Eukaryotic translation initiation machinery can operate in a prokaryotic-like mode without eif2

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1 1 Eukaryotic translation initiation machinery can operate in a prokaryotic-like mode without eif2 Ilya M. Terenin*, Sergey E. Dmitriev*, Dmitry E. Andreev & Ivan N. Shatsky A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia *These authors contributed equally to this work Unlike prokaryotes, a specialized eukaryotic initiation factor 2 (eif2), in the form of the Met ternary complex eif2*gtp*met-trna i is utilized to deliver the initiator trna to the ribosome within all eukaryotic cells 1. Phosphorylation of eif2 is known to be central to the global regulation of protein synthesis under stress conditions and infection 2. Another distinctive feature of eukaryotic translation is scanning of mrna 5 -leaders, whose origin in evolution may be relevant to the appearance of eif2 in eukaryotes. Translation initiation on the hepatitis C virus (HCV) internal ribosome entry site (IRES) occurs without scanning 3,4. Whether these unique features of the HCV IRES account for the formation of the final 80S initiation complex is unknown. Here we show that the HCV IRES-directed translation can occur without either eif2 or its GTPase activating protein eif5. In addition to the general eif2- and eif5-dependent pathway of 80S complex assembly, the HCV IRES makes use of a prokaryotic-like pathway which involves eif5b, the analogue of bacterial IF2 5,6, instead of eif2. This switch from a eukaryotic-like mode of AUG selection to a bacterial one occurs when eif2 is inactivated by phosphorylation, a way with which host cells counteract infection. The relative resistance of HCV IRES-directed translation to eif2 phosphorylation may represent one more line of defense used by this virus against host antiviral responses and can contribute to the well known resistance of HCV to interferon based therapy.

2 2 Initiation factor eif2 is a pivotal component of the translation initiation mechanism in all eukaryotic cells. It is absent from bacteria, thereby reflecting quite different modes of the start codon selection in pro- and eukaryotes. Strikingly, some viral mrnas are efficiently translated under conditions when the activity of eif2 is suppressed by phosphorylation as a result of host cells response to viral infection 2,7. This is exactly the case of HCV RNA, reported to be refractory to reduced eif2 GTP Met-tRNA Met i ternary complex availability 8. The assembly of fully functional 48S pre-initiation complexes on the HCV IRES only requires eif2 and eif3 3. Addition of conventional ribosome joining factors eif5 and eif5b along with 60S subunits led predictably to 80S complex formation 9 (Fig. 1a). Surprisingly, omitting eif5, a trigger of GTP hydrolysis on eif2, does not abolish the 80S complex formation (Fig. 1a). The possibility of contamination with eif5 within the system was ruled out by Western-blotting analysis of eif2 and eif5b preparations and by the inability of β-globin mrna to form 80S complexes when eif5, which is essential for this standard system 6, was absent (Fig.1b). This finding could be explained by eif5b functioning instead of eif5 as was suggested for 80S complex formation on AUG-triplets 6,10,11. However, control experiments in which the 48S to the 80S conversion was blocked by the presence of GMPPNP (a non-hydrolysable analogue of GTP) also resulted in 80S complex formation, although at a somewhat reduced level (Fig. 1c), indicating at least a partial independence of 80S assembly on GTP hydrolysis. The conversion of the 48S complex into the 80S is known to require GTP hydrolysis by eif2 and thus should be completely blocked by GMPPNP. Therefore, the question arises of whether there is an obligatory requirement for any eif2 if there was no strict requirement for GTP hydrolysis? Indeed, in the presence of only ribosomal subunits, eif3, eif5b, and MettRNA Met i we found that 80S translation initiation complexes were formed in the presence of GMPPNP, thereby eliminating the possibility of any residual eif2 activity. However, eif3 along with eif5b were indispensable (Fig. 1d). The functional role of eif3 in this very simplified process of 80S assembly is not yet clear.

