Eukaryotic Initiation Factors-4E and -4F Stimulate 5' cap ... - JBC

VOl. 267, No . 11, Issue of April 15, pp. 7269-7274,1992 Printed in U.S.A.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc

Eukaryotic Initiation Factors-4E and -4F Stimulate 5’ cap-dependent as Well as Internal Initiation of Protein Synthesis* (Received for publication, August 23,

1991)

Gert C. ScheperS, Harry 0. Voorma, and Adri A. M. Thomas From the Department of Molecular Cell Biology, University of Utrecht, Padunlaan 8, 3584 CH Utrecht, The Netherlands

Two mechanisms of initiation of protein synthesis are known. The 5’ cap-dependent model requires the activity of cap-binding eukaryotic initiation factors (eIF)1-4E and -4F, inducing unwinding of mRNAsecondary structures. The internal initiation model is 5‘ cap-independent and requires a ribosomal entry site formed by higher order structures of the mRNA. Ithas been proposed that this mechanism does not need eIF4E and eIF-4F. We prepared bicistronic transcripts on which both mechanisms of initiation occur, allowing the determination of the initiation factor dependence of these two mechanisms simultaneously. The unwinding factors eIF-4A, eIF-4B, and eIF-4F were found to be necessary for 5‘cap-dependent initiation as well as for internal initiation. Surprisingly, efficient translation of both cistrons on the bicistronic mRNA requhed eIF-4E. A model is presented in which assembly of eIF4E into a functional eIF-4F complex is a prerequisite for both types of initiation.

nucleotides) (13, 14). Several upstream AUGs arepresent which do not preclude efficient picornaviral translation (15). In thescanning model, upstream AUGs inhibit recognition of the initiator codon (6). The secondary, and probably tertiary structure of the picornaviral 5’-UTR forms a ribosomal landing pad (1, 2) or internal ribosomal entry site (3, 4) that allows cap-independent internal binding of the 40 S ribosomal subunit. Later it was suggested that initiation factors perform the initial recognition of the internal entry site (16). After this recognition of the entry site, the recognition of the initiator AUG takes place either directly as described for encephalomyocarditis virus (EMC) (17) or by scanning, described for poliovirus (16). Although the two mechanisms of initiation seem to differ quite extensively, up till now only one major difference in the initiation factor requirement between the two has been found. After infection with some picornaviruses, such as poliovirus and foot-and-mouth disease virus (FMDV), the p220 component of eIF-4F is proteolyzed leading to inactivation of eIF4F (18-21). Not all picornaviral infections lead to p220Protein synthesis on picornavirus RNA is initiated by a cleavage, indicating that it is not the sole mechanism by mechanism different from that of host mRNAs, in vitro as which infection of picornaviruses causes the shutoff of host well as in vivo (1-5). Therefore two different mechanisms of protein synthesis. EMC for example competes very efficiently translationinitiation exist; the mechanism formulated by for the translational apparatus (22-24), probably resulting in Kozak (6, 7) to account for initiation on host mRNA, and on host shutoff. In this article we have focussed on the two mechanisms of the other hand, the mechanism of internal initiation on the initiation with regard to initiation factor dependence. Most picornaviruses. In the model for the translation of capped host mRNAs, of the experiments were done in a reconstituted cell-free the methylated cap structure at the free 5’ end on the host protein synthesizing system that allowed manipulation of the mRNA is recognized byeukaryotic initiation factor (eIF)’-4F, composition of initiation factors and to analyze the effect of followed by unwinding of the secondary structure in the 5’- the addition of factors. This approach is not possible with the untranslated region (5”UTR) by eIF-4F or eIF-4A in coop- normally used reticulocyte lysate systems in which there is a eration with eIF-4B (8, 9). This leads to a stretch of single- fixed level of initiation factors, a level normally sufficient for stranded RNA, allowing scanning of the messenger by initi- efficient translation. ation factorsor by the ribosome to thefirst AUG in a favorable The experiments mainly focuson the initiation factors context (6), after which elongation starts. involved in cap binding and unwinding of the secondary A different mechanism has been proposed for the picorna- structure. On one hand it is interesting to see whether the viruses (10-12). The picornaviral5’-UTRs are characterized unwinding activities of eIF-4A, eIF-4B, andeIF-4F have by strong secondary structures in a long 5’-UTR (600-1200 differential effects on cap-dependent or internal initiation. *This workwas supported by the Netherlands Foundation for The 5’-UTR of EMC was used, because the ribosomal entry Chemical Research (SON). The costs of publication of this article is within a few nucleotides of the initiator AUG (17) and were defrayed in part by the payment of page charges. This article scanning is therefore hardly necessary. One would anticipate must therefore be hereby marked “advertisement” in accordance with different requirements for unwinding factors in internal ini18 U.S.C. Section 1734 solely to indicate this fact. tiation. $ To whom correspondence should be addressed. Tel.: 30-53-28-85; On the other hand a difference in the dependency on capFax: 30-51-36-55. The abbreviations used are: eIF, eukaryotic initiation factor; 5’- binding factors is expected. It has been shown that eIF-4F UTR, 5’-untranslated region; EMC, encephalomyocarditis virus; stimulated translationof a cistron, downstream of the internal FMDV, foot-and-mouth disease virus; CPMV, cowpea mosaic virus; entry siteof poliovirus (25) or of cowpea mosaicvirus (CPMV) TPT, tripartite; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate; CAT, chloramphenicol ace- (26). Therefore, eIF-4F and its cap-binding subunit eIF-4E tyltransferase; CFS, cell-free protein synthesizing system; ODC, or- were tested for their ability to stimulate cap-dependent as nithine decarboxylase. well as cap-independent translation.

