Modulation of Eukaryotic mRNA Stability via the Cap-binding Translation Complex eIF4F

Modulation of Eukaryotic mRNA Stability via theCap-binding Translation Complex eIF4F

Carmen Velasco Ramirez†, Cristina Vilela†, Karine Berthelot andJohn E. G. McCarthy*

Posttranscriptional ControlGroup, Department ofBiomolecular SciencesUniversity of ManchesterInstitute of Science andTechnology (UMIST), P.O. Box88, Manchester M60 1QD, UK

Decapping by Dcp1 in Saccharomyces cerevisiae is a key step in mRNAdegradation. However, the cap also binds the eukaryotic initiation factor(eIF) complex 4F and its associated proteins. Characterisation of therelationship between decapping and interactions involving eIF4F is anessential step towards understanding polysome disassembly and mRNAdecay. Three types of observation suggest how changes in the functionalstatus of eIF4F modulate mRNA stability in vivo. First, partial disruptionof the interaction between eIF4E and eIF4G, caused by mutations ineIF4E or the presence of the yeast 4E-binding protein p20, stabilisedmRNAs. The interactions of eIF4G and p20 with eIF4E may therefore actto modulate the decapping process. Since we also show that the in vitrodecapping rate is not directly affected by the nature of the body of themRNA, this suggests that changes in eIF4F structure could play a role intriggering decapping during mRNA decay. Second, these effects wereseen in the absence of extreme changes in global translation rates in thecell, and are therefore relevant to normal mRNA turnover. Third, atruncated form of eIF4E (D196) had a reduced capacity to inhibit Dcp1-mediated decapping in vitro, yet did not change cellular mRNA half-lives. Thus, the accessibility of the cap to Dcp1 in vivo is not simplycontrolled by competition with eIF4E, but is subject to switching betweenmolecular states with different levels of access.

q 2002 Elsevier Science Ltd. All rights reserved

Keywords: RNA–protein interactions; translation initiation; mRNAstability; eukaryotic initiation factor 4F; decapping enzyme Dcp1*Corresponding author

Introduction

For the eukaryotic translation apparatus, themRNA 50 cap is a recognition element that servesas a focal point for the assembly of initiationfactors (eIFs) and ribosomes. For the cellularmRNA degradation machinery, in contrast, thecap represents a barrier to 50 ! 30 exonucleaseactivity, and thus exerts a stabilising influence.Thus, removal of the cap (otherwise known asdecapping) is a critical event in the lifetime of anmRNA, since it both deactivates cap-dependenttranslation initiation and facilitates the decayprocess. The question addressed here is how thecell controls the interplay between the translation-promoting and degradative events at the cap.

The pathway of eukaryotic mRNA degradationseems to vary at the level of individual mRNAspecies, while at the level of the total transcriptomeorganisms may differ in the predominance of onepathway over another.1 The current model for themajor pathway in yeast2 envisages that progressivedeadenylation at the 30 end eventually triggersdecapping by virtue of an as yet unknownmechanism of coupling between events occurringat the 30 and 50 ends of the mRNA. In Saccharomycescerevisiae, decapping involves Dcp1, which in vitrocleaves capped mRNA to yield m7GDP and amonophosphate 50 end.3 –6 This cleavage rendersthe mRNA susceptible to 50 ! 30 exonucleolyticdegradation by the cytoplasmic exonucleaseXrn1.3,4 However, experiments in vitro haverevealed that access of Dcp1 to the cap is blockedby the cap-binding translation factor eIF4E aloneas well as by the cap-binding complex eIF4F.7

Schwartz & Parker8 also reported inhibition ofDcp1 by eIF4E in vitro, and observed that a

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

† C.V.R. and C.V. contributed equally to this work.

E-mail address of the corresponding author:[email protected]

Abbreviation used: eIF, eukaryotic initiation factor.

doi: 10.1016/S0022-2836(02)00162-6 available online at http://www.idealibrary.com onBw

J. Mol. Biol. (2002) 318, 951–962

decapping defect associated with a dcp1 mutation(dcp1-1) can be suppressed by a cdc33 mutationthat reduces eIF4E cap-binding affinity. Theseresults indicate that information about the relation-ship between Dcp1 and eIF4G components will becritical to our understanding of the control ofdecapping.

eIF4F comprises the cap-binding protein eIF4E(,25 kDa),9 a much larger protein, eIF4G, and theDEAD box (helicase) protein eIF4A.10 S. cerevisiaehas two versions of eIF4G, called eIF4G1 andeIF4G2 (107 kDa and 104 kDa, respectively11). Theassociation between eIF4G and eIF4A is less stablein yeast than it is in mammalian cells, but it is func-tionally significant in both organisms.12,13 eIF4Gmay act as a type of scaffolding protein, in that ithas binding sites for other translation-relatedfactors,14 – 18 including eIF4A, eIF4E, eIF3 andpoly(A) binding protein (Pab1). Moreover, yeasteIF4G binds to, and is capable of enhancing theactivity of, the decapping protein Dcp1.7

Some recent experiments using various trans-lation factor mutants have been pertinent to therole of eIF4F function in mRNA decay.19,20 Linzand colleagues20 found that the presence of par-tially defective mutant forms of eIF4E had no sig-nificant impact on mRNA decay rates. In contrast,Schwartz & Parker19 investigated a group ofmutants with strong phenotypes that suppressglobal cellular translation rates to as little as 3% ofthe wild-type level. As well as drastically reducingtranslation, these mutations caused differentdegrees of destabilisation of the mRNA speciesexamined; the MFA2 message was destabilised byup to twofold, while the PGK1 mRNA, whosedecay rate is atypically sensitive to changes intranslation rate,20 was destabilised up to fourfold.19

This work therefore demonstrated that mRNAdecay can be accelerated under conditions ofsevere translational suppression. However, it isnot known to what extent this correlation reflectsthe relationship between translation and decayprocesses on individual mRNAs during theirnormal lifetimes in wild-type cells.

The observation that eIF4G can interact bothphysically and functionally with Dcp1 suggestsone possible means by which the control of trans-lation and mRNA turnover might be coordinated.While eIF4G is potentially a recruitment factor forDcp1, the activation effect of this translation factoris blocked in the presence of eIF4E.7 This consti-tutes a striking parallel to the earlier observationthat, at least in vitro, eIF4G binding to eIF4Estabilises the eIF4E–cap interaction.21 The stabilis-ation appears to be attributable to a cooperativityeffect coupled to protein ligand binding at a siteon the dorsal face of eIF4E.21 – 23 The dorsal bindingsite for eIF4G is partially shared by the yeasteIF4E binding protein (4E-BP) called p20.21

Other proteins in the yeast cell are also thoughtto be capable of modulating Dcp1 activity. TheDCP2 gene was identified as a multi-copy sup-pressor of both a nuclear petite mutant (which

gave rise to the alternative designation PSU1) andthe growth defect of a dcp1Dski8D strain.24 One ofthe functions of Dcp2 therefore seems to be to pro-mote decapping via an as yet undefined, indirectmechanism. Decapping is also affected bymutations in MRT1, MRT3,25 SPB8,26 VPS1627 andEDC1/EDC2,28 although the mechanisms under-lying the modulatory effects of the encodedproteins are unknown. Two-hybrid screening hasidentified other potentially significant interactionswith Dcp1, including some that are involved inRNA splicing.29 Indeed, the so-called Sm-likeproteins (Lsm1-7) co-immunoprecipitate withDcp1, and lsm mutations accumulate oligo-adenylated, full-length mRNAs.30,31 Many of theseproteins may participate in networks and com-plexes that include Dcp1, and hence may influencedecapping indirectly. There is also the potentialinfluence of the poly(A)-binding protein (Pab1),which is required for stabilisation of mRNAs.32 Itis possible, but not known, that Pab1 modulatesDcp1 activity via other proteins, such as eIF4G.

