320

Nuclear RNA-binding proteins can record pre-mRNAprocessing events in the structure of messengerribonucleoprotein particles (mRNPs). During initial rounds oftranslation, the mature mRNP structure is established and ismonitored by mRNA surveillance systems. Competition for thecap structure links translation and subsequent mRNAdegradation, which may also involve multiple deadenylases.

AddressesWellcome Trust Centre for Cell Biology, ICMB, University of Edinburgh,Kings’ Buildings, Edinburgh EH9 3JR, UK*e-mail: pmitch@holyrood.ed.ac.uk†e-mail: D.Tollervey@ed.ac.uk

Current Opinion in Cell Biology 2001, 13:320–325

0955-0674/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.

AbbreviationsAREs AU-rich sequence elements CBC cap-binding complex DAN deadenylating nuclease DSEs downstream sequence elements hnRNP heterogeneous nuclear ribonucleoprotein mRNPs messenger ribonucleoprotein particles nt nucleotides PTCs premature translation termination codons STEs stabilizing elements uORFs upstream open reading frames UTR untranslated region

IntroductionmRNA turnover plays an important role in the regulation ofgene expression, affecting both the total amount of proteinthat can be synthesized from a given level of transcriptionand the time that translation continues. It has long beenappreciated that the structure of the messenger ribonucle-oprotein particle (mRNP) complex is likely to play animportant role in both translation and mRNA stability.Although the RNP structures remain poorly characterized,recent data have started to shed some light on theseprocesses. It was also clear that there is a close relationshipbetween the translational status of mRNAs and their ratesof degradation. Translation initiation factors, the decappingenzyme complex and a deadenylase have now been shownto compete for the cap structure, and multiple interactionsbetween these factors have been reported that may deter-mine the outcome of this competition. Finally, the activityin yeast that removes the poly(A) tail — the rate limitingstep in the degradation of most mRNAs — has long beensought and may now have been identified. We will attemptto provide an overview of these developments.

The burden of history: nuclear pre-mRNAprocessing imprints mRNAsRecent work has demonstrated that many diverse RNA-binding proteins associate with the nuclear pre-mRNA and

are cotransported to the cytoplasm with the mRNP. Theseproteins may preserve a record of the nuclear history of thepre-mRNA in the cytoplasmic mRNP structure. This infor-mation can strongly influence the cytoplasmic fate of themRNA and is used by mRNA surveillance systems that actas a checkpoint of mRNP integrity, particularly in the identi-fication of premature translation termination codons (PTCs).

Cotransport of nuclear mRNA-binding proteins with mRNAfrom the nucleus to the cytoplasm (nucleocytoplasmic shut-tling) was first observed for the heterogeneous nuclearribonucleoprotein (hnRNP) proteins. Some hnRNP proteinsare stripped from the mRNA at export [1], but hnRNP A1,A2, E, I and K are all exported (see [2]). Although roles forthese hnRNP proteins in transport and translation have beenreported [3•,4•], their affects on mRNA stability have beenlittle studied. More is known about hnRNP D/AUF1 andanother nuclear RNA-binding protein, HuR, which actantagonistically to modulate the stability of a range ofmRNAs containing AU-rich sequence elements (AREs)(reviewed in [2]). Several additional transcripts regulated bythese factors have been reported over the past year (see, forexample, [5,6]). A mutant form of hnRNP D that binds toAREs but does not localize to the nucleus fails to stimulatedegradation, indicating that association with the nuclear pre-mRNA is required for its effects on mRNA metabolism(A-B Shyu, personal communication).

A recently identified family of proteins that binds to HuR[7] may stimulate its interaction with nuclear AREs andpromote the nuclear export of the mRNP via CRM1. HuRexpression in trypanosomes stabilizes developmentally reg-ulated mRNAs containing AREs (L Quijada, C Hartmann,C Clayton, personal communication), suggesting that ARE-mediated degradation is conserved among eukaryotes.