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Internal Initiation of Translation Requires eIF-4E and -4F MATERIALS AND METHODS

Plasmids pTZEMCCAT and pTZ4AEMCCAT were created by digestion of pTZ4AI (pTZ18R containing the 4AI gene downstream of theT7 RNA polymerase promoter) with BamHI followed by complete (for pTZEMCCAT) or partial digestion with EcoRI. The EMCCAT fragment, obtained from pEP40EMCCAT (27) by EcoRI (partial)-BamHI digestion, was cloned into pTZ4AI EcoRI/BamHI. Digestion with EcoRI (partial) and Sac1 of pEP4OEMCSacI-U-CAT (24), followed by Klenowtreatment tocreate blunt ends and ligation, resulted in clone pEP40AEMCCAT. The SacI-BamHI fragment of pEP40EMCCAT was cloned downstream of pTZ4AI (SmaI-BamHI digestion) to create pTZ4AAEMCCAT. Construction of pYP-TPTCAT will be described elsewhere*; it contains the T7 promoter followed by the 5’ 174 nucleotides of the adenovirus tripartite (TPT) leader and theCAT gene. All manipulations were checked by DNA sequencing. Plasmids p9T70DC-71, -131,-243, and ODC-CPMV have been described (26, 28). The in vitro translation of the transcript, derived from pTC6, coding for the L-protease of FMDV (21) will bedescribed elsewhere.* Transformation of Escherichia coli was as described (29). Transcription of linearized DNA was performed as described (30) with SP6 or T7 RNA polymerase either in the presence of 0.5 mM 7mGpppG and 0.1 mM GTP to obtain capped transcripts or with 1 mM GTP for uncapped transcripts. It was shown that under these transcription conditions, i.e. a high capanalog-to-GTPratio, the majority of the transcripts were capped (30). Transcripts were purified by phenol/chloroform extraction and chromatography on a Sephadex G-50 column. Yield and integrity of the transcripts were checked by agarose electrophoresis. Translation of transcripts in a reconstitutedcell-free protein synthesizing system (31) was performed as follows. The 5-pl assays contained Hepes, dithiothreitol, 1 mM ATP, 0.4 mM GTP, creatine phosphate, creatine kinase, 120 mM potassium acetate, 2 mM magnesium acetate, 100 p M spermine, 50 p M amino acids minus methionine, 1pCi of [35S]methionine(1000 Ci/mmol), tRNA, pH 5 enzymes (32), ribosomal subunits, and the indicated amount of an A-cut of a ribosomal salt wash, always less than theamount needed for maximal translation, and 2 pgof a BC-cut. Initiation factors were added as indicated. The K’ concentration was kept the same in all assays. In the 5-pl assays 30 ng of transcript was used, approximately 3-4-fold below saturating conditions. Translation of transcripts in rabbit reticulocyte lysate was carried out as described (33). The 5-pl assays contained 1 pCi of [%I methionine and 30 ng of transcripts. The lysates were homemade. After translation 1-11 aliquots were taken to determine hot trichloroacetic acid-precipitable radioactivity, and 4-p1 samples were analyzed by SDS-polyacrylamide gel electrophoresis. The gels were prepared for fluorography by treatment with sodium salicylate. Eukaryotic initiation factors and A- and BC-cuts from ribosomal salt wash were purified from reticulocyte lysates as described (20,31). HeLa eIF-4E was purified from an A-cut of a ribosomal salt wash as described (34). Fig. 1 shows the Coomassie stain of the SDS-polyacrylamide gel containing the four initiation factors used in the translation assays. Western blots with antibodies against eIF-4A, eIF-4B, and -4F did not reveal cross-contamination of the four factors used (not shown). Furthermore, cross-linking studies with eIF-4B and eIF-4F showed that theband of approximately 80 kDa in eIF-4F (Fig. 1) was not eIF-4B (35). An essential resultis that eIF-4F antibody, reacting with p220 and eIF-4E, did not reveal cross-contamination of p220 in 0.7 pg of eIF-4E. Only 1 ng of eIF-4E was used in translation assays.

