleavage of substrate mRNAs close to the ribosome stall site. This involves Dom34 and Hbs1 and is followed by degradation of the resulting mRNA fragments by exonucleases (that is, the 5 to 3 exoribonuclease Xrn1 and the exosome) (FIG.1c).

The NGD pathway was initially identi-fied in yeast by using an artificial reporter with an RNA structure that impairs trans-lation elongation. This pathway can also be detected in insect cells, in which it requires Pelota, a Dom34 orthologue in many specie s, including fruitflies and humans11,49. Various aberrant mRNAs are thought to be physiological NGD substrates. Although initially some of those were considered to be NSD substrates, a better understanding of the mechanisms underlying their degra-dation leads us to suggest that they are NGD substrates. Indeed, in contrast to NSD substrates, the stalling sites are not located at the 3 end of mRNAs, and these mRNAs are subjected to endonucleolytic cleavage. However, the differences between some aspects of the NSD and NGD pathways are becoming less clear, and one could consider that following endonucleolytic cleavage, an NGD substrate becomes a poly(A)-less NSDsubstrate.

One class of physiological NGD sub-strates encompasses mRNAs containing internal poly(A) sequences that encode at least six consecutive positively charged resi-dues. Such stretches of positively charged residues enforce ribosomes to stop during translation, probably through interaction with the negatively charged ribosomal polypeptide exit tunnel43,50,51. In yeast, the levels of such mRNAs are at most only moderately affected (2-fold), indicating that these mRNAs are not rapidly destabilized, contrary to poly(A)-carrying non-stop mRNAs43,46. mRNAs with internal poly(A) stretches undergo endonucleolytic cleavage, and the Dom34Hbs1 complex affects the efficiency of this process but does not cata-lyse cleavage itself 50. Levels of nascent pro-teins translated from such mRNAs are lower

than expected, suggesting that stretches of basic residues result in translational repres-sion and/or protein destabilization, similar to those observed when poly(A) tails are translated44,45,50. A screen for mutations that allow higher expression of a protein derived from an mRNA containing an internal in-frame poly(A) sequence identi-fied a centra l role for yeast Asc1 (absence of growth suppressor of Cyp1; also known as RACK1 in mammals) in this process. Interestingly, this core component of the SSU binds to the ribosomal mRNA exit channel52. Evidence suggests that Asc1 stim-ulates translational arrest, thereby leading to nascent protein degradation by E3 ubiquitin ligases. Both Ltn1 and Not4, a RING finger protein associated with the CCR4NOT complex and polysomes, seem to mediate this process43,46,50,53,54.

mRNA molecules that have been chemi-cally damaged (for example, by oxidation, alkylation or depurination) have also been shown to cause translation elongation stalls, with reduced production of the correspond-ing protein55,56. Furthermore, Dom34 and Hbs1 were demonstrated to be involved in endonucleolytic cleavage and protein desta-bilization of mRNAs that contain apurinic sites within their coding sequences, prob-ably through the NGD pathway57. Owing to the difficulties in detecting these types of chemica l damage invivo, the role of NGD in the clearance of these damaged mRNAs is probably underestimated. Notably, in rare cases damaged RNA molecules can be repaired in a similar manner asDNA58.

As shown for NMD59,60, NGD may also regulate the abundance of some mRNAs. Bioinformatics searches for stable stemloops, which could induce ribosomal paus-ing, have identified a subset of yeast genes that may be NGD targets61. In a Dom34 deletion (dom34) yeast strain, the steady-state mRNA levels of some of these genes were increased62, indicating that the NGD pathway targets for degradation some cell-ular mRNAs containing particular features. Another bioinformatics screen identified five yeast genes that encode stretches of at least 10 consecutive basic residues. Among those, three genes induced translational arrest and release of partial protein prod-ucts43. One can also hypothesize that under certain physio logical conditions NGD can regulate the expression of specific mRNAs, such as the CGS1 (CYSTATHIONIN E GAMMA SYNTHETAS E 1) mRNA in Arabidopsisthaliana. Indeed, S-adenosyl-l-methionine, a downstream product in the pathway involving CGS1, regulates the

stability of this transcript through a feedback loop that involves induction of a transla-tion elongation arrest followed by mRNA endonucleolytic cleavag e, a mechanism reminiscen t ofNGD63.

Non-functional 18S-rRNA decay. Translational stalls are not only caused by erroneous mRNAs but can also result from ribosomal defects, such as mutations, chemi-cal damage or faulty biogenesis. In the yeast cytoplasm, non-functional small ribosomal subunits that remain paused as they fail to catalyse efficient translation elongation are rapidly degraded via the 18S-NRD pathway. This requires the concerted action of Ski7, Xrn1, Dom34, Hbs1 and the cytoplasmic exosome12,13 (FIG.1d).

Many 18S-NRD substrates are likely to originate from errors in ribosome pro-duction. The synthesis of ribosomes is a comple x cellular process (BOX2) that involves many checkpoints to ensure that most newly synthesized ribosomal particles are func-tional. In eukaryotes, pre-40S ribosomes are subjected to a final round of cytoplasmic maturation consisting of a translation-like cycle during which pre-40S joining the 60S is checked. Following this verification, these 80S-like ribosomes are dissociated by Rli1 and Dom34 and are then able to recruit mRNA and translation initiation factors6466.

However, some pre-40S subunits that have passed these checkpoints can initiate translation but are unable to carry out elon-gation. Such subunits are rapidly destabilized in part through the action of Dom34 and Hbs1 (REF.14). A similar late quality control mechanism validating correct ribosome biogenesis may also exist in Dictyostelium discoideum, in which efficient translation elongation is required to complete rRNA maturation67.

Key players in ribosome releaseRecent studies focusing on the mechanism and the role of the surveillance pathways dedicated to the release of stalled ribo-somes have revealed that Dom34 and Hbs1 are involved in all these pathways and are assisted byRli1 (FIG. 2).