Overall, the interactions at the interface betweentranslation and mRNA stability have yet to becharacterised. A key objective is to understand thecontrol of cap–protein interactions. On the otherhand, recognition of the dual capacity of eIF4G toact as a modulator of both eIF4E and Dcp1, atleast in vitro, has suggested a new avenue ofinvestigation. This study accordingly examinesboth aspects of the role of eIF4F in modulatingmRNA stability.

Results

Rate determination at the point of decapping

It is known that the step of decapping can beactivated at different times during the lifetime ofan mRNA. However, at the outset of this work ithad not been established whether the kinetics ofthe Dcp1-catalysed decapping reaction might be apotential source of differences in the rate ofmRNA decay. While the time-point at whichdecapping is triggered in vivo is linked by anunknown mechanism to deadenylation, thedecapping step itself might be subject to modu-lation. Previous investigations of the temporalrelationship between deadenylation and decap-ping do not rule out such potential differences indecapping rate.2 The ease with which variations indecapping rate in vivo can be detected depends onboth the resolution of the gel system in use andthe kinetics of the overall decay process. Onepotential basis for variation in decapping ratewould be that the mRNA-binding properties ofDcp1, which are likely to influence decappingrates, are modulated by mRNA structure. Thiscould mean, in turn, that the relationship betweenDcp1 activity and eIF4F might depend on themRNA species with which they are associated.Since we intended to study the influence of eIF4F

952 Modulation of Eukaryotic mRNA Stability via eIF4F

components on decapping and the decay of a sub-set of mRNAs, it was important that we establishwhether mRNA-specific factors play a role indetermining Dcp1 activity.

We therefore examined the rates of decapping invitro of two natural yeast mRNAs with different invivo half-lives. The results of these experimentswere expected to tell us about the direct relation-ship between mRNA structure and Dcp1-depen-

dent decapping. Moreover, we reasoned that anydifferences in the interactions between Dcp1 anddistinct mRNA species might manifest themselvesin changes in the ability of eIF4E to block the dec-apping reaction, and also designed experiments totest this possibility. The mRNA species studiedbelonged to the group investigated throughoutthis paper, and thus these particular assays alsoserved as direct controls for the decapping exper-iments described later in this work.

Decapping assays were performed using activeFLAG-Dcp1 purified from Escherichia coli. ThemRNAs used were YAP1 and PGK1, which in vivohave half-lives of approximately seven minutesand 35 minutes, respectively. The genes weretranscribed in vitro to yield full-length, cappedmRNAs. The results show that decapping over the60 minute period for the YAP1 and PGK1 mRNAsvaried by maximally 30% (Figure 1(a) and (b)). Wealso studied a version of the PGK1 mRNA contain-ing a poly(G) sequence in the 50 UTR. The presenceof this poly(G) tract has been found both to inhibittranslation of, and to destabilise (at least fivefold),PGK1 mRNA in vivo.33 The poly(G)-containingPGK1 mRNA was also decapped at the same ratein the in vitro assay. In conclusion, we found noevidence that mRNA structure, in itself, can signifi-cantly influence the rate of Dcp1-mediated decap-ping. Moreover, eIF4E blocked decapping of therespective mRNAs equally well (Figure 1(c)).

Dcp1 and translation initiation

Since translation is known to affect mRNAstability, one potential (indirect) route for Dcp1 toinfluence the course of mRNA decay could involveinteractions in which this protein affects the trans-lation apparatus. An earlier study of eIF4F/Dcp1–mRNA interactions using cross-linking hadsuggested that Dcp1 might affect eIF4Fconformation.7 In order to test this possibility, weexamined the relationship between Dcp1 andtranslation in vivo and in vitro. As shownpreviously,25 growth in a dcp1-1 strain is severelyimpaired. Examining this more closely, weobserved that the absence of Dcp1 causes anextended lag phase upon transfer of colonies toliquid medium as well as a slower log growthphase (Figure 2(a)). We performed polysomalgradient analysis on extracts prepared from logphase cultures. This revealed an abnormal profilein which the presence of pronounced half-mers sig-nifies serious impairment of the translation process(Figure 2(b)). There is also a marked 60 Sdeficiency. We do not know the reason for thisdeficiency, but one possible reason could be linkedto a ribosome assembly defect. This complexphenotype was suppressed by expression of wild-type DCP1 in the dcp1-1 strain (Figure 2(c)).

We then examined the effect of Dcp1 on trans-lation in a cell-free assay system. Cell-free extractswere prepared from both the Dcp1-containingstrain yRP841 and from the Dcp1-deficient strain

Figure 1. Decapping of different mRNAs in vitro. Anal-ysis of decapping activity by FLAG-Dcp1 on three differ-ent mRNAs: PGK1 (PGK1), PGK1 containing a poly(G)element in the leader sequence (PGK1pG ) and YAP1(YAP1). Dcp1-catalysed decapping activity on the differ-ent mRNAs does not show significant differences. Dec-apping by FLAG-Dcp1 was examined over a one hourtime course, with aliquots of the decapping reactionsremoved at the time points (in minutes) indicated beloweach lane after addition of the protein. The products ofthe reaction were separated by PEI-cellulose TLC; equalamounts of material were loaded on each lane. (a) Arepresentative TLC plate showing the origins. C rep-resents a sample of the mRNA incubated for 60 minutesat 30 8C without addition of FLAG-Dcp1. (b) Quanti-tation of the decapping activity in the time course exper-iment shown in (a) for each mRNA. The values given areaverages of at least three independent experiments. Dec-apping is, in all cases, strongly inhibited by the presenceof eIF4E (4E; (c)). Arrows on the respective TLC platesindicate the positions of m7GDP generated by the decap-ping reaction.

Modulation of Eukaryotic mRNA Stability via eIF4F 953

yRP1069. This strategy yields information aboutthe translation of mRNA molecules of knownstructure. Moreover, the experiments can be per-formed in the absence of the pool of capped,deadenylated mRNAs that accumulate in the cellsof a dcp1D strain. We observed that the addition ofpurified FLAG-Dcp1 exerted no significant effecton the translation of a luciferase reporter mRNAin the cell-free system (data not shown). The sumof the above in vivo and in vitro data suggests thatthe translation defect in a dcp1-deficient strain isnot directly attributable to the loss of specific inter-actions between Dcp1 and the translationapparatus. It is more likely to be caused by factorsincluding competitive inhibition of cap-dependent

translation by an excess of capped but deadenyl-ated mRNAs and deficiencies in ribosome 60 Ssubunits.

