Characterization of a novel RNA-binding region of eIF4GI critical for ribosomal scanning
2003; Springer Nature; Volume: 22; Issue: 8 Linguagem: Inglês
10.1093/emboj/cdg175
ISSN1460-2075
AutoresDéborah Prévôt, Didier Décimo, Cécile H. Herbreteau, Florence Roux‐Dalvai, Jérôme Garin, Jean‐Luc Darlix, Théophile Ohlmann,
Tópico(s)RNA Research and Splicing
ResumoArticle15 April 2003free access Characterization of a novel RNA-binding region of eIF4GI critical for ribosomal scanning Déborah Prévôt Déborah Prévôt LaboRétro, Inserm Unité de Virologie Humaine (U 412), Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, cedex 07, France Search for more papers by this author Didier Décimo Didier Décimo LaboRétro, Inserm Unité de Virologie Humaine (U 412), Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, cedex 07, France Search for more papers by this author Cécile H. Herbreteau Cécile H. Herbreteau LaboRétro, Inserm Unité de Virologie Humaine (U 412), Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, cedex 07, France Search for more papers by this author Florence Roux Florence Roux CEA-Grenoble, 17 Avenue des Martyrs, 38041 Grenoble, France Search for more papers by this author Jérôme Garin Jérôme Garin CEA-Grenoble, 17 Avenue des Martyrs, 38041 Grenoble, France Search for more papers by this author Jean-Luc Darlix Jean-Luc Darlix LaboRétro, Inserm Unité de Virologie Humaine (U 412), Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, cedex 07, France Search for more papers by this author Théophile Ohlmann Corresponding Author Théophile Ohlmann LaboRétro, Inserm Unité de Virologie Humaine (U 412), Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, cedex 07, France Search for more papers by this author Déborah Prévôt Déborah Prévôt LaboRétro, Inserm Unité de Virologie Humaine (U 412), Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, cedex 07, France Search for more papers by this author Didier Décimo Didier Décimo LaboRétro, Inserm Unité de Virologie Humaine (U 412), Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, cedex 07, France Search for more papers by this author Cécile H. Herbreteau Cécile H. Herbreteau LaboRétro, Inserm Unité de Virologie Humaine (U 412), Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, cedex 07, France Search for more papers by this author Florence Roux Florence Roux CEA-Grenoble, 17 Avenue des Martyrs, 38041 Grenoble, France Search for more papers by this author Jérôme Garin Jérôme Garin CEA-Grenoble, 17 Avenue des Martyrs, 38041 Grenoble, France Search for more papers by this author Jean-Luc Darlix Jean-Luc Darlix LaboRétro, Inserm Unité de Virologie Humaine (U 412), Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, cedex 07, France Search for more papers by this author Théophile Ohlmann Corresponding Author Théophile Ohlmann LaboRétro, Inserm Unité de Virologie Humaine (U 412), Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, cedex 07, France Search for more papers by this author Author Information Déborah Prévôt1, Didier Décimo1, Cécile H. Herbreteau1, Florence Roux2, Jérôme Garin2, Jean-Luc Darlix1 and Théophile Ohlmann 1 1LaboRétro, Inserm Unité de Virologie Humaine (U 412), Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, cedex 07, France 2CEA-Grenoble, 17 Avenue des Martyrs, 38041 Grenoble, France *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:1909-1921https://doi.org/10.1093/emboj/cdg175 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The eukaryotic translation initiation factor eIF4GI binds several proteins and acts as a scaffold to promote preinitiation complex formation on the mRNA molecule (48S). Following mRNA attachment this complex scans along the messenger in a 5′ to 3′ direction until it locates and recognizes the initiation start codon. By using a combination of retroviral and picornaviral proteases (HIV-2 and L respectively) in the reticulocyte lysate system, we have characterized a 40 amino acid (aa) region of eIF4GI (aa 642–681) that exhibits general RNA-binding properties. Removal of this domain by proteolytic processing followed by translational assays showed virtually no inhibition of internal ribosome entry on the encephalomyocarditis virus, but resulted in drastic impairment of ribosome scanning