Dom34: an eRF1 paralogue. Orthologues of Saccharomyces cerevisiae Dom34 are found in all eukaryotes and archaea but not bacterial genomes sequenced to date, which suggests an important biological function68. In budding yeast, DOM34 deletion resultsin defective meiosis and growth, decreased polysome abundance and synthetic lethalit y with deletion of several genes encodin g SSU




proteins (such as 40S ribosomal protein S30A (Rps30A))6971. The growth defect phenotype of dom34 strains can be res-cued by overexpression of Rps30A or of fruitfly Pelota70, which indicates first that the mitotic and meiotic defects observed following DOM34 deletion may result from effects on translation, and second that the function of yeast Dom34 and fruitfly Pelota is conserved. This conclusion is supported by the finding that the addition of fruitfly Pelota can restore NGD in a yeast dom34 mutant49. A similar role for Pelota has been shown in other species. In fruitflies, male pelo (pelota) mutants are sterile because of defective meiotic cell division, and female mutants are deficient in ovarian mitotic divisions72. In mice, disruption of Pelo leads to embryonic lethality and cell cycle defects73. Finally, human Pelota interacts with translation initiation factor eIF3G and the cytoskeleton; given the key role of the cytoskeleton during cell division, this may explain the cell cycle defects observed in Pelo-mutantmice74.

Dom34 and Pelota share sequence and structure similarity with the translation term ination factor eRF1 (REFS 75,76) (FIG.2a). Dom34 is composed of three domains: an N-terminal domain, a middle domain and a C-terminal domain. The middle domain and the C-terminal domain are structurally simi-lar to the corresponding domains in eRF1 (REF. 21). The Dom34 domains arrange spa-tially to mimic a tRNA, with the N-terminal domain and the middle domain correspond-ing to the anticodon stemloop and amino-acyl acceptor arm, respectively. However, Dom34 does not possess a Gly-Gly-Gln motif, which is necessary for peptide release (FIG.2a). Moreover, the Dom34 N-terminal domain structurally differs from the eRF1 N-terminal domain and adopts a Sm/Lsm fold, suggesting a role in RNA binding77. This domain was initially proposed to endow the NGD endonuclease activity, but many studies have suggested that this is not the case19,49,50,78.

The trGTPase Hbs1. Hbs1 belongs to the trGTPases family (together with eEF1A, eRF3 and Ski7). Hbs1 is found in most sequenced eukaryotic genomes, where it coexists with eEF1A and eRF3 (REFS68,71,79,80) (FIG.2b). By contrast, in archaea, a single trGTPase (aEF1) functions in translation elongation, termination and RNA quality control pathways by interacting with tRNA, the archaeal translation termi-nation factor aRF1 and archaeal Pelota81. InS.cerevisiae, Hbs1 does not complement

deletion of eRF3, eEF1A or Ski7 and does not bind eRF1, indicating that when pre-sent, these three trGTPases possess distinct functions55,80,82,83. The presence of Hbs1 and Ski7 proteins, with Hbs1 being involved in NGD and 18S-NRD and Ski7 being exosome associated and involved in NSD of poly(A)-carrying mRNAs, may be a yeast oddity. Indeed, in human cells the HBS1 orthologue co-purifies with the cytoplasmic exosome, suggesting that in metazoa, HBS1 connects the exosome to the SKI complex, similarly to Ski7 in budding yeast84.

Hbs1 was initially identified as a suppres-sor of the slow growth phenotype of yeast strains lacking Ssb1 and Ssb2 (REF. 85), two ribosome-associated heat shock protein 70 (Hsp70) chaperones that facilitate nascent protein folding. Deletion of HBS1 alone does not cause a detectable growth phenotype85. However, when combined with the deletion of several genes coding for SSU proteins, loss of Hbs1 results in slow yeast growth and cold sensitivity71,82. Finally, integrity of the Hbs1 GTP-binding site is required for its biologica l function and for its roles in surveillance pathways71,82,86. Altogether, Hbs1 seems to be important under condi-tions in which production of SSU proteins is imbalance d or limiting.

Dom34 and Hbs1 interact in a similar way to eRF1 with eRF3, and the resultin g complex also structurally mimics the elongation factor Tu (EF-Tu)tRNA complex71,75,79,82,8688 (FIG.2c). Consistently, Dom34Hbs1 binds the ribosomal A-site78(FIG.2d).

Proposed rescue mechanismThe mechanistic role of Dom34Hbs1 in surveillance pathways was unravelled recently using invitro approaches, and these proteins were shown to promote ribosome subunit dissociation together with Rli1 (REFS1820,23,89,90). This has also shed light onto the role of eRF1eRF3 and eRF1Rli1 complexes in the recy-cling of normally terminating ribosomes. This rescue mechanism relies on GTP hydrolysis by Hbs1 and ATP hydrolysis by Rli1, two activities that require the pres-ence of Dom34 and ribosomes19,20,89,90. Experimental evidence suggests that GTP hydrolysis precedes ATP hydrolysis20. Hence, from these studies, it now seems possible to propose a unified model for the molecular mechanism of these surveillance pathways. Although parts of this model remain hypothetical, its description, even in a sketchy form, allows one to delineat e important questions that remain to be solved (FIG.3).

Release of stalled ribosomes begins when Dom34 associated withHbs1GTP is recruited to the A-site of ribosomes that are stalled in translation by defective SSU or faulty mRNAs (FIG.3a). In contrast to the eRF1eRF3 complex, the recruit-ment of Dom34Hbs1 is not linked to the identity of the codon present in the A-site. Itis unclear whether recruitment depends only on a kinetic competition with other translation factors for ribosome binding or whether additional features facilitate this recruitment (for example, the presence of

Box 2 | Monitoring the synthesis of eukaryotic ribosomes

Similarly to bacteria and archaea, eukaryotic ribosomes contain two subunits. In yeast, the large 60S subunit is composed of 46 proteins and three rRNAs (25S, 5.8S and 5S), and the small 40S subunit consists of one rRNA (18S) and 33 proteins. RNA polymerase III transcribes the 5S rRNA, and RNA polymerase I synthesizes a single RNA transcript (35S), which is matured into 25S, 18Sand 5.8S rRNAs by an intricate succession of endo and exonucleolytic cleavages102.

rRNAs processing and assembly with ribosomal proteins to form active ribosomes is a highly coordinated and complex process that requires a large number of transacting factors (small nucleolar RNAs and up to 200 proteins). It occurs in different subcellular compartments, starting in the nucleolus, a specialized substructure of the nucleus, and then continues in the nucleoplasm before ending in the cytoplasm102,103.

