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RNA. Sep 2001; 7(9): 1213–1226.
PMCID: PMC1370167

IRES interaction with translation initiation factors: functional characterization of novel RNA contacts with eIF3, eIF4B, and eIF4GII.


Translation initiation promoted by picornavirus internal ribosome entry site (IRES) elements is dependent on the association of specific IRES sequences to the initiation factor eIF4G. However the RNA determinants interacting with other components of the translational machinery are still unknown. In this study, we have identified novel RNA-protein interactions between the foot-and-mouth disease virus (FMDV) IRES and three translation initiation factors. A doublet of 116/110 kDa that crosslinked to the FMDV IRES is a component of eIF3. We show here that domain 5 holds the preferential binding site for eIF3, although this complex initiation factor can establish multiple contacts with the IRES structure. We have also identified the phylogenetically conserved hairpin of domain 5 as the RNA motif responsible for eIF4B interaction. Mutation of this stem-loop structure abrogated eIF4B, but not eIF3, binding to the IRES. Remarkably, IRES mutants severely affected in their interaction with eIF4B showed a mild reduction in IRES activity when tested in the context of a bicistronic expression vector in transfected cells. Finally, we provide evidence of the interaction of eIF4GII with FMDV IRES, the RNA determinants for this interaction being shared with its functional homolog eIF4GI. The FMDV Lb protease generated a C-terminal fragment of eIF4GII that binds to the IRES as efficiently as the intact protein. Competition experiments showed that titration of eIF4B or p110/116 interaction with the FMDV IRES required a large excess of competitor relative to eIF4G, strongly suggesting that eIF4G-IRES interaction is a limiting factor to titrate the IRES. Comparative analysis of the activity of IRES mutants affected in domains 4 and 5 regarding their pattern of RNA-protein complex formation demonstrates that while binding of eIF4B with the FMDV IRES is dispensable, interaction of eIF4G is a central feature of the activity of this element.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Altmann M, Müller PP, Wittmer B, Ruchti F, Lanker S, Trachsel H. A Saccharomyces cerevisiae homologue of mammalian translation initiation factor 4B contributes to RNA helicase activity. EMBO J. 1993 Oct;12(10):3997–4003. [PMC free article] [PubMed]
  • Altmann M, Wittmer B, Méthot N, Sonenberg N, Trachsel H. The Saccharomyces cerevisiae translation initiation factor Tif3 and its mammalian homologue, eIF-4B, have RNA annealing activity. EMBO J. 1995 Aug 1;14(15):3820–3827. [PMC free article] [PubMed]
  • Aragón T, de la Luna S, Novoa I, Carrasco L, Ortín J, Nieto A. Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a translational activator of influenza virus. Mol Cell Biol. 2000 Sep;20(17):6259–6268. [PMC free article] [PubMed]
  • Asano K, Kinzy TG, Merrick WC, Hershey JW. Conservation and diversity of eukaryotic translation initiation factor eIF3. J Biol Chem. 1997 Jan 10;272(2):1101–1109. [PubMed]
  • Block KL, Vornlocher HP, Hershey JW. Characterization of cDNAs encoding the p44 and p35 subunits of human translation initiation factor eIF3. J Biol Chem. 1998 Nov 27;273(48):31901–31908. [PubMed]
  • Borman AM, Le Mercier P, Girard M, Kean KM. Comparison of picornaviral IRES-driven internal initiation of translation in cultured cells of different origins. Nucleic Acids Res. 1997 Mar 1;25(5):925–932. [PMC free article] [PubMed]
  • Buratti E, Tisminetzky S, Zotti M, Baralle FE. Functional analysis of the interaction between HCV 5'UTR and putative subunits of eukaryotic translation initiation factor eIF3. Nucleic Acids Res. 1998 Jul 1;26(13):3179–3187. [PMC free article] [PubMed]
  • Coppolecchia R, Buser P, Stotz A, Linder P. A new yeast translation initiation factor suppresses a mutation in the eIF-4A RNA helicase. EMBO J. 1993 Oct;12(10):4005–4011. [PMC free article] [PubMed]
  • Duncan R, Hershey JW. Identification and quantitation of levels of protein synthesis initiation factors in crude HeLa cell lysates by two-dimensional polyacrylamide gel electrophoresis. J Biol Chem. 1983 Jun 10;258(11):7228–7235. [PubMed]
  • Escarmís C, Toja M, Medina M, Domingo E. Modifications of the 5' untranslated region of foot-and-mouth disease virus after prolonged persistence in cell culture. Virus Res. 1992 Nov;26(2):113–125. [PubMed]
  • Fuerst TR, Niles EG, Studier FW, Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. [PMC free article] [PubMed]
  • Gamarnik AV, Andino R. Two functional complexes formed by KH domain containing proteins with the 5' noncoding region of poliovirus RNA. RNA. 1997 Aug;3(8):882–892. [PMC free article] [PubMed]
  • Gingras AC, Raught B, Sonenberg N. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem. 1999;68:913–963. [PubMed]