as demonstrated by studying poliovirus and foot-and-mouth disease virus translation. Based on these findings, we propose that this 40 aa motif of eIF4GI is critical for ribosome scanning. Introduction Translational control is a major contributor to the regulation of gene expression in eukaryotes, the initiation step being the primary determinant in controlling the rate of protein synthesis (reviewed in Preiss and Hentze, 1999). The initiation of translation in eukaryotes is a complex process involving many different eukaryotic initiation factors (eIFs) (Merrick, 1992; Gray and Wickens, 1998; Gingras et al., 1999b; Preiss and Hentze, 1999; Pestova et al., 2001). Among them, eIF4GI plays a central role in the assembly of the preinitiation complex, acting as a scaffold protein that interacts with several other initiation factors (Hentze, 1997; Morley et al., 1997; Dever, 1999). The N-terminal one-third of eIF4GI has the binding site for eIF4E, the cap-binding protein (Lamphear et al., 1995; Gingras et al., 1999a), and also interacts with the poly(A)-binding protein (PABP) (Imataka et al., 1998). The C-terminal region of eIF4GI binds the RNA helicase eIF4A (Lamphear et al., 1995; Morino et al., 2000; Korneeva et al., 2001) and also interacts with the MAPK-signal integrating kinase-1 (Mnk-1), a specific eIF4E kinase (Pyronnet et al., 1999). The central region of eIF4GI, described as the most important for translation initiation (Morino et al., 2000), contains another eIF4A-binding site (Imataka and Sonenberg, 1997), an eIF3-binding site (Lamphear et al., 1995; Korneeva et al., 2000) and one or several RNA-binding domains (Pestova et al., 1996; Lomakin et al., 2000). Thus, eIF4GI, eIF4E and eIF4A assemble the eIF4F complex (Gingras et al., 1999b), which interacts with the 40S ribosomal subunit via eIF3 (Trachsel et al., 1977) to form the 43S complex (also containing eIF5, eIF2, GTP and Met-tRNAi; Asano et al., 2000). The 43S complex then binds the mRNA molecule at the 5′ capped extremity to become the 48S complex and moves along in a 5′ to 3′ direction (scanning process) until it encounters the initiation codon. Eukaryotic cells can use an alternative mechanism of translation initiation that was first reported for members of the picornavirus family such as poliovirus (PV), encephalomyocarditis virus (EMCV) and foot-and-mouth disease virus (FMDV). This mechanism, called internal initiation, is dependent upon an RNA structure named internal ribosome entry segment (IRES) that allows translation by direct ribosome binding within the 5′-untranslated region (UTR) (Jackson and Wickens, 1997; Sachs and Varani, 2000). Following internal entry at the AUG triplet at the 3′ end of the IRES, ribosomes initiate translation at this site for cardiovirus IRES (e.g. EMCV), while in the case of entero/rhinovirus IRES (e.g. PV) initiation takes place at the next AUG codon, which is reached by ribosome scanning from the original entry site. Initiation of translation on aphthovirus IRES occurs both at the landing site and the next downstream AUG (Jackson and Kaminski, 1995). During translation of IRES-containing mRNAs, eIF4GI directly interacts with mRNA (Pestova et al., 1996; Kolupaeva et al., 1998; Lopez de Quinto and Martinez-Salas, 2000; Saleh et al., 2001) and associates with eIF4A promoting 48S complex assembly (Lomakin et al., 2000). Infection of cells with FMDV or PV results in a rapid inhibition of host cell protein synthesis. This inhibition has been attributed, at least in part, to the rapid cleavage of eIF4GI and the delayed cleavage of eIF4GII (Etchison et al., 1982; Gradi et al., 1998), by L (from FMDV) and 2A (from PV) picornaviral proteases. Proteolysis of eIF4GI occurs between amino acids (aa) 634–635 (L protease) and 641–642 (2A protease), which segregates the N-terminal one-third of eIF4GI, containing the eIF4E-binding site, from the C-terminal two-thirds of the molecule, thereafter named p100 (635 or 642 to 1560; Lamphear et al., 1995). Translation of capped mRNAs is strongly inhibited upon this cleavage (Etchison et al., 1982), whereas the C-terminal two-thirds of eIF4GI (p100) is able to support and even stimulate translation of IRES-driven mRNAs (Borman et al., 1997) by direct binding to the IRES (Pestova et al., 1996). Besides IRES initiation, translation of uncapped mRNA has also been studied in vitro (Ohlmann et al., 1995; De Gregorio et al., 1998) and in vivo (Gunnery and Mathews, 1995; Thoma et al., 2001). Very little is known about this mechanism, except that it involves binding of the 43S complex at the 5′ end of the mRNA followed by linear scanning of the 5′-UTR (Gunnery et al., 1997; De Gregorio et al., 1998). The factors mediating ribosome attachment and progression of the 48S complex on the uncapped RNA have not been clearly defined. However, this process is strongly enhanced by cleavage of endogenous eIF4GI by picornaviral proteases or by addition of recombinant p100 fragment (Ohlmann et al., 1995; Borman et al., 1997; De Gregorio et al., 1998) suggesting that the C-terminal part of eIF4GI plays a role in mediating ribosome binding or ribosome scanning or both. In this report, we use the protease (PR) from HIV-2 and show that, like HIV-1 PR, it can process eIF4GI into two C-termini fragments due to recognition of two cleavage sites. The cleavage site yielding the larger fragment (named Ch-1 thereafter) is located 47 aa downstream of the L proteolytic site; this fragment is further cleaved by addition of higher doses of protease. Thus, we have used the HIV-2 PR as a tool to investigate the role of these fragments in the translation of capped, uncapped and IRES-driven mRNAs. Our results show that, while capped and uncapped mRNAs translation was severely inhibited by HIV-2 PR-mediated cleavage of eIF4GI, translation driven by the EMCV IRES was marginally affected. By using UV-crosslinking assays we were able to show that a 40 aa region which is present on p100 but absent on the Ch-1 fragment binds RNA. Moreover, in vitro translation with RNA constructs driven by IRESes from picornaviral origin revealed that this eIF4GI RNA-binding domain is critical in the progression of the 48S complex to the AUG codon. Taken together, these results suggest that eIF4GI is not only involved in 43S complex formation on the mRNA but has a critical role in ribosome scanning. Results Recombinant HIV-2 PR cleaves eIF4GI from rabbit reticulocyte lysate We have previously shown that the HIV-1 PR was able to cleave eIF4GI resulting in an inhibition of translation in the rabbit reticulocyte lysate (RRL; Ohlmann et al., 2002). Thus, we have investigated the cleavage of eIF4GI by HIV-2 PR. Incubation of RRL with recombinant HIV-1 PR or HIV-2 PR showed that eIF4GI was cleaved by the two enzymes, in a dose-dependent manner (Figure 1). In both cases, eIF4GI was fully processed with 2.5 ng/μl retroviral protease (lanes 3 and 8), leading to the appearance of two fragments recognized by C-terminus-directed antibodies (epitope E, see Figure 2B). These fragments migrate at ∼100 kDa (fragment named Ch-1 for C-terminal HIV PR-resulting fragment 1) and 55 kDa (Ch-2) on SDS–PAGE, suggesting that HIV-1 PR and HIV-2 PR share the same cleavage sites on the C-terminal region of eIF4GI. Interestingly, the amount of Ch-1 generated upon cleavage of eIF4GI was much higher with the HIV-2 PR (compare lanes 2–5 with lanes 7–9), with Ch-2 being detected only with a high amount of enzyme added (compare lanes 6 and 9). This could be due to a weaker activity of the HIV-2 recombinant protease, but complete processing of eIF4GI and the fact that the Ch-1 fragment is still present at high doses of HIV-2 PR seem to rule out this possibility. Figure 1.eIF4GI is cleaved by recombinant HIV-1 and HIV-2 proteases. RRL (10 μl) was incubated with buffer (lane 1) or increasing amounts of recombinant HIV-1 PR (lanes 2–5: 0.25, 1.25, 5 and 12.5 ng/μl) or HIV-2 PR (lanes 6–9: 0.5, 2.5, 10 and 25 ng/μl) for 1 h at 30°C in a final volume of 20 μl. A 1 μl aliquot was resolved on 10% SDS–PAGE, proteins transferred to PVDF and the membrane was incubated with antibodies specific to the C-terminal part of eIF4GI (serum E, see Figure 2B). The resulting fragments and molecular weight markers (in kDa) are indicated on the figure. Download figure Download PowerPoint Figure 2.Characterization of the C-terminal cleavage sites of eIF4GI. (A) RRL (10 μl) was incubated without (lane 1) or with L protease (1 μl, lanes 2–4) or HIV-2 PR (10 ng/μl, lanes 5 and 6), for 