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Page 1: Nonsense-MediatedmRNADecayBeginsWhere Translation Ends€¦ · Nonsense-MediatedmRNADecayBeginsWhere Translation Ends Evangelos D. Karousis and Oliver Mühlemann Department of Chemistry

Nonsense-MediatedmRNADecay BeginsWhereTranslation Ends

Evangelos D. Karousis and Oliver Mühlemann

Department of Chemistry and Biochemistry, University of Bern, CH-3012 Bern, Switzerland

Correspondence: [email protected]

Nonsense-mediated mRNA decay (NMD) is arguably the best-studied eukaryotic messengerRNA (mRNA) surveillance pathway, yet fundamental questions concerning the molecularmechanism of target RNA selection remain unsolved. Besides degrading defective mRNAsharboring premature termination codons (PTCs), NMD also targets many mRNAs encodingfunctional full-length proteins. Thus, NMD impacts on a cell’s transcriptome and is implicat-ed in a range of biological processes that affect a broad spectrum of cellular homeostasis.Here, we focus on the steps involved in the recognition of NMD targets and the activation ofNMD. We summarize the accumulating evidence that tightly links NMD to translation ter-mination and we further discuss the recruitment and activation of the mRNA degradationmachinery and the regulation of this complex series of events. Finally, we review emergingideas concerning themechanistic details of NMDactivation and the potential role of NMDasa general surveyor of translation efficacy.

Originally conceived as a quality controlpathway that recognizes and specifically

degrades aberrant messenger RNAs (mRNAs)with premature termination codons (PTCs), ithas meanwhile become clear that nonsense-mediated mRNA decay (NMD) contributes toposttranscriptional gene regulation in a waythat goes far beyond quality control. Exploringin which biological contexts NMD-mediatedgene regulation plays an important role is a rel-atively new but rapidly expanding area of re-search that has been covered in recent reviews(Nasif et al. 2017; Nickless et al. 2017). Here, wesummarize our current understanding regard-ing the molecular mechanism of NMD, with thefocus on data obtained from mammalian sys-tems. Despite more than 25 years of research

and a wealth of biochemical data characterizinginteractions between different NMD factors,their enzymatic functions and posttranslationalmodifications, the mechanism and criteria forselection of an mRNA for the NMD pathwayare still not well understood. The slow progressin deciphering the mechanism of NMD canat least partially be attributed to the lack of asuitable in vitro system that faithfully recapitu-lates the key steps of NMD. Nevertheless, workfrom many laboratories during the last fewyears has provided compelling evidence thatNMD is tightly coupled to the process oftranslation termination. During translation ter-mination, it is decided whether the translatedmRNA shall remain intact and serve as a tem-plate for additional rounds of translation or

Editors: Michael B. Mathews, Nahum Sonenberg, and John W.B. HersheyAdditional Perspectives on Translation Mechanisms and Control available at www.cshperspectives.org

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whether it shall be degraded by the NMD path-way (He and Jacobson 2015). In a nutshell, thecurrent view is that NMD ensues when ribo-somes at nonsense codons (hereafter called ter-mination codon [TC]) fail to terminate correct-ly. Because of the tight link between NMD andtranslation termination, we begin this reviewwith a brief overview of eukaryotic translationtermination. For a more detailed review of themechanism of translation termination, see Hel-len (2018).

THE MECHANISM OF EUKARYOTICTRANSLATION TERMINATION ANDRIBOSOME RECYCLINGTranslation termination is signaled by the pres-ence of one of the three TCs in the A site of theribosome. Canonical translation termination ismarked by three key events: (1) proper recogni-tion of the termination signal, (2) hydrolysis ofthe terminal peptidyl-tRNA bond and release ofthe nascent peptide, and (3) dissociation of theribosome into its 60S and 40S subunits (Fig. 1A)

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Figure 1. Schematic illustration of the sequential events taking place in normal translation termination andaberrant translation termination resulting in the activation of nonsense-mediatedmRNA decay (NMD). (A) Thepresence of a termination codon (TC) in the A site of the ribosome provides the signal for translation terminationthat is marked by recognition of the TC by eukaryotic release factor (eRF)1 and eRF3, a conformational change ofeRF1 that facilitates hydrolysis of the terminal peptidyl-transfer RNA (tRNA) bond and the release of the nascentpeptide. Subsequently, release of the ribosome from the messenger RNA (mRNA) is facilitated by ABCE1. (B)When translation termination occurs in an unfavorable environment, UPF1 is activated by UPF2 and/or UPF3that are free in the cytoplasm or positioned nearby on an exon junction complex (EJC). UPF1 activation involvesits phosphorylation by SMG1, whose kinase activity is controlled by SMG8 and SMG9. The fate of the stalledribosomes in the context of NMD activation remains unclear. Phosphorylated UPF1 (indicated by red dots)recruits the NMD-specific SMG6 endonuclease, which cleaves the targetedmRNAnearby the TC and the SMG5/SMG7 heterodimer that stimulates the exonucleolytic degradation of the mRNA by recruiting the CCR4/NOTcomplex. For clarity, a part of the NMD-activating complex (SMG1, UPF2, and UPF3) is depicted in gray tohighlight the factors triggering mRNA degradation in the lower part of the panel.

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(Dever and Green 2012; Simms et al. 2017). Incomparison to the initiation and elongationsteps, the step of translation termination is lesswell studied and accordinglymuch less is knownregarding its molecular mechanism. Insteadof cognate aminoacylated (aa) transfer RNAs(tRNAs) that get recruited to the A site of theribosome during elongation, eukaryotic releasefactor 1 (eRF)1 binds the A site when it harborsone of the three TCs. On terminating ribosomes,eRF1 is found as a ternary complex with theGTPase eRF3 and GTP. After its recruitmentto the A site, GTP hydrolysis by eRF3 stimulatesa large conformational change in eRF1 thatenhances polypeptide release by engaging theactive site of the ribosome. Despite the extendedconformational change of the middle and car-boxy-terminal parts of eRF1, the amino-termi-nal part of the protein interacts stably with theTC throughout the process (Alkalaeva et al.2006; Becker et al. 2012; Eyler et al. 2013; Brownet al. 2015; Shao et al. 2016).

Following GTP hydrolysis, eRF3 dissociatesfrom the termination complex, allowing for thesubsequent interaction of eRF1 with the ABC-type ATPase ABCE1 (Rli1 in yeast), a factor thatstimulates the recycling of the ribosome by split-ting the ribosomal subunits. ABCE1 containstwo nucleotide-binding domains (NBDs) anda unique amino-terminal FeS cluster domainaligned by two diamagnetic [4Fe–4S]2+ clusters.ATP hydrolysis by ABCE1 causes extended con-formational changes that provide the mechani-cal force leading to the dissociation of the 60Sfrom the 40S ribosomal subunit (Pisarev et al.2010; Becker et al. 2012). Structural studiesshowed that an initial closure of the NBDspositions the FeS cluster domain toward eRF1,exerting an immediate force that destabilizes theintersubunit interactions (Heuer et al. 2017).Cross-linking and mass spectrometry ap-proaches showed that after ribosomal splitting,ABCE1 remains bound to the translationalGTPase-binding site of the small ribosomalsubunit, establishing major contacts with theS24e ribosomal protein mainly through NBD1and FeS cluster domains. Structural data suggestthat ABCE1 performs a tweezer-like movementthat positions the FeS cluster domain in a cleft

between S12 and rRNA on the small subunit(Kiosze-Becker et al. 2016). Additionally, ABCE1associates with the 43S preinitiation complex(consisting of eIF1A, eIF2, eIF3, and initiatortRNA) but its role in translation initiation re-mains to be elucidated (Heuer et al. 2017). Bio-chemical data suggest thatABCE1alsoacceleratesthe rate of the peptide release by eRF1 in anATP-independentmanner, providing a function-al link between translation termination and ribo-some recycling (Shoemaker and Green 2011).

Most of the information concerning trans-lation termination originates from structuralstudies and in vitro assays. The isolation oftranslation termination intermediates can beachieved by using purified components in thepresence of nucleotide analogs that trap specificsteps (Shao et al. 2016) or mutated release fac-tors (Alkalaeva et al. 2006; Brown et al. 2015).The biochemical dissection of translation termi-nation was facilitated by the establishment of areconstituted system with mammalian compo-nents containing purified ribosomal subunits,translation factors, and aminoacylated tRNAs.This in vitro translation system made possiblethe stepwise investigation of the configuration oftermination complexes by using the toeprintingtechnique, which allows the identification ofribosomal positions on a reporter mRNA atsingle-nucleotide resolution (Alkalaeva et al.2006). However, the establishment of similarapproaches by using mammalian cell lysates,as well as studies of translation termination inliving cells, remains a challenge in the field. Thedevelopment of techniques that allow studies inliving cells such as ribosome profiling and livecell imaging already provides valuable insightinto the dynamics of translation termination(Wang et al. 2016; Shirokikh et al. 2017).

TRANSLATION-COUPLED RNADEGRADATION PATHWAYS

Apart from synthesizing proteins, translationalso acts as a quality-control checkpoint fortemplate mRNAs, newly synthesized proteins,and the translation machinery itself. As dis-cussed above, accurate translation terminationoccurs in the presence of an in-frame TC and

NMD Begins Where Translation Ends

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requires the engagement of eRF1, which acts as aTC decoding factor, and of the translationalGTPase eRF3 in the A site of the ribosome.For mRNAs lacking a TC (nonstop decay) andfor those on which ribosomes stall inside anopen reading frame (ORF) caused by strong sec-ondary structures or the lack of a cognate tRNA(no-go decay), the rescue of otherwise trappedribosomes is essential (see Heck and Wilusz2018). In such cases, the roles of the decodingfactor and the GTPase are performed by theHBS1-like protein (HBS1L; Uniprot Q9Y450)and protein Pelota homolog (PELO; UniprotQ9BRX2; Dom34 in yeast), respectively. Whenthe HBS1L/PELO complex binds a stalled ribo-some, degradation of the corresponding mRNAis induced by an endonucleolytic cleavage.These ribosome rescue mechanisms may occurat any point during the translation cycle andrequire the recruitment of specific factors intothe A site of the ribosome. Unlike no-go andnonstop mRNA decay that depend on special-ized termination factors, NMD relies on thecanonical termination factors eRF1 and eRF3(Naeger et al. 1992; Dever and Green 2012;Simms et al. 2017).