3 3 Recently, a factorless translation initiation process has been reported for the HCV IRES at elevated Mg 2+ concentrations 12. Although we performed the assays for 80S complex formation at physiological ionic concentrations, sucrose gradients employed to analyze translation initiation complexes routinely contained elevated concentrations of Mg 2+ (6 mm). However, decreasing the Mg 2+ concentration to 2 mm during centrifugation did not abolish eif2-independent 80S assembly (Fig. 1e). We failed to reproduce factorless 80S complex formation 12 either under our experimental conditions or at elevated Mg 2+ concentrations (data not shown). The functionality of the 80S initiation complexes assembled from 40S and 60S ribosomal subunits, eif3, eif5b, and Met-tRNA Met i, i.e. their ability to be engaged into the subsequent step of translation, the elongation of the polypeptide chain, had to be proven. To this end, we changed the 7th triplet of the HCV ORF to a UAA stop codon and then reconstituted the translation elongation process from totally purified components 13. The position of the stop codon was chosen in such a way that the ribosomes did not reach a putative frameshift site 14 previously identified within the coding sequence of HCV RNA. The location of elongating ribosomes at the nucleotide triplet preceding the termination codon was identified by toeprint assay. This technique is based on the primer extension inhibition of reverse transcription from an oligodeoxynucleotide, which is hybridized 3 to the codon of an mrna positioned in the P-site of the ribosome. The arrest of reverse transcriptase always occurs at the same position, +16 to +18 nt 3 of the first nucleotide residue of the codon occupying the P-site of the ribosome. Identical elongation factor-dependent arrests of the ribosomes can be clearly seen in Fig. 2 regardless of whether the 80S initiation complexes were formed with the complete set of eifs (including eif2) or with just eif3 and eif5b (compare lanes 3 and 7). Thus, the 80S initiation complex formed in the absence of eif2 was totally functional and not an artifact. Together these data might suggest that the eukaryotic initiation factor eif2 may be dispensable if scanning of a 5 UTR does not occur, i.e. when the 40S subunit immediately binds at or very close to the initiator codon. This situation is analogous to that

4 4 observed for prokaryotes; like bacterial IF2 15, eif5b plays in this case two roles: promotes binding of the initiator trna to ribosomes and couples the ribosomal subunits with formation of the 80S (70S in the case of bacteria) translation initiation complex. Notably, in our in vitro experiments the presence of eif2 in the reconstitution system strongly inhibited GTP hydrolysis-independent formation of the 80S complex (data not shown). This could be due to a higher affinity of the ternary complex for the 40S subunit, implying that eif2 outcompetes eif5b from the 40S. It also means that eif2 inactivation should result in a switch of the translation initiation modes utilized by HCV from eif2-dependent to eif2-independent rather than in a severe inhibition of its translation. To address the physiological relevance of these data we inactivated eif2 via phosphorylation both in vitro and in vivo and studied the effect on HCV IRES-driven translation. Rabbit reticulocyte lysate (RRL) was treated with dsrna to induce PKR 16 and supplemented with GMPPNP to block the eif2-dependent pathway at the 48S stage. Control assays were not treated with dsrna but also contained GMPPNP. It was found that no 80S complexes were assembled on the HCV IREScontaining mrna in control lysates (Fig. 3a). In contrast, in dsrna treated RRL 80S complexes were formed on the HCV IRES transcript. This was not the case for the ß-globin mrna, a typical cellular mrna which can only use the canonical eif-2 dependent pathway of translation initiation (Fig. 3a, b). It is clear that eif2 had been inactivated, at least partially, since the amount of 48S complexes assembled on the ß-globin mrna was reduced (Fig. 3b). The phosphorylation of eif2 in the presence of dsrna was confirmed by Western-blotting with anti phosphor-eif2α(ser51) antibodies (Fig. 3c). These data may explain why in the presence of GMPPNP in cell extracts no 80S complexes have been observed previously 3,17,18. They indicate that under normal conditions, i.e. when eif2 is fully active, the translation initiation on the HCV IRES proceeds primarily through the eif2-dependent pathway.