1 h at 30°C in a final volume of 20 μl. HIV-2 PR (20 ng/μl; lane 4) or 10 ng/μl HIV-1 PR (lanes 3 and 6) were then added and the mixture further incubated for 1 h. The samples were analysed by SDS–PAGE and western blotting as described in Figure 1. (B) The eIF4GI molecule is schematically represented with its different interaction domains for PABP and eIF4E (Gingras et al., 1999b), eIF4A (Lomakin et al., 2000; Morino et al., 2000), eIF3 (Korneeva et al., 2000), Mnk-1 (Morino et al., 2000) and the EMCV IRES (Lomakin et al., 2000). Cleavage sites for L/2A and HIV proteases, the obtained C-terminal fragments and the E epitope used for the western blot analysis are represented. Download figure Download PowerPoint Mapping the HIV-2 PR cleavage sites on eIF4GI These results suggest that HIV-2 PR could present a stronger affinity for the cleavage site yielding Ch-1 rather than Ch-2 (HIV-1 PR would have the opposite preferences). However, it is also possible that cleavage of eIF4GI by HIV-2 PR would lead to a Ch-1 fragment protected in some way from further digestion, this protection being inefficient when the cleavage is generated by HIV-1 PR. Therefore, in order to better characterize the fragments of eIF4GI generated upon cleavage by HIV-2 PR, we have used a combination of three different viral proteases in the RRL: FMDV L, HIV-1 PR and HIV-2 PR. As shown in Figure 2A, the p100 fragment obtained with L protease can be cleaved further in Ch-1 and Ch-2 by HIV proteases (compare lanes 3 and 4 with lane 2). This shows that the HIV-2 PR cleavage site is downstream of the L protease site and that the p100 fragment is not protected from further digestion by HIV proteases. It is important to note that, at this dose of HIV-1 PR, the Ch-1 species is barely detectable (lane 3). When RRL was first incubated with HIV-2 PR and then with HIV-1 PR, the Ch-1 fragment was further processed into Ch-2 (Figure 2A, compare lanes 5 and 6), indicating that the Ch-1 fragment generated by HIV-2 PR was not protected from further proteolysis by HIV-1 PR. This indicates that HIV-1 PR and HIV-2 PR present different affinities for the two cleavage sites of the eIF4GI substrate. We and others have previously characterized the 55 kDa fragment (Ch-2) resulting from HIV-1 PR cleavage (Ventoso et al., 2001; Ohlmann et al., 2002), which corresponds to the very C-terminal domain of eIF4GI (aa 1087–1560). In addition, Ventoso and colleagues have also located two cleavage sites at positions 678–679 and 681–682 that yield the Ch-1 fragment (Ventoso et al., 2001). Thus, cleavage products generated by the HIV-2 PR were subjected to a mass spectrometry analysis. This analysis set out that the cleavage site leading to Ch-2 was located between aa 1086–1087, as already established for HIV-1 PR. Although we were unable to precisely identify the cleavage site leading to Ch-1, peptide analysis indicated that the latter was located between aa 670 and 707. Given that (i) this cleavage site is situated downstream of that of L protease, (ii) processing occurs between aa 670 and 707, (iii) cleavage of eIF4GI by HIV-1 PR and HIV-2 PR yields fragments migrating at the same size on SDS–PAGE, and (iv) HIV-1 PR and HIV-2 PR share the same cleavage site at position 1086; it is very likely that HIV-2 PR and HIV-1 PR both proteolyse eIF4GI at the same sites (aa 678–679 and 681–682) to yield Ch-1. At the highest amount of protease Ch-1 is further proteolysed into a middle fragment (Mh aa 679 or 682–1085) and Ch-2 (aa 1086–1560). It should be noted that this study was carried out with antibodies specific to the C-terminal region of eIF4GI (epitope 1139–1166) thus we could not detect the Mh fragment in western blot analysis, but we were able to visualize it by cleaving radiolabelled in vitro translated eIF4GI (data not shown). Figure 2B shows a representation of the eIF4GI molecule with its functional domains and the positions of the different cleavage sites. Impact of HIV-2 PR on translation in the RRL Our previous study in RRL showed that eIF4GI cleavage by the HIV-1 PR resulted in the inhibition of capped, uncapped and IRES-driven mRNA translation (Ohlmann et al., 2002). Although both HIV-1 PR and HIV-2 PR cleave eIF4GI at the same sites, the relative amount