NMD IS LINKED TO INEFFICIENTTRANSLATION TERMINATION

That NMD depends on translation is welldocumented (Carter et al. 1995; Thermannet al. 1998). It was initially suggested that onlymRNAs associatedwith the nuclear cap-bindingcomplex (CBC) were sensitive to NMD, leadingto the idea that NMD can target mRNAs onlyduring the first round of translation, the so-called pioneer round of translation (Maquatet al. 2010). However, it was later shown thatNMD also targets eIF4E-bound mRNAs (Du-rand and Lykke-Andersen 2013; Rufenerand Mühlemann 2013), consistent with datafrom Saccharomyces cerevisiae (Maderazo et al.2003; Gao et al. 2005). This led to a revised,unified NMD model, according to whichNMD can be triggered at any aberrant transla-tion termination event during any round oftranslation (He and Jacobson 2015). This modelposits that the likelihood for a ribosome to ter-

minate correctly primarily depends on the avail-ability of termination stimulating factors inthe immediate vicinity of the TC. For classicalNMD targets with a PTC located in a messengerribonucleoprotein particle (mRNP) environ-ment lacking such factors needed for propertranslation termination, the first ribosome at-tempting to terminate at this PTC will mostlikely trigger NMD. However, the probabilityfor a terminating ribosome to activate NMD atmany other TCs located in more termination-friendly environments would be smaller and theNMD may therefore ensue only after severalribosomes have already terminated properly.This latter scenariomight apply formost endog-enous NMD targets and explain their mostlymoderate NMD-dependent mRNA level reduc-tion (Colombo et al. 2017).

In contrast to no-go and nonstop mRNAdecay, which both use the specialized releasefactors HBS1L and PELO to free the stalledribosomal subunits, translation termination onNMD-sensitive mRNAs appears to operate withthe common release factors eRF1 and eRF3.What, then, is the difference between a termina-tion event that activates NMD and one that doesnot? Evidence from S. cerevisiae and rabbit re-ticulocyte lysate indicates that ribosomes residelonger at the TC of NMD-sensitive comparedwith NMD-insensitive mRNAs (Amrani et al.2004; Peixeiro et al. 2011). Based on thesedata, it was proposed that kinetically slow trans-lation termination caused by the lack of termi-nation-promoting factors may be the signaltriggering NMD (reviewed in Kervestin and Ja-cobson 2012; He and Jacobson 2015). The so-farbest-characterized termination-promoting factoris poly(A)-binding protein (PABP). Experimentsusing a fully reconstituted mammalian in vitrotranslation termination system showed that un-der limiting concentrations of release factors,PABP directly stimulates translation terminationby enhancing the recruitment of eRF3 and eRF1to the ribosome in a GTP- and peptide release-independent manner (Ivanov et al. 2016). Evi-dence from both yeast andmammalian cells sug-gests that NMD ensues when the terminationoccurs in an mRNP environment that is spatiallydistant from the PAPB. Accordingly, NMD can

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be antagonized when cytoplasmic PABP (Pab1pin yeast, PABPC1 in mammals) is artificiallytethered into the vicinity of an otherwise NMD-triggering TC (Amrani et al. 2004; Behm-Ans-mant et al. 2007; Eberle et al. 2008; Ivanov et al.2008; Silva et al. 2008; Singh et al. 2008) and theefficiency of suppression is correlated withthe physical distance between the TC and thetethered PABP. Somewhat unexpectedly, giventhe reported interaction of eRF1 with the car-boxy-terminal domain of PABP, it was furthershown that the carboxyl terminus of PABPC1is dispensable for suppression of NMD, whereasRRMs 1–3 are sufficient and RRMs 1–2 are nec-essary. Furthermore, abrogation of the PABPC1–eIF4G interaction reduces NMD suppression byPABPC1 and tethering of eIF4G downstreamfrom an NMD-eliciting TC also suppressesNMD (Fatscher et al. 2014; Joncourt et al.2014). Although the nature of the termination-promoting activity is still unknown, this findingsuggests that it might lie in the formation of thecircular structure that the mRNP is thought toadopt by eIF4G interacting with both the 50 cap-bound eIF4E and the 30 poly(A) tail-boundPABP (Wells et al. 1998).

UPF1, one of the key NMD factors, has beenreported to associate with eRF1 and eRF3 (Cza-plinski et al. 1998; Wang et al. 2001; Kashimaet al. 2006), fueling the hypothesis that UPF1antagonizes PABPC1 at termination eventsthat trigger NMD (Kervestin and Jacobson2012). There is evidence from coimmunopre-cipitation experiments that UPF1 and PABPC1compete for association with eRF3 (Singh et al.2008). However, a very recent study usingrecombinant proteins in in vitro experimentsshowed that this interaction is most likely indi-rect, bridged by UPF3B (Neu-Yilik et al. 2017).In addition, interactions of eIF3 with hyper-phosphorylated UPF1 (Isken et al. 2008) andwith UPF2 (Morris et al. 2007) have also beendetected, but their roles in NMD are not clear.

KEY EVENTS IN NMD ACTIVATION:THE PIVOTAL ROLE OF UPF1

NMD activation relies on the formation of dy-namic protein complexes on the target mRNA

that eventually lead to the degradation of themRNA. The first NMD factors were identifiedby genetic screens in S. cerevisiae (Leeds et al.1991, 1992) and Caenorhabditis elegans (Pulakand Anderson 1993), and mammalian homo-logs have been subsequently identified byhomology searches (Applequist et al. 1997;Lykke-Andersen et al. 2000; Denning et al.2001; Serin et al. 2001; Chiu et al. 2003; Reichen-bach et al. 2003). More recently, additionalscreens in C. elegans and human cells have iden-tified several additional proteins that appear tobe needed for NMD (Longman et al. 2007;Anastasaki et al. 2011; Alexandrov et al. 2017;Hoque et al. 2017); but, apart from the RNAhelicase DHX34 (Longman et al. 2013; Hugand Caceres 2014; Melero et al. 2016), theirrole in NMD is not yet known. Three of theinitially identified NMD factors are conservedfrom yeast to humans: UPF1, UPF2, and UPF3(of whichmammals have two very similar genes,UPF3A and UPF3B) (Maquat 2004). SMG1,SMG5, SMG6, and SMG7, in contrast, appearto be present only in metazoans (Chen et al.2008). As outlined below, some of the NMDfactors are constituents of more than one ofthe biochemically characterized protein com-plexes that form along the pathway of NMDand also have multiple functions in NMD,which complicates the determination of thetemporal order of events and ultimately the un-derstanding themolecular mechanism of NMD.

UPF1, an ATP-Dependent RNA Helicase,Is at the Heart of NMD

UPF1 is an ATP-dependent RNA helicase of thehelicase superfamily 1 (Fairman-Williams et al.2010) and a key factor in NMD. UPF1 seems toplay a central role in substrate selection and itforms the hub for assembling other NMD fac-tors and recruiting the RNA decay factors. Itshelicase activity is tightly regulated and essentialfor NMD in both yeast and humans (Weng et al.1998; Franks et al. 2010). ATP binding to theATP-binding pocket located between the twoRecA domains (Rec1A and Rec2A) reduces theaffinity of UPF1 for RNA and induces the cata-lytically active conformation (Bhattacharya et

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al. 2000; Cheng et al. 2007). Mammalian UPF1contains three domains that regulate its helicaseactivity: an amino-terminal domain rich in cys-teines and histidines (CH domain); 1B and 1Csubdomains embedded within the Rec1A do-main; and a low-structured carboxy-terminalSQ-rich domain (Cheng et al. 2007; Chakrabartiet al. 2011; Fiorini et al. 2013). Additionally,UPF1 is regulated by phosphorylation of multi-ple SQ and TQmotifs located in the amino- andcarboxy-terminal parts of the protein and thisphosphorylation/dephosphorylation cycle is es-sential for NMD in metazoans (Yamashita etal. 2001; Ohnishi et al. 2003; Okada-Katsuhataet al. 2012). UPF1 phosphorylation is catalyzedby SMG1, a phosphatidylinositol 3-kinase-re-lated protein kinase (Yamashita 2013).

Is UPF1 Binding Sufficient to Mark mRNAs forDegradation?

The questions of when and where UPF1 bindsmRNAs and whether mere binding of UPF1 issufficient to mark RNAs for degradation havelong been investigated and have given rise tocontradictory findings. In support of specificUPF1 recruitment to NMD-targeted tran-scripts, UPF1 has been found significantly en-riched on PTC-containing mRNAs comparedwith PTC-less mRNAs at steady state (Johans-son et al. 2007; Johns et al. 2007; Silva et al. 2008;Hwang et al. 2010; Kurosaki et al. 2014; Lee et al.2015). On the other hand, CLIP-seq and im-munoprecipitation experiments in human andmouse cells showed that UPF1 is considerablyenriched on the 30 untranslated region (UTR) ofmRNAs independently of whether or not theyare targeted by NMD (Hurt et al. 2013; Zundet al. 2013), suggesting that UPF1 binding toRNA is not the discriminatory step in NMDtarget identification. Furthermore, it appearsthat UPF1 associates with mRNA before trans-lation starts at seemingly random positions onthe RNA and that translating ribosomes subse-quently displace UPF1 from the ORF, resultingin the majority of UPF1 being detected on30UTRs at steady state (Fig. 2) (Hogg and Goff2010; Hurt et al. 2013; Zund et al. 2013). Basedon the available data, it is possible that transla-

tion affects UPF1-RNA association in two in-dependent ways: (1) translation removes UPF1from the CDS; and (2) UPF1 gets recruited byrelease factors (Ivanov et al. 2008) and in yeastcells also by ribosomal proteins (Min et al. 2013)to the 30UTR of NMD targets. The ATPase ac-tivity of UPF1 was shown to be required forreleasing UPF1 from nontarget mRNAs (Leeet al. 2015), and there is evidence that UPF1phosphorylation occurs specifically on NMDtargets (Kurosaki et al. 2014). Thus, a plausiblescenario is that UPF1 initially binds all mRNAsand that improper/delayed translation termina-tion triggers phosphorylation of the remainingRNA-bound UPF1, eventually activating NMD,whereas ATP hydrolysis results in the release ofUPF1 from mRNAs that undergo proper trans-lation termination. In this working model,UPF1 functions as a time bomb that destroysthe mRNA if not removed quickly enough, anidea that was postulated some time ago (Hillerenand Parker 1999).

