the c-terminal domain of eukaryotic protein synthesis initiation factor

The EMBO Journal vol.15 no.6 pp.1371-1382, 1996

The C-terminal domain of eukaryotic proteinsynthesis initiation factor (elF) 4G is sufficient tosupport cap-independent translation in the absenceof elF4E

Theophile Ohimann, Michael Rau,Virginia M.Pain and Simon J.Morley1Department of Biochemistry. School of Biological Sciences.University of Sussex. Falmer. Brighton BNI 9QG. UK

'Corresponding author

The foot and mouth disease virus, a picornavirus,encodes two forms of a cysteine proteinase (leader orL protease) that bisects the eIF4G polypeptide of theinitiation factor complex eIF4F into N-terminal (Nt)and C-terminal (Ct) domains. Previously we showedthat, although in vitro cleavage of the translationinitiation factor, eIF4G, with L protease decreases cap-dependent translation, the cleavage products them-selves may directly promote cap-independent proteinsynthesis. We now demonstrate that translation ofuncapped mRNAs normally exhibits a strong require-ment for eIF4E. However, this dependence is abolishedwhen eIF4G is cleaved, with the Ct domain capable ofsupporting translation in the absence of the Nt domain.In contrast, the efficient translation of the secondcistron of bicistronic mRNAs, directed by two distinctInternal Ribosome Entry Segments (IRES), exhibitsno requirement for eIF4E but is dependent uponeither intact eIF4G or the Ct domain. These resultsdemonstrate that: (i) the apparent requirement foreIF4F for internal initiation on IRES-driven mRNAscan be fulfilled by the Ct proteolytic cleavage product;(ii) when eIF4G is cleaved, the Ct domain can alsosupport cap-independent translation of cellularmRNAs not possessing an IRES element, in the absenceof eIF4E; and (iii) when eIF4G is intact, translation ofcellular mRNAs, whether capped or uncapped, isstrictly dependent upon eIF4E. These data complementrecent work in other laboratories defining the bindingsites for other initiation factors on the eIF4G molecule.Keywords: cap-independent translation/eIF4E/initiationfactor/IRES element

IntroductionThe translation of eukaryotic, cellular mRNA into proteinis a complex process involving the co-ordinate interactionof many RNA and protein components in a regulatedmanner (reviewed by Morley, 1994). A common featureof viral infection is a diversion of the cell's translationalcapacity from the synthesis of normal cellular proteinstowards the preferential translation of viral products.Some, but not all, members of the picornavirus familyencode proteases that cleave the eIF4G polypeptide of theinitiation factor complex, eIF4F. Cleavage of this factorinhibits the translation of the capped mRNAs encoding

host cell proteins without preventing that of the picorna-virus RNA, and thus provides the basis of a strategyutilized by enteroviruses, rhinoviruses and foot-and-mouthdisease virus (FMDV) to impose conditions in infectedcells that favour translation of their own mRNAs overthat of host cell mRNAs (Etchison et al., 1982; Devaneyet al., 1988). Picornavirus RNAs are naturally uncappedand possess highly structured sequences within their 5'untranslated regions (5' UTRs) which direct 40S ribosomalsubunits to bind internally rather than at the extreme 5'end (reviewed by Jackson et al., 1990, 1994, 1995).These elements, called Internal Ribosome Entry Segments(IRESes), have been found in all picornavirus RNAsstudied to date, although there are considerable structuraldifferences between IRESes of enteroviruses and rhino-viruses on one hand and cardioviruses and aphthoviruseson the other. Although IRES elements were first discoveredin the RNAs of picornaviruses (Jackson et al., 1990,1995), they have now been identified in other mRNAs,including those of hepatitis C virus (Tsukyama-Koharaet al., 1992; Wang et al., 1993) and members of thepestivirus family (Poole et al., 1995). In addition,sequences directing internal initiation have been reportedin a number of cellular mRNAs, although these bear littlestructural resemblance to picornavirus IRESes (Macejakand Sarnow, 1991; OH et al., 1992; Vagner et al., 1995).

Cap-dependent initiation of translation involves theassembly of initiation factors at the 5' end of mRNA,including the cap-binding protein, eIF4E, the ATP-dependent RNA helicase, eIF4A, and the eIF4G polypep-tide (p220) to form the eIF4F complex (reviewed inHershey, 1989; Merrick, 1992; Rhoads, 1993; Morley,1994). This complex is believed to promote the unwindingof mRNA secondary structure to facilitate the binding ofthe 40S ribosomal subunit. The exact function of eIF4Gis not known, but translation of capped mRNAs is disruptedwhen this polypeptide is cleaved by viral proteases suchas 2A of poliovirus, coxsackie virus and human rhinovirus(Etchison et al., 1982; Liebig et al., 1993) or the leader(L) protease of FMDV (Devaney et al., 1988; Belshamand Brangwyn, 1990; Ohlmann et al., 1995). While thiscorrelation exists, the observation that cleavage of eIF4Gis not sufficient for total shut-off of host protein synthesisin vivo suggests that other events may play a role inthe inhibition of cap-dependent initiation (Bonneau andSonenberg, 1987; Perez and Carrasco, 1992). However,mRNAs possessing an IRES element in the 5' UTR canstill be translated when the eIF4G subunit is cleavedproteolytically. An important question concerning theinternal initiation mechanism is its requirement for thecanonical initiation factors promoting mRNA binding toribosomes. eIF4A was originally identified as a factorrequired for translation of a picornavirus RNA (Dahl andBlair, 1979) and rapidly recognized as a general initiation

1371( Oxford University Press

T.Ohlmann et al.

factor, which appears to be involved in translation of allmRNAs so far examined (Altmann et al., 1990; Pauseet al., 1994a). In addition, eIF4B, which is believed toaugment the activity of eIF4A, has been shown to cross-link to the IRES element of FMDV (Meyer et al., 1995).As one would predict, translation of IRES-directed mRNAis observed in eIF4E-deficient extracts from mutant yeastcells (Altmann et al., 1990) and in mammalian celltranslation systems in which the factor has been seques-tered by addition of a specific binding protein (Pauseet al., 1994b). However, internal initiation can be stimu-lated in vitro by the addition of the purified eIF4F complex(Anthony and Merrick, 1991; Thomas et al., 1991), oreven purified eIF4E (Scheper et al., 1992), suggesting apossible role in this mechanism. Moreover, several reportshave suggested that proteolytic cleavage of eIF4G, ratherthan merely discouraging translation of competing cellularmRNAs, may exert a positive role in promoting internalinitiation on picornavirus RNAs in infected cells (Buckleyand Ehrenfeld, 1987; Hambidge and Sarnow, 1992;Scheper et al., 1992; Macadam et al., 1994; Pause et al.,1994a; Lamphear et al., 1995). More direct studies within vitro systems have suggested that cleavage of eIF4Genhances translation driven by enterovirus or rhinovirusIRES elements (Liebig et al., 1993; Ziegler et al., 1995)and even translation of uncapped transcripts encodingcellular proteins (Ohlmann et al., 1995; Ziegler et al.,1995), while translation of coding sequences downstreamof a cardiovirus IRES element was not affected (Thomaset al., 1992; Ohlmann et al., 1995; Ziegler et al., 1995).Thus, although eIF4G clearly plays a role in cap-dependenttranslation by virtue of its ability to bind eIF4E (Lamphearet al., 1995; Mader et al., 1995), it has been suggestedbut not directly proven, that eIF4G could have one ormore additional functions that extend to cap-independentmechanisms (Lamphear et al., 1995; Ohlmann et al., 1995).Lamphear et al. (1995) have now reported that primary

cleavage of eIF4G by picornaviral proteases divided thepolypeptide into two domains; the N-terminal domainbinds eIF4E (Mader et al., 1995), while the C-terminaldomain binds eIF4A and eIF3. They propose that theN-terminal domain is involved, via eIF4E, in cap binding,whereas the C-terminal domain is responsible for ribosomebinding (via eIF3) and, in complex with eIF4A, formspart of the RNA helicase apparatus. Their findings predictthat the C-terminal domain alone should fulfil the functionsof eIF4F in cap-independent translation. In this paper, wepresent direct evidence for this, using reticulocyte lysatesmanipulated in different ways to effect selective depletionof eIF4E and/or the N-terminal domain of eIF4G.