Modulation of eIF4F function

An important potential source of modulation ofdecapping rate is the interplay between cap-associated translation factors and Dcp1. It wasdemonstrated that while eIF4E blocks the enhance-ment of Dcp1 activity by eIF4G in vitro, decappingcan be enhanced by eIF4G in the absence ofeIF4E.7 We asked the question whether mutationsor ligands that interfere with the ability of eIF4Eto interact with eIF4G, and thus perturb the normalprocess of eIF4F assembly, affect the control ofDcp1 activity. We were aware of the possibilitythat mutations that strongly compromise the func-tion of individual eIFs could exert multiple indirecteffects on mRNA turnover by virtue of their drasticimpairment of translation. Indeed, in initial experi-ments with a haploid yeast strain in which trans-lation is dependent on cdc33E72D, we observedlarge decreases in the levels of cellular mRNAs atthe non-permissive temperature with increasingtime of incubation subsequent to the temperatureshift (data not shown). The cells rapidly ceasedgrowing, rendering analysis of the mRNA levelsof dubious value.

We therefore chose to avoid experiments inhaploid cdc33 mutant backgrounds with strongphenotypes. Instead, we introduced mutant geneson expression plasmids into wild-type host cells.This allowed us to modify the behaviour of theeIF4F components in a more stable system. Weinvestigated the effects on translation of twoproteins that bind to the dorsal binding site ofS. cerevisiae eIF4E: p20 and the eIF4E-bindingdomain of eIF4G1.21 We chose p20 as a proteinligand that is known to bind to eIF4E34 at the dorsalface21 and is therefore capable of competing witheIF4G. The eIF4E-binding domain of eIF4G1 isreferred to in this paper as 4G-BD4E. It lacks anybinding sites other than that for eIF4E, and there-fore can only form an inhibitory “dead-end” com-plex with this factor.

The mutant version of eIF4E (E72D) was co-synthesised together with the above proteinligands. The E72D mutation was shown previouslyto cause a recessive temperature-sensitive pheno-type in which eIF4E binding to eIF4G (4G-BD4E),but apparently not to p20, is weakened.21 In thepresent context, it was important to demonstratethat the E72D protein retains the p20-bindingproperties of wild-type eIF4E and still allows p20to exert a regulatory influence on eIF4F function.We therefore prepared a cell-free extract from ahaploid yeast strain in which translation is depen-dent on the cdc33 allele encoding this mutant ver-sion of eIF4E. Using this extract, we were able toconfirm that the inhibitory potential of p20 isenhanced for the mutant form of eIF4E relative tothe wild-type eIF4E extract (Figure 3(f)). A mutant

Figure 2. A mutation in dcp1 impairs growth and pre-vents normal translation. The dcp1-1 mutation stronglyimpairs growth in vivo (a). The mutation dcp1-1 also hasdrastic consequences for translation in vivo, as mani-fested by the polysome profiles distinguishable uponsucrose gradient analysis (b). Expression of the YCpSU-PEX2-DCP1 plasmid containing the wild-type DCP1gene suppresses the slow growth phenotype (a) and thepolysome defects (c). The positions of the 40 S, 60 S, and80 S peaks are indicated in (b) and (c). The arrows showthe half-mers present in (b).


form of p20 in which the binding motif for eIF4Ewas deleted (Dmp20) did not inhibit translation ineither of the cell-free extracts tested (data notshown). In further work, we observed that CAF20over-expression caused synthetic lethality in a hap-loid strain dependent on cdc33E72D for translation(M. Ptushkina & J.E.G.M., unpublished data).These results suggest that enhanced complex for-mation between p20 and the E72D form of eIF4Eallows the putative regulatory protein to exert amarked inhibitory effect on posttranscriptionalgene expression.

Polysome profiles were prepared for the wild-type strain JDSþS transformed with expressionplasmids bearing CDC33 alleles and/or genesencoding eIF4E binding partners (Figure 3). Weused these profiles to monitor the overall status ofthe translation system, thus demonstrating that itwas not seriously compromised in any of the

expression experiments. The synthesis ofeIF4EE72D from a galactose-inducible expressionvector caused a small shift in the polysome profilein favour of the 80 S peak (Figure 3(c)) and nodetectable change in growth rate (Table 1). Thesimultaneous overproduction of p20 together witheIF4EE72D increased the shift to monosomes,while there were minor effects on growth andtranslation initiation/polysome structure (Figure3(d) and Table 1). In this case, the complex of p20and the E72D mutant version of eIF4E would beexpected to act as a competitive inhibitor of eIF4F.We also found that the combination of eIF4EE72Dand 4G-BD4E gave a similar level of inhibition(Figure 3(e) and Table 1). Here, we would expectthe observed inhibitory effect to be attributable tothe combination of interference by eIF4EE72D,binding the cap either alone or in combinationwith (chromosomally encoded) p20, and of the

Table 1. Average polysome sizes and growth rates for transformants of strain JDSþS

Strainsa Ratio (%) polysomes:monosomesb Doubling times (hour)

– 100 4.2 ^ 0.10eIF4E 94 4.1 ^ 0.07eIF4EE72D 72 4.2 ^ 0.03eIF4EE72D þ p20 42 5.2 ^ 0.07eIF4EE72D þ 4G-BD4E 58 5.2 ^ 0.02

a This indicates which proteins were encoded by expression plasmids in the transformed derivatives of JDSþS.b The ratio of the areas of polysomal fractions to monosomal fractions in the respective gradients.

Figure 3. Effects of cdc33 dorsal site mutations and eIF4E ligands on polysome profiles and in vitro translation. Poly-some profiles are shown for the wild-type strain JDSþS containing no expression plasmid (a), or containing YCpSU-PEX2 expressing wild-type CDC33 (b) or cdc33E72D (c). Further profiles are shown for the same strain co-expressingpFL39-cdc33E72D and YCpSUPEX-CAF20 (p20; (d)) or pFL39-cdc33E72D and YCpSUPEX-4G-BD4E (e). The positionsof the 40 S, 60 S and 80 S peaks are indicated in (a). (f) The inhibitory effect of p20 on translation in vitro. Addition ofp20 to cell-free extracts prepared from haploid strains expressing either wild-type CDC33 (X) or the mutant formE72D (W) (see Methods) resulted in inhibition of luciferase mRNA translation.


blocking effect of 4G-BD4E, which will bind to (chro-mosomally encoded) wild-type eIF4E in the cell.