SMG1 Complex Regulates UPF1Phosphorylation

In mammalian cells, the SMG1-mediated UPF1phosphorylation is essential for NMD (Yama-shita et al. 2001; Okada-Katsuhata et al. 2012)and appears to occur in a translation-dependentmanner (Yamashita et al. 2001, 2009; Yamashita2013). At steady state, most UPF1 of an immor-talized cell is hypophosphorylated (Chiu et al.2003; Okada-Katsuhata et al. 2012). The kinaseactivity of SMG1 is regulated by SMG8 andSMG9, two proteins forming a complex withSMG1 that keeps SMG1 in a kinase-inactive con-formation (Yamashita et al. 2009; Arias-Palomoet al. 2011; Fernandez et al. 2011; Melero et al.2014). SMG8 inhibits SMG1 activity in vitro butenhances UPF1 phosphorylation in vivo (Yama-shita et al. 2009), indicating that in addition toregulating the kinase activity of SMG1, SMG8also functions in recruiting the SMG1 complexto UPF1. UPF1 can be coimmunoprecipitatedfrom human cells in a complex with SMG1,eRF1, and eRF3 (the so-called SURF complex[Kashima et al. 2006]). These interactions supportthe view that UPF1 phosphorylation occurs in

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Aberrant translation terminationand NMD activation

Figure 2.Distribution andmolecular architecture of factors that affect nonsense-mediated mRNA decay (NMD)onNMD-sensitive andNMD-insensitivemessenger RNAs (mRNAs) at various stages. NMD-insensitive (green)and NMD-sensitive (red) messenger ribonucleoprotein particles (mRNPs) are loaded with UPF3 bound to exonjunction complexes (EJCs) before export to the cytoplasm, and it is possible (although not known) that someUPF1 also associates with mRNA in the nucleus. In the cytoplasm, UPF1 is bound to all mRNAs independent oftheir sensitivity toNMD.UPF2 interacts withUPF3 and the nuclear cap-binding complex (CBC) and the nuclearpoly(A)-binding protein (PABPN)1 are replaced by their cytoplasmic counterparts eIF4F (composed of eIF4E, G,and A) and PABPC1, respectively. During translation, UPF1 and EJCs are displaced from the coding regions ofthe mRNA by elongating ribosomes, while they remain associated on the 30 untranslated region (UTR). In thecontext of normal translation termination, ribosome release is very efficient, and the corresponding mRNAspersist. When translation termination is not efficient, however, NMD ensues by UPF1 activation and/or recruit-ment of additional UPF1 molecules to the site of termination. The presence of an EJC downstream from thetermination codon (TC) increases the concentration of UPF2 andUPF3 in the vicinity of UPF1, which is thoughtto facilitate the activation of NMD.

NMD Begins Where Translation Ends

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the context of translation termination (Fig. 1B)(Kashima et al. 2006; Kurosaki et al. 2014).UPF2 (see below) and DHX34 (Hug andCaceres 2014) contribute to the activation ofSMG1-catalyzed UPF1 phosphorylation.

UPF2 and UPF3 Are Involved in NMDActivation

UPF2 is conserved in eukaryotes and functionsas a ring-like adaptor protein betweenUPF1 andUPF3 (Serin et al. 2001; Chamieh et al. 2008;Melero et al. 2012). It consists of three MIF4G(middle fragment of eIF4G) domains, with thefirst two domains providing structural support(Clerici et al. 2009) and the third domain inter-acting with UPF3B (Kadlec et al. 2004). UPF2’sinteraction with the CH domain of UPF1 in-duces a large conformational change of this do-main that triggers the helicase activity of UPF1by switching it from an RNA-clamping to anRNA-unwinding mode (Fig. 1B) (Chakrabartiet al. 2011). In addition to its interaction withUPF1 and UPF3B, UPF2 has been reported tobind to the DEAD-box helicase Dbp6 in yeast(DDX51 in Homo sapiens) (Fourati et al. 2014)and to SMG1 and ribosomal proteins of bothsubunits (Lopez-Perrote et al. 2016). UPF2also directly interacts with eRF3 through a car-boxy-terminal region that is essential for UPF3Bbinding but does not interfere with UPF1 bind-ing (Lopez-Perrote et al. 2016). Furthermore,UPF2 forms a complex with SURF, allowingits direct association to ribosomes in an exonjunction complex (EJC)-independent manner(Lopez-Perrote et al. 2016). The fact that UPF2appears to associate with some NMD-relatedcomponents simultaneously and with others ina mutually exclusive way implies an importantrole for UPF2 in orchestrating the order ofevents leading to NMD activation (Lopez-Per-rote et al. 2016). Somewhat surprisingly, givenits central role in NMD, degradation of a subsetof NMD targets has been reported to occur in-dependently of UPF2 (Gehring et al. 2005).

UPF3 is the least conserved of the three coreNMD factors (Culbertson and Leeds 2003). Atsteady state, it predominately resides in thenucleus (Serin et al. 2001), but it is thought to

shuttle with the mRNP to the cytoplasm inwhich NMD takes place (Trcek et al. 2013).Rather than binding RNA, the conserved ami-no-terminal putative RNA recognition motif(RRM) appears to interact with UPF2 (Kadlecet al. 2004). In addition, the three EJC corefactors eIF4A3, MAGOH, and Y14 interactthrough a composite binding site with a shortmotif located at the carboxyl terminus of UPF3(Chamieh et al. 2008). Thus, UPF3 and UPF2bridge UPF1 to the EJC, providing a molecularlink between NMD and the EJC (Chamiehet al. 2008). UPF3 and UPF2 together werealso shown to stimulate the helicase activityof UPF1 (Chamieh et al. 2008). Mammals con-tain two UPF3 paralogs, UPF3A and UPF3B(UPF3B is located on the X chromosome andis also called UPF3X). UPF3A and UPF3B aresimilar in their amino acid sequence and bothassociate with other NMD factors but UPF3Ais less efficient in destabilizing NMD reportermRNAs in tethering assays (Lykke-Andersenet al. 2000; Kunz et al. 2006; Chan et al. 2009;Hug and Caceres 2014). Inmice, the conditionalknockout of UPF3A even enhanced the NMDresponse of a fraction of endogenous NMD tar-gets, suggesting that UPF3A may function as anantagonist of UPF3B by sequestering UPF2from the NMD machinery (Shum et al. 2016).

Degradation of NMD-Targeted mRNAs

As mentioned above, UPF1 phosphorylationseems to be a prerequisite for the direct or indi-rect recruitment of nucleases that degrade thetargeted mRNA (Lejeune et al. 2003; Iskenet al. 2008). Endonucleolytic cleavage in thevicinity of the NMD-eliciting TC is mediatedby SMG6 and the SMG5/SMG7 heterodimerrecruits exonucleolytic decay activities (Mühle-mann and Lykke-Andersen 2010). SMG6 is ametazoan-specific NMD factor that confers itsendonuclease activity through a triad of catalyt-ically active Asp residues in its carboxy-terminalPIN domain (Huntzinger et al. 2008; Eberle et al.2009). In immortalized human cells (HeLa andHEK-293), the bulk of NMD targets appears tobe degraded by SMG6 (Boehm et al. 2014; Lykke-Andersen et al. 2014; Schmidt et al. 2014). The

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unprotected RNA ends generated by the endo-nucleolytic cleavage are then subjected to exonu-cleolytic degradation by the 50–30exonucleaseXRN1 and by 30–50 endonucleases (Gatfieldand Izaurralde 2004; Huntzinger et al. 2008;Eberle et al. 2009; Franks et al. 2010). Notably,SMG6 interacts with phosphorylated T28 ofUPF1 (Okada-Katsuhata et al. 2012) and addi-tionally in a phosphorylation-independent man-ner with the unique stalk protruding from theRec1A domain of UPF1 (Chakrabarti et al.2014; Nicholson et al. 2014). In tethering exper-iments, SMG6 requires UPF1 and SMG1 to de-grade a reporter transcript, implying that theSMG6 endonuclease might adopt its active con-formation only when bound to phosphorylatedUPF1 (Nicholson et al. 2014).

SMG5 and SMG7 form a highly stable het-erodimer (Jonas et al. 2013) that binds to phos-phorylated S1096 of UPF1 in vivo (Ohnishi et al.2003; Okada-Katsuhata et al. 2012) and triggersRNA degradation when tethered to reportertranscripts (Unterholzner and Izaurralde 2004;Cho et al. 2013). This activity requires thecarboxy-terminal proline-rich region of SMG7,which was shown to interact with CNOT8(POP2), the catalytic subunit of the CCR4-NOT deadenylase complex (Loh et al. 2013).Tethered SMG7 has further been reported toinduce reporter transcript decay by DCP2-me-diated decapping and XRN1-mediated 50-to-30

exonucleolytic degradation (Unterholzner andIzaurralde 2004). These factors are presumablyrecruited indirectly via the CCR4-NOT com-plex. There is ample evidence that SMG5/SMG7 and SMG6 RNA degradation pathwaysact in, at least partially, a redundant way. Co-depletion of SMG6 with SMG5 or SMG7 inhib-its NMD much more effectively than depletionof each of the factors alone (Luke et al. 2007;Jonas et al. 2013; Metze et al. 2013; Colomboet al. 2017). A transcriptome-wide approach re-vealed that SMG6 and SMG7 target essentiallythe same sets of transcripts (Colombo et al.2017), even though individual mRNAs mayhave preferences for either SMG6- or SMG7-mediated decay (Ottens et al. 2017).