of cleavage products (Ch-1 and Ch-2) generated is rather different. Therefore, the next step was to investigate the impact of HIV-2 PR on in vitro translation. Translation was programmed with various mRNAs, including natural capped globin and an uncapped bicistronic construct coding for cyclin B2 and the NS protein of influenza driven by the EMCV IRES. As shown in Figure 3, addition of HIV-2 PR resulted in a dramatic inhibition of translation of capped (A) and uncapped (B) mRNAs, but only had a moderate effect on EMCV IRES-driven translation (B). Strikingly, translation of capped globin and uncapped cyclin B2 was almost abolished with the highest amount of protease (Figure 3A and B, lanes 4), while synthesis of NS was only partially impaired (Figure 3B, lane 4). At the lowest dose of protease used, the contrast between uncapped and IRES-driven translation was clear since uncapped cyclin translation was inhibited by 50%, whereas EMCV IRES translation was only slightly, if at all, impaired (Figure 3B, lane 2). To detect any non-specific effects due to general damage to the translation machinery, we have also employed a bicistronic mRNA construct containing the IRES of hepatitis C virus (HCV), which does not require eIF4GI for activity (Pestova et al., 1998b). Addition of HIV-2 PR strongly inhibited translation of the first cistron but did not affect HCV translation (Figure 3C), indicating no alteration of other key components of the translational apparatus at the amount of protease used. HCV-driven translation even shows a slight stimulation, which may reflect an increased availability of general translation factors after cleavage of eIF4GI by HIV-2 PR. Figure 3.HIV-2 PR abolishes capped and uncapped mRNAs translation, but moderately inhibits EMCV IRES-driven translation. (A, B and C) A RRL under full translation conditions (see Materials and methods) was pre-incubated without (lanes 1) or with different amounts of HIV-2 PR (lanes 2–4: 3.5, 7 and 14 ng/μl). After 1 h at 30°C, Palinavir (10 μM final concentration) was added to the reactions. Different transcripts (schematically represented on the upper part of each panel) including (A) natural capped globin mRNA (2.5 ng/μl), (B) XL–EMCV mRNA (10 ng/μl) and (C) DC–HCV mRNA (10 ng/μl) were translated under these conditions. Samples were processed on 15% SDS–PAGE, submitted to autoradiography, the relative intensities of the bands were quantified and the results are presented at the bottom of each panel. (D) Aliquots of the samples 1–4 from (A) were resolved on 10% SDS–PAGE, proteins transferred to PVDF and the membranes were incubated with antibodies specific to the C-terminal part of eIF4GI (serum E). The resulting fragments and molecular weight markers (in kDa) are indicated on the figure. Download figure Download PowerPoint Western blot analysis was performed to visualize the impact of HIV-2 PR on eIF4GI in these translation reactions (Figure 3D). As expected, inhibition of capped globin mRNA correlated with the disappearance of intact eIF4GI (Figure 3A and D, compare lanes 2 and 3). However, uncapped cyclin translation was inhibited at low amounts of protease, when eIF4GI was only partially cleaved into the Ch-1 fragment (Figure 3B and D, compare lanes 1 and 2). This is rather surprising given the similarities between Ch-1 and the p100 fragment (see Figure 2B), which has been previously shown to stimulate translation of uncapped mRNAs (Ohlmann et al., 1995). On the contrary, inhibition of IRES-driven translation was only marginal upon cleavage of eIF4GI into Ch-1 (Figure 3B and D, compare lane 1 with 2 and 3), but was increased upon further cleavage of eIF4GI and detection of the Ch-2 species (lanes 3 and 4). These results suggest that cleavage of eIF4GI by the HIV-2 PR has different consequences on translation of capped, uncapped and IRES-driven mRNAs. While capped and uncapped translation is dramatically inhibited by processing of eIF4GI, it appears that IRES-driven initiation is not. The inhibition of translation by HIV-2 PR is due to eIF4GI cleavage We wondered if HIV-2 PR could cleave other factors implicated in the general translation mechanism but unnecessary to HCV IRES initiation. To rule out this possibility, we incubated RRL with