Mutations in eIF4E alter Dcp1 decappingactivity in vivo

Parallel experiments were performed to deter-mine the effects of the mutations and ligands ofeIF4E on mRNA stability in vivo (Figure 4). Half-life measurements were performed on threemRNAs that are representative of the full breadthof stabilities seen in S. cerevisiae. We observed astabilisation effect when the E72D mutant form ofeIF4E was synthesised in vivo (Figure 4). This resultis consistent with a model in which eIF4G pro-motes the efficient decapping of yeast mRNAs,7

since on this basis disruption of the eIF4E–eIF4Ginteraction would be expected to interfere withDcp1 recruitment, and thus decapping. In a controlexperiment, synthesis of the W75R derivative ofeIF4E21 had no effect on the stability of the threemRNAs (data not shown). The W75R mutationweakens binding to both eIF4G and to p20, andwould therefore not be expected to participate inany form of stable cap-binding complex. This, inturn, means that eIF4EW75R will not competeeffectively with wild-type eIF4E for assembly intothe cap-binding complex. The co-expression of thegenes encoding eIF4EE72D and 4G-BD4E resultedin more marked stabilisation of the test mRNAs(Figure 4). Earlier work had already shown thatthe coordinated synthesis of 4G-BD4E and wild-type eIF4E in the strain JDSþS causes a markedstabilisation.7

p20 can modulate mRNA stability

In previous work, it has not been possible toidentify a specific function for p20 in vivo.35 It istherefore particularly notable that co-productionof eIF4EE72D and p20 resulted in stabilisation ofthe tested mRNAs (Figure 4). Indeed, stabilisationcan be induced by expression of only thecdc33E72D allele in a wild-type strain, presumablybecause of the presence of normal levels of p20,which are bound preferentially by the mutanteIF4E protein. Moreover, a modified form of p20,in which the eIF4E-binding site had been deleted(Dmp20), was not capable of stabilising themRNAs (data not shown). The results presentedin Figures 3 and 4 suggest that p20 is capable ofinhibiting translation and stabilising mRNA. Inthis sense, it can function analogously to the“dead-end” 4G-BD4E domain. Evidently thepresence of eIF4EE72D in the cell enables even thewild-type level of p20 to shift the balance awayfrom functional eIF4F complexes. At this point itis noteworthy that immunological analyses oftranslation factor levels in yeast cells haverevealed that overexpression of eIF4E isaccompanied by increased detectable levels ofintact p20 (T. von der Haar, unpublished obser-vations). One potential reason for this obser-vation is that p20 is protected from proteolyticdegradation by its association with eIF4E. Over-all, the above experiments with both 4G-BD4Eand p20 demonstrate the significance of thespecific binding functions of the eIF4E dorsalsite.

Figure 4. Effects of cdc33 mutation E72D and of eIF4E ligands on mRNA half-lives. Half-life measurements were per-formed for three different mRNAs (MATa1, YAP1 and ACTIN ) by means of Northern blotting. The numbers aboveeach lane indicate the time (in minutes) following transcriptional repression. A representative gel for each experimentis shown and the average value (minutes ^ standard deviation) of the half-life for the full-length transcript from atleast three experiments is given for each type of experiment. The half-lives were performed using either JDSþS (con-trol) or the strain producing wild-type eIF4E or the mutant form eIF4EE72D (see left-hand margin). The protein ligandsp20 and 4G-BD4E were co-expressed with eIF4EE72D (see Methods).


eIF4E dorsal site mutants and Dcp1 activity invitro

Having observed stabilisation of mRNAs, wethen asked the question whether the dorsal sitemutations affect the ability of eIF4E to blockdecapping in vitro. We performed further in vitrodecapping assays using purified recombinantmutant forms of eIF4E (Figure 5). Neither theE72D mutation nor the W75R mutation impairedthe inhibitory function of eIF4E in these assays.This result is consistent with the interpretationthat the eIF4E dorsal site mutations affectdecapping by virtue of their influence on theincorporation of eIF4E into stable cap-bindingcomplexes with the protein ligands eIF4G and p20.

Control over Dcp1 access to the mRNA cap

In the above we have explored the role of eIF4Edorsal site interactions in modulating decapping.These interactions not only influence the assemblyof an active eIF4F complex but can also modulatethe binding of eIF4E to the cap. Understandingthe relationship between the strength of eIF4E-capbinding and control of the decapping reaction invivo is of key importance, and we therefore investi-gated this further. If the affinity of eIF4E for the capplays an important role in controlling Dcp1 accessto the cap, it would be expected that reducing thisaffinity should enhance the rate of decapping, andthus destabilise mRNAs, in the cell. We tested thisrelationship using a C-terminal deletion mutantform of eIF4E (D196) that shows markedly reducedaffinity for the cap structure and manifests arelatively mild translation deficiency phenotype inyeast.36 The D196 protein was considerably lessinhibitory of the decapping reaction in vitro thanwild-type eIF4E (Figure 5(a) and (b)). However,further in vivo studies demonstrated that the YAP1mRNA half-life in a haploid strain dependent oneIF4E D196 for translation is indistinguishablefrom those in a wild-type strain (Figure 5(c)). Thisresult is consistent with earlier determinations ofthe PGK1 and luciferase mRNA half-lives in thesame strain.20 Overall, therefore, the above datademonstrate that the time-point at which decap-ping of an mRNA is initiated must be determinedby factors other than eIF4E-cap affinity.

Discussion

In this study we have examined the role thatinteractions involving eIF4F might play in control-ling decapping. In our biochemical experimentsusing purified Dcp1, but no translation factors, wehave observed that the decapping rate isapparently not dictated directly by any specificproperties of individual mRNAs. This means thatthe influence of structural features in individualmRNAs is likely to be mediated by other factors.Modulation of the structural and functionalproperties of eIF4F clearly affects Dcp1 function.

Figure 5. Effects of mutant forms of eIF4E on Dcp1activity in vitro. Analysis of decapping activity byFLAG-Dcp1 in the presence of wild-type and mutant-eIF4E proteins (eIF4E, E72D, W75R, D196) is shown in(a). A representative TLC plate generated by theseexperiments is shown. In each lane the same amount ofPGK1 mRNA was incubated with equimolar amounts ofDcp1 and the form of eIF4E indicated (see Methods) for60 minutes. The arrow indicates the position of m7GDPgenerated by the decapping reaction. (b) The quanti-tation of the decapping activity for the experimentshown in (a). The values given are averages of at leastthree independent experiments (plus error bars equival-ent to the respective standard deviations). The mutantforms E72D and W75R have the same inhibitory effecton decapping as the wild-type eIF4E protein. A trun-cated form of eIF4E (D196), which shows reduced affinityfor the cap structure,36 does not significantly affect theactivity of Dcp1. (c) Half-life determinations for YAP1mRNA in a cdc33<LEU2 disruption strain (4-2) carryingthe D196 allele compared to strain 4-2 transformed withthe same expression vector carrying the wild-typeCDC33 gene (control). The sample time points (inminutes) are indicated above the Northern blot lanes.The estimated half-life values (^standard deviations)represent averages of measurements performed using atleast three independent sets of RNA preparations.


On the other hand, the ability of eIF4F to competewith Dcp1 does not vary intrinsically from mRNAto mRNA. We conclude that the events controllingthe triggering of decapping must occur upstreamof the competitive relationship between theseproteins.

Our investigation of the relationship between theactivities of eIF4F and Dcp1 has revealed thatmodulation of the eIF4E–eIF4G interaction isrelevant to mRNA decay under experimental con-ditions that deviate only minimally from the stateof translation in wild-type cells. The results suggestthat, during the normal lifetime of an mRNA, ashift in the relationship between eIF4E and eIF4Gmight allow the latter factor to promote Dcp1 bind-ing to the cap-proximal part of the mRNA whiledisplacing the stabilised association of eIF4E withthe cap. This provides a mechanism by whichdecapping can be triggered, although more workwill be needed to determine how the timing ofthis event is controlled. For many yeast mRNAs,deadenylation is normally a required step prior todecapping,2 and in future work it will be importantto focus on the temporal control of the couplingbetween these two processes.