Human UPF1 also interacts with mRNAdecay factors independently of SMG5/SMG7,

namely, with the decapping factors DCP1,DCP2, and PNRC2 (Lykke-Andersen 2002; Le-jeune et al. 2003; Fenger-Gron et al. 2005; Iskenet al. 2008; Cho et al. 2009; Lai et al. 2012).PNRC2 binds directly to DCP1 and UPF1 (Laiet al. 2012; Cho et al. 2013). The published dataregarding whether or not SMG5 interacts withPNRC2 and is involved in the PNRC2-depen-dent decay pathway are contradictory and fur-ther investigation is needed (Cho et al. 2013; Lohet al. 2013). It is noteworthy that many NMDfactors colocalize with decapping and 50–30 exo-nucleolytic degradation components in process-ing bodies (P-bodies) (Fukuhara et al. 2005;Durand et al. 2007; Franks et al. 2010) but theformation of detectable P-bodies in cells is notrequired for NMD (Stalder and Mühlemann2009).

NMD-INDUCING FEATURES

As outlined above, the current model posits thatNMD is stimulated when the TC occurs in amicroenvironment of the mRNP that is unfa-vorable for translation termination. Assumingthat aberrant mRNAs with PTCs are degradedby the same mechanism as endogenous NMDtargets with full-length ORFs, one would expectto find common features that render mRNAssusceptible to NMD. Transcriptome-wide ap-proaches to identify NMD targets in cells ofdifferent species revealed that the majorityof NMD-sensitive transcripts do not containPTCs but are ordinarymRNAs coding for seem-ingly full-length functional proteins (Rehwinkelet al. 2006; Lykke-Andersen and Jensen 2015;Colombo et al. 2017). Despite the fact thatin most of these studies it was not possible todistinguish between direct and indirect targets,there is ample evidence that NMD can targetboth normal and erroneous transcripts. Amongthe NMD-inducing features, the presence ofthe 30-most exon–exon junction >50 nt down-stream from the TC is the feature with the stron-gest predictive value for NMD susceptibility(Colombo et al. 2017). PTCs resulting frommutations in the ORF or from aberrant or alter-native splicing, as well as genes with an intron inthe 30UTR mostly belong to this class of NMD

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targets. In addition, long 30UTRs (>1000 nt inmammalian cells) (Buhler et al. 2006), the pres-ence of actively translated short upstream ORFs(uORFs) (Hurt et al. 2013), or selenocysteinecodons (UGA) in cells grown in the absence ofselenium (Moriarty et al. 1998) are also featuresthat can—but not always do—trigger NMD(Fig. 3). uORF translation often inhibits trans-lation of the main ORF, either constitutively orin response to stress (Young and Wek 2016).Under such circumstances, ribosomes terminateat the TC of the uORF with usually severalEJCs remaining bound further downstream onthe mRNA, which creates an NMD-promotingtranslation termination environment. Despiteall these empirically determined features, it hasso far remained impossible to computationallypredict NMD targets with high confidence.

Based on the aforementioned ability of PABPto suppress NMD, it has been suggested thatthe physical distance between the TC and thepoly(A) tail might be the crucial feature deter-mining NMD sensitivity (Stalder and Mühle-mann 2008). Because such information aboutthe 3D architecture of mRNPs is currently notavailable, this TC-to-poly(A) tail distance isvirtually impossible to measure and predict.

The EJC as an NMD Enhancer

EJCs are multiprotein complexes organizedaround a central core of four proteins: eIF4A3,MLN51, and MAGOH/Y14, a heterodimer thatinteracts with UPF3B (Buchwald et al. 2010).The EJC core binds tightly to RNA withoutapparent sequence specificity, generally ∼24 ntupstream of spliced exon–exon junctions duringthe course of splicing (Le Hir et al. 2000a,b).However, a transcriptome-wide mapping ofeIF4A3 in human cells indicated that not allsplicing events result in the deposition of anEJC and a significant fraction of eIF4A3 wasdetected on mRNA in noncanonical positions(Sauliere et al. 2012; Singh et al. 2012). EJCs aredeposited onmRNA in the nucleus during splic-ing and accompany the mature mRNAs to thecytoplasm, in which those EJCs located in ORFsare thought to be removed by the first ribosometranslating the mRNA (Fig. 2). EJCs located inthe 30UTR of an mRNP >30 nt downstreamfrom the TC in contrast will remain bound tothe mRNA (Le Hir et al. 2016). The presence ofEJCs in mRNPs was shown to activate transla-tion mediated by an interaction of MLN51 witheIF3 subunits a and d (Chazal et al. 2013). ThoseEJCs that remain bound to the mRNA aftertranslation has begun (i.e., those bound >30 ntdownstream of the TC) strongly promote theactivation of NMD on the correspondingmRNAs (Buhler et al. 2006; Metze et al. 2013)by recruiting NMD factors UPF3, UPF2, and/orSMG6 to the mRNP (Le Hir et al. 2001; Gehringet al. 2005; Kashima et al. 2010). A similarNMD-promoting effect was observed by tether-ing EJC components to the 30UTR of reportertranscripts (Lykke-Andersen et al. 2000; Gehr-ing et al. 2003; Palacios et al. 2004). The presence

PTC-containing NMD-sensitive mRNAs

AUG

A

B

TCAAAAAAAAA

PTCEJC

EJC

ORF3′UTR

AUG

AUGORF

TC

TCAAAAAAAAA

AAAAAAAAA

PTC

ORF3′UTR

3′UTR

AUGORF

uORF

TCAAAAAAAAA

3′UTR

AUGORF

TCAAAAAAAAA

3′UTR

Physiological NMD-sensitive mRNAs

Figure 3. Features that can render aberrant and phys-iological mRNAs sensitive to nonsense-mediatedmRNA decay (NMD). (A) Messenger RNAs(mRNAs) in which the open reading frame (ORF) istruncated caused by the presence of a premature ter-mination codon (PTC) are typically sensitive toNMD. The presence of one or several exon junctioncomplexes (EJCs) downstream from the PTC is animportant NMD-enhancing feature. (B) In mRNAswith an intact ORF coding for a full-length protein,long 30 untranslated regions (UTRs), EJCs located inthe 30UTR, or short upstreamORFs (uORFs) can alsotrigger NMD.

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of EJCs in 30UTRs was reported to enhance theefficiency of SMG6-mediated endonucleolysiswithout altering the cleavage site (Boehm et al.2014). This enhanced SMG6-mediated NMDactivity can be attributed to the interaction ofSMG6 with the EJC via two conserved EJC-binding motifs (EBMs) in the amino-terminalportion of SMG6 (Kashima et al. 2010). Inter-estingly, the SMG6 EBMs interact with the samesurface of the EJC as UPF3B (Kashima et al.2010), suggesting that these interactions aremutually exclusive and may therefore occursequentially.

Long 30UTRs

That mRNAs with a long 30UTR can be targetedby NMD has been observed in yeast, plants, andanimals (Pulak and Anderson 1993; Muhlradand Parker 1999; Buhler et al. 2006; Kerteszet al. 2006; Eberle et al. 2008; Singh et al. 2008;Hogg and Goff 2010; Hurt et al. 2013; Boehmet al. 2014; Colombo et al. 2017) and, according-ly, insertion of long 30UTRs into reporter con-structs can render themNMD-sensitive (Huanget al. 2011; Yepiskoposyan et al. 2011). Becausethe physical distance between TC and PABPstrongly affects the half-life of NMD reportertranscripts (Eberle et al. 2008), it is very likelythat those long 30UTRs that render mRNAssensitive to NMD adopt an extended 30UTRstructure in which the poly(A) tail is too faraway from the TC to promote proper translationtermination. Consistent with this idea, intramo-lecular secondary structures that bring a distalpoly(A) tail into close proximity with an other-wise NMD-triggering TC effectively antagonizeNMD, even in the context of EJC-enhancedNMD (Eberle et al. 2008). Although the NMD-suppressing effect of PABP is well documented(Amrani et al. 2004; Behm-Ansmant et al. 2007;Eberle et al. 2008; Ivanov et al. 2008; Silva et al.2008; Singh et al. 2008), the underlying mecha-nism remains elusive. Interestingly, several en-dogenous mRNAs with long 30UTRs werefound to be immune to NMD (Weil and Bee-mon 2006; Hogg and Goff 2010; Yepiskoposyanet al. 2011; Hurt et al. 2013). A subset of theseNMD-insensitive mRNAs with long 30UTRs

contains AU-rich elements within the first200 nt downstream from the TC, which wereshown to be necessary and sufficient to inhibitNMD (Toma et al. 2015). However, the mecha-nism by which this NMD protection is con-ferred is not known. Another well-studied ex-ample of an NMD-resistant transcript with along 30UTR is the unspliced viral RNA of Roussarcoma virus (RSV). RSV harbors a 400-ntRNA stability element (RSE) located immedi-ately downstream from the gag TC, which isrequired to confer NMD immunity on the viralRNA (Weil and Beemon 2006). It has beenfound that the polypyrimidine tract-bindingprotein 1 (PTBP1) binds to the RSE and therebyblocks UPF1 association with this region (Geet al. 2016). Insertion of PTBP1-binding sitesinto NMD reporter constructs downstream ofthe TC rendered these transcripts NMD-insen-sitive (Ge et al. 2016).

OPEN QUESTIONS

Despite the fact that NMD has been an area ofintensive research for the past two decades,many points still remain unclear. A long-stand-ing open question is whether all eukaryotes haveNMD. Trypanosomes have homologs of UPF1and UPF2 but UPF1 depletion showed no effecton the RNA level of genes that were predictedNMD targets, leading the investigators to con-clude that a classical NMD pathway might beabsent in Trypanosoma brucei (Delhi et al.2011). In contrast, NMD appears to functioninGiardia lamblia, another parasitic protist, de-spite the lack of recognizable homologs of UPF2and UPF3 (Chen et al. 2008). Although UPF3seems to be absent from most protists, a UPF3homolog has been found in Paramecium tet-raurelia and it was shown to play a role inNMD (Contreras et al. 2014). Interestingly,however, this UPF3 variant lacks the motifs re-quired for interacting with the EJC, indicatingthat in these early-branched eukaryotes, NMDmight function independently of EJCs, similarto S. cerevisiae, which lacks EJCs altogether.Consistently, NMD in Tetrahymena thermo-phila has recently been shown to be EJC-in-dependent (Tian et al. 2017). In addition, this

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study identified a novel protozoa-specific nucle-ase that is responsible for degradingmanyNMDtargets, a Smg6-like bacterial YacP nuclease(NYN) domain-containing protein (Smg6L).These recent findings corroborate the view thatNMD in different species can occur by severaldifferent RNA decay pathways and that the evo-lutionarily conserved part may be confinedto the substrate RNA selection, in which UPF1plays a key function.