The recognition of PTCs during mRNA surveillance inmammalian cells is dependent upon pre-mRNA splicing.Introns are rarely found in the 3′ untranslated regions(3′UTRs) of transcripts, and an exon junction (the positionof a former intron) positioned more than about 50nucleotides (nt) downstream from the translation termina-tion codon causes this to be read as premature terminationsite, triggering rapid mRNA degradation. Thus, for exonjunctions to be recognized during translation, splicing mustmark them in some way [8]. A good candidate marker is thelarge (~335 kDa) (also known as the 20–24 complex) thatassociates with the mRNA 20 to 24nt upstream from thesplice junction, probably at a late stage during the splicingreaction [9••,10•,11••,12,13•]. This complex includes thesplicing factors SRm160, DEK and RNPS1, the shuttlingprotein Y14 and the mRNA export factor REF/Aly. It is notclear whether all these proteins accompany the mRNP intothe cytoplasm, but both Y14 and REF/Aly are exported.

mRNA turnoverPhilip Mitchell* and David Tollervey†

mRNA turnover Mitchell and Tollervey 321

Pre-mRNA splicing requires other shuttling proteins,including the U2AF35–U2AF65 heterodimer and membersof the family of SR-proteins [14,15]. SR-proteins bind tosites, including exonic splicing enhancer (ESE) sequences,located close to the splice site. ‘Fail-safe’ sequences locat-ed around splice sites can trigger mRNA surveillance even

when the intron is deleted [15,16]. SR-proteins and otherfactors bound to nearby ESEs are good candidates for thisfail-safe signal.

Analyses in yeast have identified three proteins —Upf1p, Upf2p and Upf3p — that play a key role in

Figure 1

80S

PTC mRNA

80S80S

Wild-type mRNA

Nucleocytoplasmic export

Pre-mRNA splicing

Translation initiation

Translation elongation

Current Opinion in Cell Biology

Replacement of CBC by eIF4ETranslation reinitationmRNP circularisation

mRNA surveillance detectsdownstream exon junctionmarker

eIF4G

eIF4G eIF4G

eIF4G

eIF4G

Mark Mark Mark

Mark Mark Mark

AAAAA

AAA

A

AUG

AUG

AUG

AUG

AUG

UAA

AAAAAAAAA

AAAAAAAAA

SCUAA

RF SC

SC

UAARF

AUG

SC

Decappingcomplex

AAAAAA

AAA

Decapping and degradation

PABP

PABP

AAAAAAAAAPABP

AAAAAAAAA

AAAAAAAAA

AAAAAAAAAPABP

PABP

PABP

PABP

eIF4G

CBCm7G

CBCm7G

CBCm7G

4Em7G

CBCm7G

CBCm7G

CBCm7G

m7G

(a)

(b)

(c)

(d) (e)

Models for mRNA surveillance. (a) Nuclear assembly and processinggenerates complex RNPs. Exon junction markers are indicated.(b) Upon export from the nucleus, eIF4G binds to the CBC andtranslation is initiated. (c) During elongation, marker proteins locatedwithin the ORF are dissociated. Upon translation termination, Upf1p isrecruited by the release factors (RF) and a surveillance complex (SC)that includes Upf1p then translocates through the 3′ UTR. (d) If only

correct marker proteins are encountered the mature mRNP structure isformed. eIF4E replaces CBC and the interaction between PABP andeIF4G circularizes the mRNA. (e) Interaction of the surveillancecomplex with inappropriate proteins triggers decapping and rapiddegradation. Upf2 and 3p may assemble with Upf1p before scanningor only after scanning is inhibited at the mark.

322 Nucleus and gene expression

mRNA surveillance. Functional homologues have recent-ly been characterized in humans (hUpf1p/RENT,hUpf2p and hUpf3p) and Caenorhabditis elegans (smg-2,smg-3 and smg-4) [17••,18,19•]. Upf1p is more abundantthan Upf2p and Upf3p and functions directly in the ter-mination of translation [20]. Unlike yeast, mammalianUpf1p appears to be essential for the viability of bothembryos and isolated blastocysts [21].