ResultsPreparation of in vitro translation systemsdepleted of elF4E and with reduced levels of elF4GThe foot and mouth disease virus L protease cleaves theeIF4G polypeptide between Gly479 and Arg480 to givetwo primary fragments (Kirchweger et al., 1994). In ourprevious study (Ohlmann et al., 1995) we examined theeffect of L protease treatment of a reticulocyte lysate onthe translation of three types of mRNA. Translationof capped, cellular mRNAs was, as expected, severelyinhibited, whereas internal initiation driven by a cardio-

pXLCycAlT7

A

pXLIODA

T7

pXL40-372.NS'T7

C |Cyclin B2cistroDHCVIRES NS' cistron

HCVcoding region

Fig. 1. Constructs used in these studies. (A) pXLcycAl encodingXenopus laevis cyclin A. (B) pXLJODA, the bicistronic constructencoding X.laevis cyclin B2 (first cistron) and influenza virus NS'(second cistron) with the IRES from TMEV in the intercistronicspacer. (C) pXL40-372.NS', the bicistronic construct also encodingX.Iaevis cyclin B2 (first cistron) and influenza virus NS' (secondcistron) but with the IRES (nucleotides 40-340) from HCV in theintercistronic spacer and also containing 32 nucleotides (nucleotides340-372) of the HCV coding region, as indicated on the figure.

virus IRES was unaffected. However, the translation ofuncapped transcripts encoding cellular proteins-whichlack an IRES element-was stimulated over control levelsunder these conditions. Utilizing the transcripts describedin Figure 1, the aim of the present work was to elucidateand compare the requirements for eIF4E and eIF4G inthe translation of capped, uncapped cellular and IRES-driven mRNAs and to investigate which of the functionsof eIF4G can be fulfilled by the separate proteolyticcleavage products.

The N-terminal fragment (Nt) of the cleaved elF4Gpolypeptide remains attached to elF4EPrevious observations from several laboratories have indi-cated that at least one fragment of cleaved eIF4G remainsassociated with eIF4E in picornavirus-infected cells(Buckley and Ehrenfeld, 1987; Etchison and Smith, 1990).We have established conditions for the batchwise additionof the affinity matrix, m7GTP-Sepharose, to the reticulo-cyte lysate such that virtually all the eIF4E will bind tothe resin and the resultant unbound material can beemployed in translation assays (summarized in Figure 2).We have applied these conditions to control and L protease-treated lysates to compare the association of the proteolyticfragments of eIF4G with eIF4E, using Sepharose 4Bwithout the m7GTP affinity ligand as the control resin.Figure 2B-D shows immunoblots detecting the eIF4G,eIF4A and eIF4E remaining in the lysates after exposureto the two resins, while Figure 3 depicts the polypeptidesrecovered from these resins. In all cases, the immunoblotsignal was in the linear range of response with theseantisera (data not shown). Treatment of either control orL protease-treated lysate with m7GTP-Sepharose removed>70% of the eIF4E (Figure 2D; see quantification detailsin legend). This could subsequently be recovered fromthe resin following elution in the presence of m7GTP(Figure 3C, lanes 1 and 3; see legend). As can be seen inFigure 3C, lanes 2 and 4, a small amount of eIF4E wasalso removed non-specifically by the parallel treatment of

1372

cap-independent translation and elF4G

Fig. 2. m7GTP-Sepharose depletion of a Messenger Dependent Lysate(MDL). (A) Experimental design. Reticulocyte MDL (90 g1) waspreincubated in the absence of (1 and 2) or presence (3 and 4) of Lprotease for 60 min on ice, as described in Materials and methods. Atthe end of the preincubation, the protease inhibitor Elastatinal wasadded to the lysate to a final concentration of 300 p.M. The lysate wassubjected to batch adsorption using either m7GTP-Sepharose (1 and 3)or Sepharose 4B control resin (2 and 4), as described. (B-D) Aliquots(2 p.l) of the different unbound fractions (1-4) were resolved bySDS-PAGE, and proteins transferred to PVDF and visualized byimmunoblotting with the following antisera recognizing: (B) the Ntpart (NI-N4) or the Ct part (C1-C4) of the eIF4G polypeptide;(C) eIF4A; (D) eIF4E. Quantification of the eIF4E immunoblots bydensitometric scanning gave the following data (in arbitrary units):lane 1, 0.15; lane 2, 1.00; lane 3, 0.29; lane 4, 1.28. Migrationpositions of the molecular weight markers (in kDa) are indicated onthe sides. These data are representative of those obtained in at leastfive separate experiments.

control samples with Sepharose 4B. In the absence of Lprotease treatment, ~40-50% of the intact eIF4G was alsoremoved concomitant with that of eIF4E; this was shownusing either N-terminal (Ni) or C-terminal (Ct)-directedspecific antiserum for detection (Figure 2B, lanes NI, N2and Cl, C2). After cleavage of eIF4G, almost the entirepool of N-terminal products was removed by treatmentwith m7GTP-Sepharose resin (Figure 2B, lanes N3 andN4) and could be recovered in association with eIF4Eupon elution of the resin (Figure 3A, lane N3). In contrast,however, the C-terminal proteolytic fragment was notretained by the resin during m7GTP-Sepharose treatment(Figure 2B), indicating that the C-terminal domain ofeIF4G no longer associates stably with eIF4E. This wasconfirmed by the finding that the Ct domain was notrecovered following elution of the m7GTP-Sepharose

Fig. 3. Both eIF4E and the Nt domain of eIF4G are specificallyretained by m7GTP-Sepharose chromatography. Reticulocyte MDL(90 il) was preincubated in the absence of (1 and 2) or presence (3and 4) of L protease for 60 min on ice, as described in Materials andmethods. At the end of the preincubation, the protease inhibitorElastatinal was added to the lysate to a final concentration of 300 gM.The lysate was subjected to batch adsorption using either m7GTP-Sepharose (1 and 3) or Sepharose 4B control resin (2 and 4), asdescribed. Following removal of the supernatant, the resin was washedwith four volumes of Buffer A, the bound proteins eluted with gelsample buffer, and proteins resolved by SDS-PAGE, transferred toPVDF and visualized by immunoblotting with the following antiserarecognizing: (A) the N, part (N1-N4) or the Ct part (C1-C4) of theeIF4G polypeptide; (B) eIF4A; (C) eIF4E. Quantification of the eIF4Eimmunoblots by densitometric scanning gave the following data (inarbitrary units): lane 1, 0.88; lane 2, 0.28; lane 3, 1.08; lane 4, 0.35.Migration positions of the molecular weight markers (in kDa) areindicated on the sides. These data are representative of those obtainedin at least five separate experiments.

resin (Figure 3A, lane C3). The ability of the Ct-directedantiserum (but not the Nt-directed antiserum) to detectvery low amounts of intact eIF4G following cleavagereflects the greater sensitivity of the Ct-directed antiserumat the levels employed in these studies (see also Lamphearet al., 1995). These data confirm recent reports that thebinding site of the eIF4E subunit is located in theN-terminal domain of the eIF4G polypeptide while theC-terminus seems to be responsible, directly or indirectly,for ribosomal attachment (Lamphear et al., 1995; Maderet al., 1995). Lamphear et al. (1995) also identified abinding domain for eIF4A in the C-terminal part of theeIF4G molecule. On this basis, one might have predictedthat eIF4A from the L protease-treated lysates would failto bind to m7GTP-Sepharose, i.e. would behave in thesame manner as the C-terminal degradation product ofeIF4G. However, we were unable to detect any changesin the binding behaviour of eWF4A (Figures 2C and 3B).It is possible that meaningful changes could be maskedby the substantial background of non-specific binding ofthis factor to Sepharose resin (Figure 3, lane 2). In thiscontext it is important to note that eIF4A is present atvery high concentrations in mammalian cells relative toother initiation factors; hence the total eIF4A signal (seenin Figures 2C and 3B), probably represents a 10-foldmolar excess over the corresponding fractions of eIF4E(Pause et al., 1994a).