Recent ribosome profiling data in S. cerevi-siae revealed that NMD-targeted mRNAs thatlack PTCs according to their annotated se-quences tend to show elevated rates of out-of-frame translation (Celik et al. 2017a). Such out-of-frame translation would lead to prematuretranslation termination, which could explainwhy these mRNAs are NMD targets. It hastherefore been proposed that NMD should beconsidered as a probabilistic quality-controlpathway that continually detects errors through-out the translation process (Celik et al. 2017b).This is an interesting novel concept that needs tobe further investigated.

Apart from understanding the mechanisticdetails that govern NMD activation in the con-text of inefficient translation termination, itwould also be interesting to assess whether in-terconnections between NMD and other trans-lation-dependent mRNA degradation mecha-nisms exist. Preliminary evidence supports thenotion of such cross talk at least with regard tothe mRNA degradation machinery involved.When endonucleolytic cleavage occurs upstreamof a PTC, the resulting 50RNA fragment was sta-bilized on depletion of nonstop decay compo-nents Pelota and Hbs1 in Drosophila cells (Ha-shimoto et al. 2017). Along the same lines, it wasshown inC. elegans that the degradation of PTC-containing mRNAs as well as endogenous NMDtargets is coupled to nonstop decay (Arribere andFire 2018). This implies that the same mRNAcould be targeted by more than one translation-dependent RNA surveillance pathway, allowingfor more efficient RNA degradation.

It is evident from this review that manyfundamental questions regarding the NMDphenomenon are still unsolved. Notably, it iscurrently not even clear whether what is com-

monly described as NMD constitutes onedefined biochemical pathway or maybe severaldifferent pathways with requirements for anoverlapping set of proteins. Thus, we are stillfar away from a comprehensive understandingof the biological functions of NMD, its under-pinning molecular mechanism(s), and theevolutionary forces that led to the emergenceof NMD.

ACKNOWLEDGMENTS

The research in the laboratory of O.M. is sup-ported by the National Center of Competence inResearch (NCCR) RNA&Disease funded by theSwiss National Science Foundation (SNSF), bythe SNSF Grant 31003A-162986, and by thecanton of Bern.

REFERENCES�Reference is also in this collection.

Alexandrov A, Shu MD, Steitz JA. 2017. Fluorescence am-plification method for forward genetic discovery of fac-tors in humanmRNA degradation.Mol Cell 65: 191–201.

Alkalaeva EZ, Pisarev AV, Frolova LY, Kisselev LL, PestovaTV. 2006. In vitro reconstitution of eukaryotic translationreveals cooperativity between release factors eRF1 andeRF3. Cell 125: 1125–1136.

Amrani N, Ganesan R, Kervestin S, Mangus DA, Ghosh S,Jacobson A. 2004. A faux 30-UTR promotes aberrant ter-mination and triggers nonsense-mediated mRNA decay.Nature 432: 112–118.

Anastasaki C, Longman D, Capper A, Patton EE, Caceres JF.2011. Dhx34 andNbas function in theNMDpathway andare required for embryonic development in zebrafish.Nu-cleic Acids Res 39: 3686–3694.

Applequist SE, Selg M, Raman C, Jack HM. 1997. Cloningand characterization of HUPF1, a human homolog ofthe Saccharomyces cerevisiae nonsense mRNA-reducingUPF1 protein. Nucleic Acids Res 25: 814–821.

Arias-Palomo E, Yamashita A, Fernandez IS, Nunez-Ramirez R, Bamba Y, Izumi N, Ohno S, Llorca O. 2011.The nonsense-mediated mRNA decay SMG-1 kinase isregulated by large-scale conformational changes con-trolled by SMG-8. Genes Dev 25: 153–164.

Arribere JA, Fire AZ. 2018. Nonsense mRNA suppressionvia nonstop decay. eLife 7: e33292.

Becker T, Franckenberg S, Wickles S, Shoemaker CJ, AngerAM, Armache JP, Sieber H, Ungewickell C, Berninghau-sen O, Daberkow I, et al. 2012. Structural basis of highlyconserved ribosome recycling in eukaryotes and archaea.Nature 482: 501–506.

Behm-Ansmant I, Gatfield D, Rehwinkel J, Hilgers V, Izaur-ralde E. 2007. A conserved role for cytoplasmic poly(A)-

E.D. Karousis and O. Mühlemann

12 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032862

on June 11, 2018 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from

Page 13: Nonsense-MediatedmRNADecayBeginsWhere Translation Ends€¦ · Nonsense-MediatedmRNADecayBeginsWhere Translation Ends Evangelos D. Karousis and Oliver Mühlemann Department of Chemistry

binding protein 1 (PABPC1) in nonsense-mediatedmRNA decay. EMBO J 26: 1591–1601.

Bhattacharya A, Czaplinski K, Trifillis P, He F, Jacobson A,Peltz SW. 2000. Characterization of the biochemicalproperties of the human Upf1 gene product that is in-volved in nonsense-mediated mRNA decay. RNA 6:1226–1235.

Boehm V, Haberman N, Ottens F, Ule J, Gehring NH. 2014.30UTR length and messenger ribonucleoprotein compo-sition determine endocleavage efficiencies at terminationcodons. Cell Rep 9: 555–568.

Brown A, Shao S, Murray J, Hegde RS, Ramakrishnan V.2015. Structural basis for stop codon recognition in eu-karyotes. Nature 524: 493–496.

Buchwald G, Ebert J, Basquin C, Sauliere J, Jayachandran U,Bono F, Le Hir H, Conti E. 2010. Insights into the recruit-ment of the NMDmachinery from the crystal structure ofa core EJC-UPF3b complex. Proc Natl Acad Sci 107:10050–10055.

Buhler M, Steiner S, Mohn F, Paillusson A, Mühlemann O.2006. EJC-independent degradation of nonsense immu-noglobulin-mu mRNA depends on 30UTR length. NatStruct Mol Biol 13: 462–464.

Carter MS, Doskow J, Morris P, Li S, Nhim RP, Sandstedt S,Wilkinson MF. 1995. A regulatory mechanism that de-tects premature nonsense codons in T-cell receptor tran-scripts in vivo is reversed by protein synthesis inhibitorsin vitro. J Biol Chem 270: 28995–29003.

Celik A, Baker R, He F, Jacobson A. 2017a. High-resolutionprofiling of NMD targets in yeast reveals translationalfidelity as a basis for substrate selection. RNA 23: 735–748.

Celik A, He F, Jacobson A. 2017b. NMD monitors transla-tional fidelity 24/7. Curr Genet 63: 1007–1010.

Chakrabarti S, Jayachandran U, Bonneau F, Fiorini F, Bas-quin C, Domcke S, Le Hir H, Conti E. 2011. Molecularmechanisms for the RNA-dependent ATPase activity ofUpf1 and its regulation by Upf2. Mol Cell 41: 693–703.

Chakrabarti S, Bonneau F, Schussler S, Eppinger E, Conti E.2014. Phospho-dependent and phospho-independent in-teractions of the helicase UPF1 with the NMD factorsSMG5-SMG7 and SMG6. Nucleic Acids Res 42: 9447–9460.

Chamieh H, Ballut L, Bonneau F, Le Hir H. 2008. NMDfactors UPF2 andUPF3 bridge UPF1 to the exon junctioncomplex and stimulate its RNA helicase activity. NatStruct Mol Biol 15: 85–93.

ChanWK, Bhalla AD, Le Hir H, Nguyen LS, Huang L, GeczJ, Wilkinson MF. 2009. A UPF3-mediated regulatoryswitch that maintains RNA surveillance. Nat Struct MolBiol 16: 747–753.

Chazal PE, Daguenet E, Wendling C, Ulryck N, TomasettoC, Sargueil B, Le Hir H. 2013. EJC core componentMLN51 interacts with eIF3 and activates translation.Proc Natl Acad Sci 110: 5903–5908.

Chen YH, Su LH, Sun CH. 2008. Incomplete nonsense-mediated mRNA decay in Giardia lamblia. Int J Parasitol38: 1305–1317.

Cheng Z, Muhlrad D, Lim MK, Parker R, Song H. 2007.Structural and functional insights into the human Upf1helicase core. EMBO J 26: 253–264.

Chiu SY, Serin G, Ohara O, Maquat LE. 2003. Characteriza-tion of human Smg5/7a: A protein with similarities toCaenorhabditis elegans SMG5 and SMG7 that functionsin the dephosphorylation of Upf1. RNA 9: 77–87.

Cho H, Kim KM, Kim YK. 2009. Human proline-rich nu-clear receptor coregulatory protein 2 mediates an interac-tion between mRNA surveillance machinery and decap-ping complex. Mol Cell 33: 75–86.

Cho H, Han S, Choe J, Park SG, Choi SS, Kim YK. 2013.SMG5-PNRC2 is functionally dominant compared withSMG5-SMG7 in mammalian nonsense-mediated mRNAdecay. Nucleic Acids Res 41: 1319–1328.

Clerici M, Mourao A, Gutsche I, Gehring NH, Hentze MW,Kulozik A, Kadlec J, Sattler M, Cusack S. 2009. Unusualbipartite mode of interaction between the nonsense-me-diated decay factors, UPF1 and UPF2. EMBO J 28: 2293–2306.

ColomboM, Karousis ED, Bourquin J, Bruggmann R, Müh-lemann O. 2017. Transcriptome-wide identification ofNMD-targeted human mRNAs reveals extensive redun-dancy between SMG6- and SMG7-mediated degradationpathways. RNA 23: 189–201.

Contreras J, Begley V,Macias S, Villalobo E. 2014. AnUPF3-based nonsense-mediated decay in Paramecium. Res Mi-crobiol 165: 841–846.