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4A 48 4E 4F FIG. 1. Polyacrylamide gel analysis of purified initiation factors. The initiation factors used to measure their effect on 5’ capdependent and internal initiation are shown as a Coomassie stain of a 12.5% SDS-polyacrylamide gel. Lanes 1 and 6, molecular mass markers (molecular masses indicated in the left margin); lane 2, 1 pg of eIF-4A; lane 3 , l pg of eIF-4B; lane 4,0.5 pg of eIF-4E lane 5,4 pg of eIF-4F.

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FIG.2. Schematic representation of the transcripts used. 4A (gray box) encodes for 4AI (41) and contains a 5’-UTRconsisting of GGGAATTCCACC. The EMC element (white box) is the 5’-UTR from nucleotide 260 to 834 (3). In the deleted form the 3‘ 83 nucleotides of the 5‘-UTR of EMC are present. CAT (black box) is the gene encoding chloramphenicol acetyltransferase. It is used to determine the efficiency of translation of the second cistron. The plasmids were digested with BamHI and transcribed with T7 RNA polymerase.The open triangles represent the start codons and the black triangles the stop codons.

coding region followed by the EMC 5’-UTR (nucleotides 260834, for numbering see Ref. 3), and the chloramphenicol acetyltransferase gene (CAT). To show that the complete picorna 5’UTR is neededforCAT translation, a control construct was prepared in which the 5’ terminal 490 nucleotides of the EMC 5’-UTR were deleted (Fig. 2). T7 RNA polymerase-derived transcripts of the linearized DNAs weretranslated ina fully reconstituted cell-free protein synthesizing system (CFS) that gave the possibility to measure the effect of the addition of certain eukaryotic initiation factors. In the normally used reticulocyte lysate system it is only possible to measure a stimulatory effect on translation caused by the addition of initiation factors, because sufficient amounts of the initiation factors for efficient translation are present. RESULTS As can be seen in Fig. 3,the transcriptsbehaved as expected. The construction of bicistronic messengers has been de- Capped 4A translation resulted in a band of approximately scribed by various authors (1, 3, 5). These RNAs have been 44 kDa (lane2 ) and translation of EMCCAT in a protein of used to show internal initiation on picornaviral5’-UTRs and 26 kDa (lane6). The bicistronic messenger gave both proteins to show the importance of certain regions of these 5’-UTRs. (lane 4 ) when a capped transcript was used. Uncapped bicisIt was found that a large part of the 5’-UTR is needed, tronic 4AEMCCAT gavealmost exclusively translation of the approximately 500 nucleotides, to allow translation of the CAT protein (lane 9 ) . Deletion of most of the EMC 5’-UTR led in the monocistronic (lane5) as well as in the bicistronic downstream cistron in bicistronic transcripts. We created a bicistronic construct containing the 4AI- (lane3) transcript to analmost complete lossof CAT synthesis. Uncapped 4A and 4AAEMCCAT only led to thesynthesis *A. A. M. Thomas, G. C. Scheper, M. Kleijn, M. De Boer, and H. of a small amount of 4A, reflecting the need fora cap structure for efficient 5’ cap-dependent translation. This cap depend0. Voorma, submitted for publication.