The three Upf proteins copurify from extracts of yeast andhuman cell extracts, and the yeast mutants have identicalphenotypes in mRNA surveillance, indicating that theyfunction as a complex. Consistent with this, tethering anyof the hUpf proteins within the 3′ UTR of wild-type β-glo-bin mRNA, more than 50nt beyond the termination codon,triggered mRNA surveillance [17••]. Surprisingly, thehuman Upf proteins did not colocalize using immunofluo-rescence [17••,19••]. hUpf1p is cytoplasmic, whereas hUpf2is restricted to a region closely surrounding the nucleus.hUpf3p is a nucleocytoplasmic-shuttling protein that asso-ciates with nuclear mRNA in a splicing-dependent manner[17••,] and is therefore another candidate marker for exonjunctions. The Upf complex that mediates mRNA surveil-lance is likely to be present at the overlap of thesedistributions, in the perinuclear region. Here, hUpf2p mayinteract with hUpf3p bound to newly exported mRNP andwith hUpf1p recruited to the mRNA by the translationrelease factors. This newly synthesized mRNA may remainclose to, or attached to, the nuclear pores or associated fib-rils, providing a possible explanation for the reportednuclear association of mRNA surveillance mechanisms [8].

The mRNP structure also provides signals for other surveillance checkpoints. PTC-containing mammalianmRNAs that escape initial mRNA surveillance are degrad-ed inefficiently on cytoplasmic polysomes by a mechanismthat also recognizes marks deposited in the nucleus [22].In addition, aberrant mRNA precursors are degraded bydistinct pathways within the nucleus [23•,24].

Alternative marks to splicing in mRNP assemblyMost yeast genes lack introns, and recognition of PTCsdepends on poorly conserved downstream sequence elements (DSEs). mRNA destabilization by a DSE in thePGK1 gene is mediated by binding of Hrp1p/Nab4p, anucleocytoplasmic shuttling protein with homology tohnRNP D [25••]. Hrp1p interacts with Upf1p, and thisinteraction may be involved in triggering rapid degrada-tion. Hrp1p also binds to pre-mRNA polyadenylation sites[26], and incorrect positioning of polyadenylation triggersmRNA surveillance in yeast, but not in a mammalian system [27,28]. The role of Hrp1p in the recognition ofincorrect polyadenylation has not been reported.

Many eukaryotic mRNAs have small, regulatory, upstreamopen reading frames (uORFs) 5′ to the main protein-coding ORF. The uORFs in yeast GCN4 and YAP1 reducetranslation but do not elicit RNA degradation, owing to the

presence of stabilizing elements (STEs) located betweenthe uORF and the major coding region [29,30]. TheseSTEs bind to Pub1p [31••], an HuR-related protein thatpresumably antagonizes DSE/Hrp1p-mediated decappingand degradation. Pub1p is a major nuclear RNA-bindingprotein and is likely to be transported to the cytoplasmwith the mRNA. Even in human mRNAs, not all markersarise through splicing. An hnRNP complex bound withinthe c-fos ORF inhibits deadenylation until displaced bytranslation [32•].

Never like the first time: a privileged initialround of translationNewly exported mRNAs have an RNP structure distinctfrom the ‘mature’ mRNP and may undergo an initial roundof translation that is substantially different from the sub-sequent translation. During this initial round, the7-methylguanosine cap carries the nuclear cap-bindingcomplex (CBC), a Cbp20p–Cbp80p dimer, which may beexported with the mRNA [33•]. Furthermore, the mRNAis decorated by many protein complexes marking splicesites and other sites of hnRNP assembly. CBC interactswith the translation initiation factor eIF4G and can promote initiation of translation [33•]. During the initialtranslation, the mRNP structure is substantially remod-eled; nuclear proteins bound within the ORF are displacedand rapidly reimported, allowing the major protein signalswithin the mature mRNP to be discerned by the transla-tion and degradation machinery.