This process is errorprone, and any mistakes in this assembly line may result in nonfunctional or incomplete ribosomes. Therefore, cells have evolved several quality control mechanisms to monitor correct ribosome synthesis. One pathway relies on nuclear polyadenylation of incorrect rRNAs by the TRAMP (TrfAirMtr4 polyadenylation) complex followed by 3 to 5 degradation by the nuclear exosome104. Nonfunctional ribosomal subunits can also be rapidly degraded in the cytoplasm by the nonfunctional rRNA decay surveillance pathways12,105. Interestingly, molecular analyses revealed that deficient 60S or 40S subunits are rapidly degraded by two independent mechanisms12,106. A nonfunctional 60S subunit is degraded by ubiquitylation of ribosomeassociate d proteins, which is mediated by the ubiquitin E3 ligase Rtt101 and its partner Mms1. Decay of the 25S rRNA requires core exosome activity but neither the associated cytoplasmic factor super killer 7 (Ski7) nor the 5 to 3 cytoplasmic exoribonuclease Xrn1 (REFS 106,107). Degradation of defective 18S rRNA is detailed in the main text.





a Dom34

c PelotaaEF1 complex

d A/T state e A/A state

EF-TutRNA complex

b Hbs1eRF1

Middle domain

Carboxy- terminaldomain

GTPasedomain

Amino-terminaldomain

Domain II

Domain III

aEF1 EF-Tu

Pelota

tRNA

GDP

a partly unfolded protein attached to the ribosome for a long period of time, modi-fication of specific ribosomal proteins or a specific ribosome conformation). When bound to stalled ribosomes in the presence of Hbs1, Dom34 adopts a distorted A/T state conformation, which is reminiscent of the conformation of the tRNA in the r ibosome-EF-TutRNA complex78,91 (FIG.2d).

Dom34Hbs1 recruitment to stalled ribosomes is not essential for mRNA endonucleolytic cleavage during NGD but strongly stimulates it. The precise loca-tion of this cleavage site is not clear, as it has been mapped both downstream11 and upstream of the stalled ribosome48. However, if cleavage occurs downstream of the stalled ribosome, the ribosome will be stalled close to the 3end of an mRNA that most likely does not contain a stop codon and resembles an NSD substrate. It is thus tempting to speculate that the NGD and poly(A)-less NSD pathways merge at this point48. Interestingly, the length of the mRNA that is downstream of the P-site seems to be important for ribosome dis-sociation, and this process is optimal at a location up to 23 or 9 nucleotides after the P-site codon for yeast or human Dom34Hbs1, respectively18,20. This length depend-ency relies on Hbs1 and could be mediated by the Hbs1 N-terminal domain, which interacts with the ribosome at the mRNA entry site according to cryo-electron microscopy studies78 (FIG.2d). This obser-vation could explain the endonucleolytic cleavage observed invivo during NGD, as this cleavage near the stall site would remove the 3 mRNA fragment, possibly stimulating ribosome dissociation and rapid degradation of the 5 mRNA frag-ment from the 3 end by the exosome11,48. Endonucleolytic cleavage of mRNAs occurs before GTP hydrolysis by Hbs1, as only GTP binding is required for this cleavage activity in NGD. This suggests that the conformation of the Hbs1Dom34 complex induced by GTP binding is sufficient for NGD71,82,86.

Following mRNA cleavage, GTP hydroly-sis leads to Hbs1 dissociation from the ribo-some. This is likely to be accompanied by a large conformational change of the Dom34 middle domain, which stimulates GTP but probably not GDP binding by Hbs1. This Dom34 domain would interact more loosely withHbs1GDP and could reorient towards the ribosome peptidyl transferase centre, thereby adopting an A/A state conforma-tion (FIGS2e,3c). Hence, similarly to tRNAs during translation elongation and probably

Figure 2 | Structures of Dom34 and Hbs1. a | Ribbon representation of Saccharomyces cerevisiae Dom34 (left) and human eukaryotic release factor 1 (eRF1; right) crystal structures. The different domains are highlighted with different colours. b | Ribbon representation of the crystal structure of S.cerevisiae Hbs1 bound to GDP. c | Comparison of the crystal structure of the Aeropyrumpernix Pelotaelongation factor 1 (aEF1) complex (left) and the bacterial EF-TutRNA complex (right). Thesame colour codes as for Dom34 in panel a and Hbs1 in panel b are used to depict Pelota and the different translational GTPase (trGTPase) domains. d | Representation of the cryo-electron microscopy structure of the Dom34Hbs1 complex bound to the 80S ribosome with Dom34 in the A/T state78. ThePsite tRNA is depicted in green and Dom34 is shown in pink. The Hbs1 aminoterminal domain is shown in red and the remaining region (GTPase domain, domains II and III) in turquoise. The 40S and 60S ribosomal subunits are shown in yellow and grey, respectively. e | Representation of the cryo-electro n microscopy structure of the Dom34Rli1 complex bound to the 80S ribosome with Dom34 in the A/A state92. Same colour code is used as in panel d. Rli1 is shown in blue.




a b

cde

E P A

60S

40S

AUG (A)nStalling site

Nascentpeptide

E P AAUG (A)n

Dom34(A/T state)

Hbs1

E P AAUG +

E P AAUG

mRNAcleavage

(A)n

Xrn1

E P AAUG

ATP hydrolysis,subunit dissociation

60S 40S

AUG

Exosome

Proteasome

Ltn1- or Not4-mediateddegradation of nascent peptide

Degradationof mRNA 5fragment

Ribosomestalling


GTP hydrolysis,release of Hbs1GDP

Dom34 conformationalchange (A/A state)