1373

I.

T.Ohimann et aL

-

.,Fig. 4. The N-terminal domain of eIF4G is not required for efficient cap-independent initiation of translation. Capped (A) or uncapped (B) versionsof the pXLcycAl transcripts and the uncapped versions of the bicistronic constructs pXLJODA (C) and pXL40-372.NS' (D) were translated inassays based on the reticulocyte lysate subjected to the pre-treatments described in Figure 2A and as described in the Materials and methods:(1) m7GTP-Sepharose batch adsorption; (2) Sepharose 4B adsorption; (3) L protease treatment followed by m7GTP-Sepharose batch adsorption;(4) L protease treatment followed by Sepharose 4B batch adsorption. Aliquots (2 gl) were removed at the times indicated and processed to measureincorporation of [35S]methionine into protein (A and B). The uncapped bicistronic constructs pXLJODA (C) and pXL40-372.NS' (D) were translatedfor 40 min, aliquots (I wl) were removed and analysed by SDS-PAGE and the autoradiograms are presented. The migration of marker proteins(kDa) is indicated and the arrows indicate the position of cyclin B2 and NS' translation products. The IRES-driven NS' products from pXLJODA(E) and pXL40-372.NS' (F) were quantified by densitometric scanning; the data presented are expressed in arbitrary units as a percentage with theSepharose 4B control (condition 2) set as 100%. These data are representative of those obtained in at least three separate experiments.

Effect of depletion of eIF4E and partial depletion ofeIF4G on the translation of uncapped mRNAsUtilizing the unbound fractions from the proceduredescribed in Figure 2, translation assays were performedto determine the effect of depletion of eIF4E and theN-terminal domain of eIF4G on the translation of tran-scripts encoding Xenopus cyclin A (see Figure 1). As

shown in Figure 4A, relative to the non-depleted system,the efficiency of translation of capped cyclin A mRNAwas substantially reduced, but not totally inhibited, byremoval of eIF4E; the remaining activity was probablydue to the retention by the lysate of part of the eIF4Ebound to ribosomes, which is not easily removed bytreatment with the affinity resin (Figure 2D; C.Fraser,

1374


unpublished results). Prior cleavage of eIF4G by L proteasealso severely impaired the ability of the lysate to translatecapped cyclin A mRNA, with a combination of depletionof eIF4E and eIF4G cleavage reducing translation stillfurther. Figure 4B shows the effect of these manipulationson the ability of the derived lysates to translate Xenopuscyclin A mRNA when this was uncapped. In this case,the inhibitory effect of the m7GTP-Sepharose treatmentalone was, if anything, even greater than that seen withthe capped transcript. However, relative to the non-treatedsystem, lysates preincubated with L protease showedelevated translational activity with uncapped mRNA, inde-pendent of m7GTP-Sepharose treatment. In some experi-ments the activity of the lysate treated with both affinityresin and L protease did not quite reach the level seenwith that treated with L protease alone. However, in allcases the L protease treatment effectively negated theinhibitory effects of depletion with m7GTP-Sepharose.Since the lysate resulting from the combined treatmentswas severely depleted of both eIF4E and the N-terminalcleavage product of eIF4G (Figure 2B and D), these datasuggest that the C-terminal domain of eIF4G alone is ableto support translation of uncapped transcripts. In agreementwith the model predicted by Lamphear et al. (1995), thecleavage of eIF4G somehow liberates this translation fromits dependence on eIF4E.We have also examined the effect of such depletions

upon the translation of IRES-driven mRNAs in the bicis-tronic constructs, pXLJODA and pXL40-372.NS' (seeFigure 1). Figure 4C and E shows the results of translationof the uncapped pXLJODA bicistronic mRNA [of whichthe upstream cistron encodes Xenopus cyclin B2 andthe downstream cistron encodes the influenza virus NS'protein, driven by the TMEV IRES element (Hunt et al.,1993)] in the depleted lysate system. Only the translationof the downstream cistron is readily detectable in theexposure of the autoradiogram presented, as that of theupstream cistron in this uncapped transcript is very ineffi-cient (Borman et al., 1995). Translation of the IRES-driven downstream cistron was substantially impaired inthe lysate treated with m7GTP-Sepharose (compare lanesI and 2); as shown in Figure 4E, densitometric scanningindicates a 50% inhibition relative to the control lysatetreated with Sepharose 4B. However, it seems likely thatthis effect can be attributed to the partial depletion of theeIF4G polypeptide rather than to the removal of eIF4E(see later). As seen for the uncapped cyclin A mRNA(Figure 4B), translation of the NS' product is enhancedfollowing cleavage of eIF4G, independent of m7GTP-Sepharose depletion, suggesting that any requirement foreIF4F can be supplied by the C-terminal fragment ofeIF4G. Essentially the same results were obtained witha second uncapped bicistronic mRNA, pXL40-372.NS',encoding the same proteins but with an insert in theintercistronic space that includes the HCV IRES (seeFigure 1), which is poorly utilized by the reticulocytelysate (Borman et al., 1995). In this case the upstreamcistron was translated much more actively than in thecase of pXLJODA. The distinction probably reflects thedifferent extent to which translation of the downstreamcistron exerts a competitive effect on that of the upstreamcistron. As previously described by Borman et al. (1995),the presence of a cardiovirus IRES directs very efficient

translation of the downstream cistron, at the expense ofthat of the upstream cistron; on the other hand, thepresence of a downstream cistron directed by the HCVIRES is much less inhibitory to the translation of itsupstream partner. The data for the translation of theuncapped cyclin B2 cistron (Figure 4D) follow thoseobtained with the uncapped, monocistronic cyclin AmRNA (Figure 4B). The data for the downstream cistron,whose translation is driven by the HCV IRES (Figure 4Dand F), confirm those obtained for the TMEV IRES inindicating that although IRES-driven protein synthesisoccurs independently of eIF4E, it does require either theintact eIF4G or its Ct domain for efficient translation.Moreover, the need for the intact eIF4G polypeptide seemsto be quantitatively significant, as some impairment oftranslation was seen when only 40-50% of the eIF4G wasremoved (Figure 4E and F). These results confirm thatthe Nt of eIF4G, which is removed with the eIF4E subunitby the affinity matrix, does not play a significant role incap-independent translation.