Culbertson MR, Leeds PF. 2003. Looking at mRNA decaypathways through the window of molecular evolution.Curr Opin Genet Dev 13: 207–214.

Czaplinski K, Ruiz-Echevarria MJ, Paushkin SV, Han X,Weng Y, PerlickHA, Dietz HC, Ter-AvanesyanMD, PeltzSW. 1998. The surveillance complex interacts with thetranslation release factors to enhance termination anddegrade aberrant mRNAs. Genes Dev 12: 1665–1677.

Delhi P, Queiroz R, Inchaustegui D, Carrington M, ClaytonC. 2011. Is there a classical nonsense-mediated decaypathway in trypanosomes? PLoS ONE 6: e25112.

Denning G, Jamieson L, Maquat LE, Thompson EA, FieldsAP. 2001. Cloning of a novel phosphatidylinositol kinase-related kinase: Characterization of the human SMG-1RNA surveillance protein. J Biol Chem 276: 22709–22714.

Dever TE, Green R. 2012. The elongation, termination, andrecycling phases of translation in eukaryotes. Cold SpringHarb Perspect Biol 4: a013706.

Durand S, Lykke-Andersen J. 2013. Nonsense-mediatedmRNA decay occurs during eIF4F-dependent translationin human cells. Nat Struct Mol Biol 20: 702–709.

Durand S, Cougot N, Mahuteau-Betzer F, Nguyen CH,Grierson DS, Bertrand E, Tazi J, Lejeune F. 2007. Inhibi-tion of nonsense-mediated mRNA decay (NMD) by anew chemical molecule reveals the dynamic of NMD fac-tors in P-bodies. J Cell Biol 178: 1145–1160.

Eberle AB, Stalder L, Mathys H, Orozco RZ, Mühlemann O.2008. Posttranscriptional gene regulation by spatial rear-rangement of the 30 untranslated region. PLoS Biol 6: e92.

Eberle AB, Lykke-Andersen S, Mühlemann O, Jensen TH.2009. SMG6 promotes endonucleolytic cleavage of non-sense mRNA in human cells. Nat Struct Mol Biol 16:49–55.

Eyler DE, Wehner KA, Green R. 2013. Eukaryotic releasefactor 3 is required for multiple turnovers of peptide

NMD Begins Where Translation Ends

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032862 13

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Page 14: Nonsense-MediatedmRNADecayBeginsWhere Translation Ends€¦ · Nonsense-MediatedmRNADecayBeginsWhere Translation Ends Evangelos D. Karousis and Oliver Mühlemann Department of Chemistry

release catalysis by eukaryotic release factor 1. J Biol Chem288: 29530–29538.

Fairman-Williams ME, Guenther UP, Jankowsky E. 2010.SF1 and SF2 helicases: Family matters. Curr Opin StructBiol 20: 313–324.

Fatscher T, Boehm V, Weiche B, Gehring NH. 2014. Theinteraction of cytoplasmic poly(A)-binding protein witheukaryotic initiation factor 4G suppresses nonsense-me-diated mRNA decay. RNA 20: 1579–1592.

Fenger-Gron M, Fillman C, Norrild B, Lykke-Andersen J.2005.Multiple processing body factors and theAREbind-ing protein TTP activate mRNA decapping. Mol Cell 20:905–915.

Fernandez IS, Yamashita A, Arias-Palomo E, Bamba Y, Bar-tolome RA, Canales MA, Teixido J, Ohno S, Llorca O.2011. Characterization of SMG-9, an essential compo-nent of the nonsense-mediated mRNA decay SMG1Ccomplex. Nucleic Acids Res 39: 347–358.

Fiorini F, Boudvillain M, Le Hir H. 2013. Tight intramolec-ular regulation of the human Upf1 helicase by its N- andC-terminal domains. Nucleic Acids Res 41: 2404–2415.

Fourati Z, Roy B, Millan C, Coureux PD, Kervestin S, vanTilbeurghH, He F, Uson I, JacobsonA, GrailleM. 2014. Ahighly conserved region essential for NMD in the Upf2N-terminal domain. J Mol Biol 426: 3689–3702.

Franks TM, SinghG, Lykke-Andersen J. 2010. Upf1ATPase-dependent mRNP disassembly is required for completionof nonsense- mediated mRNA decay. Cell 143: 938–950.

Fukuhara N, Ebert J, Unterholzner L, Lindner D, IzaurraldeE, Conti E. 2005. SMG7 is a 14-3-3-like adaptor in thenonsense-mediated mRNA decay pathway. Mol Cell 17:537–547.

Gao Q, Das B, Sherman F, Maquat LE. 2005. Cap-bindingprotein 1-mediated and eukaryotic translation initiationfactor 4E-mediated pioneer rounds of translation in yeast.Proc Natl Acad Sci 102: 4258–4263.

Gatfield D, Izaurralde E. 2004. Nonsense-mediated messen-ger RNA decay is initiated by endonucleolytic cleavage inDrosophila. Nature 429: 575–578.

Ge Z, Quek BL, Beemon KL, Hogg JR. 2016. Polypyrimidinetract binding protein 1 protects mRNAs from recognitionby the nonsense-mediatedmRNA decay pathway. eLife 5:e11155.

Gehring NH, Neu-Yilik G, Schell T, Hentze MW, KulozikAE. 2003. Y14 and hUpf3b form an NMD-activatingcomplex. Mol Cell 11: 939–949.

Gehring NH, Kunz JB, Neu-Yilik G, Breit S, Viegas MH,Hentze MW, Kulozik AE. 2005. Exon-junction complexcomponents specify distinct routes of nonsense-mediatedmRNAdecaywith differential cofactor requirements.MolCell 20: 65–75.

Hashimoto Y, Takahashi M, Sakota E, Nakamura Y. 2017.Nonstop-mRNA decay machinery is involved in theclearance of mRNA 50-fragments produced by RNAiand NMD inDrosophila melanogaster cells. Biochem Bio-phys Res Commun 484: 1–7.

He F, Jacobson A. 2015. Nonsense-mediated mRNA decay:Degradation of defective transcripts is only part of thestory. Annu Rev Genet 49: 339–366.

� Heck AM, Wilusz J. 2018. The interplay between the RNAdecay and translation machinery in eukaryotes. Cold

Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032839.

� Hellen CUT. 2018. Translation termination and ribosomerecycling in eukaryotes. Cold Spring Harb Perspect Bioldoi: 10.1101/cshperspect.a032656.

Heuer A, Gerovac M, Schmidt C, Trowitzsch S, Preis A,Kotter P, Berninghausen O, Becker T, Beckmann R,Tampe R. 2017. Structure of the 40S-ABCE1 post-split-ting complex in ribosome recycling and translation initi-ation. Nat Struct Mol Biol 24: 453–460.

Hilleren P, Parker R. 1999. mRNA surveillance in eukary-otes: Kinetic proofreading of proper translation termina-tion as assessed by mRNP domain organization? RNA 5:711–719.

Hogg JR, Goff SP. 2010. Upf1 senses 30UTR length to po-tentiate mRNA decay. Cell 143: 379–389.

Hoque M, Park JY, Chang YJ, Luchessi AD, Cambiaghi TD,Shamanna R, Hanauske-Abel HM, Holland B, Pe’ery T,Tian B, et al. 2017. Regulation of gene expression bytranslation factor eIF5A: Hypusine-modified eIF5A en-hances nonsense-mediated mRNA decay in human cells.Translation (Austin) 5: e1366294.

Huang L, Lou CH, Chan W, Shum EY, Shao A, Stone E,Karam R, Song HW, Wilkinson MF. 2011. RNA homeo-stasis governed by cell type-specific and branched feed-back loops acting on NMD. Mol Cell 43: 950–961.

HugN, Caceres JF. 2014. The RNAhelicaseDHX34 activatesNMD by promoting a transition from the surveillance tothe decay-inducing complex. Cell Rep 8: 1845–1856.

Huntzinger E, Kashima I, Fauser M, Sauliere J, Izaurralde E.2008. SMG6 is the catalytic endonuclease that cleavesmRNAs containing nonsense codons in metazoan. RNA14: 2609–2617.

Hurt JA, Robertson AD, Burge CB. 2013. Global analyses ofUPF1 binding and function reveal expanded scope ofnonsense-mediated mRNA decay. Genome Res 23:1636–1650.

Hwang J, Sato H, Tang Y, Matsuda D, Maquat LE. 2010.UPF1 association with the cap-binding protein, CBP80,promotes nonsense-mediated mRNA decay at two dis-tinct steps. Mol Cell 39: 396–409.

Isken O, Kim YK, Hosoda N, Mayeur GL, Hershey JW,Maquat LE. 2008. Upf1 phosphorylation triggers transla-tional repression during nonsense-mediated mRNA de-cay. Cell 133: 314–327.

Ivanov PV, Gehring NH, Kunz JB, HentzeMW, Kulozik AE.2008. Interactions between UPF1, eRFs, PABP and theexon junction complex suggest an integrated model formammalian NMD pathways. EMBO J 27: 736–747.

Ivanov A, Mikhailova T, Eliseev B, Yeramala L, Sokolova E,Susorov D, Shuvalov A, Schaffitzel C, Alkalaeva E. 2016.PABPenhances release factor recruitment and stop codonrecognition during translation termination.Nucleic AcidsRes 44: 7766–7776.

Johansson MJ, He F, Spatrick P, Li C, Jacobson A. 2007.Association of yeast Upf1p with direct substrates of theNMD pathway. Proc Natl Acad Sci 104: 20872–20877.

Johns L, Grimson A, Kuchma SL, Newman CL, Anderson P.2007. Caenorhabditis elegans SMG-2 selectively marksmRNAs containing premature translation terminationcodons. Mol Cell Biol 27: 5630–5638.

E.D. Karousis and O. Mühlemann

14 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032862

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Page 15: Nonsense-MediatedmRNADecayBeginsWhere Translation Ends€¦ · Nonsense-MediatedmRNADecayBeginsWhere Translation Ends Evangelos D. Karousis and Oliver Mühlemann Department of Chemistry

Jonas S, Weichenrieder O, Izaurralde E. 2013. An unusualarrangement of two 14-3-3-like domains in the SMG5-SMG7 heterodimer is required for efficient nonsense-me-diated mRNA decay. Genes Dev 27: 211–225.