Internal Initiation 5

of Translation Requires eIF-4E -4F and

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Addition of increasing amounts of A-cut resulted in an increase of both gene products as expected. 4A -m One of the initiation factors that plays a role in unwinding is eIF-4B (8, 9). This factor was tested in the CFS omitting CAT + the A-cut and supplementing eIF-3 and eIF-4F. As can be seen in Fig. 4 4 both cistronswere translated more efficiently FIG.3. Translation of mono- and bicistronic messengers. under conditions with higher eIF-4B concentrations. No difTranslations were performed with 30 ng of transcript and 0.5 pg of ferential effects were measured on 4A and CAT translation. A-cut/assay as described under “Materials and Methods.” RNA ad- The results show that both types of initiation have similar ditions were: lane 1, no RNA; lane 2, capped 4A; lane 3, capped 4AAEMCCAT; lane 4, capped 4AEMCCAT; lane 5, capped requirements for eIF-4B. It has been suggested that theinternal ribosomal entry site AEMCCAT; lane 6, capped EMCCAT; lane 7, uncapped 4A; lane 8, uncapped 4AAEMCCAT; lane 9, uncapped 4AEMCCAT; lane 10, of EMC allowed binding of the ribosomes close to the start uncapped EMCCAT. Molecular mass markers are indicated on the codon and therefore abolished the need for scanning (17). right side of the figure. Whether unwinding of the mRNA by eIF-4A was still necessary was tested in the CFS using an A- and BC-cut that were A B purified by elution from a Sepharose-heparin column (36). elF-4B A-cut eIF-4A is the only initiation factor (next to eIF-4D, which 1 2 3 4 5 1 2 3 4 5 - 45 has no effect in theseassays) that does not bind to theheparin 4A I - 40 resin (36). Therefore, translation with this A-cut and BC-cut - 29 was eIF-4A-dependent. If initiationon the EMC 5’-UTR CAT I wouldbe independent of eIF-4A, one should expect CAT c synthesis from the bicistronic transcript in the absence of 45 4A I - 40 added eIF-4A. However, lane 1 in Fig. 4C shows hardly any - 29 CAT product in the absence of eIF-4A, suggesting that the CAT1 EMC 5’-UTR actually did need eIF-4A for efficient transla1 2 3 4 5 6 7 8 9 1 0 tion. Increasing the amount of eIF-4A (lanes 2-5) resulted in elF-4A increased 4A synthesis as well as in increased CAT synthesis. FIG. 4. Effect of different initiation factor additions on the The eIF-4A titration on the uncapped bicistronic transcript Translation and fluorography demonstrates that almost no 4A is synthesized from the translation of capped 4AEMCCAT. was performed as described under “Materials and Methods.” Molec- uncapped transcript (Fig. 4C, lane 6-9). CAT synthesis inular mass markers are indicated on the right side of the figure. A , effect of an A-cut of a ribosomal salt wash. Amounts of A-cut added creased upon increasing amounts of eIF-4A, whereas 4A synper 5-pl assay: lane 1, none; lane 2, 0.05 pg; lane 3, 0.15 pg; lane 4, thesis only occurred at high eIF-4A concentrations (lane 10). 