Effect of specific depletion of eIF4E with PHAS-l onmRNA translation in a non-fractionatedreticulocyte lysateAlthough treatment of the reticulocyte lysate with anm7GTP-Sepharose affinity matrix is an effective meansof eIF4E depletion, it is not possible, as shown above, toavoid the concomitant removal of some of the intacteIF4G. In order to investigate more specifically the effectof eIF4E depletion on cap-independent translation, weused the physiological eIF4E binding protein, PHAS-I.This protein was recently described as a protein whichbinds specifically to eIF4E and precludes it from inter-acting with eIF4G to form the eIF4F cap binding complex(Lin et al., 1994; Pause et al., 1994; Mader et al., 1995).Recent work in our laboratory is consistent with thismechanism; immunoprecipitation from a reticulocytelysate with antibodies recognizing either PHAS-I or eIF4Gresults in co-precipitation of eIF4E, but PHAS-I andeIF4G are never found in the same immunoprecipitate,suggesting that their interaction with eIF4E is mutuallyexclusive (M.Rau, T.Ohlmann, S.Morley and V.M.Pain,manuscript submitted). This indicates that addition ofPHAS-I protein can be used to sequester the eIF4E in alysate without concomitant removal of eIF4G. Figure 5shows the effect on translation of adding recombinant,bacterially expressed PHAS-I to control and L protease-treated reticulocyte lysates at a concentration shown inpreliminary experiments to be extremely inhibitory fortranslation of capped transcripts (T.Ohlmann andS.J.Morley, unpublished observation). In agreement withour previous findings (Ohlmann et al., 1995), L proteasetreatment of the lysate in the absence of PHAS-I resultedin a substantial stimulation of protein synthesis fromuncapped mRNA (Figure SA; compare 'Buffer + L' with'Buffer'). In the absence of L protease, addition of PHAS-Ireduced the translation rate of uncapped cyclin A mRNAby >70%. In contrast, however, in the presence of Lprotease, addition of PHAS-I resulted in an inhibition oftranslation by only 25% at 60 min. These data suggestthat although eIF4E may play an essential role in thetranslation of uncapped mRNAs in the presence of theintact eIF4G polypeptide, this requirement is eliminated

1375

T.Ohlmann et al.

Fig. 5. The PHAS-I protein has no effect on internally initiated mRNAs but severely inhibits translation of uncapped cellular mRNA. (A) UncappedpXLcycAI rnRNA was translated in a MDL in the absence (buffer) or presence of PHAS-I, or L protease as indicated in the figure. Buffer, PHAS-I1and L protease (1 pl//10 l) were added at the start of the incubation and aliquots (2 tl) were removed at the times indicated and processed tomeasure [35SImethionine incorporation into protein as described. Uncapped pXLJODA (B) and uncapped pXL40-372.NS' (C) were translated in theabsence (1) or presence of (2) PHAS-I or of both L protease and PHAS-I (3). After 40 min, aliquots (I fl) were removed and analysed by SDS-PAGE and the autoradiograms are presented. The migration of mal-ker proteins (kDa) is indicated and the arrows show cyclin B2 and NS' products.The IRES-driven NS' products from pXLJODA (D) and pXL40-372.NS' (E) were quantified by densitometr-ic scanning: the data presented areexpressed in arbitrary units as a percentage with the buffer control set as 100%. Quantification of the translation of cyclin B2 in a similar manner,ave the following data (in arbitrary units): (B) lane 1, 0.053; lane 2, 0.032:; lane 3. 0.081; (C) lane 1, 0.192; lane 2, 0.103; lane 3. 0.331. These dataare representative of those obtained in at least five separate experiments.

when eIF4G is cleaved. Thus, there might be alternative,and distinct, translational mechanisms employed byuncapped RNAs. Translation of the uncapped upstream(cyclin B2) cistron of the bicistronic constructs (Figure 5;quantified in the figure legend) shows a similar trend.However, in the case of the downstream cistrons, theeffect of PHAS-I on translation was negligible, irrespectiveof L protease treatment (Figure 5B-E). The lack of

effect of PHAS-I alone on IRES-driven translation is inagreement with the findings of Pause et al. (1 994b). Thesedata confirm the lack of requirement for eIF4E in theIRES-driven internal initiation process, and indicate thatthe impairment of translation of these mRNAs in lysatestreated with m7GTP-Sepharose (Figure 4) is most likelyto be due to partial loss of eIF4G than to depletionof eIF4E.

1376


The C-terminal domain of the eIF4G polypeptide isboth necessary and sufficient to supporttranslation of uncapped and IRES-driven mRNAUltracentrifugation of a lysate permits the separationi ofthe N- anid C-terminial cleavage products of eIF4G. Ourprevious work has demonstrated that translation ofuncapped cellular RNAs in the standard reticulocyte lysatesystem is stimulated by the proteolytic cleavage of eIF4Gby L protease. Kinetic considerations have suggested thatthis phenomenon may be induced by the appearance ofone or both of the degradation products of eIF4G (Ohlmannet al., 1995). Recently, Lamphear et al. (1995) have alsosuggested that the C-terminal proteolytic fragment ofeIF4G may play a general role in translation of all mRNAs,including uncapped transcripts. Therefore, a primary aimof these studies was to devise a cell-free translation systemin which to test this hypothesis directly. As an alternativeapproach to the use of m7GTP-Sepharose to separate theN- and C-terminal domains of eIF4G, we investigatedtheir subcellular distribution as an initial step in designingsuch a translation system. To this end, an mRNA-dependent lysate was incubated either in the presence orin the absence of recombinant L protease for 30 min at30°C, before being centrifuged at 435 000 g (100 000r.p.m.) for 40 min in the Beckman TL-100 ultracentrifuge(Figure 6A). The post-ribosomal supernatant fraction(designated PRS) was removed and the ribosomes resus-pended in Buffer A, as described in Matenrals and methods.Fractions were then analysed by SDS-PAGE and thedistribution of the N- and C-terminal cleavage productsof eIF4G, and that of eIF4E analysed by immunoblotting(Figure 6B). Intact eIF4G is mainly found in the ribosomalfraction (lanes N2, C2), with little remaining in the PRS(lanes Ni, Cl). However, after L protease treatment,the N- and C-terminal proteolytic fragments show quitedistinct distribution patterns (lanes 3 and 4); virtually allof the Ct domain remains associated with the ribosomes(lane C4), with little detectable in the PRS (lane C3). Incontrast, the Nt domain was mainly released into the PRS(lane N3), with little or none of this fragment associatedwith the ribosome fraction (lane N4). In agreement withrecent published data (Lamphear et al., 1995), these datasuggest that the main site responsible for the binding ofeIF4G to the 40S ribosomal subunit lies within the Ctproteolytic fragment. We have also monitored the effectof L protease on the distribution of eIF4E (Figure 6C). Inthe control lysate -25% of the eIF4E was found in theribosome pellet (compare lane 2 with lane 1); after Lprotease treatment most of the ribosome-bound eIF4E wasreleased (lane 4), so that virtually all the eIF4E was foundin the PRS (lane 3).

Effects of manipulating levels of eIF4E, eIF4G anid eIF4Gcleavage products on in vitro tracnslation of differentmRNAs. To investigate further the potential role of theindividual degradation products of eIF4G on translation,we have exploited the above changes in the subcellulardistribution of the N1 and Ct products after cleavage by Lprotease to modify the fractionated, reconstituted reticulo-cyte lysate translation system described by Morley andHershey (1990). Figure 7A shows the construction of a

translation system containing the C-terminal domain ofeIF4G but severely depleted of both the N-terminal domain

Fig. 6. L protease cleavage of eIF4G localizes the C, domain to theribosomes and releases ribosome-associated eIF4E. (A) An mRNA-dependent lysate (MDL) was incubated either in the presence of buffer(I and 2) or L protease (3 and 4). for 40 min at 30°C. Lysates werethen centrifuged in a Beckman TL-100 ultracentrifuge for 40 min at100 000 r.p.m. (435 000 g). The post-ribosomal supematant (PRS) wasremoved and the ribosomal pellet was resuspended in Buffer A ( 1/10of the oriczinal volume of lIsate). PRS (2 111; 1.3) and I pl of theribosomal suspension (2 and 4) were resolved by SDS-PAGE andproteins were transferred to PVDF and visualized by immunoblottingh,with antibodies recognizing: (B) the Nt domain (NI-N4) or the Ctdomain (CI-C4) of the eIF4G polypeptide; (C) elF4E. Migration ofthe molecular weight markers (kDa) is indicated on the sides. Thesedata are representative of those obtained in at least five separateexperiments. Note that in order to facilitate the detection of the releaseof eIF4E and the N, domain of eIF4G from the ribosomiies. we haveanalysed correspondingly large amounts of the ribosomal fractionrelative to the PRS (equivalent to 10 pl of the original MDL for theribosomes and 2 .1l for the PRS).