Joncourt R, Eberle AB, Rufener SC, Mühlemann O. 2014.Eukaryotic initiation factor 4G suppresses nonsense-me-diated mRNA decay by two genetically separable mecha-nisms. PLoS ONE 9: e104391.

Kadlec J, Izaurralde E, Cusack S. 2004. The structural basisfor the interaction between nonsense-mediated mRNAdecay factors UPF2 and UPF3. Nat Struct Mol Biol 11:330–337.

Kashima I, Yamashita A, Izumi N, Kataoka N, Morishita R,Hoshino S, Ohno M, Dreyfuss G, Ohno S. 2006. Bindingof a novel SMG-1-Upf1-eRF1-eRF3 complex (SURF) tothe exon junction complex triggers Upf1 phosphorylationand nonsense-mediated mRNA decay. Genes Dev 20:355–367.

Kashima I, Jonas S, Jayachandran U, Buchwald G, Conti E,Lupas AN, Izaurralde E. 2010. SMG6 interacts with theexon junction complex via two conserved EJC-bindingmotifs (EBMs) required for nonsense-mediated mRNAdecay. Genes Dev 24: 2440–2450.

Kertesz S, Kerenyi Z, Merai Z, Bartos I, Palfy T, Barta E,Silhavy D. 2006. Both introns and long 30-UTRs operateas cis-acting elements to trigger nonsense-mediated decayin plants. Nucleic Acids Res 34: 6147–6157.

Kervestin S, Jacobson A. 2012. NMD: A multifaceted re-sponse to premature translational termination. Nat RevMol Cell Biol 13: 700–712.

Kiosze-Becker K, Ori A, Gerovac M, Heuer A, Nurenberg-Goloub E, Rashid UJ, Becker T, Beckmann R, Beck M,Tampe R. 2016. Structure of the ribosome post-recyclingcomplex probed by chemical cross-linking and massspectrometry. Nat Commun 7: 13248.

Kunz JB, Neu-Yilik G, Hentze MW, Kulozik AE, GehringNH. 2006. Functions of hUpf3a and hUpf3b in non-sense-mediated mRNA decay and translation. RNA 12:1015–1022.

Kurosaki T, Li W, Hoque M, Popp MW, Ermolenko DN,Tian B, Maquat LE. 2014. A post-translational regulatoryswitch on UPF1 controls targeted mRNA degradation.Genes Dev 28: 1900–1916.

Lai T, Cho H, Liu Z, Bowler MW, Piao S, Parker R, Kim YK,Song H. 2012. Structural basis of the PNRC2-mediatedlink between mRNA surveillance and decapping. Struc-ture 20: 2025–2037.

Lee SR, Pratt GA, Martinez FJ, Yeo GW, Lykke-AndersenJ. 2015. Target discrimination in nonsense-mediatedmRNA decay requires Upf1 ATPase activity. Mol Cell59: 413–425.

Leeds P, Peltz SW, Jacobson A, Culbertson MR. 1991. Theproduct of the yeastUPF1 gene is required for rapid turn-over of mRNAs containing a premature translational ter-mination codon. Genes Dev 5: 2303–2314.

Leeds P, Wood JM, Lee BS, Culbertson MR. 1992. Geneproducts that promotemRNA turnover in Saccharomycescerevisiae. Mol Cell Biol 12: 2165–2177.

Le Hir H, Izaurralde E, Maquat LE, Moore MJ. 2000a. Thespliceosome depositsmultiple proteins 20–24 nucleotidesupstream of mRNA exon–exon junctions. EMBO J 19:6860–6869.

LeHir H,MooreMJ,Maquat LE. 2000b. Pre-mRNA splicingalters mRNP composition: Evidence for stable associa-tion of proteins at exon–exon junctions. Genes Dev 14:1098–1108.

Le Hir H, Gatfield D, Izaurralde E, Moore MJ. 2001. Theexon–exon junction complex provides a binding platformfor factors involved in mRNA export and nonsense-me-diated mRNA decay. EMBO J 20: 4987–4997.

Le Hir H, Sauliere J, Wang Z. 2016. The exon junction com-plex as a node of post-transcriptional networks. Nat RevMol Cell Biol 17: 41–54.

Lejeune F, Li X, Maquat LE. 2003. Nonsense-mediatedmRNA decay in mammalian cells involves decapping,deadenylating, and exonucleolytic activities. Mol Cell12: 675–687.

Loh B, Jonas S, Izaurralde E. 2013. The SMG5–SMG7 het-erodimer directly recruits the CCR4–NOT deadenylasecomplex to mRNAs containing nonsense codons via in-teraction with POP2. Genes Dev 27: 2125–2138.

Longman D, Plasterk RH, Johnstone IL, Caceres JF. 2007.Mechanistic insights and identification of two novelfactors in the C. elegans NMD pathway. Genes Dev 21:1075–1085.

Longman D, Hug N, Keith M, Anastasaki C, Patton EE,Grimes G, Caceres JF. 2013. DHX34 and NBAS formpart of an autoregulatory NMD circuit that regulates en-dogenous RNA targets in human cells, zebrafish andCae-norhabditis elegans. Nucleic Acids Res 41: 8319–8331.

Lopez-Perrote A, Castano R, Melero R, Zamarro T, Kurosa-wa H, Ohnishi T, Uchiyama A, Aoyagi K, Buchwald G,Kataoka N, et al. 2016. Human nonsense-mediatedmRNA decay factor UPF2 interacts directly with eRF3and the SURF complex.Nucleic Acids Res 44: 1909–1923.

Luke B, Azzalin CM, HugN, Deplazes A, PeterM, Lingner J.2007. Saccharomyces cerevisiae Ebs1p is a putative ortho-log of human Smg7 and promotes nonsense-mediatedmRNA decay. Nucleic Acids Res 35: 7688–7697.

Lykke-Andersen J. 2002. Identification of a human decap-ping complex associated with hUpf proteins in nonsense-mediated decay. Mol Cell Biol 22: 8114–8121.

Lykke-Andersen S, Jensen TH. 2015. Nonsense-mediatedmRNA decay: An intricate machinery that shapes tran-scriptomes. Nat Rev Mol Cell Biol 16: 665–677.

Lykke-Andersen J, Shu MD, Steitz JA. 2000. Human Upfproteins target an mRNA for nonsense-mediated decaywhen bound downstream of a termination codon. Cell103: 1121–1131.

Lykke-Andersen S, Chen Y, Ardal BR, Lilje B, Waage J, San-delin A, Jensen TH. 2014. Human nonsense-mediatedRNA decay initiates widely by endonucleolysis and tar-gets snoRNA host genes. Genes Dev 28: 2498–2517.

Maderazo AB, Belk JP, He F, Jacobson A. 2003. Nonsense-containing mRNAs that accumulate in the absence of afunctional nonsense-mediated mRNA decay pathway aredestabilized rapidly upon its restitution.Mol Cell Biol 23:842–851.

Maquat LE. 2004. Nonsense-mediated mRNA decay: Acomparative analysis of different species. Current Geno-mics 5: 175–190.

Maquat LE, Tarn WY, Isken O. 2010. The pioneer round oftranslation: Features and functions. Cell 142: 368–374.

NMD Begins Where Translation Ends

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032862 15

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Page 16: Nonsense-MediatedmRNADecayBeginsWhere Translation Ends€¦ · Nonsense-MediatedmRNADecayBeginsWhere Translation Ends Evangelos D. Karousis and Oliver Mühlemann Department of Chemistry

Melero R, Buchwald G, Castano R, Raabe M, Gil D, LazaroM, Urlaub H, Conti E, Llorca O. 2012. The cryo-EMstructure of the UPF–EJC complex shows UPF1 poisedtoward the RNA 30 end. Nat Struct Mol Biol 19: 498–505.

Melero R, Uchiyama A, Castano R, Kataoka N, Kurosawa H,Ohno S, Yamashita A, Llorca O. 2014. Structures ofSMG1–UPFs complexes: SMG1 contributes to regulateUPF2-dependent activation of UPF1 in NMD. Structure22: 1105–1119.

Melero R, HugN, Lopez-Perrote A, Yamashita A, Caceres JF,Llorca O. 2016. The RNA helicase DHX34 functions as ascaffold for SMG1-mediated UPF1 phosphorylation. NatCommun 7: 10585.

Metze S, Herzog VA, Ruepp MD, Mühlemann O. 2013.Comparison of EJC-enhanced and EJC-independentNMD in human cells reveals two partially redundant deg-radation pathways. RNA 19: 1432–1448.

Min EE, Roy B, Amrani N, He F, Jacobson A. 2013. YeastUpf1 CH domain interacts with Rps26 of the 40S ribo-somal subunit. RNA 19: 1105–1115.

Moriarty PM, Reddy CC, Maquat LE. 1998. Selenium defi-ciency reduces the abundance ofmRNA for Se-dependentglutathione peroxidase 1 by a UGA-dependent mecha-nism likely to be nonsense codon-mediated decay of cy-toplasmic mRNA. Mol Cell Biol 18: 2932–2939.

Morris C, Wittmann J, Jack HM, Jalinot P. 2007. HumanINT6/eIF3e is required for nonsense-mediated mRNAdecay. EMBO Rep 8: 596–602.

MühlemannO, Lykke-Andersen J. 2010. How andwhere arenonsense mRNAs degraded in mammalian cells? RNABiol 7: 28–32.

MuhlradD, Parker R. 1999. AberrantmRNAswith extended30UTRs are substrates for rapid degradation by mRNAsurveillance. RNA 5: 1299–1307.

Naeger LK, Schoborg RV, ZhaoQ, Tullis GE, PintelDJ. 1992.Nonsense mutations inhibit splicing of MVM RNA in ciswhen they interrupt the reading frame of either exon ofthe final spliced product. Genes Dev 6: 1107–1119.

Nasif S, Contu L, Mühlemann O. 2017. Beyond quality con-trol: The role of nonsense-mediatedmRNAdecay (NMD)in regulating gene expression. Semin Cell Dev Biol 75:78–87.