GTP

GTP GTP

GDP

ATP ATP

ATPRli1

eRF1 during translation termination, Dom34 undergoes significant conforma-tional changes within the ribosomal A-site. Following Hbs1 dissociation, Rli1-ATP binds to the same ribosomal region as trGTPases and interacts with a region at the Dom34 C-terminal domain that over-laps with the surface recognized by Hbs1 (REF.92) (FIG.3d). These observations ration-alize the mechanistic differences observed for Dom34 and Hbs1 mutants disrupting theDom34Hbs1 complex71. Hbs1 mutants that are unable to interact with Dom34 are strongly affected in NGD but only modestly in 18S-NRD, whereas Dom34 mutants that cannot interact with Hbs1 are defective in both NGD and 18S-NRD71. Interestingly, based on available structural informa-tion92, these Dom34 mutants should also impede the Dom34Rli1 interaction. This confirms that the formation of the Dom34Hbs1 complex is important for mRNA endonucleolytic cleavage during NGD but not for 18S-NRD, in which formation of the Dom34Rli1 complex would be most important, probably owing to its role in stalled ribosome dissociation.

Dissociation of stalled ribosomes requires ATP hydrolysis by Rli1, probably accom-panied by a conformational change of Rli1, similar to that observed for other ABC enzymes. The ADP-bound Rli1 conforma-tion would then not be compatible with the structure of Dom34 bound in the A/A state in the ribosomal A-site and would induce further conformational changes in Dom34, thereby resulting in subunit dissociation20,90 (FIG.3e). Ribosome dissociation stimulates the decay of the 5 mRNA intermediate fragment from its 3end by recruiting the exosome, thus inhibiting further translation initiation of these faulty mRNA fragments48. This observation questions the fate of the mRNA during 18S-NRD. Are only non-functional or immature SSUs degraded, or is the mRNA engaged in translation also targeted for degradation even if it is fully functional?

The fate of the peptidyl-tRNA associate d to these stalled ribosomes still remains unclear. Because it lacks a Gly-Gly-Gln motif, Dom34 cannot catalyse the release of the nascent peptide, which is also not trig-gered by translocation of the peptidyl-tRNA

from the ribosomal P-site to the E-site19. Inthe case of ribosomes stalled at, or shortly after, initiation, the nascent peptide contains only a few amino acids, and the peptidyl-tRNA can easily diffuse away from the ribo-some19. However, when translation stops after incorporation of many amino acids, a long nascent peptide is likely to be irrevers-ibly engaged into the ribosomal tunnel from which it may emerge as a partially folded protein, possibly bound by other factors (such as chaperones, ligands or other sub-units of a protein complex). In this case, it remains to be determined whether the bond that connects the tRNA to the newly synthe-sized, incomplete peptide is hydrolysed by a peptidyl hydrolase, similarly to what occurs during bacterial NSD93. Recent results indi-cate that this process does not involve the Pth1 or Pth2 peptidyl-tRNA hydrolase in yeast48. Alternatively, the tRNA and/ orthe ribosomal subunit could be degraded. Thenascent peptide is likely to be targeted for degradation by the proteasome through the action of the E3 ubiquitin ligases Ltn1 or Not4 before, or after, detachment from thetRNA.

Figure 3 | A model for eukaryotic quality control pathways rescuing ribosomes stalled in translation. a | Ribosome stalling induces the recruitment of Dom34 (in the A/T state) and Hbs1 (loaded with GTP) by theribosome. This recruitment possibly occurs because of disfavoured binding of competing translation elongation factors and charged tRNAs. In this example, the mRNA is considered to be responsible for the translational stall. b | Endonucleolytic cleavage of faulty mRNAs (not shown) forces ribo-somes to stall. The enzyme responsible for this cleavage has not yet been identified. Whether the mRNAs translated by non-functional ribosomes are subjected to endonucleolytic cleavage during non-functional 18S-rRNA decay (18S-NRD) remain unknown. The 3 mRNA fragment (the one located downstream of the ribosome) can be degraded by the cytoplasmic 5 to 3 exoribonuclease Xrn1, while the 5 fragment remains associated to stalled

ribosomes. c|Dom34- and ribosome-dependent hydrolysis of the GTP mol-ecule bound to Hbs1 is followed by a rearrangement of the Dom34 central domain towards the peptidyl transferase centre (A/A state) and release of Hbs1GDP. d|Rli1ATP binds to the stalled ribosome on the same region as translational GTPases (trGTPases) such as eukaryotic release factor 3 (eRF3) and Hbs1 and contacts a Dom34 surface overlapping with the Hbs1-binding site. e | Dom34- and ribosome-dependent hydrolysis of the ATP molecule bound to Rli1 and global conformational changes of Rli1, and most probably Dom34, occur, which results in ribosomal subunit dissociation. The 5 mRNA fragment is degraded by the exosome and the nascent peptide is degraded by the proteasome. It remains unknown whether the nascent peptide is released from the peptidyl-tRNA before degradation and whether the tRNA itself is degraded or recycled.




ConclusionsThe past decade has seen the identification of many translation surveillance pathways that contribute to gene expression accuracy. Here, we focus on cytoplasmic surveillance pathways that detect translationally stalled ribosomes and subsequently degrade asso-ciated RNA molecules. Beyond mRNAs and ribosomes, several factors have been implicated in these diverse surveillance pathways, all dedicated to the rescue of stalled ribosomes. Our proposed model for the action of Dom34, Hbs1 and Rli1 aims to reconcile the latest results, which have also revealed a considerable wealth of information on the mechanism of normal translatio n termination by eRF1 andeRF3.