5 5 The in vitro experiments were complemented with those performed using transfected cells. HEK293T cells were first treated with various reagents which elicited eif2 phosphorylation either by activation of various eif2 kinases (PKR by dsrna transfection, PERK by DTT 19, or HRI by sodium arsenite 20 ) or by inhibition of PP1 phosphatase (by okadaic acid 21 ) and then transfected with a mixture of a standard cap-dependent mrna and the HCV IRES containing monocistronic mrna. As seen in Fig. 4, all these treatments affected much less the HCV IRES-driven translation than that directed by a standard 5 -UTR. Importantly, the translation directed by certain picornavirus IRESes, like HRV or EMCV, were inhibited by these treatments to the same extent as cap-dependent translation (Fig. 4, data not shown). Interestingly, treatment with IFNα, widely used in HCV therapy, also lead to relative resistance of the HCV RNA translation. Thus, consistent with other previously reported data 7,8,22, eif2-inactivation does not lead to severe inhibition of HCV IRES-driven translation. The data described above present for the first time the mechanistic explanation of this phenomenon, demonstrating the existence of the alternative, eif2-independent, pathway of translation initiation for the HCV RNA. The switch from a eukaryotic-like mode of AUG selection to a bacterial one occurs when eif2 is inactivated by phosphorylation, a way with which host cells counteract infection. Thus, presented data throw a bridge over evolutionary different modes of translation initiation. Methods Summary Purification of ribosome subunits, initiation factors, and Met-tRNA i Met. Ribosomal subunits, initiation and elongation factors were purified from RRL or Krebs-2 ascites cells or were expressed in E.coli as described 13,23,24. Assembly and analysis of translation initiation complexes. Ribosomal complexes were assembled and analyzed by sucrose gradient centrifugation or toeprinting as described 3,23. For in vitro translation elongation experiments factors eef1h, eef2, and total aminoacylated trna were

6 6 added to preformed 80S complexes 13. For in vitro eif2 inhibition, 20 ng of synthesized dsrna 13 were added to 14 μl of RRL (Promega) and incubated for 15 min at 30 o C, then lysate was supplemented with GMPPNP and ribosomal complexes were assembled as described previously 23. Plasmids and in vitro transcription. Plasmids encoded for mrnas with β-globin 23 or HCV UTRs for ribosomal complexes assembly have been described. For cells transfection polyadenylated mrnas were synthesized using PCRs product as a template, T7-promoter and 50 nt poly(a)-tail being introduced by corresponding primers 25. In vivo studies. Cell culture manipulations and RNA transfection procedures were performed as described 25. Exponentially growing HEK293T cells were replated to 24-well plates at densities 1:2, IFNα was added where indicated to the final concentration 1000 ME/ml. After 12 hours of growth, 2 μg/ml dsrna with Lipofectamine2000 (Invitrogen) was added for another 1 hour, or else 2,5 mm DTT, 2 mm NaAsO 2 or 1 μm okadaic acid (Calbiochem) were added for 30 minutes, followed by transfection of reporter mrnas. The reporter expression was measured 2 hours after transfection. Western-blotting. Cells for Western-blotting were grown in parallel with cells for transfection, similarly treated with the effectors, and harvested 30 min after (or 1 h in the case of dsrna treatment). The equal amounts of cell lysates were resolved by SDS-PAGE and probed with the anti-phosphor-eif2α(ser51) antibodies (Cell Signaling) or anti-eif2α antibodies (a kind gift from W.C.Merrick). 1. Hershey, J.B.W. & Merrick, W.C. The Pathway and Mechanism of Initiation of Protein Synthesis. in Translational Control of Gene Expression (eds. Sonenberg, N., Hershey, J. & Merrick, W.C.) (Cold Spring Harbor Press, Cold Spring Harbor, New York, 2000).

7 7 2. Ron, D. & Harding, H.P. eif2α Phosphorylation in Cellular Stress Responses and Desease. in Translational Control in Biology and Medicine (eds. Mathews, M.B., Sonenberg, N. & Hershey, J.W.B.) (Cold Spring Harbor Press, Cold Spring Harbor, New York, 2006). 3. Pestova, T.V., Shatsky, I.N., Fletcher, S.P., Jackson, R.J. & Hellen, C.U. A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. Genes Dev 12, (1998). 4. Ji, H., Fraser, C.S., Yu, Y., Leary, J. & Doudna, J.A. Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA. Proc Natl Acad Sci U S A 101, (2004). 5. Choi, S.K., Lee, J.H., Zoll, W.L., Merrick, W.C. & Dever, T.E. Promotion of mettrnaimet binding to ribosomes by yif2, a bacterial IF2 homolog in yeast. Science 280, (1998). 6. Pestova, T.V. et al. The joining of ribosomal subunits in eukaryotes requires eif5b. Nature 403, (2000). 7. Rivas-Estilla, A.M. et al. PKR-dependent mechanisms of gene expression from a subgenomic hepatitis C virus clone. J Virol 76, (2002). 8. Robert, F. et al. Initiation of protein synthesis by hepatitis C virus is refractory to reduced eif2 GTP Met-tRNA Met i ternary complex availability. Mol Biol Cell 17, (2006). 9. Locker, N., Easton, L.E. & Lukavsky, P.J. HCV and CSFV IRES domain II mediate eif2 release during 80S ribosome assembly. EMBO J 26, (2007). 10. Peterson, D.T., Safer, B. & Merrick, W.C. Role of eukaryotic initiation factor 5 in the formation of 80 S initiation complexes. J Biol Chem 254, (1979).