HIV-2 PR and then performed western blot analysis using antibodies against eIF1A, eIF3, eIF4A, eIF4B, eIF4E and the C-terminal region of eIF4GI. As shown in Figure 4A, no proteolysis could be detected with HIV-2 PR except the expected cleavage of eIF4GI at the doses of proteases we used. In this figure, it is important to note that we observed a doublet with the anti-eIF4A antibody, which may correspond to eIF4AI and eIF4II as suggested previously (Belsham et al., 2000). Moreover, as the p170 subunit of eIF3 was detected with an antibody directed against the whole eIF3 complex and eIF4GI, the cleavage of the latter can be observed on the blot. As our antibody against eIF1 recognized only the human protein, we performed a similar analysis with a lysate of HeLa cells. As shown in Figure 4B, no cleavage of eIF1 could be observed at a dose of HIV-2 PR where no intact eIF4GI can be detected (lane 5). Figure 4.Inhibition of translation observed with HIV-2 PR is due to cleavage of eIF4GI (A and B). (A) RRL (10 μl) or (B) HeLa lysate (127.5 μg) was incubated with buffer (lane 1) or increasing amounts of recombinant HIV-2 PR (lanes 2–5: 2.5, 5, 10 and 25 ng/μl) for 1 h at 30°C in a final volume of 20 μl. Aliquots were resolved on SDS–PAGE, proteins transferred to PVDF and the membranes were incubated with antibodies specific to eIF1, eIF1A, eIF3, eIF4A, eIF4B, eIF4E and the C-terminal part of eIF4GI, as indicated on the left side of each panel. For eIF4GI, resulting fragments and molecular weight markers (in kDa) are indicated on the figure. (C) RRL under full translation conditions was pre-incubated without (lanes 1 and 2) or with (lanes 3 and 4) 7 ng/μl HIV-2 PR. After 30 min at 30°C, Palinavir (10 μM final concentration) was added to the reactions with 1 ng/μl recombinant p100 fragment (lanes 2 and 4) or buffer (lanes 1 and 3). Uncapped XL–EMCV mRNA (10 ng/μl) was then translated under these conditions. Samples were processed on 15% SDS–PAGE, submitted to autoradiography, the relative intensities of the bands were quantified and the results are presented at the bottom of the figure. Download figure Download PowerPoint In addition to checking the integrity of several initiation factors, we have also performed rescue experiments of uncapped and IRES-driven mRNA translation with a recombinant p100 C-terminal fragment. This fragment has been shown to support, and even stimulate, uncapped and IRES-driven translation (Ohlmann et al., 1995; Borman et al., 1997). For this, RRL was first submitted to HIV-2 PR, then the action of the enzyme was neutralized by the peptidomimetic Palinavir, a specific inhibitor of HIV proteases (Lamarre et al., 1997). Recombinant p100 fragment was added and translation of bicistronic mRNA encoding cyclin B2 (uncapped 5′-UTR-driven) and NS (EMCV IRES-driven) was monitored (Figure 4C). Whereas inhibition of translation was effective after pre-incubation with the HIV-2 PR (lane 3), addition of p100 restored translation of uncapped cyclin B2 as well as the EMCV IRES-driven cistron (lane 4). These results show that the translation inhibition of uncapped mRNA can be relieved by addition of recombinant p100 fragment, suggesting that this inhibition is due to eIF4GI processing. 'Clipping' of p100 to Ch-1 by HIV-2 PR leads to an inhibition of cap-independent translation At this stage, it appears that Ch-1 and p100 exhibit very different properties on uncapped and IRES-driven mRNAs translation. This is particularly interesting as Ch-1 and p100 are very similar in sequence except for the very N-terminal end of the fragment (aa 635–681). In an attempt to understand the role of this region, we have examined the effects of sequential cleavage of eIF4GI by both L and HIV-2 proteases. For this, endogenous eIF4GI from the RRL was processed by L protease to yield the p100 fragment, which was then further cleaved by HIV-2 PR. Translation of a bicistronic mRNA coding for cyclin B2 (uncapped 5′-UTR-driven) and NS (EMCV IRES-driven) was performed in RRL (Figure 5A), and western blot analysis was carried out in parallel to monitor conversion of p100 into the shorter Ch-1 fragment (Figure 5B). Whereas cleavage of eIF4GI into the p100 fragment by the FMDV L protease induced activation of uncapped mRNA