Much of our current understanding of these mechanisms originates from studies of various mutants of these yeast proteins by several groups that have used different reporters. Hence, validation of the proposed model will probably require assaying the stability of reporters degraded by different pathways in a homogeneous set of well-characterized mutants. Several points still remain to be clarified, including the fates ofthe tRNAnascent peptide complex andof the functional non-targeted molecule (that is, released ribosomes in NGD and NSD or released mRNA in 18S-NRD). How is the peptidyl-tRNA released from the ribosomal tunnel during NGD and NSD? Are mRNAs that are associated with defective ribosomes degraded? Mechanisms leading to 18S rRNA degradation during 18S-NRD are much less clear. It is also questionable whether the entire SSU is degraded or whether some SSU proteins are recycled into new ribosomes. Mutant analysis indicates that the mRNAs and ribosomal subunits engaged in the stalled translation complex are not always degraded simultaneously; thus, it remains to be seen how molecules are effectively selected for degradation.

Finally, current studies have mainly addressed the mechanistic role of Dom34 and Hbs1 in these surveillance pathways using artificial reporters. The strong evolu-tionary conservation of Dom34 and Hbs1 proteins in eukaryotes and archaea, which share similar translation machineries, com-bined with the diverse phenotypes observed in cognate mutants, such as lethality of Pelo-null mice73, support important bio-logical roles for these proteins. In this respect, numerous physiological substrates are emerging, and there is no doubt that in the future the role of these quality control pathways in cell biology will be addressed using cutting-edge technologies such as

ribosome profiling. Indeed, one can imag-ine that, similarly to NMD, which beyond degrading mRNAs that harbour premature stop codons also regulates the expression of a subset (around 10%) of genes in yeast and human cells59,60, the NGD and NSD path-ways may have been co-opted to regulate the expression of somegenes.

Marc Graille is at the Institut de Biochimie et Biophysique Molculaire et Cellulaire (IBBMC), Centre

National de la Recherche Scientifique (CNRS), UMR8619, Bat 430, Universit Paris Sud, F-91405

Orsay Cedex, France, and at the Laboratoire de Biochimie, CNRS, UMR 7654, Ecole Polytechnique,

F-91128 Palaiseau Cedex, France.

Bertrand Sraphin is at the Equipe Labellise La Ligue, Institut de Gntique et de Biologie Molculaire et Cellulaire (IGBMC), Illkirch F-67400, France, at the

CNRS, UMR7104, Illkirch F-67404, France, at Inserm, U964, Illkirch F-67400, France, and at Universit de

Strasbourg, Strasbourg F-67000, France.

e-mails:[email protected]; [email protected]

doi:10.1038/nrm3457 Published online 17 October 2012

1. Doma, M.K. & Parker, R. RNA quality control in eukaryotes. Cell 131, 660668 (2007).

2. Garneau, N.L., Wilusz, J. & Wilusz, C.J. The highways and byways of mRNA decay. Nature Rev. Mol. Cell Biol. 8, 113126 (2007).

3. Isken, O. & Maquat, L.E. Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function. Genes Dev. 21, 18331856 (2007).

4. Nicholson, P. & Muhlemann, O. Cutting the nonsense: the degradation of PTC-containing mRNAs. Biochem.Soc. Trans. 38, 16151620 (2010).

5. Rebbapragada, I. & Lykke-Andersen, J. Execution of nonsense-mediated mRNA decay: what defines a substrate? Curr. Opin. Cell Biol. 21, 394402 (2009).

6. Kervestin, S. & Jacobson, A. NMD: a multifaceted response to premature translational termination. Nature Rev. Mol. Cell Biol. 17 Oct 2012 (doi:10.1038/nrm3454).

7. Kim, Y.K., Furic, L., Desgroseillers, L. & Maquat, L.E. Mammalian Staufen1 recruits Upf1 to specific mRNA 3UTRs so as to elicit mRNA decay. Cell 120, 195208 (2005).

8. Frischmeyer, P.A. etal. An mRNA surveillance mechanism that eliminates transcripts lacking termination codons. Science 295, 22582261 (2002).

9. Keiler, K.C., Waller, P.R. & Sauer, R.T. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science271, 990993 (1996).

10. van Hoof, A., Frischmeyer, P.A., Dietz, H.C. & Parker,R. Exosome-mediated recognition and degradation of mRNAs lacking a termination codon. Science 295, 22622264 (2002).

11. Doma, M.K. & Parker, R. Endonucleolytic cleavage of eukaryotic mRNAs with stalls in translation elongation. Nature 440, 561564 (2006).

12. Cole, S.E., LaRiviere, F.J., Merrikh, C.N. & Moore,M.J. A convergence of rRNA and mRNA quality control pathways revealed by mechanistic analysis of nonfunctional rRNA decay. Mol. Cell 34, 440450 (2009).

13. LaRiviere, F.J., Cole, S.E., Ferullo, D.J. & Moore,M.J. A late-acting quality control process for mature eukaryotic rRNAs. Mol. Cell 24, 619626 (2006).

14. Soudet, J., Gelugne, J.P., Belhabich-Baumas, K., Caizergues-Ferrer, M. & Mougin, A. Immature small ribosomal subunits can engage in translation initiation in Saccharomyces cerevisiae. EMBO J. 29, 8092 (2010).

15. Alkalaeva, E.Z., Pisarev, A.V., Frolova, L.Y., Kisselev,L.L. & Pestova, T.V. In vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3. Cell 125, 11251136 (2006).

16. Frolova, L. etal. A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor. Nature 372, 701703 (1994).

17. Zhouravleva, G. etal. Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. EMBO J. 14, 40654072 (1995).

18. Pisareva, V.P., Skabkin, M.A., Hellen, C.U., Pestova,T.V. & Pisarev, A.V. Dissociation by Pelota, Hbs1 and ABCE1 of mammalian vacant 80S ribosomes and stalled elongation complexes. EMBO J. 30, 18041817 (2011).

19. Shoemaker, C.J., Eyler, D.E. & Green, R. Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay. Science330, 369372 (2010).

20. Shoemaker, C.J. & Green, R. Kinetic analysis reveals the ordered coupling of translation termination and ribosome recycling in yeast. Proc. Natl Acad. Sci. USA 108, E1392E1398 (2011).