Upf1p is a member of an ATPase superfamily referred to ashelicases, although analysis of the DNA helicase PcrA suggests that the conserved domain functions primarily as atranslocase [34]. Following the first round of translation,Upf1p associates with the translation release factors, and itmay ‘walk’ 5′ to 3′ along the mRNA, scanning for the pres-ence of bound hUfp3p, markers of exon junctions and othercomplexes that have not been displaced by the translatingribosome. The Upf complex provokes mRNA decappingand degradation (see Figure 1), possibly via an interactionbetween hUpf2p and initiation factors [18]. It is probablethat not all 3′ UTRs are scanned during the subsequenttranslation, and the RNP context of the termination codonis proposed to influence this decision [35].

The CBC–eIF4G interaction is weakened by binding ofthe cytoplasmic cap-binding protein, eIF4E, to eIF4G.This may promote the exchange of CBC for eIF4E follow-ing or during the initial round of translation. Theinteraction between eIF4E–eIF4G and the poly(A) tailbinding protein (Pab1p in yeast, PABP in mammals) willthen circularize the now mature mRNP (see Figure 1).

Coupling translation and mRNA turnovereIF4E has much lower intrinsic affinity for the cap structure than does the decapping enzyme Dcp1p [36•].However, the association of eIF4E with the cap is greatlystabilized by its interaction with eIF4G and by binding of

mRNA turnover Mitchell and Tollervey 323

eIF4G to Pab1p [37,38•]. Co-overexpression of botheIF4E and the interacting domain from eIF4G increasesmRNA stability [38•]. Conversely, an eIF4E mutationincreased decapping by a defective Dcp1p mutant [36•].These results indicate direct competition for the capbetween Dcp1p and eIF4E bound to eIF4G. However,Dcp1p also binds directly to both eIF4G and Pab1p andcan modulate the interactions of eIF4E and eIF4G withthe mRNA [38•]. Binding of eIF4G with Dcp1p enhancesdecapping in vitro, and this effect is negated by eIF4E.Decapping rates may therefore reflect competitionbetween Dcp1p and eIF4E, both of which bind to theeIF4G-Pab1p complex. A testable prediction of this modelis that of eIF4G mutants would be defective in decappingowing to loss of Dcp1p binding.

A complex set of additional signals can also regulatedecapping activity. The in vivo decapping complex alsocontains the decapping activator Dcp2p and two homolo-gous proteins, Edc1p and Edc2p [39,40], which stimulatedecapping under suboptimal conditions. Dcp1p is phos-phorylated [41] and the phosphorylation state potentiallyregulates its activity. Furthermore, several factors requiredfor efficient decapping have been identified, includingMrt3p and Vsp16p (see [42]). Decapping is also inhibitedby mutations in a complex comprising seven Sm-like(Lsm) proteins, Lsm1–7p, and Pat1p/Mrt1p [43•,44••].Pat1p is required for stable association of the 40S riboso-mal subunit with the mRNP during initiation of translation[45], whereas the Lsm proteins probably facilitate alter-ations in RNP structure [46]. However, unlike the Dcp1pmutation, decapping defects owing to mutations in theLsm–Pat1 complex are not suppressed by an eIF4E muta-tion [36•]. Alterations in mRNP structure during initiationof translation by the Lsm–Pat1 complex may thereforerender the cap accessible to both the translation anddecapping machinery. Finally, the rapid decappingobserved for transcripts subject to mRNA surveillance isnot sensitive to mutations in Lsm–Pat1 or the decappingcofactors Mrt3p and Vsp16p (see [42]); whether this pathway acts through eIF4E has not been determined.

Mutants defective in the eIF4E–eIF4G complex or ineIF3, which facilitates 40S binding to the mRNP, showincreased rates of both deadenylation and decapping,whereas strains defective in translation elongation showreduced rates of deadenylation and decapping (see [42]).This difference in mRNA stability may reflect a ‘windowof opportunity’ for mRNA degradation during the initia-tion of translation.

Dcp1p interacts with both the cap and part of the RNA[41], and this property was exploited to unmask a decap-ping activity in HeLa cell extracts by competing outinhibitory cap-binding proteins with a cap analogue [47•].The in vitro decapping activity was stimulated by an ARE,suggesting that ARE-binding proteins can promote accessof the decapping machinery to the cap structure. This

mechanism may allow the translation-independentturnover that has been reported for some mRNAs, as wellas the observed destabilization of snRNAs by AREs [48],presumably during their transit through the cytoplasm.