0.30 pg; lane 5, 0.50 pg. B, effect of eukaryotic initiation factor 4B. It can be concluded that both types of translation need eIFTranslation was performed in the absence of A-cut and in the pres- 4A. The requirement of the EMC 5’UTR for eIF-4A does not ence of 0.18 pg of eIF-4F and 0.8 pg of eIF-3 (per 5-pl CFS assay). contradict the result of Kaminski et ul. (17) as will be disAmount of eIF-4B added lane 1, none; lane 2, 0.03 pg; lane 3, 0.08 cussed later. A similar assay as described in Fig. 4 for eIF-4B pg; lane 4, 0.16 pg; lane 5, 0.27 pg. C, effect of eukaryotic initiation factor 4A on translation of capped and uncapped 4AEMCCAT. and for eIF-4A was performed with eIF-3. This factor also Translation was performed as described under “Materials and Meth- stimulated 4A and CAT synthesis equally well (not shown). eIF-4F is a three-subunit complex that has been shown to ods” with 0.8 pg of A-cut and 0.8 pg of BC-cut (per 5-pl assay), isolated by Sepharose-heparin purification (36). In lanes 1-5 capped play an important role in cap recognition and therefore in 4AEMCCAT was added, in lanes 6-10 the uncapped transcript (30 cap-dependent translation. Some picornaviruses block host ng/5-pl assay). Amount of eIF-4A added lanes 1 and 6 none; lanes 2 mRNA translation by cleaving p220 (37), the largest subunit and 7: 0.04 pg; lanes 3 and 8, 0.13 pg; lanes 4 and 9, 0.25 pg; lanes 5 of eIF-4F, leading to inactivation of its mRNA cap binding and 10, 0.42 pg. ability (19, 38, 39). The effect of eIF-4F on cap-dependent and -independent translation was tested in the CFS in the ence was also measured in reticulocyte lysates. In our work, presence of low amounts of an A-cut as source of the other under salt conditions optimal for globin mRNA translation, cap dependence is found for almost every mRNA or transcript. necessary factors eIF-3 and eIF-4B (Fig. 5). As expected the translation of the capped 4A transcript was The strong increase of translation induced by capping sugstimulated by the addition of eIF-4F (lunes 1-3). A similar gests that the majority of thetranscriptsare capped, in stimulation of 4A synthesis was found when this transcript accordance with previous data (30). Translation of uncapped AEMCCAT (not shown) is as was present as a monocistronic messenger together with inefficient as translationof the capped AEMCCAT transcript EMCCAT (lanes 7-9) or when the bicistronic transcripts were (lane 5 ) . Capping of the EMCCAT transcript did not change used (lanes 10-12 and 13-15). Surprisingly, eIF-4F had a clear thetranslation efficiency conferred by the EMC 5’-UTR stimulatory effect on the translation of (uncapped) EMCCAT (lanes 4-6). Apparently, eIF-4F does have a general stimula(lanes 6 and 10). In agreement with previous data (30)) the downstream tory effect on translation of capped and uncapped mRNAs. cistron was translated when the EMC 5’-UTR was used as This will be discussed in more detail below. In Fig. 5 it was also shown that thecombination of the two intercistronic region. The use of a bicistronic transcript gave the advantage that the effect of the initiation factors, on monocistronic messengers, 4A and EMCCAT (lanes 7-9), similar way asthe bicistronic messenger internal as well as on 5’ cap-dependent initiation, could be behaved ina 4AEMCCAT (lanes 10-12). The small difference in 4A synmeasured simultaneously. The A-cut of a ribosomal salt wash contains two initiation thesis is apparently due to the higher competitive ability of factors important for unwinding, eIF-4B and eIF-4F (8, 9). the EMC 5’-UTR for the translational apparatus (24) when Besides these two, also eIF-3 is present in this fraction. Fig. it is present in amonocistronic transcript. Translation of4AAEMCCAT (lanes 13-15) showed that 4A shows that the absence of A-cut led to a total absence of 4A translation and to a very low level of CAT synthesis. internal initiation needed the complete EMC 5’-UTR and 1