21. Song, H. etal. The crystal structure of human eukaryotic release factor eRF1 mechanism of stop codon recognition and peptidyl-tRNA hydrolysis. Cell100, 311321 (2000).

22. Frolova, L. etal. Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase. RNA 2, 334341 (1996).

23. Barthelme, D. etal. Ribosome recycling depends on a mechanistic link between the FeS cluster domain and a conformational switch of the twin-ATPase ABCE1. Proc. Natl Acad. Sci. USA 108, 32283233 (2011).

24. Mayer, S.A. & Dieckmann, C.L. Yeast CBP1 mRNA 3end formation is regulated during the induction of mitochondrial function. Mol. Cell. Biol. 11, 813821 (1991).

25. Pan, Z. etal. An intronic polyadenylation site in human and mouse CstF-77 genes suggests an evolutionarily conserved regulatory mechanism. Gene 366, 325334 (2006).

26. Sparks, K.A. & Dieckmann, C.L. Regulation of poly(A) site choice of several yeast mRNAs. Nucleic Acids Res. 26, 46764687 (1998).

27. Dziembowski, A., Lorentzen, E., Conti, E. & Sraphin,B. A single subunit, Dis3, is essentially responsible for yeast exosome core activity. NatureStruct. Mol. Biol. 14, 1522 (2007).

28. Lebreton, A., Tomecki, R., Dziembowski, A. & Sraphin, B. Endonucleolytic RNA cleavage by a eukaryotic exosome. Nature 456, 993996 (2008).

29. Schaeffer, D. etal. The exosome contains domains with specific endoribonuclease, exoribonuclease and cytoplasmic mRNA decay activities. Nature Struct. Mol. Biol. 16, 5662 (2009).

30. Schaeffer, D. & van Hoof, A. Different nuclease requirements for exosome-mediated degradation of normal and nonstop mRNAs. Proc. Natl Acad. Sci.USA 108, 23662371 (2011).

31. Schneider, C., Leung, E., Brown, J. & Tollervey, D. TheN-terminal PIN domain of the exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast core exosome. Nucleic Acids Res. 37, 11271140 (2009).

32. Seminara, S.B. etal. The GPR54 gene as a regulator of puberty. N.Engl. J.Med. 349, 16141627 (2003).

33. Taniguchi, A. etal. A germline mutation abolishing the original stop codon of the human adenine phosphoribosyltransferase (APRT) gene leads to complete loss of the enzyme protein. Hum. Genet. 102, 197202 (1998).

34. Anquetil, V. etal. Polypyrimidine tract binding protein prevents activity of an intronic regulatory element that promotes usage of a composite 3-terminal exon. J.Biol. Chem. 284, 3237032383 (2009).

35. Di Segni, G., Gastaldi, S., Zamboni, M. & Tocchini-Valentini, G.P. Yeast pheromone receptor genes STE2 and STE3 are differently regulated at the transcription and polyadenylation level. Proc. Natl Acad. Sci. USA 108, 1708217086 (2011).

36. Grec, S., Wang, Y., Le Guen, L., Negrouk, V. & Boutry,M. Cryptic polyadenylation sites within the coding sequence of three yeast genes expressed in tobacco. Gene 242, 8795 (2000).

37. Kaida, D. etal. U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation. Nature 468, 664668 (2010).

38. Eberle, A.B., Lykke-Andersen, S., Muhlemann, O. & Jensen, T.H. SMG6 promotes endonucleolytic cleavage of nonsense mRNA in human cells. NatureStruct. Mol. Biol. 16, 4955 (2009).




39. Gatfield, D. & Izaurralde, E. Nonsense-mediated messenger RNA decay is initiated by endonucleolytic cleavage in Drosophila. Nature 429, 575578 (2004).

40. Meaux, S. & Van Hoof, A. Yeast transcripts cleaved by an internal ribozyme provide new insight into the role of the cap and poly(A) tail in translation and mRNA decay. RNA 12, 13231337 (2006).

41. Wyers, F. etal. Cryptic pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase. Cell 121, 725737 (2005).

42. Thompson, D.M. & Parker, R. Cytoplasmic decay of intergenic transcripts in Saccharomyces cerevisiae. Mol. Cell. Biol. 27, 92101 (2007).

43. Dimitrova, L.N., Kuroha, K., Tatematsu, T. & Inada, T. Nascent peptide-dependent translation arrest leads to Not4p-mediated protein degradation by the proteasome. J.Biol. Chem. 284, 1034310352 (2009).

44. Inada, T. & Aiba, H. Translation of aberrant mRNAs lacking a termination codon or with a shortened 3-UTR is repressed after initiation in yeast. EMBO J. 24, 15841595 (2005).

45. Ito-Harashima, S., Kuroha, K., Tatematsu, T. & Inada,T. Translation of the poly(A) tail plays crucial roles in nonstop mRNA surveillance via translation repression and protein destabilization by proteasome in yeast. Genes Dev. 21, 519524 (2007).

46. Bengtson, M.H. & Joazeiro, C.A. Role of a ribosome-associated E3 ubiquitin ligase in protein quality control. Nature 467, 470473 (2010).

47. Wilson, M.A., Meaux, S. & van Hoof, A. A genomic screen in yeast reveals novel aspects of nonstop mRNA metabolism. Genetics 177, 773784 (2007).

48. Tsuboi, T. etal. Dom34:hbs1 plays a general role in quality-control systems by dissociation of a stalled ribosome at the 3 end of aberrant mRNA. Mol. Cell 46, 518529 (2012).

49. Passos, D.O. etal. Analysis of Dom34 and its function in no-go decay. Mol. Biol. Cell 20, 30253032 (2009).

50. Kuroha, K. etal. Receptor for activated C kinase 1 stimulates nascent polypeptide-dependent translation arrest. EMBO Rep. 11, 956961 (2010).

51. Lu, J., Kobertz, W.R. & Deutsch, C. Mapping the electrostatic potential within the ribosomal exit tunnel. J.Mol. Biol. 371, 13781391 (2007).