8 8 11. Peterson, D.T., Merrick, W.C. & Safer, B. Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex formation. J Biol Chem 254, (1979). 12. Lancaster, A.M., Jan, E. & Sarnow, P. Initiation factor-independent translation mediated by the hepatitis C virus internal ribosome entry site. RNA 12, (2006). 13. Andreev, D.E., Terenin, I.M., Dunaevsky, Y.E., Dmitriev, S.E. & Shatsky, I.N. A leaderless mrna can bind to mammalian 80S ribosomes and direct polypeptide synthesis in the absence of translation initiation factors. Mol Cell Biol 26, (2006). 14. Choi, J., Xu, Z. & Ou, J.H. Triple decoding of hepatitis C virus RNA by programmed translational frameshifting. Mol Cell Biol 23, (2003). 15. Gualerzi, C.O. et al. Initiation factors in the early events of mrna translation in bacteria. Cold Spring Harb Symp Quant Biol 66, (2001). 16. Farrell, P.J., Balkow, K., Hunt, T., Jackson, R.J. & Trachsel, H. Phosphorylation of initiation factor elf-2 and the control of reticulocyte protein synthesis. Cell 11, (1977). 17. Otto, G.A. & Puglisi, J.D. The pathway of HCV IRES-mediated translation initiation. Cell 119, (2004). 18. Costa-Mattioli, M., Svitkin, Y. & Sonenberg, N. La autoantigen is necessary for optimal function of the poliovirus and hepatitis C virus internal ribosome entry site in vivo and in vitro. Mol Cell Biol 24, (2004). 19. Harding, H.P., Zhang, Y., Bertolotti, A., Zeng, H. & Ron, D. Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol Cell 5, (2000).

9 9 20. McEwen, E. et al. Heme-regulated inhibitor kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure. J Biol Chem 280, (2005). 21. Brush, M.H., Weiser, D.C. & Shenolikar, S. Growth arrest and DNA damage-inducible protein GADD34 targets protein phosphatase 1 alpha to the endoplasmic reticulum and promotes dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2. Mol Cell Biol 23, (2003). 22. Honda, M. et al. Cell cycle regulation of hepatitis C virus internal ribosomal entry sitedirected translation. Gastroenterology 118, (2000). 23. Dmitriev, S.E., Pisarev, A.V., Rubtsova, M.P., Dunaevsky, Y.E. & Shatsky, I.N. Conversion of 48S translation preinitiation complexes into 80S initiation complexes as revealed by toeprinting. FEBS Lett 533, (2003). 24. Terenin, I.M. et al. A cross-kingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry. Mol Cell Biol 25, (2005). 25. Dmitriev, S.E. et al. Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mrna is strictly cap dependent rather than internal ribosome entry site mediated. Mol Cell Biol 27, (2007). Acknowledgements We thank L.P.Ovchinnikov for the kind gift of eef2 and W.C.Merrick for anti-eif2α antibodies. We are also very grateful to G.Belsham for critical reading of the manuscript and valuable suggestions. This work was supported by grants from the Russian Foundation for Basic Research (RFBR) to I.N.S. and I.M.T.

10 10 Author contributions: I.M.T. and S.E.D. contributed equally to this work. I.M.T. and D.E.A. performed in vitro experiments whereas S.E.D. carried out cell transfection studies. I.N.S., and I.M.T. wrote the article. All authors discussed the results and commented on the manuscript. Correspondence and requests for materials should be addressed to I.N.S. ( Figure 1. Assembly of 80S translation initiation complexes on the HCV IRES and β- globin mrnas. Sucrose density gradient centrifugation of 80S complexes reconstitution on (a) the HCV IRES with GTP, Met-tRNA Met i, 40S, 60S, eif2, eif3 with omission of eif5 or eif5b as indicated and (b) the β-globin mrna with GTP, Met-tRNA Met i, 40S, 60S, eif1, eif1a, eif2, eif3, eif4a, eif4b and eif4f with or without eif5 or eif5b as indicated. (c) 80S complexes assembled on the HCV mrna with the complete set of factors, GTP being substituted for its nonhydrolysable analog GMPPNP. (d) 80S complexes formed with GMPPNP, 40S, 60S, eif3 and eif5b or with omission of one of these factors. (e) analysis of 80S complexes reconstituted on the HCV IRES RNA in sucrose gradients containing 2 or 5mM Mg 2+ (see text). Figure 2. 80S complexes formed without eif2 are competent for translational elongation. 80S complexes were assembled from 40S, 60S, eif3, eif5b, eif2 and eif5 as indicated on the HCV mrna bearing a termination codon at the 7 th amino acid position, and then elongation factors were added. Initiation and termination codons and relevant reverse transcription stops are indicated by arrows.