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Internal Initiation

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of the ODC protein (Fig. 6, lane 3 ) , and this synthesiscould be stimulated 2.5-fold by addition of eIF-4F (Fig. 6, lanes 3 and 4 ) . These assays were carried out in reticulocyte lysates, suggesting that for initiation on the CPMV 5'-UTR sufficient " amounts of eIF-4F were not present. ODC-71, a construct 1 2 3 4 5 6 ' 7 " 8' 9 with only 71 nucleotides of the 5'-UTR, resulting in a de4A + creasedsecondary structure and therefore in an increased ,e"- 4 5 - 40 translation efficiency (28), was also stimulated by the addition of eIF-4F in theseassays (Fig. 6, lanes 1 and 2 ) ,but toa lesser - 29 extent (1.5-fold). In conclusion, the 5'sequence of CPMV CAT + mRNA is another example of a 5'-UTR on which internal initiation is enhanced by the additionof extra eIF-4F. Besides the cap-binding subunit eIF-4E, eIF-4F contains FIG. 5. Dependence on eukaryotic initiation factor 4F of an eIF-4A-like subunit (eIF-4AI and eIF-4AII (41)) with an translation of mono- and bicistronic transcripts. Translation was performed with 0.2 pg of A-cut/5-pl assay as described under unwinding activity (8, 9) and a p220 subunit with unknown "Materials and Methods." Transcripts were added at 30 ng/5-p1 as function. It isexpected that the stimulatoryeffect was due to unknown p220 activity. follows. Lanes 1-3, capped 4A; lanes 4-6, uncapped EMCCAT, lanes either theunwinding activity or to an 7-9, capped 4A and uncapped EMCCAT; lanes 10-12, capped eIF-4E has been shown to be the cap-binding protein (424AEMCCAT; lanes 13-15, capped 4AAEMCCAT. The symbols under 44). It was expected that addition of eIF-4E to the capped the figure indicate the amount of eIF-4F added to the 5 pl assay; -, bicistronic transcript 4AEMCCAT would only lead to inno eIF-4F; +, 0.13 pgof eIF-4F; ++, 0.42pgof eIF-4F. Molecular creased synthesis of the 4A product. However, more surprismass markers are indicated on the right side of the figure. ingly than the eIF-4Fresults, eIF-4E addition resulted in an increase of the translation of the downstream cistron aswell 5 6 as of the upstream one (Fig. 7). The assays were performed with a small amount of A-cut (Fig. 7, lanes 1-6), because a t higher A-cutconcentrations(and thereforehigher eIF-4E concentrations), translation could not be stimulated by eIF4E addition (lanes 7 and 8 ) . At a low A-cut concentration, eIF-4E had a stimulatory effect on both gene-products (lanes 1-6). The possible mechanism by which eIF-4E resulted in an increased CAT synthesis will be discussed below. cap analogs inhibit cap-dependent translation (45). TransFIG. 6. Stimulation of internal initiation of tr anslation on lation of capped 4AEMCCAT in the presence of the cap the cowpea mosaic virus 5'-UTR by eIF-4F. Translation was done in reticulocyte lysates as described under "Materials and Meth- analog 7mGpppG led to a strong decrease of 4A synthesis (Fig. ods.'' In lanes 2, 4 , and 6 0.21 pg of eIF-4F per 5 pl assay was added 8, lane 2 ) and a slight increase of CAT synthesis from the as indicated. Transcripts (100 ng/5 pl) were added as follows: lanes 1 bicistronic messenger. Apparently, the addition of the cap and 2,ODC-71; lanes 3 and 4 , ODC-CPMV; lunes 5 and 6,ODC-243. analog led to an eIF-4Eincapable of initiating translation on The ODC bands were cut out from the gel and radioactivity deter- the cap, as amply documented (2, 6, 12, 45). It seems rather mined. Lane 1, 3644 cpm; lane 2, 5566 cpm; lane 3, 613 cpm; lune 4, 1541 cpm; lane 5, 185 cpm; lane 6, 178 cpm. The arrow at the right unlikely that aneIF-4E-cap analog complex is more active in side of the figure indicates the position of the ODC protein. Molecular internal initiation than eIF-4E itself, we therefore suggest that the stimulation of CAT synthesis is caused by an inmass markers are indicated on the left side. creased availability of the other factors to initiate translation thatreinitiationdidnottake place. The inability of the on the EMC 5'-UTR.As already shown in Fig. 7 the addition of both products deleted 5'-UTR to initiate translation (as Fig. in 3) could not of eIF-4E resulted in an enhanced translation (Fig. 8, lane 3 ) . Under conditions where the cap analog is be overcome by increasing amounts of eIF-4F. Apparently, present (Fig. 8, lane 4 ) , addition of eIF-4E no longer had a the 83 nucleotides of the EMC 5'-UTR remaining afterdeletion, present as the intercistronic region, do not respond to stimulatory effect on translationof either cistron.More than eIF-4F asa noncapped form of a cellular mRNA, as described 95% of the added eIF-4E will be bound by the 50 ~ L Mcap for globin mRNA (40). I t should be noted that translation of analog, based on association equilibrium constants obtained uncapped 4A transcripts could be enhanced by increasing amounts of eIF-3, eIF-4B, eIF-4F (not shown), eIF-4A or (Fig. 4C) in accordance with the results of Fletcher et al. (40) - 45 4 A -b obtained with uncappedglobin mRNA. - 40 Our result with eIF-4Fsuggests that this factorplays a role in internal initiation on EMC RNA. A similar conclusion was drawn for the poliovirus 5'-UTR (25). Also, we have shown -29 that translation initiation on CPMV mRNA occurs by a capCAT -+ independentinternalentrymechanism (26). Instead of a bicistronic messenger, a construct was used containing the elF-4E -+ - + -+ - + major part of the ornithinedecarboxylase (ODC)5'-UTR (28) FIG. 7. eIF-4E stimulates translation of both cistrons on upstream of a part of the CPMV mRNA 5' sequence (nucleotides 161-512), followed by the ODC coding region. This capped 4AEMCCAT. Translation was done in 5-pl CFS assays as under "Materials andMethods." Amount of A-cut per assay: ODC 5'-UTRhas a very stable secondary structure(28), described lanes 1 and 2: none; lanes 3 and 4 , 0.1 pg; lanes 5 and 6, 0.3 pg; lanes preventing translation initiation directed bya 5' cap structure 7 and 8,0.5 pg. In the lanes indicated with +, 1 ng of eIF-4E was (Fig. 6, lane 5). The insertionof the CPMVsequence between added; - indicates no addition of eIF-4E. Molecular mass markers the ODC 5'-UTR and the coding region led to the synthesis are indicated on the right side of the figure. L

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FIG. 8. Effect of cap-analog addition on the translation of capped 4AEMCCAT. Translations were performed in CFS with 30 ng of transcript in the presence of 0.1 pg ofA-cut/5-pl assay. Additions were: lane 1, none; lane 2, 50 p~ ‘mGpppG; lane 3, 1 ng of eIF-4E; lane 4, 50 p~ ‘mGpppG 1 ng of eIF-4E. Molecular mass markers are indicated on the right side of the figure.