52. Ben-Shem, A. etal. The structure of the eukaryotic ribosome at 3.0 resolution. Science 334, 15241529 (2011).

53. Albert, T.K. etal. Identification of a ubiquitinprotein ligase subunit within the CCR4NOT transcription repressor complex. EMBO J. 21, 355364 (2002).

54. Panasenko, O. etal. The yeast Ccr4Not complex controls ubiquitination of the nascent-associated polypeptide (NACEGD) complex. J.Biol. Chem. 281, 3138931398 (2006).

55. Shan, X., Chang, Y. & Lin, C.L. Messenger RNA oxidation is an early event preceding cell death and causes reduced protein expression. FASEB J. 21, 27532764 (2007).

56. Wurtmann, E.J. & Wolin, S.L. RNA under attack: cellular handling of RNA damage. Crit. Rev. Biochem. Mol. Biol. 44, 3449 (2009).

57. Gandhi, R., Manzoor, M. & Hudak, K.A. Depurination of Brome mosaic virus RNA3 in vivo results in translation-dependent accelerated degradation of the viral RNA. J.Biol. Chem. 283, 3221832228 (2008).

58. Aas, P.A. etal. Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. Nature 421, 859863 (2003).

59. He, F. etal. Genome-wide analysis of mRNAs regulated by the nonsense-mediated and 5 to 3 mRNA decay pathways in yeast. Mol. Cell 12, 14391452 (2003).

60. Mendell, J.T., Sharifi, N.A., Meyers, J.L., Martinez-Murillo, F. & Dietz, H.C. Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise. Nature Genet. 36, 10731078 (2004).

61. Jacobs, J.L., Belew, A.T., Rakauskaite, R. & Dinman,J.D. Identification of functional, endogenous programmed-1 ribosomal frameshift signals in the genome of Saccharomyces cerevisiae. Nucleic Acids Res. 35, 165174 (2007).

62. Belew, A.T., Advani, V.M. & Dinman, J.D. Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast. Nucleic Acids Res. 39, 27992808 (2010).

63. Onouchi, H. etal. Nascent peptide-mediated translation elongation arrest coupled with mRNA

degradation in the CGS1 gene of Arabidopsis. GenesDev. 19, 17991810 (2005).

64. Lebaron, S. etal. Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits. Nature Struct. Mol. Biol. 19, 21942204 (2012).

65. Strunk, B.S. etal. Ribosome assembly factors prevent premature translation initiation by 40S assembly intermediates. Science 333, 14491453 (2011).

66. Strunk, B.S., Novak, M.N., Young, C.L. & Karbstein,K.A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits. Cell 150, 111121 (2012).

67. Mangiarotti, G., Chiaberge, S., & Bulfone, S. rRNA maturation as a quality control step in ribosomal subunit assembly in Dictyostelium discoideum. J.Biol. Chem. 272, 2781827822 (1997).

68. Atkinson, G.C., Baldauf, S.L. & Hauryliuk, V. Evolution of nonstop, no-go and nonsense-mediated mRNA decay and their termination factor-derived components. BMC Evol. Biol. 8, 290 (2008).

69. Bhattacharya, A., McIntosh, K.B., Willis, I.M. & Warner, J.R. Why Dom34 stimulates growth of cells with defects of 40S ribosomal subunit biosynthesis. Mol. Cell. Biol. 30, 55625571 (2010).

70. Davis, L. & Engebrecht, J. Yeast dom34 mutants are defective in multiple developmental pathways and exhibit decreased levels of polyribosomes. Genetics 149, 4556 (1998).

71. van den Elzen, A.M. etal. Dissection of Dom34Hbs1 reveals independent functions in two RNA quality control pathways. Nature Struct. Mol. Biol. 17, 14461452 (2010).

72. Eberhart, C.G. & Wasserman, S.A. The pelota locus encodes a protein required for meiotic cell division: an analysis of G2/M arrest in Drosophila spermatogenesis. Development 121, 34773486 (1995).

73. Adham, I.M. etal. Disruption of the Pelota gene causes early embryonic lethality and defects in cell cycle progression. Mol. Cell. Biol. 23, 14701476 (2003).

74. Burnicka-Turek, O. etal. Pelota interacts with HAX1, EIF3G and SRPX and the resulting protein complexes are associated with the actin cytoskeleton. BMC Cell Biol. 11, 28 (2010).

75. Graille, M., Chaillet, M. & van Tilbeurgh, H. Structure of yeast Dom34: a protein related to translation termination factor Erf1 and involved in no-go decay. J.Biol. Chem. 283, 71457154 (2008).

76. Lee, H.H. etal. Structural and functional insights into Dom34, a key component of no-go mRNA decay. Mol. Cell 27, 938950 (2007).

77. Wilusz, C.J. & Wilusz, J. Eukaryotic Lsm proteins: lessons from bacteria. Nature Struct. Mol. Biol. 12, 10311036 (2005).

78. Becker, T. etal. Structure of the no-go mRNA decay complex Dom34Hbs1 bound to a stalled 80S ribosome. Nature Struct. Mol. Biol. 18, 715720 (2011).

79. Chen, L. etal. Structure of the Dom34Hbs1 complex and implications for no-go decay. Nature Struct. Mol. Biol. 17, 12331240 (2010).

80. Wallrapp, C. etal. The product of the mammalian orthologue of the Saccharomyces cerevisiae HBS1 gene is phylogenetically related to eukaryotic release factor 3 (eRF3) but does not carry eRF3-like activity. FEBS Lett. 440, 387392 (1998).

81. Saito, K. etal. Omnipotent role of archaeal elongation factor 1 (EF1) in translational elongation and termination, and quality control of protein synthesis. Proc. Natl Acad. Sci. USA 107, 1924219247 (2010).

82. Carr-Schmid, A., Pfund, C., Craig, E.A. & Kinzy, T.G. Novel G-protein complex whose requirement is linked to the translational status of the cell. Mol. Cell. Biol. 22, 25642574 (2002).