11 11 Figure 3. eif2 phosphorylation activates the alternative pathway for 80S formation on the HCV IRES in RRL. The HCV mrna (a) and β-globin mrna (b) were incubated in RRL first treated with dsrna and GMPPNP, and then ribosome complexes were resolved by sucrose gradient centrifugation. (c) eif2 phosphorylation was confirmed by Western blotting with antibodies specific for eif2(ser51)-p. Lanes are: 1 untreated, 2 dsrnatreated. Figure 4. HCV IRES directed translation is relatively resistant to eif2 inactivation in vivo. HEK293T cells were treated with 2 μg/ml dsrna, 2.5 mm DTT, 2 mm sodium arsenite, 1 μm okadaic acid or 1000 ME/ml IFNα as indicated, and then either (a) transfected with the mixture of capped Rluc and HCV-Fluc or HRV-Fluc mrnas, harvested 2 hrs later and assayed for reporter protein expression, or (b) lysed and probed for eif2 phosphorylation or total eif2 amount (lanes are: 1 untreated, 2 dsrna, 3 DTT, 4 arsenite, 5 - OA, 6 - IFNα). Results are means ± s.d. of three representative experiments performed in triplicate. Methods Purification of ribosome subunits, initiation factors, and Met-tRNA i Met. 40S, 60S, eif4f, eif5, eif5b and eef1h were isolated from RRL, eif2 and eif3 were purified from Krebs-2 ascites cells. eif1, eif1a, eif4a, eif4b, and MetRS were expressed in E.coli. Rabbit eef2 was a kind gift from L.P.Ovchinnikov. Met-tRNA i Met was prepared as described 13. The S100 supernatant from ascites cells, freed from nucleic acids by passing through DEAE-cellulose at 0.25 M KCl and ammonium sulfate precipitation, was used as a source of mammalian aminoacyl-trna synthetases. Total calf liver trna (Novagen) was aminoacylated using a protocol similar to that which was employed for the aminoacylation of trna Met i.

12 12 Assembly and analysis of translation initiation complexes. 48S complexes were assembled on the β-globin mrna by incubating 0.5 pmol of mrna for 10 min at 30 o C in a 20-μl reaction volume that contained the reconstitution buffer (20 mm Tris-HCl, ph 7.5, 120 mm KOAc, 2.5 mm Mg(OAc) 2, 0.1 mm EDTA, 1 mm DTT), 0.4 mm GTP or GMPPNP, 1 mm ATP or AMPPNP, 0.25 mm spermidine-hcl, 5 pmol of Met-tRNA Met i, eif1 (10 pmol), eif1a (10 pmol), eif2 (8 pmol), eif3 (5 pmol), eif4a (10 pmol), eif4b (6 pmol), eif4f (2.3 pmol), 40S ribosomal subunits (2,5 pmol). In the case of the 80S assembly, preformed 48S complexes were supplemented with 60S subunits (2.5 pmol), eif5b (3 pmol), eif5 (3 pmol) and incubated for additional 10 minutes. In the case of the HCV mrna, 48S and 80S complexes were assembled similarly, except eif1, 1A, 4A, 4B, 4F, and ATP were omitted (also see text). For in vitro translation elongation experiments factors eef1h (4 µg), eef2 (1 µg), and total aminoacylated trna (2 µg) were added to preformed 80S complexes as described 13. For sucrose gradient centrifugation analysis mrnas were labeled cotranscriptionally with α-[ 32 P]-UTP and assembled complexes were centrifuged through 5-20% linear sucrose gradient for 3.5 h at 33,000 rpm in SW41 rotor (Beckman). For toeprinting 5'- GGGATTTCTGATCTCGGCG-3' oligonucleotide was used 3. Assembled complexes were analyzed by primer extension using AMV RT (Promega) essentially as described 23. cdna products were analyzed by electrophoresis through 6% polyacrylamide sequencing gel.

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