The deadenylating nuclease (DAN) is the major deadeny-lase activity in HeLa cell extracts. DAN is a cap-bindingprotein, and its in vitro deadenylase activity is stimulated bythe presence of a cap [49•,50•,51]. DAN activity is inhibit-ed by eIF4E or cap analogues, suggesting that it may alsocompete with eIF4E and Dcp1p for the mRNA cap. DANis not detectably associated with polysomes [49•], and itsdeadenylase activity is inhibited by Pab1p, presumablyowing to competition for binding to the poly(A) tail.

Recent analyses identified the Ccr4p–Caf1p andPan2p–Pan3p complexes as major yeast deadenylases as[52••]. Proteins with homology to DAN are present in manyeukaryotes (although not Saccharomyces cerevisiae), and theCcr4p–Caf1p and Pan2p–Pan3p complexes are highly conserved. Eukaryotic cells therefore contain multiple deadenylase activities that may target different mRNP substrates, perhaps in response to specific regulatory signals.Yeast Puf3p and Mpt5p bind to the 3’ UTRs of the COX17and HO mRNAs, respectively, and Puf3p stimulates dead-enylation [53•,54•]. Puf3p and Mpt5p are members of thePUF family of 3′ UTR-binding proteins that regulate mRNAtranslation and poly(A) status during early development (see[55]). It is very likely that further transcript-specific regulatorsof mRNA turnover will be found.

ConclusionsPre-mRNAs associate with a wide range of nuclear RNA-binding proteins to yield complex, and at least partlymRNA-specific, RNP structures. Some of this structure ispreserved in the cytoplasmic mRNP where it can be ‘read’by RNA surveillance systems and probably by transportand translation factors. Given the heterogeneity and lowabundance of individual mRNPs, understanding this indetail will be a substantial challenge. Recent work hasrevealed a complex interplay between the factors involvedin mRNA turnover and translation. The challenge is nowto understand how these interactions are integrated in vivo.The identification of yeast deadenylases and regulatoryproteins offers the opportunity to understand how mRNA-specific deadenylation rates are established, a keyparameter in mRNA turnover.

AcknowledgementsWe would like to thank Christine Clayton, Lynne Maquat, Roy Parker, Ann-Bin Shyu and Jeff Wilusz for sharing data prior to publication. This workwas supported by the Wellcome Trust.

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45. Wyers F, Minet M, Dufour ME, Vo LTA, Lacroute F: Deletion of thePAT1 gene affects translation initiation and suppresses a PAB1gene deletion in yeast. Mol Cell Biol 2000, 20:3538-3549.

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degradation rates in vitro. Mol Cell 2000, 5:479-488.The major deadenylase activity in HeLa extracts is identified asDAN/PARN. DAN is a cap-binding protein, and this interaction is importantfor deadenylation activity.

51. Martínez J, Ren Y-G, Thuresson A-C, Hellman U, Åström J, Virtanen A:A 54-kDa fragment of the poly(A)-specific ribonuclease is anoligomeric, processive, and cap-interacting poly(A)-specific 3′′exonuclease. J Biol Chem 2000, 275:24222-24230.

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Caf1p, are components of the major cytoplasmic mRNAdeadenylase in Saccharomyces cerevisiae. Cell 2001, in press.

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53. Olivas W, Parker R: The Puf3 protein is a transcript-specific• regulator of mRNA degradation in yeast. EMBO J 2000,

19:6602-6611.Yeast has five members of the PUF protein family, which includes the translational regulators Pumilio and FBF. Puf3p promotes mRNA-specific deadenylation of COX17 mRNA through binding to the 3′ UTR.

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Pumilio and FBF. EMBO J 2001, 20:552-556Another yeast PUF protein, Mpt5p, acts post-transcriptionally to reduce the abundance of the HO mRNA. Mpt5p binds to the 3′ UTR at a site homologous to the binding sites of Pumilio and FBF.

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