This raises the question whether translation rateper se is a general determinant of decapping (Figure6(a)). Results from earlier studies are relevant tothis question. Investigations in which the trans-lation rates of individual mRNAs have beenattenuated by means of structural alterations haveindicated that even a decrease of 95% in translationrate does not affect the stability of most mRNAstested.20,37,38 Consideration of this theme is compli-cated by the fact that there is more than one modeof response of mRNA decay to changes in the rateof translation. For example, in contrast to othermRNAs, the much-studied PGK1 mRNA ismarkedly destabilised when its translation rate isinhibited by the insertion of an inhibitory structurein the 50 UTR.33 It has been suggested that thelong half-life of PGK1 is partially attributable tostabilisation afforded by the normally efficienttranslation of this mRNA, whereas the stability ofless well translated mRNAs in yeast is already at areduced level.19 However, since variations in thetranslation rate of most other yeast mRNAs thathave been studied do not affect their half-lives signi-ficantly, it seems likely that the behaviour of PGK1mRNA is not representative of most mRNAs.20

Figure 6. Models for the control of mRNA decay. (a) A flow diagram indicating theoretical pathways linkingdeadenylation with later steps in the decay process. A key step will be the remodulation of the eIF4F–cap interactionsin order to allow access for Dcp1 to the cap. For example, deadenylation will exert a negative influence on translationinitiation and this might represent one possible route to rendering the cap accessible to Dcp1. Alternatively, deadenyla-tion might influence cap-accessibility for Dcp1 via restructuring (or recompartmentalisation) of the polysome. Finally,there could be coupling between deadenylation and decapping that is enabled by molecular signalling to the50-associated translation proteins. As discussed in this paper, there is no evidence that changes in the translation rateson individual mRNAs constitute, in themselves, triggers for decapping. This suggests that direct signalling and/orpolysome restructuring are more likely routes. (b) A simple testable model that could explain how deadenylationcould be coupled to decapping via the eIF4F complex. It is assumed that the system has to be “switched” between astate of restricted cap-access for Dcp1 and one in which decapping can proceed. Restructuring of eIF4F–cap inter-actions may be triggered by signalling from the deadenylation complex. If eIF4E and eIF4G separate, eIF4G couldhelp recruit Dcp1 to the “allowed access” state of the 50 complex. The roles of the other proteins that have been associ-ated with the decapping process are unknown, but for example the Lsm proteins and other factors1 might contribute tothe rearrangement that allows Dcp1 access to the cap.


Moreover, the means by which the translationrate of an mRNA is limited is also important. Forexample, only barely detectable changes in theturnover rate of PGK1 are observed in cells whosetranslation is strongly restricted by virtue ofdeletion mutations in eIF4E.19,20 This result con-trasts with the very significant destabilisation ofPGK1 mRNA observed when its translation isinhibited to similar levels by structural alterationsto the 50 UTR.20,33 Thus, the effects on mRNAdecay caused by structure inserted into the 50 UTRwithin a subset of polysomes are distinct from theeffects caused by limitation of the activity of thewhole cellular translation apparatus by an eIF defect.Added to this is the complication that the impact ofan eIF defect can vary, depending on the eIF affectedand the nature of the defect (see below).

Experiments that have used a range of eIFmutants with highly disruptive phenotypes havebeen interpreted to mean that there is a generallink between translation rate and global mRNAstability.19 However, where there is a major changein the activity of a component of the translationapparatus, mRNA stability can potentially beaffected via a number of mechanisms, includingindirect effects. A further layer of complexity isadded by the observation that the degree of changein mRNA stability caused by an imposed degree oftranslational inhibition differs markedly fordifferent eIF mutants.19 It follows that the effectsassociated with mutations in eIFs that cause majorchanges in global protein synthesis will not necess-arily relate meaningfully to the series of events onindividual mRNAs that take place during thenormal process of degradation (Figure 6(a)).

In this study we have also observed a new prop-erty of the eIF4E-binding protein p20. This putativeregulatory protein has been regarded as an enigmasince its first description39 because of the absence ofany clearly defined physiological role.21 Like mam-malian 4E-BPs, p20 is a phosphoprotein,40 and invitro experiments have established its potential asa competitive inhibitor of eIF4G binding toeIF4E,34 yet unequivocal evidence for a regulatoryfunction in vivo has been lacking.22 One limitationfor experimental investigations of this protein invivo is its proteolytic sensitivity, which hasmediated against the successful execution ofdosage-effect studies. However, eIF4E is capableof protecting p20 from degradation. This effect isexpected to be particularly strong in the presenceof eIF4EE72D, since this mutant form greatlyreduces the ability of eIF4G to compete for bindingto eIF4E. Overall, our data constitute in vivoevidence that p20 is capable of acting as a regulatorof eIF4E function, thereby influencing both trans-lation and mRNA decay, although the physiologi-cal significance of this remains to be elucidated.

In this context it is noteworthy that a previousreport described an increase in growth rate associ-ated with the disruption of CAF20.34 This effect isevidently small under standard laboratory growthconditions, but may be considerably enhanced

under more restrictive growth conditions. Afurther potential reason why detectable mani-festations of the in vivo function of p20 have beenelusive relates to the presence of more than one4E-BP in yeast. One candidate is the Eap1 protein,which also binds to the eIF4E dorsal face and hasbeen implicated in translational regulation relatedto the TOR signalling cascade.41 The functionaloverlap between the respective 4E-BPs may havecontributed to obscuring the functional impact ofchanges in p20 activity in past experiments.

We have concluded that direct competitionbetween Dcp1 and eIF4E for cap binding isunlikely to be a variable for distinct mRNA species,and thus unlikely to act as the source of differenthalf-lives. However, the other findings describedhere lead us to suggest a new model that canexplain how the decapping phase of a givenmRNA can be activated. This testable workingmodel, which may function in combination withother processes that influence decapping, assumesthat deadenylation induces restructuring of partor all of the polysome, thus switching the cap-associated translation complex into a low cap-affinity mode, possibly by dissociating eIF4G fromeIF4E (Figure 6(b)). As demonstrated by ourexperiments with the eIF4E mutant D196, the inter-action of eIF4E with the cap would need to be con-siderably weakened to allow access of Dcp1. Keycomponents of the cytoplasmic deadenylase haverecently been identified as Ccr4, Caf1, Pan2 andPan3,42 and one or more of these factors couldpotentially be involved in signalling the state ofdeadenylation to the 50 end of the polysome. Itnow seems likely that this signalling is mediatedby a network of proteins, possibly including thecytoplasmic Lsm complex.1 The resulting liberationof eIF4G from the cap-binding complex wouldallow it to promote Dcp1 binding to the mRNA byvirtue of its RNA-binding properties. This effec-tively incorporates earlier conclusions concerningeIF4G–Dcp1 interactions7 into a unifying workingmodel. An additional feature of the proposed path-way is that the eIF4F rearrangement could occurwithin an active polysome structure; this wouldthen simultaneously switch off cap-dependentinitiation and commit the mRNA to the final phaseof rapid exonucleolytic degradation in one combinedprocess. The dying moments of the mRNA could,however, be productive, since Xrn1 might chase thelast translating ribosome as it moves through thebody of the mRNA to its 30 UTR.