and eIF4E. An untreated reticulocyte lysate (i.e. withoutremoval of endogenous mRNAs) was centrifuged to yielda PRS fraction (Figure 7A); previous experiments in ourlaboratory have shown that the PRS from such a lysatecontains ~80% of the total eIF4E but only .20% ofthe eIF4G (M.Rau, T.Ohlmann, S.Morley and V.M.Pain,manuscript submitted). Separate samples of this PRS werethen treated with either m7GTP-Sepharose (SD) or controlSepharose 4B (SC) and the unbound material retained.Ribosomal fractions were prepared in parallel from aMDL that had been preincubated either with L protease(LR) or with buffer (CR) before centrifugation. Fromthese fractions, reconstituted in vitro systems could thenbe set up by mixing the supernatants (SC or SD) with theribosomes (CR or LR) in four different combinations: thecontrol system (SC + CR) contains both factors at levelssimilar to those seen in an untreated lysate; the (SD +

CR) combination yields a system with a similar contentof eIF4G, but with reduced eIF4E; the systems that includeribosomes from the L protease-treated lysate contain lower(SC + LR) or very low (SD + LR) amounts of intact

eIF4G, but retained levels of the Ct cleavage productapproximately equal to those seen in the L protease-treated, unfractionated lysate and the SD + LR mixture

1377

T.Ohimann et al

Fig. 7. The Ct domain of eIF4G alone is sufficient to promote the translation of uncapped mRNA. (A) A non-nuclease treated reticulocyte lysate(RRL) was centrifuged for 40 min at 100 000 r.p.m. in the Beckman TL-100 to yield a PRS fraction. This was then exposed to either m7GTP-Sepharose (SD) or Sepharose 4B (SC) as described in Materials and methods and the unbound fractions retained. Ribosomal fractions were preparedin parallel by using an MDL that had been preincubated for 60 min on ice either with L protease (LR) or with buffer (CR) and resuspended asdescribed above. Supernatants (4 ,l; SC or SD) were then mixed with 1 ,ul of ribosomes and 5 gl of translation mix to give the final (10 ul)translation assay as described in Materials and methods. Capped (B) or uncapped (C) transcripts of pXLcycA1 were translated for up to 60 min inthis fractionated assay system, using the four different combinations as indicated. Aliquots (2 ,ul) were removed at the times indicated and processedto measure [35S]methionine incorporation into protein. Uncapped pXLJODA (D) and uncapped pXL40-372.NS' (E) transcripts were translated in theindicated fractionated systems for 40 min. Aliquots (I gl) were removed and analysed by SDS-PAGE and the autoradiogram is presented. Themigration of marker proteins (kDa) is indicated and the arrows show the position of cyclin B2 and NS' products. The IRES-driven NS' productsfrom pXLJODA (F) and pXL40-372.NS' (G) were quantified by densitometric scanning; the data presented are expressed in arbitrary units as apercentage, with the supernatant control (SC) mixed with the control ribosomes (CR) set as 100%. The cyclin B2 product was quantified in a similarmanner to give the following data (expressed in arbitrary units): (D), lane 1, 0.025; lane 2, 0.093; lane 3, 0.020; lane 4, 0.038; (E) lane 1, 0.171;lane 2, 0.328; lane 3, 0.181; lane 4, 0.276. These data are representative of those obtained in at least five separate experiments.

1378


was severely depleted of eIF4E (Figures 2 and 6; alsodata not shown).

In previous studies we have found the in vitro translationof transcripts encoding Xenopus cyclin A to be stronglycap-dependent (Ohlmann et al., 1995). Figure 7B demon-strates that, as expected, in this reconstituted system eIF4Edepletion decreased translation of capped cyclin A by25% (compare SD + CR with SC + CR); most of theprotein synthesis in this case was presumably supportedby the eIF4E bound to the control ribosomes. However,translation of capped cyclin A mRNA was decreased insystems utilizing ribosomes prepared from L protease-treated lysates, irrespective of whether they were supple-mented by PRS fractions containing (SC + LR) or depletedof (SD + LR) eIF4E. As discussed by Ziegler et al.(1995), the unexpectedly high level of translation ofcapped cyclin in the apparent absence of eIF4E and eIF4Gprobably reflects the stimulation of translation of low levelsof contaminating uncapped mRNA in the preparation(Ohlmann et al., 1995). The results of a parallel translationexperiment with the fractionated system employinguncapped cyclin A mRNA, are shown in Figure 7C. Inthis instance, translation seen in the control system (SC +CR) was poor relative to the capped transcript (comparepanels A and B, note different scale), with depletion ofeIF4E (SD + CR) having little additional effect. However,the use of ribosomes derived from an L protease-treatedlysate resulted in a pronounced stimulation of translation,which showed very slight, but reproducible, attenuationfollowing eIF4E depletion. This demonstrates that a systemseverely depleted in eIF4E, intact eIF4G and the N,domain of eIF4G, but enriched in the Ct cleavage product,retained the ability to stimulate translation of an uncappedtranscript. Thus, these data also suggest that eIF4E, intacteIF4G and the N, domain of eIF4G may exert a negativeinfluence on the translation of uncapped mRNA.We have also utilized this assay system to examine the

effect of the C-terminal domain of eIF4G on IRES-driventranslation. Figure 7D-G shows the translation of thetwo uncapped bicistronic mRNAs in these reconstitutedsystems. In both cases the translation of the uncappedcyclin B2 cistron confirms our findings with the uncapped,monocistronic cyclin A mRNA above (see quantificationin the figure legend). However, no consistent differenceswere seen between any of the assay systems in their abilityto translate the IRES-driven cistrons. These data indicatethat IRES-driven initiation is resistant to severe depletionof both eWF4E and the N-terminal domain of eIF4G,provided that the C-terminal cleavage product is present.

DiscussionThe aim of this work was to examine directly the rolesof eIF4E, and the N- and C-terminal domains of the eIF4Gpolypeptide on the translation of capped and uncappedmRNAs. This was prompted by our observations thattranslation of uncapped mRNA could occur actively wheneIF4G was cleaved by viral proteinases (Ohlmann et al.,1995). Several authors have reported that the productsof proteolysis of eIF4G may specifically stimulate thetranslation in vitro of uncapped, virally-encoded mRNAbearing an IRES (Buckley and Ehrenfeld, 1987; Hambidgeand Sarnow, 1992; Scheper et al., 1992; Macadam et al.,

1994; Whetter et al., 1994). This was confirmed by Ziegleret al. (1995), who further demonstrated stimulation oftranslation of mRNAs driven by either enterovirus orrhinovirus IRES elements in reticulocyte lysate translationsystems supplemented with extracts from HeLa cells.Similar data, but from a system based completely on HeLacells, had been presented previously by the same group(Liebig et al., 1993) using the rhinovirus 2A proteinase,which has a different specificity from the L protease butcleaves at a site very close in the eIF4G molecule(Lamphear et al., 1993). However, the exact requirementof IRES-driven and uncapped mRNA translation forcanonical initiation factors required for binding mRNA toribosomes has remained confused. There is evidence inthe literature that the intact eIF4F complex (eIF4E.eIF4G.eIF4A) may play a role even in IRES-driven internalinitiation (Anthony and Merrick, 1991; Thomas et al.,1991; Scheper et al., 1992), and there are also observationssuggesting that translation of uncapped cellular mRNAsis hypersensitive to inhibition by m7GTP cap analogues(Fletcher et al., 1990). Recent biochemical studies byLamphear et al. (1995) have suggested, but not directlyshown, that the Ct cleavage product of eIF4G may stimulatecap-independent initiation through interaction with otherinitiation factors or with mRNA.