83. van Hoof, A. Conserved functions of yeast genes support the duplication, degeneration and complementation model for gene duplication. Genetics 171, 14551461 (2005).

84. Tomecki, R. etal. The human core exosome interacts with differentially localized processive RNases: hDIS3 and hDIS3L. EMBO J. 29, 23422357 (2010).

85. Nelson, R.J., Ziegelhoffer, T., Nicolet, C., Werner-Washburne, M. & Craig, E.A. The translation machinery and 70 kd heat shock protein cooperate in protein synthesis. Cell 71, 97105 (1992).

86. Kobayashi, K. etal. Structural basis for mRNA surveillance by archaeal Pelota and GTP-bound EF1 complex. Proc. Natl Acad. Sci. USA 107, 1757517579 (2010).

87. Cheng, Z. etal. Structural insights into eRF3 and stop codon recognition by eRF1. Genes Dev. 23, 11061118 (2009).

88. Nissen, P. etal. Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. Science 270, 14641472 (1995).

89. Khoshnevis, S. etal. The ironsulphur protein RNase L inhibitor functions in translation termination. EMBO Rep. 11, 214219 (2010).

90. Pisarev, A.V. etal. The role of ABCE1 in eukaryotic posttermination ribosomal recycling. Mol. Cell 37, 196210 (2010).

91. Schmeing, T.M. etal. The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA. Science 326, 688694 (2009).

92. Becker, T. etal. Structural basis of highly conserved ribosome recycling in eukaryotes and archaea. Nature 482, 501506 (2012).

93. Gagnon, M.G., Seetharaman, S.V., Bulkley, D. & Steitz, T.A. Structural basis for the rescue of stalled ribosomes: structure of YaeJ bound to the ribosome. Science 335, 13701372 (2012).

94. Keiler, K.C. Biology of trans-translation. Annu. Rev. Microbiol. 62, 133151 (2008).

95. Andreev, D. etal. The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome. RNA 14, 233239 (2008).

96. Hayes, C.S. & Sauer, R.T. Cleavage of the A site mRNA codon during ribosome pausing provides a mechanism for translational quality control. Mol. Cell 12, 903911 (2003).

97. Neubauer, C. etal. The structural basis for mRNA recognition and cleavage by the ribosome- dependent endonuclease RelE. Cell 139, 10841095 (2009).

98. Sunohara, T., Jojima, K., Tagami, H., Inada, T. & Aiba, H. Ribosome stalling during translation elongation induces cleavage of mRNA being translated in Escherichia coli. J.Biol. Chem. 279, 1536815375 (2004).

99. Felden, B. & Gillet, R. SmpB as the handyman of tmRNA during trans-translation. RNA Biol. 8, 440449 (2011).

100. Neubauer, C., Gillet, R., Kelley, A.C. & Ramakrishnan, V. Decoding in the absence of a codon by tmRNA and SmpB in the ribosome. Science 335, 13661369 (2012).

101. Hayes, C.S. & Keiler, K.C. Beyond ribosome rescue: tmRNA and co-translational processes. FEBS Lett. 584, 413419 (2010).

102. Henras, A.K. etal. The post-transcriptional steps of eukaryotic ribosome biogenesis. Cell. Mol. Life Sci. 65, 23342359 (2008).

103. Strunk, B.S. & Karbstein, K. Powering through ribosome assembly. RNA 15, 20832104 (2009).

104. LaCava, J. etal. RNA degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell 121, 713724 (2005).

105. Lafontaine, D.L. A garbage can for ribosomes: how eukaryotes degrade their ribosomes. Trends Biochem. Sci. 35, 267277 (2010).

106. Fujii, K., Kitabatake, M., Sakata, T., Miyata, A. & Ohno, M. A role for ubiquitin in the clearance of nonfunctional rRNAs. Genes Dev. 23, 963974 (2009).

107. Fujii, K., Kitabatake, M., Sakata, T. & Ohno, M. 40S subunit dissociation and proteasome-dependent RNA degradation in nonfunctional 25S rRNA decay. EMBO J. 31, 25792589 (2012).

AcknowledgementsM.G. acknowledges funding from the Agence Nationale pour la Recherche (grant ANR 11 BSV8 009 02), the CNRS, the University Paris-Sud and the Human Frontier Science Program (grant RGP0018/2009-C). B.S. is supported by the CERBM-IGBMC, the CNRS, the Ligue Contre le Cancer (Equipe Labellise 2011) and Agence Nationale pour la Recherche (grant ANR 11 BSV8 009 02). The authors apolo-gize for the many studies that were not cited owing to space constraints.

Competing interests statementThe authors declare no competing financial interests.

FURTHER INFORMATIONBertrand Sraphin s homepage: http://www.igbmc.fr/research/2/team/27Marc Grailles homepage: http://bioc.polytechnique.fr/spip.php?rubrique117&lang=en

ALL LINKS ARE ACTIVE IN THE ONLINE PDF




Abstract | Living cells require the continuous production of proteins by the ribosomes. Any problem enforcing these protein factories to stall during mRNA translation may then have deleterious cellular effects. To minimize these defects, eukaryotic cells Stalled ribosomes need to be releasedBox 1 | Degradation of bacterial mRNAs forcing ribosome stalling during elongationFigure 1 | Releasing ribosomes at the termination codon or when stalled during translation elongation.a | During normal translation termination, a stop codon entering the ribosome A-site recruits eukaryotic release factor 1 (eRF1) and eRF3GTP. GTP hydroKey players in ribosome releaseProposed rescue mechanismBox 2 | Monitoring the synthesis of eukaryotic ribosomesFigure 2 | Structures of Dom34 and Hbs1.a | Ribbon representation of Saccharomyces cerevisiae Dom34 (left) and human eukaryotic release factor 1 (eRF1; right) crystal structures. The different domains are highlighted with different colours. b | Ribbon reConclusions

Date post:	03-Dec-2016
Category:	Documents
Upload:	bertrand
View:	214 times
Download:	1 times

Surveillance pathways rescuing eukaryotic ribosomes lost in translation

Documents