Materials and Methods

Yeast and bacterial strains

Yeast and bacterial transformations were performedaccording to standard procedures.43 The strain JDSþS(Mata ura3-52 trp1-D1 his4-38 leu2-1 rpb1-1) was used forgrowth rate determination, half-life measurements andpolysomal profile experiments. The strain yRP1104(Mata ura3-52 dcp1-1 rpb1-1) was used in growth curve


determinations and polysomal profile experiments. Thestrains were cultured on media lacking uracil or trypto-phan containing 2% (w/v) galactose, to select and main-tain the plasmids used in these studies, and grown at26 8C. The diploid strain GEX136 was used as the basisfor generating haploid strains containing the plasmid-borne wild-type eIF4E gene (CDC33) or the mutant formcdc33E72D for the preparation of cell-free extracts (Figure3). The plasmids used to create these haploid strainswere YCpSUPEX2-CDC33 and YCpSUPEX2-cdc33E72D(see below). The haploid cdc33D196 strain has beendescribed36 and was used in mRNA half-life experiments(Figure 6). The strains yRP841 (Mata trp1 ura3-52 leu2-3,112 lys22-201 cup1<LEU2(PM)) and yRP1069 (Mata trp1ura3-52 leu2-3, 112 lys22-201 cup1<LEU2(PM) dcp1<URA3) were used in the preparation of cell-free extracts.The E. coli strains BL21 hsdS gal (lcIts857 ind1 Sam7 nin5lacUV5-T7 gene 1) and CAG629 (lon rpoHam165zhg<Tn10 lacZam trpam phoam supCts malam rpsL ) wereused for protein production.

Plasmids

DNA cloning and sequencing were performed usingstandard methods.44 For the purpose of producingeIF4E, W75R, E72D, 4G-BD4E, p20, Dmp20 and Dcp1 inS. cerevisiae, the respective genes were subcloned intoYCpSUPEX2 (PGPF promoter7,45). Dmp20 is a truncatedform of p20 created by deleting the binding site foreIF4E by PCR using the oligonucleotides 50-GGTTTCAT-ATGAAGCCAAGTTTAACTTTGG-30 and 50-GGGAATT-CTTAGTGGTGATGGTGATGGTGTG-30 that was sub-sequently inserted into YCpSUPEX2 as an Nde I/Eco RIfragment. YCpSUPEX2-cdc33E72D was restricted withHindIII and the resulting fragment, containing the PGPF

promoter and the gene, subcloned into pFL39 (pFL39-cdc33E72D ).46 These TRP1-marker expression vector con-structs were used in co-expression experiments with theURA3-marker YCpSUPEX2 plasmids. For the YAP1mRNA half-life studies in the haploid cdc33D19636 strain,the YAP1 gene was subcloned into the YCpSUPEX2 as aNde I/Xba I fragment, generating YCpSUPEX2-YAP1.This plasmid was subsequently restricted with HindIIIand the resulting fragment inserted into pFL39 (pFL39-YAP1).46 pCYTEXP1 was used for production of thedifferent forms of eIF4E (eIF4E, W75R, E72D and D196),p20-His6 and Dmp20-His6

21,36 in the E. coli CAG629strain, whereas production of FLAG-Dcp1 was per-formed using the expression plasmid PET5A7 in theBL21 strain.

The in vitro transcription vector used in the decappingstudies was pHST7 (derived from pHST047). The Bam HIand Xba I fragments from YCp22FP and YCp22PGP,encoding the PGK1 mRNA either with the original leadersequence or with a poly(G) element, containing 18residues of G, were inserted into the Bgl II and Xba Isites of the pHST7 vector. Similarly, the YAP1 DNA con-taining the leader sequence and the gene was digestedBam HI/Xba I and cloned into the Bgl II/Xba I sites of thesame plasmid, immediately downstream of the T7polymerase promoter.

mRNA half-lives

Half-life analysis of endogenous mRNAs was per-formed using JDSþS transformed with either YCpSU-PEX2-CDC33 or YCpSUPEX2-cdc33E72D, oralternatively, with both YCpSUPEX2-CAF20 and pFL39-

cdc33E72D, or YCpSUPEX2-4G-BD4E and pFL39-cdc33E72D. RNA extractions48 and mRNA decay rates20

were performed using standard procedures, and theresults of these experiments were quantified using aTyphoon 8600 Imager. The mRNA abundance was nor-malised using 18 S rRNA as a reference for the amountof total RNA isolated.

Decapping assays

Synthetic uncapped mRNAs were generated in vitrousing T7 RNA polymerase (New England Biolabs)according to the manufacturer’s specifications. Briefly,the pHST7 vectors containing the two PGK1 forms andthe YAP1 gene were cleaved with Xba I and the resultinglinear fragments were used as templates in the transcrip-tion reaction. The resulting uncapped transcripts werepurified by phenol–chloroform extraction and SephadexG-50 chromatography. The mRNAs were capped in thepresence of [32P]GTP following the method described byKnapp49 and decapping reactions were assayed at 30 8Cas described by Stevens.6 The reactions contained 66 ngof Dcp1 (and where indicated, equimolar amounts ofpurified wild-type eIF4E or the mutant forms E72D,W75R and D196), 0.2 pmol of each m7G[32P]mRNA,20 mM Hepes (pH 7.5), 1 mM MgCl2, one unit RNasin(Promega) and 1 mM DTT in a volume of 15–20 ml. Theproducts of the reaction were separated using PEI-cellu-lose thin layer chromatography developed in 0.45 M(NH4)2SO4 and detected using a Typhoon 8600 imager.

Protein purification

The preparation of recombinant FLAG-Dcp1 was per-formed using an anti-FLAGM2 monoclonal antibodyimmunoaffinity column (SIGMA) following the protocoldescribed by Vilela et al.7 Recombinant wild-type andmutant forms of eIF4E were purified using m7GTP-Sepharose according to methods used previously.21 Thep20 forms were purified by Ni-affinity chromatography,as described.50

Sucrose density gradients

Yeast cultures (100 ml) overexpressing the varioustranslation initiation factors were grown to an A600 of0.4. After addition of cycloheximide (10 mg/ml), thecells were harvested for the production of extracts. Theextracts were subsequently loaded onto 12 ml 15%–40%(w/v) sucrose gradients. Gradients were centrifuged at22,000 rpm for 14 hours at 4 8C in a Beckman SW40Tirotor, and subsequently drawn through a spectro-photometer to monitor the A260 profile.