In an attempt to clarify these requirements, we haveused the FMDV L protease to cleave the endogenous eIF4Gin the reticulocyte lysate, and employed two alternativestrategies to prepare translation systems that would allowus to investigate the separate roles of the N- and C-terminalportions of the molecule. The first approach was to depletethe lysate of eIF4E by incubation with m7GTP-Sepharoseaffinity resin. In the intact system this resulted in theremoval of ~75% of the eIF4E, together with ~40-50%of the eIF4G (Figure 2), which was recovered upon elutionof the resin (Figure 3). As well as impairing translationof capped mRNA, this treatment was detrimental totranslation of uncapped mRNAs both possessing and,particularly, lacking an IRES element (Figure 4). For eachclass of mRNA, these effects could be explained by eitherthe depletion of eIF4E or by the partial removal of eIF4G.To distinguish between these alternatives, we examinedthe effect of adding the eIF4E binding protein, PHAS-I,which does not bind eIF4G (Mader et al., 1995). At aconcentration that completely inhibited translation ofcapped mRNA (not shown) this protein exerted a differen-tial effect on the translation of the two types of uncappedmRNA (Figure 5). Translation of the IRES-driven down-stream cistron of a bicistronic construct was virtuallyunaffected, as previously shown by Pause et al. (1994b).This is also consistent with the observation that in vitrosystems derived from mutant yeast cells devoid of eIF4Eactivity were capable of IRES-driven translation (Altmannet al., 1990). In contrast, translation of an uncappedmonocistronic transcript encoding cyclin A was inhibitedby ~70% in the PHAS-I-treated system (Figure 5). Thesedata indicate that eIF4E is normally necessary for thetranslation of this type of transcript, whereas the decreasedtranslational efficiency of IRES-driven mRNAs inm7GTP-Sepharose-treated lysates (Figure 4) is more likelyto be attributable to partial loss of eIF4G. When thesemanipulations were applied to lysates in which the endo-genous eIF4G had first been cleaved by L protease,

1379

T.Ohlmann et aL

completely different results were obtained. In the case ofIRES-driven translation, the effects of m7GTP-Sepharosetreatment on translation were attenuated (Figure 4).Immunoblotting indicated that the resulting lysate wasseverely depleted of eIF4E and the Nt domain of eIF4G,but that it retained virtually all its original content of theCt domain (Figures 2 and 3). This indicates that, whileIRES-driven initiation is normally sensitive to partialdepletion of intact eIF4G, the requirement for this moleculecan be fulfilled by the Ct domain alone. In the case ofuncapped transcripts lacking an IRES, the effects of Lprotease treatment were more pronounced, in that transla-tion was stimulated above control levels. The result withthe m7GTP-Sepharose-treated system (Figure 4) againsuggests an important role for the Ct domain of eIF4G,both in permitting protein synthesis under conditions ofeIF4E depletion and in mediating the stimulatory effectof L protease treatment observed previously (Ohlmannet al., 1995; Ziegler et al., 1995). Furthermore, prior Lprotease treatment prevented the severe loss of transla-tional activity in response to PHAS-I (Figure 5), suggestingthat cleavage of eIF4G greatly reduced the dependence ofuncapped mRNA translation on eIF4E.

These data suggest that there may be two alternativemechanisms for translating uncapped mRNAs lacking anIRES element. When eIF4G is intact, such mRNAs aretranslated at a very low efficiency, and appear to utilize amechanism that is dependent on eIF4E. The dramaticinhibition of their translation in eIF4E-depleted or PHAS-I-treated systems is consistent with earlier work (Fletcheret al., 1990) indicating a greater sensitivity to inhibitionby cap analogues. Cleavage of eIF4G, however, seems toallow a switchover to a more active, eIF4E- and Ntdomain-independent mechanism, presumably involvingthe Ct fragment localized to the 40S ribosome. This doesnot appear to involve a significant shift towards utilizationof downstream AUG codons (of which cyclin A mRNAhas several), since the pattern of translation products wasnot altered (not shown).The main features of these data were reproduced when

we used an alternative procedure to separate the N- andC-terminal cleavage fragments of eIF4G, based on theassociation of the latter with ribosomes (Figures 6 and 7).The findings are entirely consistent with recent predictionsof Lamphear et al. (1995); they suggest a domain model forthe function of eIF4G in cap-dependent and independenttranslation, whereby the Nt domain interacts with eIF4Eand the Ct domain localizes the eIF4G, either directly orindirectly, to the 40S ribosome. Our data also suggest thatthe function of eIF4F in internal initiation, identified byothers (Anthony and Merrick, 1991; Thomas et al., 1991;Scheper et al., 1992) can be attributed to an activity ofthe C-terminal domain of eIF4G. These findings wouldbe consistent with the idea that this part of eIF4G, likeeIF4A, plays a fundamental role in the translation on alltypes of mRNA rather than being confined to a functionin cap-dependent initiation. Since the C-terminal cleavagefragment is almost entirely ribosome-bound in the reticulo-cyte lysate (this study and M.Rau, T.Ohlmann, S.Morleyand V.M.Pain, manuscript submitted), and has beenreported to associate with eIF4A and eIF3 (Lamphearet al., 1995), a likely function could be to bring eIF4A tothe ribosome. Further study is needed to elucidate its

role in both cap-dependent and independent initiation inmore detail.

Materials and methodsPurification of the L protease and PHAS-1The construct for the recombinant Lb form of the L protease (kindlyprovided by G.Kirchweger and T.Skern, Institute of Biochemistry,University of Vienna) was expressed in BL21(DE3) bacteria and theprotein purified as described previously (Kirchweger et al., 1994). Itsactivity was characterized by the ability to cleave -95% of the eIF4Gpolypeptide in a nuclease-treated rabbit reticulocyte lysate during a 60min incubation on ice. The concentration of the purified Lb found tofulfil these conditions was 1 ,ul (0.03 gg) of the Lb protein preparationper 10 gl of messenger RNA-dependent reticulocyte lysate (MDL).Preincubations of MDL with purified L protease (1 ,ul/10 gl) werecarried out for 60 min on ice and terminated by addition of 300 ,uM ofthe protease inhibitor Elastatinal (Sigma) before in vitro translationreactions. The purified Lb preparation is referred to as L protease.Recombinant (His)6-tagged PHAS-I protein (0.15 tg/4tl) was isolatedfrom BL21(DE3) bacteria and purified as described by Haystead et al.(1994). During the final step of both L protease and PHAS-I preparation,an overnight dialysis was carried out against Buffer A (20 mM MOPS-KOH, pH 7.2, 10 mM NaCl, 25 mM KCI, 1.1 mM MgCl2, 0.1 mMEDTA, 7 mM f-mercaptoethanol); therefore, Buffer A was used forparallel control incubations throughout the experiments described.

Transcription reactionsPlasmids pXLcycA, encoding Xenopus laevis cyclin A, and the bicistronicconstructs pXLJODA (referred to as pXLJODA 1099 by Hunt et al.,1993) and pXL340-372.NS' (Figure 1), were kindly donated by DrsN.Standart, R.J.Jackson and J.Reynolds, University of Cambridge, UK.Plasmid DNA from pXLcycAl was linearized with BamHI and bothpXLJODA and pXL340-372.NS' were linearized with EcoRI as describedpreviously (Ohlmann et al., 1995). For synthesis of capped transcripts,GTP concentrations were reduced to 0.48 mM and m7GpppG capanalogue (New England BioLabs) added at a concentration of 1.92 mM.