Neu-Yilik G, Raimondeau E, Eliseev B, Yeramala L, AmthorB, Deniaud A, Huard K, Kerschgens K, Hentze MW,Schaffitzel C, et al. 2017. Dual function of UPF3B in earlyand late translation termination. EMBO J 36: 2968–2986.

Nicholson P, Josi C, KurosawaH, Yamashita A,MühlemannO. 2014. A novel phosphorylation-independent interac-tion between SMG6 and UPF1 is essential for humanNMD. Nucleic Acids Res 42: 9217–9235.

Nickless A, Bailis JM, You Z. 2017. Control of gene expres-sion through the nonsense-mediated RNA decay path-way. Cell Biosci 7: 26.

Ohnishi T, Yamashita A, Kashima I, Schell T, Anders KR,Grimson A, Hachiya T, Hentze MW, Anderson P, OhnoS. 2003. Phosphorylation of hUPF1 induces formation ofmRNA surveillance complexes containing hSMG-5 andhSMG-7. Mol Cell 12: 1187–1200.

Okada-Katsuhata Y, Yamashita A, Kutsuzawa K, Izumi N,Hirahara F, Ohno S. 2012. N- and C-terminal Upf1 phos-phorylations create binding platforms for SMG-6 and

SMG-5:SMG-7 during NMD. Nucleic Acids Res 40:1251–1266.

Ottens F, Boehm V, Sibley CR, Ule J, Gehring NH. 2017.Transcript-specific characteristics determine the contri-bution of endo- and exonucleolytic decay pathways dur-ing the degradation of nonsense-mediated decay sub-strates. RNA 23: 1224–1236.

Palacios IM, Gatfield D, St Johnston D, Izaurralde E. 2004.An eIF4AIII-containing complex required for mRNA lo-calization and nonsense-mediated mRNA decay. Nature427: 753–757.

Peixeiro I, Inacio A, Barbosa C, Silva AL, Liebhaber SA,Romao L. 2011. Interaction of PABPC1 with the transla-tion initiation complex is critical to theNMDresistance ofAUG-proximal nonsense mutations. Nucleic Acids Res40: 1160–1173.

Pisarev AV, Skabkin MA, Pisareva VP, Skabkina OV, Rako-tondrafara AM, Hentze MW, Hellen CU, Pestova TV.2010. The role of ABCE1 in eukaryotic postterminationribosomal recycling. Mol Cell 37: 196–210.

Pulak R, Anderson P. 1993. mRNA surveillance by the Cae-norhabditis elegans smg genes. Genes Dev 7: 1885–1897.

Rehwinkel J, Raes J, Izaurralde E. 2006. Nonsense-mediatedmRNA decay: Target genes and functional diversificationof effectors. Trends Biochem Sci 31: 639–646.

Reichenbach P, Hoss M, Azzalin CM, Nabholz M, Bucher P,Lingner J. 2003. A humanhomolog of yeast est1 associateswith telomerase and uncaps chromosome ends whenoverexpressed. Curr Biol 13: 568–574.

Rufener SC, Mühlemann O. 2013. eIF4E-bound mRNPs aresubstrates for nonsense-mediated mRNA decay in mam-malian cells. Nat Struct Mol Biol 20: 710–717.

Sauliere J, Murigneux V, Wang Z, Marquenet E, Barbosa I,Le Tonqueze O, Audic Y, Paillard L, Roest Crollius H, LeHir H. 2012. CLIP-seq of eIF4AIII reveals transcriptome-wide mapping of the human exon junction complex. NatStruct Mol Biol 19: 1124–1131.

Schmidt SA, Foley PL, Jeong DH, Rymarquis LA, Doyle F,Tenenbaum SA, Belasco JG, Green PJ. 2014. Identifica-tion of SMG6 cleavage sites and a preferred RNA cleavagemotif by global analysis of endogenous NMD targets inhuman cells. Nucleic Acids Res 43: 309–323.

Serin G, Gersappe A, Black JD, Aronoff R, Maquat LE. 2001.Identification and characterization of human orthologuesto Saccharomyces cerevisiae Upf2 protein and Upf3 pro-tein (Caenorhabditis elegans SMG-4). Mol Cell Biol 21:209–223.

Shao S, Murray J, Brown A, Taunton J, Ramakrishnan V,Hegde RS. 2016. Decodingmammalian ribosome-mRNAstates by translational GTPase complexes.Cell 167: 1229–1240.e1215.

Shirokikh NE, Archer SK, Beilharz TH, Powell D, Preiss T.2017. Translation complex profile sequencing to study thein vivo dynamics of mRNA-ribosome interactions duringtranslation initiation, elongation and termination. NatProtoc 12: 697–731.

Shoemaker CJ, Green R. 2011. Kinetic analysis reveals theordered coupling of translation termination and ribo-some recycling in yeast. Proc Natl Acad Sci 108: E1392–E1398.

E.D. Karousis and O. Mühlemann

16 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032862

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Page 17: Nonsense-MediatedmRNADecayBeginsWhere Translation Ends€¦ · Nonsense-MediatedmRNADecayBeginsWhere Translation Ends Evangelos D. Karousis and Oliver Mühlemann Department of Chemistry

Shum EY, Jones SH, Shao A, Dumdie J, Krause MD, ChanWK, Lou CH, Espinoza JL, Song HW, Phan MH, et al.2016. The antagonistic gene paralogs Upf3a and Upf3bgovern nonsense-mediated RNA decay. Cell 165: 382–395.

Silva AL, Ribeiro P, Inacio A, Liebhaber SA, Romao L. 2008.Proximity of the poly(A)-binding protein to a prematuretermination codon inhibits mammalian nonsense-medi-ated mRNA decay. RNA 14: 563–576.

Simms CL, Thomas EN, Zaher HS. 2017. Ribosome-basedquality control of mRNA and nascent peptides. WileyInterdiscip Rev RNA doi: 10.1002/wrna.1366.

Singh G, Rebbapragada I, Lykke-Andersen J. 2008. A com-petition between stimulators and antagonists of Upf com-plex recruitment governs human nonsense-mediatedmRNA decay. PLoS Biol 6: e111.

SinghG, Kucukural A, CenikC, Leszyk JD, Shaffer SA,WengZ, Moore MJ. 2012. The cellular EJC interactome revealshigher-order mRNP structure and an EJC-SR proteinnexus. Cell 151: 750–764.

Stalder L, Mühlemann O. 2008. The meaning of nonsense.Trends Cell Biol 18: 315–321.

Stalder L, Mühlemann O. 2009. Processing bodies are notrequired for mammalian nonsense-mediated mRNA de-cay. RNA 15: 1265–1273.

Thermann R, Neu-Yilik G, Deters A, Frede U, Wehr K,Hagemeier C, Hentze MW, Kulozik AE. 1998. Binaryspecification of nonsense codons by splicing and cyto-plasmic translation. EMBO J 17: 3484–3494.

Tian M, Yang W, Zhang J, Dang H, Lu X, Fu C, Miao W.2017. Nonsense-mediated mRNA decay in Tetrahymenais EJC independent and requires a protozoa-specific nu-clease. Nucleic Acids Res 45: 6848–6863.

Toma KG, Rebbapragada I, Durand S, Lykke-Andersen J.2015. Identification of elements in human long 30UTRsthat inhibit nonsense-mediated decay. RNA 21: 887–897.

Trcek T, Sato H, Singer RH,Maquat LE. 2013. Temporal andspatial characterization of nonsense-mediatedmRNAde-cay. Genes Dev 27: 541–551.

Unterholzner L, Izaurralde E. 2004. SMG7 acts as a molec-ular link between mRNA surveillance and mRNA decay.Mol Cell 16: 587–596.

Wang W, Czaplinski K, Rao Y, Peltz SW. 2001. The role ofUpf proteins in modulating the translation read-throughof nonsense-containing transcripts. EMBO J 20: 880–890.

WangC,HanB, ZhouR, ZhuangX. 2016. Real-time imagingof translation on single mRNA transcripts in live cells.Cell 165: 990–1001.

Weil JE, Beemon KL. 2006. A 30UTR sequence stabilizestermination codons in the unspliced RNA of Rous sar-coma virus. RNA 12: 102–110.

Wells SE, Hillner PE, Vale RD, Sachs AB. 1998. Circulariza-tion of mRNA by eukaryotic translation initiation factors.Mol Cell 2: 135–140.

Weng Y, Czaplinski K, Peltz SW. 1998. ATP is a cofactor ofthe Upf1 protein that modulates its translation termina-tion and RNA binding activities. RNA 4: 205–214.

Yamashita A. 2013. Role of SMG-1-mediated Upf1 phos-phorylation in mammalian nonsense-mediated mRNAdecay. Genes Cells 18: 161–175.

Yamashita A, Ohnishi T, Kashima I, Taya Y, Ohno S. 2001.Human SMG-1, a novel phosphatidylinositol 3-kinase-related protein kinase, associates with components ofthe mRNA surveillance complex and is involved in theregulation of nonsense-mediated mRNA decay. GenesDev 15: 2215–2228.

Yamashita A, Izumi N, Kashima I, Ohnishi T, Saari B, Kat-suhata Y, Muramatsu R, Morita T, Iwamatsu A, HachiyaT, et al. 2009. SMG-8 and SMG-9, two novel subunits ofthe SMG-1 complex, regulate remodeling of the mRNAsurveillance complex during nonsense-mediated mRNAdecay. Genes Dev 23: 1091–1105.

YepiskoposyanH, Aeschimann F, NilssonD,OkoniewskiM,Mühlemann O. 2011. Autoregulation of the nonsense-mediated mRNA decay pathway in human cells. RNA17: 2108–2118.

Young SK, Wek RC. 2016. Upstream open reading framesdifferentially regulate gene-specific translation in the in-tegrated stress response. J Biol Chem 291: 16927–16935.

Zund D, Gruber AR, Zavolan M, Mühlemann O. 2013.Translation-dependent displacement of UPF1 from cod-ing sequences causes its enrichment in 30UTRs.Nat StructMol Biol 20: 936–943.

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