Cell-free translation

Cell-free translation extracts were prepared fromhaploid products of the S. cerevisiae strain GEX1 carryingeither the wild-type eIF4E gene CDC33 or the mutantform E72D, as well as from the Dcp1-deficient strainyRP1069 and the corresponding isogenic DCP1 strain(yRP841). Extract preparation followed the general pro-cedures described.51 – 53 In short, S. cerevisiae cells weregrown to an A600 of 0.8 in YEP galactose medium andsubsequently broken using glass beads. Cappedluciferase transcripts were prepared in vitro usingm7GpppG and T7 RNA polymerase according to themanufacturer’s instructions (Promega). The template


used was pHST7-LUC linearised with Nsi I. CappedmRNAs were further purified from unincorporatednucleotides and m7GpppG by means of phenol/chloro-form/isoamylalcohol extractions, followed by gelfiltration (NAP-5 column from Pharmacia) and precipi-tations with ammonium acetate and sodium acetate.Translation was performed for 60 minutes at 20 8C with100 ng capped luciferase RNAs and 15 ml of cell-freeextract in a final volume of 30 ml.

Acknowledgments

C.V.R. and K.B. were supported by the Biotechnologyand Biological Sciences Research Council (BBSRC, UK).C.V. was supported by a fellowship from the PortugueseFundacao para a Ciencia e Tecnologia (FCT). We thankDr Audrey Atkin (Lincoln, Nebraska) and Dr Roy Parker(Tucson, Arizona) for strains.

References

1. Tucker, M. & Parker, R. (2000). Mechanisms and con-trol of mRNA decapping in Saccharomyces cerevisiae.Annu. Rev. Biochem. 69, 571–595.

2. Caponigro, G. & Parker, R. (1996). Mechanisms andcontrol of mRNA turnover in Saccharomyces cerevisiae.Microbiol. Rev. 60, 233–249.

3. Beelman, C. A., Stevens, A., Caponigro, G.,LaGrandeur, T. E., Hatfield, L., Fortner, D. M. &Parker, R. (1996). An essential component of thedecapping enzyme required for normal rates ofmRNA turnover. Nature, 382, 642–646.

4. LaGrandeur, T. E. & Parker, R. (1998). Isolation andcharacterization of Dcp1p, the yeast mRNA decap-ping enzyme. EMBO J. 17, 1487–1496.

5. Stevens, A. (1980). An mRNA decapping enzymefrom ribosomes of Saccharomyces cerevisiae exoribo-nuclease which yields 50-mononucleotides by a 50-30 mode of hydrolysis. J. Biol. Chem. 255, 3080–3085.

6. Stevens, A. (1988). mRNA-decapping enzyme fromribosomes of Saccharomyces cerevisiae. Biochem.Biophys. Res. Commun. 81, 656–661.

7. Vilela, C., Velasco, C., Ptushkina, M. & McCarthy, J. E.G. (2000). The eukaryotic mRNA decapping proteinDcp1 interacts physically and functionally with theeIF4F translation initiation complex. EMBO J. 19,4372–4382.

8. Schwartz, D. C. & Parker, R. (2000). mRNA decap-ping in yeast requires dissociation of the cap bindingprotein, eukaryotic translation initiation factor 4E.Mol. Cell Biol. 20, 7933–7942.

9. Raught, B., Gingras, A.-C. & Sonenberg, N. (2000).Translational Control of Gene Expression (Sonenberg,N., Hershey, J. W. B. & Mathews, M. B., eds), pp.245–293, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, NY.

10. de la Cruz, J., Kressler, D. & Linder, P. (1999).Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families. Trends Biochem.Sci. 24, 192–198.

11. Goyer, C., Altmann, M., Lee, H. S., Blanc, A.,Deshmukh, M., Woolford, J. L., Jr et al. (1993).TIF4631 and TIF4632: two yeast genes encoding thehigh-molecular-weight subunits of the cap-bindingprotein complex (eukaryotic initiation factor 4F) con-tain an RNA recognition motif-like sequence and

carry out an essential function. Mol. Cell Biol. 13,4860–4874.

12. Dominguez, D., Altmann, M., Benz, J., Baumann, U.& Trachsel, H. (1999). Interaction of translationinitiation factor eIF4G with eIF4A in the yeastSaccharomyces cerevisiae. J. Biol. Chem. 274,26720–26726.

13. Neff, C. L. & Sachs, A. B. (1999). Eukaryotic trans-lation initiation factors 4G and 4A from Saccharo-myces cerevisiae interact physically and functionally.Mol. Cell Biol. 19, 5557–5564.

14. Lamphear, B. J., Kirchweger, R., Skern, T. & Rhoads,R. E. (1995). Mapping of functional domains ineukaryotic protein synthesis initiation factor 4G(eIF4G) with picornaviral proteases. Implications forcap-dependent and cap-independent translationalinitiation. J. Biol. Chem. 270, 21975–21983.

15. Mader, S., Lee, H., Pause, A. & Sonenberg, N. (1995).The translation initiation factor eIF-4E binds to acommon motif shared by the translation factor eIF-4gamma and the translational repressors 4E-bindingproteins. Mol. Cell Biol. 15, 4990–4997.

16. Morley, S. J., Curtis, P. S. & Pain, V. M. (1997). eIF4G:translation’s mystery factor begins to yield itssecrets. RNA, 3, 1085–1104.

17. Pyronnet, S., Imataka, H., Gingras, A.-C., Fukunaga,R., Hunter, T. & Sonenberg, N. (1999). Humaneukaryotic translation initiation factor 4G (eIF4G)recruits mnk1 to phosphorylate eIF4E. EMBO J. 18,270–279.

18. Tarun, S. Z., Jr & Sachs, A. B. (1996). Association of theyeast poly(A) tail binding protein with translationinitiation factor eIF-4G. EMBO J. 15, 7168–7177.

19. Schwartz, D. C. & Parker, R. (1999). Mutations intranslation initiation factors lead to increased ratesof deadenylation and decapping of mRNAs inSaccharomyces cerevisiae. Mol. Cell Biol. 19, 5247–5256.

20. Linz, B., Koloteva, N., Vasilescu, S. & McCarthy, J. E.G. (1997). Disruption of ribosomal scanning on the50-untranslated region, and not restriction of transla-tional initiation per se, modulates the stability of non-aberrant mRNAs in the yeast Saccharomyces cerevisiae.J. Biol. Chem. 272, 9131–9140.

21. Ptushkina, M., von der Haar, T., Vasilescu, S., Frank,R., Birkenhager, R. & McCarthy, J. E. G. (1998).Cooperative modulation by eIF4G of eIF4E-bindingto the mRNA 50 cap in yeast involves a site partiallyshared by p20. EMBO J. 17, 4798–4808.

22. McCarthy, J. E. G. (1998). Posttranscriptional controlof gene expression in yeast. Microbiol. Mol. Biol. Rev.62, 1492–1553.

23. von der Haar, T., Ball, P. D. & McCarthy, J. E. G.(2000). Stabilization of eukaryotic initiation factor 4Ebinding to the mRNA 50-cap by domains of eIF4G.J. Biol. Chem. 275, 30551–30555.

24. Dunckley, T. & Parker, R. (1999). The DCP2 protein isrequired for mRNA decapping in Saccharomycescerevisiae and contains a functional MutT motif.EMBO J. 18, 5411–5422.

25. Hatfield, L., Beelman, C. A., Stevens, A. & Parker, R.(1996). Mutations in trans-acting factors affectingmRNA decapping in Saccharomyces cerevisiae. Mol.Cell Biol. 16, 5830–5838.