Translation reactionsRabbit reticulocyte lysates were prepared in the laboratory as describedpreviously (Jackson and Hunt, 1983). In all cases, translation reactionscontained 50% (v/v) reticulocyte lysate with the addition of the following(final concentrations): 12.5 ,uM haemin, 75 mM KCI, 0.8 mM magnesiumacetate, 50 gM each amino acid (except methionine and leucine), 200 ,uMleucine, 3 mM D-glucose, S mg/ml creatine phosphate, 25 ,ug/ml creatinephosphokinase, 15 mM 2-aminopurine, 2 mM dithiothreitol, 50 ,ug/mlcalf liver tRNA, 300 U/mI human placental RNase inhibitor and25 ,ug/ml mRNA. Translation reactions were performed with 200 lCi[35S]methionine/ml assay; at the times indicated in the individual figurelegends, aliquots (2 gl) were removed and processed to measuretricholoroacetic acid-precipitable radioactivity. In each experiment, anincubation containing no mRNA was run using the most complete system.

m7GTP-Sepharose chromatographyBefore use, m7GTP-Sepharose 4B (Pharmacia) resin was washed exten-sively in Buffer A, pretreated with cytochrome c [100 ,ug/mI in a 50%(v/v) suspension of resin in buffer] and Buffer A was removed byaspiration using a needle attached to a vacuum line. Batch depletions ofeIF4E were carried out by adding 1 vol of m7GTP-Sepharose resin to3 volumes of MDL, the mix gently agitated for 10 min on ice, centrifugedfor 30 s in a microfuge and the unbound fraction removed and utilizedfor in vitro translation. In each case, a parallel incubation was carriedout with the control matrix Sepharose 4B.

Fractionation of the reticulocyte lysateFractionation and reconstitution of rabbit reticulocyte lysates was asdescribed (Morley and Hershey, 1990). Briefly, a 10 tl assay wasobtained by mixing 4 pl of the post-ribosomal supematant (PRS), I ,lof the ribosomes resuspended in Buffer A (1/10 of the original volumeof lysate) and 5 gl of the translation mix to yield the final assayconditions described above for the unfractionated lysate.

SDS-PAGE analysis and immunoblotting12% polyacrylamide gel electrophoresis (SDS-PAGE) and immuno-blotting were as described previously (Morley and Pain, 1995). Anti-

1380


bodies used for iiiiniutnoblotting*were: polvclonal antiseruum directedagainst either peptide 7 (Morley and Pain. 1995). or peptide 6 (a kindeift from Dr R.E.Rhoads. Louisiana State Universitv. USA) of the eIF4Gpolypeptide Ifollowing cleavage with L protease. peptide 7 is in the Ntpart and peptide 6 in the Ct part of the p220 molecule. as describedpreviously (Yan et of.. 1992)]: elF4E. polvclonal rabbit anti-peptideantiserum (Mo-rley anid Pain. 1995): eIF4A. mouse monoclonal antiserum.kindly provided by; M.Altmann. University of Berne. Switzerland.Inmmunoblots were visualized with an alkaline phosphatase-coupledsecond antibody (Sigma). as per the manufacturer's instructions. Analysisof translation products derived from the bicistronic mRNAs was per-formed oni a 2'§, polvacrylamide gel. the gel dried and exposed toHyperfilm 3-milax (Amileisham).

AcknowledgementsWe would like to thank Drs Richard J.Jackson. Ann Kaminski. NancyStandart. Jo Reynolds. Gina KirchwveEer. Tim Skern and John LawrenceJr for the plasmids used in this w,ork: Dr Robert E.Rhoads for theC-terminal-specific antiserum to eIF4G and Dr Nlichael Altmann for themonoclonal antiserum specific for eIF4A. T.Ohlmann is supported by aBursarv from the University of Sussex and S.Morlev is a Senior ResearchFellow of The Wellcome Trust. This work was funded by grants (034710/Z/91/ZI.5 and 040800/Z/94/Z/040) from The Wellcomiie Trust.

ReferencesAAltmann.M.. Blum.S.. Pelletier.J.. Sonenber-.N.. Wilson.T.M.A. and

Trachsel.H. ( 1990) Translation initiation factor-dependent extractsfrom Sacchorooinvces cerevisiae. Bioch1ioii. Bioph7Ys. ACtM. 1050. 155-159.

Anthonv.D.D. and Merrick.W.C. (1991) Eukarvotic initiation factor(elF)4F. Implications for a role in internal initiation. J. Biol. Chem..266. 10218-10226

Belsham.G.J. and Brangw-yn.J.K. (1990) A reeion of the 5' noncodinceregion of foot-and-mouth disease virus RNA directs efficient ininternal initiation of protein synthesis within cells: involvement withthe role of L protease in translational control. J. Virol.. 64. 5389-5395.

Bonneau.A.-M. and Sonenberg.N. (1987) Proteolysis of the p220component of the cap-binding, protein complex is not sufficient forcomplete inhibition of host cell protein synthesis after poliovirusinfection. J. Vir-ol.. 61. 986-991.

Borman.A.M.. Bailly.J.-L.. Girard.M. and Kean.K.M. (1995)Picornavirus internal ribosome entry segments: comparison oftranslation efficiency and the requirements for optimal internalinitiation of tranislation in7 vitro. Nulucleic Acids Res.. 23. 3656-3663.

BucklevyB. and Ehrenfeld.E. (1987) The cap-binding protein complexin uninfected and poliovirus-infected HeLa cells. J. Biol. Chem.. 262.13599-13600.

Dahl.H.H.M. and Blair.G.E. (1979) Purification of four eukarvoticinitiation factors required for natural mRNA translation. MethodsEnzwnol.. 60, 87-101.

Devaney.M.A., Vakharia.V.N.. Lloyd.R.E.. Ehrenfeld.E. and Grubman.M.J. ( 1988) Leader protein of foot-and-mouth disease virus is requiredfor cleavage of the p220 component of the cap-binding complex.J. Virol.. 62. 4407-4409.

Etchison.D. and Smith.K. (1990) Variations in cap-bindina complexesfrom uninfected and poliovirus-infected HeLa-cells. J. Biol. Chem..265. 7492-750)).

Etchison.D.E.. Milburn.S.C.. Edery.I.. Sonenberg.N. and Hershey.J.W.B.(1982) Inhibition of HeLa cell protein synthesis follow'ving poliovirusinfection correlates with the proteolvsis of a 220.000-daltonpolypeptide associated with eukaryotic initiation factor 3 and a capbinding protein complex. J. Biol. Chew7.. 257. 14806-14810.

Fletcher.L.. Corbin.S.D.. Browning,K.G. and Ravel.J.M. (1990) Theabsence of a m7GTP cap on 3-globin mRNA and alfalfa mosaic virus4 increases the amounts of initiation factor 4F required for translation.J. Biol. Cheil7.. 265. 19582-19587.

Hambidge.S.J. and Sarnow.P. (1992) Translational enhancement of thepoliovirus 5' noncoding region mediated by virus-encoded polypeptide2A. Proc. Nat! Acad. Sci. USA. 89. 10272-10276.

Haystead.T.A.J.. Haystead.C.M.M., Hus.C.. Lin.T.-A. and Lawrence.J.C.Jr (1994) Phosphorylation of PHAS-I by mitogen-activated protein(MAP) kinase- identification of a site phosphorylated by MAP kinasein vitr-o and in response to insulin in rat adipocytes. J. Biol. Clieiii..269. '185-'3191

Hershev.J.W.B. (1989) Protein phosphorylation controls translation rates.J. Biol. C/wmn.. 264. 20823-20826.

Hunt.S.L.. Kaminski.A. and Jackson.R.J. (1993) The influence of viralcoding sequences on the efficiency of internal initiation of translationof cardiovirus RNAs. Virology. 197. 801-807.

Jackson.R.J. and Hunt.T. (1983) Preparation and use of nuclease-treatedrabbit reticulocyte lysates for the translation of eukaryotic messengerRNA. Methods Enzvonol.. 96. 50-74.