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Capped ODC-131 and globin mRNA are examples of hosttype messengers which were p220-dependent as expected (Fig. 9, lanes 1-4). Also thetranslation of 4A, in capped 4AEMCCAT (lanes 7 and 8), was sensitive to p220 cleavage as was capped TPT-CAT (lanes 9 and 10).This last result with theTPT 5’-UTR of adenovirus shows that theTPT 5’UTR behaved like a host-type mRNA in in uitro translations? The two 5’-UTRs capable of internalinitiation,EMCin capped 4AEMCCAT (lanes 7-8) and CPMV in ODC-CPMV (lanes 11-12), were resistant to p220 proteolysis as expected from internal initiation studies with the EMC 5’-UTR in poliovirus-infected cells (4). Although the EMC and CPMV 5’-UTRs require eIF-4F for efficient translation (Figs. 5 and 6), these 5’UTRs were not dependent on intactp220 (Fig. 9). DISCUSSION

The experiments described herein were performed to find differences and similarities between cap-dependent and -independent initiation of protein synthesis. Although, theoretically, the two mechanisms seem to differ significantly, especially in the need for cap-binding initiation factors, we did not find any major differences. All of the known initiation 1 2 3 4 5 6 7 S 9 1 0 1112 1 3 1 4 factors present in an A-cut of a ribosomal salt wash, eIF-3 (not shown), eIF-4B(Fig. 4B), and eIF-4F(Fig. 5), stimulated protein synthesis of the upstream gene in a bicistronic mes+ ODC senger as well as of the downstream coding region. Also eIF+ 4A 4A, the protein with RNA-unwinding activity (8, 9), stimulated both initiations(Fig. 4C). Most surprisingly, eIF-4E also stimulated translationof the cistron,downstream of the EMC 5’-UTR (Fig. 7). eIF-4E is known as the cap-binding protein 4- CAT and has as far as known no other functions in translation 4-L initiation. eIF-4A and eIF-4B areactive in unwinding the mRNA (8, 9) to give rise to a stretch of single-strandedRNA that facilitates binding of the ribosome and recognition of the +globin initiator AUG. Kaminski et al. (17) showed that the EMC - t - t - + - t - + - + + start-codon is not selected bya scanning mechanism and they proposed that the ribosome “landed” on the start codon or FIG. 9. p220 dependence of translation of different messengers. Transcripts were translated in rabbit reticulocyte lysate as very close to it. If unwinding of the mRNA is not necessary described under “Materials and Methods.” In lanes I , 3, 5, 7, 9, 11, for this direct binding of the ribosomes on or close to the and 14, lysates were preincubated without exogenous RNA for 25 initiator AUG, this would abolish the need for eIF-4B and min. In lanes 2, 4, 6, 8, 10, 12, and 13, preincubation was with L eIF-4A. In our experiments it was shown clearly (Figs. 4, B transcript for 25 min to allow synthesis of L-protease and cleavage of and C) that the two factors stimulated protein synthesis of p220. The following messengers were added after the 25-min preincubations. Lanes I and 2, capped ODC-131; lanes 3 and 4 , globin both genes. Apparently, although no ribosomal scanning ocmRNA; lanes 5 and 6, EMCCAT; lanes 7 and 8, capped 4AEMCCAT; curs at the EMC 5’-UTR,unwinding factors are stillneeded lanes 9 and 10, capped TPT-CAT, lanes 11 and 12, capped ODC- to create a single-stranded region on the mRNA that allows CPMV; lanes 13 and 14, no mRNA. Forunknown reasons, the internal entryof the ribosome on or close to the startcodon. translation of L transcript alone (lane 13) was less efficient than It hasbeen shown that eIF-4F can stimulate translation of when another transcript was included in the assay. Migration of the uncapped satellite tobacco necrosis virus mRNA (40). In our proteins is indicated on the right side of the figure. Globin migrated slightly slower than the frontof the gel into a diffuse band (lane 3). experiments eIF-4F not only stimulated translation of the uncapped transcripts (not shown) butalso stimulated transwith fluorescent studies (44). Addition of cap analog as well lation of the downstream cistron (Fig. 5). Recently was reas eIF-4E resulted in a decreased 4A synthesis andagain in a ported that internal initiation on TKIPPCAT (1) was also slight stimulation of CAT production (as in lane 2). The stimulated by eIF-4F (25). In those experiments no stimulatory effect was found for eIF-4A and eIF-4B, but this may be absence of eIF-4E stimulation on internal initiation in the due to the fact that rabbitreticulocyte lysates were used, in presence of cap analogwill be discussed below. The effect of eIF-4F and eIF-4E on translation was shown which these factors are present in nonlimiting amounts. The in Figs. 5 and 7. An in uitro assay was developed to investigate stimulation caused by eIF-4F might be due to an unknown the role of p220 and itscleavage products in translation. This function of p220 or may be caused by the unwinding activity assay will be described in detail elsewhere.’ In short, rabbit presentineIF-4F (9). Moreover, eIF-4E was also able to reticulocyte lysate was preincubated withoutadded transcript stimulate both types of initiation (Fig. 7). The cap binding or with a transcript that encodes the L-protease of foot-and- activity of eIF-4E iswell documented, but apparentlyeIF-4E mouth disease virus (21). Subsequent addition of different can also have a function in the internal initiationof translakinds of transcripts allows the determinationof the influence tion. Putting all these translation results together we suggest of L-induced p220 proteolysis on translationof these mRNAs that translation occurs in its most efficient way when p220, (Fig. 9). ~