translation and no change or slight stimulation of IRES-driven translation (Figure 5A, lane 5), the conversion of p100 into Ch-1 by HIV-2 PR led to a dramatic reduction of uncapped cyclin mRNA translation (20-fold inhibition), with only a moderate effect on IRES-dependent translation (2-fold inhibition; Figure 5A compare lanes 5 and 8). Interestingly, levels of translation observed with the highest dose of HIV-2 PR are the same whether RRL was first incubated with L protease or not (Figure 5A, compare lanes 4 and 8), corresponding to complete processing of either eIF4GI or p100 by HIV-2 PR (Figure 5B, compare lanes 4 and 8). An interesting feature is that uncapped translation only collapsed when processing of the p100 fragment was complete (Figure 5A and B, compare lanes 7 and 8), suggesting that small amounts of p100 fragment are sufficient to promote uncapped mRNA translation. Figure 5.'Clipping' of p100 by HIV-2 PR inhibits uncapped translation. (A) RRL under full translation conditions was pre-incubated without (lanes 1–4) or with (lanes 5–8) 0.5 μl of L protease for 20 min at 30°C. Either buffer (lanes 1 and 5) or 1.75 ng/μl (lanes 2 and 6), 3.5 ng/μl (lanes 3 and 7) and 7 ng/μl (lanes 4 and 8) of HIV-2 PR were then added to the reactions. After 1 h at 30°C, Palinavir (10 μM final concentration) was added and translation of XL–EMCV mRNA (10 ng/μl) was performed under these conditions. Samples were analysed as described in Figure 5. (B) Western blot analysis of samples 1–8 of (A) was performed as described in Figure 1. A longer exposition of the upper part of the blot, corresponding to proteins of high molecular weight including intact eIF4GI is presented at the bottom of the panel. Download figure Download PowerPoint Interestingly, moderate inhibition of IRES-driven translation occurs only at the highest dose of HIV-2 PR used (Figure 5A, lanes 4 and 8), and this also corresponds to partial conversion of Ch-1 into Ch-2 (Figure 5B, lanes 4 and 8), raising different possible explanations for this inhibition of IRES-driven translation: (i) an intrinsic reduced ability of Ch-1 to support this type of initiation; (ii) a reduction in the amount of Ch-1 fragment; or (iii) a dominant negative effect of the Ch-2 species. Once again, comparison of the two cistrons in Figure 5A shows that, at the highest dose of HIV-2 PR, inhibition of uncapped mRNA is 7–10 times stronger than IRES-driven translation (lanes 8), confirming that p100 and Ch-1 exhibit different properties, the latter being unable to support translation of uncapped mRNAs. Ch-1 fragment can support translation of IRESs, but not that of uncapped mRNAs In order to investigate in more detail the biochemical properties of the eIF4GI cleavage products, we have analysed their distribution on ribosomes isolated from RRL. To this aim, lysate was incubated with FMDV L or HIV-2 proteases, and centrifuged after inhibition of the enzymatic activities. During this manipulation, the ribosomes and associated factors are pelleted, while other components remain in the supernatant. Figure 6A is a schematic representation of the supernatant (S) and ribosomal (R) fractions obtained from control RRL (CS and CR), RRL incubated with L protease (LS and LR), and RRL incubated with HIV-2 PR (HS and HR). Figure 6B shows a western blot analysis of these different fractions using antibodies against the C-terminal region of eIF4GI (E epitope, see Figure 2B), the eIF4E protein and the p48 subunit of eIF3. As shown previously (Rau et al., 1996), the p100 fragment resulting from cleavage of eIF4GI by FMDV L protease was almost entirely found associated with the ribosomes, with very little remaining in the supernatant (Figure 6B, top, LS and LR). In contrast, Ch-1 was distributed in both the ribosomal and supernatant fractions (lanes HS and HR), like intact eIF4GI (lanes CS and CR). It should be noted that we reproducibly observed that material from the ribosomal fraction appears to run slightly slower than samples from the S100 fraction (compare CS with CR, LS with LR, and HS with HR); the reason for this remains unknown. As expected, Ch-2 was exclusively found in the supernatant fraction (Figure 6B, lane HS), confir
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