Jackson.R.J.. Howell.M.T. and Kaminski.A. (1990) The novel mechanismof initiation of picornavirus RNA translation. Trenicds Bioch1ewtl. Sci..15. 477-483.

Jackson.R.J.. Hunt.S.L.. Gibbs.C.L. and Kaminski.A. (1994) Internalinitiation of translation of picornavirus RNAs. Mol. Biol. Rep.. 19.147-159.

Jackson.R.J.. Hunt.S.L.. Reynolds.J.E. and Kaminski.A. (1995) Cap-dependent and cap-independent translation: operational distinctionsand mechanistic interpretations. Clurtr Topics Mict-obiol. Izutnllitiol..203. 1-29.

Kirchweger.R. et of. ( 1994) Foot-and-mouth disease virus leaderproteinase: purification of the Lb form and determination of itscleavare site on eIF-4y. J. Virol.. 68. 5677-5684.

Lamphear.B.J.. Yan.R.. Waters.D.. Liebig.H.-D.. Klump.H.. Kuechler.E..SkernmT. and Rhoads.R.E. (1993) Mapping the cleavage site in proteinsynthesis initiation factor eIF-4y of the 2A proteases from humancoxsackievirus and rhinoviirus. J. Biol. Clhewti.. 268. 19200-19203.

Lamphear.B.J.. Kirchweger.R.. Skern.T. and Rhoads.R.E. (1995)Mapping of functional domains in eukaryotic protein synthesisinitiation factor 4G (eIF4G) with picornavirus proteases. J. Biol.Chiewti.. 270. 21975-21983.

Liebig.H.-D. et of. (1993) Purification of two picornaviral 2A proteinases:interaction with eIF-4y and influence on in vitro translation.Biochenistry. 32. 7581-7588.

Lin.T.-A.. Kong.X.. Havstead.T.A.J.. Pause.A.. Belsham.G.J..Sonenberg.N. and Lawrence.J.C..Jr (1994) PHAS-I as a link betweenmitogen-activated protein kinase and translation initiation. Scienice.266. 653-656

Macadam.A.J.. Ferguson,G._ Fleming,T., Stone,D.M.. Almond,J.W. andMinor.P.D. (1994) Role for poliovirus protease 2A in cap independenttranslation. EMBO J., 13. 924-927.

Macejak.D.G. and Sarnow.P. (1991) Internal initiation of translationmediated by the 5' leader of a cellular mRNA. Natlure, 353. 90-94.

Mader,S.. Lee.H., Pause,A. and Sonenberg.N. (1995) The translationinitiation factor eIF4E binds to a common motif shared by translationfactor eIF4G and the translational repressor 4E-binding proteins. Mol.Cell. Biol.. 15. 4990-4997.

Merrick,W.C. (1992) Mechanism and regulation of eukaryotic proteinsvnthesis. Microhiol. Rev.. 56, 291-315.

Mever.K.. Petersen.A., Niepman.M. and Beck,E. (1995) Interactionof eukaryotic initiation factor eIF-4B with a picornavirus internaltranslation initiation site. J. Virol., 69. 2819-2824.

Morley,S.J. (1994) Signal transduction mechanisms in the regulation ofprotein synthesis. Mol. Biol. Rep.. 19, 221-232.

Morley,S.J. and Hershey.J.W.B. (1990), A fractionated reticulocyte lysateretains high efficiency for protein synthesis. Biochiimnie, 72, 259-264

Morley,S.J. and Pain,V.M. (1995) Hormone-induced meiotic maturationin Xetnopuis oocytes occurs independently of p70%SK activation and isassociated with enhanced initiation factor (eIF)-4F phosphorylationand complex formation. J. Cell Sci., 108, 1751-1760.

OH,S.K.. Scott.M.P. and Sarnow.P. (1992) Homeotic gene Antennopediamessenger RNA contains 5' noncoding sequences that confertranslational initiation by internal ribosome binding. Genies Dev.. 6.1643-1653.

Ohlmann.T.. Rau.M., Morley.S.J. and Pain.V.M. (1995) Proteolyticcleavace of initiation factor eIF-4y in the reticulocvte lysate inhibitstranslation of capped mRNAs but enhances that of uncapped mRNAs.Nucleic Acids Res.. 23, 334-340.

Pause,A., Methot,N., Svitkin.Y.. Merrick.W.C. and Sonenberg.N. (1994a)Dominant negative mutants of mammalian translation initiation factoreIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation. EMBO J.. 13, 1205-1215.

Pause.A.. Belsham.G.J., Gingras.A.-C.. Donze.O.. Lin.T.-A.,Lawrence,J .C..Jr and Sonenberg.N. (1994b) Insulin-dependentstimulation of protein synthesis by phosphorylation of a regulator of5'-cap function. Nature. 371. 762-767.

Perez.L. and Carrasco.L. (1992) Lack of direct correlation between p220cleava2e and the shut-off of host translation after poliovirus infection.Virology. 189. 178-186.

1381

T.Ohimann et aL

Poole,T.L., Wang,C., Popp,R.A., Potgieter,L.N.D., Sidiqui,A. andCollet,M.S. (1995) Pestivirus translation initiation occurs by internalribosome entry. Virology, 206, 750-754.

Rhoads,R.E. (1993) Regulation of eukaryotic protein synthesis byinitiation factors. J. Biol. Chem., 268, 3017-3020.

Scheper,G.C., Voorma,H.O. and Thomas,A.A.M. (1992) Eukaryoticinitiation factors-4E and -4F stimulate 5' cap-dependent as well asinternal initiation of protein synthesis. J. Biol. Chem., 267, 7269-7274.

Thomas,A.A.M., ter Haar,E., Wellink,J. and Voorma,H.O. (1991) Cowpeamosaic virus middle component RNA contains a sequence that confersinternal binding of ribosomes and that requires eukaryotic initiationfactor 4F for optimal translation. J. Virol., 65, 2953-2959.

Thomas,A.A.M, Scheper,G.S., Kleijn,M., De Boer,M. and Voorma,H.O.(1992) Dependence of the adenovirus tripartite leader on the p220subunit of eukaryotic initiation factor 4F during in vitro translation.Eur J. Biochem., 207, 471-477.

Tsukiyama-Kohara,K., lizuka,N., Kohara,M. and Nomoto,A. (1992)Internal ribosome entry site within hepatitis C virus RNA. J. Virol.,66, 1476-1483.

Vagner,S., Gensac,M.-C., Maret,A., Bayard,F., Almaric,F., Prats,H. andPrats,A.-C. (1995) Alternative translation of human fibroblast growth-factor-2 messenger-RNA occurs by internal entry of ribosomes. Mol.Cell. Biol., 15, 35-44.

Wang,C., Sarnow,P. and Siddiqui,A. (1993) Translation of humanhepatitis C virus mRNA in cultured cells is mediated by an internalribosome-binding mechanism. J. Virol., 67, 3338-3344.

Whetter,L.E., Day,S.P., Elroy-Stein,O., Brown,E.A. and Lemon,S.M.(1994) Low efficiency of the 5' nontranslated region of hepatitis-Avirus-RNA in directing cap-independent translation in permissivemonkey kidney-cells. J. Virol., 68, 5253-5263.

Yan,R., Rychlik,W., Etchison,D. and Rhoads,R.E. (1992) Amino acidsequence of human protein synthesis initiation factor eIF-4 gamma.J. Biol. Chem., 267, 23226-23231.

Ziegler,E., Borman,A.M., Kirchweger,R., Skern,T. and Kean,K.M. (1995)Foot-and-mouth disease virus Lb proteinase can stimulate rhinovirusand enterovirus IRES-driven translation and cleave several proteinsof cellular and viral origin. J. Virol., 69, 3465-3474.

Received on September 11, 1995; revised on November 20, 1995

1382

the c-terminal domain of eukaryotic protein synthesis initiation factor

Documents