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7274

Internal Initiation of Translation Requires eIF-4E and -4F

eIF-4A, and eIF-4E assemble in a eIF-4F complex, in accordance with the model of Rhoads (46). Omissionof eIF-4E leads to a decreased activity of p220 and eIF-4A, possibly by the inability of the lattertwo to perform their function. Therefore, addition of eIF-4E will lead to a more efficient assembly of this complex. Apparently, an added eIF-4E cap analog complex (Fig. 8, lane 4 ) was unable to assemble into a functional initiation complex. Furthermore, the requirement for eIF-4F in internal initiation shows that assembly of eIF-4E into eIF4F is not dependent on the presence of a capped mRNA. Whether other initiationfactors, such as eIF-3, bind to eIF4F to form a larger multifunctional complex wassuggested by the experiments of others (37, 47-49). In uiuo, p220 cleavage leads to the loss of eIF-4E from a fast sedimenting complex of eIF-3 andproteolyzed eIF-4F (37). Our results suggest that translation of all mRNAs would be affected because of this loss of eIF-4E. Nonetheless, sucrose gradient centrifugation will probably destroy the interaction between eIF-3 and proteolyzed eIF-4F. Moreover, others have isolated eIF-4F containing eIF-4Eand thep220 degradation products, from poliovirus-infected cells (20). It follows from these remarks that we suggest that the degradation products of p220 remain bound to eIF-4A and eIF-4E to exert a function in internal initiation. Although p220 cleavage abrogates the cap binding activity of eIF-4F, the cleavage does not inactivate other eIF4F functions. Apparently, the intactness of p220 determines whether the cap binding by eIF-4E results in protein synthesis. Besides the sensitivity toward addition of capanalog (Fig. 8),the only difference found between the two mechanisms is the sensitivity to p220 cleavage(Fig. 9). The major difference between cap-dependent and -independent initiation is that entry of the ribosome oncapped mRNA is directed by the cap binding activity of eIF-4F which stimulates mRNA unwinding by eIF-4A and eIF-4B; in internal initiation, direct binding of unwinding factors, including eIF-4F, occurs. These modes of entry of the ribosome on the mRNA are in agreement with a recently proposed theory (16). Whether the peculiar secondary structure of picornaviral 5’-UTRs allows direct binding of unwinding factors or that trans-acting factors like p52 (50) or p57 (51) induce this binding has yet to be established. Acknowledgments-We thank Marcelle Kasperaitis and Cor van der Mastfor their help with the purification of some of the initiation factors and the protein synthesis assay, Miranda Kleijn and Lotte van Aalzum for their contribution to Figs. 6 and 9, Berwyn Clarke (Wellcome, Beckenham, Great Britain) for his gift of pTC6, and Peter Nielsen (Freiburg, Federal Republic of Germany) for pTZ4AI.

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