26. Boeck, R., Lapeyre, B., Brown, C. E. & Sachs, A. B.(1998). Capped mRNA degradation intermediatesaccumulate in the yeast spb8-2 mutant. Mol. CellBiol. 18, 5062–5072.

27. Zhang, S., Williams, C. J., Hagan, K. & Peltz, S. W.(1999). Mutations in VPS16 and MRT1 stabilize


mRNAs by activating an inhibitor of the decappingenzyme. Mol. Cell Biol. 19, 7568–7576.

28. Dunckley, T., Tucker, M. & Parker, R. (2001). Tworelated proteins. Edc1p and Edc2p, stimulate mRNAdecapping in Saccharomyces cerevisiae. Genetics, 157,27–37.

29. Uetz, P., Giot, L., Cagney, G., Mansfield, T. A.,Judson, R. S., Knight, J. R. et al. (2000). A compre-hensive analysis of protein–protein interactions inSaccharomyces cerevisiae. Nature, 403, 623–627.

30. Bouveret, E., Rigaut, G., Shevchenko, A., Wilm, M. &Seraphin, B. (2000). A Sm-like protein complex thatparticipates in mRNA degradation. EMBO J. 19,1661–1671.

31. Tharun, S., He, W., Mayes, A. E., Lennertz, P., Beggs,J. D. & Parker, R. (2000). Yeast Sm-like proteinsfunction in mRNA decapping and decay. Nature,404, 515–518.

32. Caponigro, G. & Parker, R. (1995). Multiple functionsfor the poly(A)-binding protein in mRNA decappingand deadenylation in yeast. Genes Dev. 9,2421–2432.

33. Muhlrad, D., Decker, C. J. & Parker, R. (1995). Turn-over mechanisms of the stable yeast PGK1 mRNA.Mol. Cell Biol. 15, 2145–2156.

34. Altmann, M., Schmitz, N., Berset, C. & Trachsel, H.(1997). A novel inhibitor of cap-dependent trans-lation initiation in yeast: p20 competes with eIF4Gfor binding to eIF4E. EMBO J. 16, 1114–1121.

35. de la Cruz, J., Iost, I., Kressler, D. & Linder, P. (1997).The p20 and Ded1 proteins have antagonistic rolesin eIF4E-dependent translation in Saccharomycescerevisiae. Proc. Natl Acad. Sci. USA, 94, 5201–5206.

36. Vasilescu, S., Ptushkina, M., Linz, B., Muller, P. P. &McCarthy, J. E. G. (1996). Mutants of eukaryoticinitiation factor eIF-4E with altered mRNA capbinding specificity reprogram mRNA selection byribosomes in Saccharomyces cerevisiae. J. Biol. Chem.271, 7030–7037.

37. Beelman, C. A. & Parker, R. (1994). Differentialeffects of translational inhibition in cis and in transon the decay of the unstable yeast MFA2 mRNA.J. Biol. Chem. 269, 9687–9692.

38. Vega Laso, M. R., Zhu, D., Sagliocco, F., Brown, A. J.,Tuite, M. F. & McCarthy, J. E. G. (1993). Inhibition oftranslational initiation in the yeast Saccharomycescerevisiae as a function of the stability and positionof hairpin structures in the mRNA leader. J. Biol.Chem. 268, 6453–6462.

39. Altmann, M., Krieger, M. & Trachsel, H. (1989).Nucleotide sequence of the gene encoding a 20 kDaprotein associated with the cap binding proteineIF-4E from Saccharomyces cerevisiae. Nucl. Acids Res.17, 7520.

40. Zanchin, N. I. & McCarthy, J. E. G. (1995). Character-ization of the in vivo phosphorylation sites of themRNA cap-binding complex proteins eukaryoticinitiation factor-4E and p20 in Saccharomycescerevisiae. J. Biol. Chem. 270, 26505–26510.

41. Cosentino, G. P., Schmelzle, T., Haghighat, A.,Helliwell, S. B., Hall, M. N. & Sonenberg, N. (2000).

Eap1p, a novel eukaryotic translation initiation factor4E-associated protein in Saccharomyces cerevisiae. Mol.Cell Biol. 20, 4604–4613.

42. Tucker, M., Valencia-Sanchez, M. A., Staples, R. R.,Chen, J., Denis, C. L. & Parker, R. (2001). The tran-scription factor associated Ccr4 and Caf1 proteinsare components of the major cytoplasmic mRNAdeadenylase in Saccharomyces cerevisiae. Cell, 104,377–386.

43. Schiestl, R. H. & Gietz, R. D. (1989). High efficiencytransformation of intact yeast cells using singlestranded nucleic acids as a carrier. Curr. Genet. 16,339–346.

44. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).Molecular Cloning: A Laboratory Manual, 2nd edit,Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY.

45. Oliveira, C. C., van den Heuvel, J. J. & McCarthy, J. E.G. (1993). Inhibition of translational initiation inSaccharomyces cerevisiae by secondary structure: theroles of the stability and position of stem-loops inthe mRNA leader. Mol. Microbiol. 9, 521–532.

46. Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E.,Li, J., Hieter, P. & Boeke, J. D. (1998). Designerdeletion strains derived from Saccharomyces cerevisiaeS288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.Yeast, 14, 115–132.

47. Jobling, S. A., Cuthbert, C. M., Rogers, S. G., Fraley,R. T. & Gehrke, L. (1988). In vitro transcription andtranslational efficiency of chimeric SP6 messengerRNAs devoid of 50 vector nucleotides. Nucl. AcidsRes. 16, 4483–4498.

48. Schmitt, M. E., Brown, T. A. & Trumpower, B. L.(1990). A rapid and simple method for preparationof RNA from Saccharomyces cerevisiae. Nucl. AcidsRes. 18, 3091–3092.

49. Knapp, G. (1989). RNA processing. Methods Enyzmol.180, 192–212.

50. Hughes, J. M., Ptushkina, M., Karim, M. M.,Koloteva, N., von der Haar, T. & McCarthy, J. E. G.(1999). Translational repression by human 4E-BP1 inyeast specifically requires human eIF4E as target.J. Biol. Chem. 274, 3261–3264.

51. Gasior, E., Herrera, F., McLaughlin, C. S. & Moldave,K. (1979). The analysis of intermediary reactionsinvolved in protein synthesis, in a cell-free extract ofSaccharomyces cerevisiae that translates naturalmessenger ribonucleic acid. J. Biol. Chem. 254,3970–3976.

52. Gerstel, B., Tuite, M. F. & McCarthy, J. E. G. (1992).The effects of 50-capping, 30-polyadenylation andleader composition upon the translation andstability of mRNA in a cell-free extract derived fromthe yeast Saccharomyces cerevisiae. Mol. Microbiol. 6,2339–2348.

53. Iizuka, N. & Sarnow, P. (1997). Translation-competentextracts from Saccharomyces cerevisiae: effects of L-ARNA, 50 cap, and 30 poly(A) tail on translationalefficiency of mRNAs. Methods, 11, 353–360.

Edited by K. Nagai

(Received 5 December 2001; received in revised form 25 February 2002; accepted 4 March 2002)


Modulation of Eukaryotic mRNA Stability via the Cap-binding Translation Complex eIF4F

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