  • Goldstaub D, Gradi A, Bercovitch Z, Grosmann Z, Nophar Y, Luria S, Sonenberg N, Kahana C. Poliovirus 2A protease induces apoptotic cell death. Mol Cell Biol. 2000 Feb;20(4):1271–1277. [PMC free article] [PubMed]
  • Gradi A, Imataka H, Svitkin YV, Rom E, Raught B, Morino S, Sonenberg N. A novel functional human eukaryotic translation initiation factor 4G. Mol Cell Biol. 1998 Jan;18(1):334–342. [PMC free article] [PubMed]
  • Gradi A, Svitkin YV, Imataka H, Sonenberg N. Proteolysis of human eukaryotic translation initiation factor eIF4GII, but not eIF4GI, coincides with the shutoff of host protein synthesis after poliovirus infection. Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11089–11094. [PMC free article] [PubMed]
  • Guo J, Hui DJ, Merrick WC, Sen GC. A new pathway of translational regulation mediated by eukaryotic initiation factor 3. EMBO J. 2000 Dec 15;19(24):6891–6899. [PMC free article] [PubMed]
  • Jang SK, Kräusslich HG, Nicklin MJ, Duke GM, Palmenberg AC, Wimmer E. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol. 1988 Aug;62(8):2636–2643. [PMC free article] [PubMed]
  • Kieft JS, Zhou K, Jubin R, Doudna JA. Mechanism of ribosome recruitment by hepatitis C IRES RNA. RNA. 2001 Feb;7(2):194–206. [PMC free article] [PubMed]
  • Kirchweger R, Ziegler E, Lamphear BJ, Waters D, Liebig HD, Sommergruber W, Sobrino F, Hohenadl C, Blaas D, Rhoads RE, et al. Foot-and-mouth disease virus leader proteinase: purification of the Lb form and determination of its cleavage site on eIF-4 gamma. J Virol. 1994 Sep;68(9):5677–5684. [PMC free article] [PubMed]
  • Kolupaeva VG, Pestova TV, Hellen CU. Ribosomal binding to the internal ribosomal entry site of classical swine fever virus. RNA. 2000 Dec;6(12):1791–1807. [PMC free article] [PubMed]
  • Kolupaeva VG, Pestova TV, Hellen CU, Shatsky IN. Translation eukaryotic initiation factor 4G recognizes a specific structural element within the internal ribosome entry site of encephalomyocarditis virus RNA. J Biol Chem. 1998 Jul 17;273(29):18599–18604. [PubMed]
  • Lamphear BJ, Kirchweger R, Skern T, Rhoads RE. Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. Implications for cap-dependent and cap-independent translational initiation. J Biol Chem. 1995 Sep 15;270(37):21975–21983. [PubMed]
  • Lomakin IB, Hellen CU, Pestova TV. Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation. Mol Cell Biol. 2000 Aug;20(16):6019–6029. [PMC free article] [PubMed]
  • López de Quinto S, Martínez-Salas E. Conserved structural motifs located in distal loops of aphthovirus internal ribosome entry site domain 3 are required for internal initiation of translation. J Virol. 1997 May;71(5):4171–4175. [PMC free article] [PubMed]
  • López de Quinto S, Martínez-Salas E. Parameters influencing translational efficiency in aphthovirus IRES-based bicistronic expression vectors. Gene. 1998 Sep 14;217(1-2):51–56. [PubMed]
  • López de Quinto S, Martínez-Salas E. Involvement of the aphthovirus RNA region located between the two functional AUGs in start codon selection. Virology. 1999 Mar 15;255(2):324–336. [PubMed]
  • López de Quinto S, Martínez-Salas E. Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo. RNA. 2000 Oct;6(10):1380–1392. [PMC free article] [PubMed]
  • Luz N, Beck E. Interaction of a cellular 57-kilodalton protein with the internal translation initiation site of foot-and-mouth disease virus. J Virol. 1991 Dec;65(12):6486–6494. [PMC free article] [PubMed]
  • Marcotrigiano J, Lomakin IB, Sonenberg N, Pestova TV, Hellen CU, Burley SK. A conserved HEAT domain within eIF4G directs assembly of the translation initiation machinery. Mol Cell. 2001 Jan;7(1):193–203. [PubMed]
  • Martínez-Salas E, Ramos R, Lafuente E, López de Quinto S. Functional interactions in internal translation initiation directed by viral and cellular IRES elements. J Gen Virol. 2001 May;82(Pt 5):973–984. [PubMed]
  • Martínez-Salas E, Regalado MP, Domingo E. Identification of an essential region for internal initiation of translation in the aphthovirus internal ribosome entry site and implications for viral evolution. J Virol. 1996 Feb;70(2):992–998. [PMC free article] [PubMed]
  • Martínez-Salas E, Sáiz JC, Dávila M, Belsham GJ, Domingo E. A single nucleotide substitution in the internal ribosome entry site of foot-and-mouth disease virus leads to enhanced cap-independent translation in vivo. J Virol. 1993 Jul;67(7):3748–3755. [PMC free article] [PubMed]
  • Mengod G, Trachsel H. Eukaryotic protein synthesis initiation factor eIF-3: determination of concentration and association with ribosomes in rabbit reticulocyte and HeLa cell lysates. Biochim Biophys Acta. 1985 Jun 24;825(2):169–174. [PubMed]
  • Méthot N, Pause A, Hershey JW, Sonenberg N. The translation initiation factor eIF-4B contains an RNA-binding region that is distinct and independent from its ribonucleoprotein consensus sequence. Mol Cell Biol. 1994 Apr;14(4):2307–2316. [PMC free article] [PubMed]
  • Methot N, Pickett G, Keene JD, Sonenberg N. In vitro RNA selection identifies RNA ligands that specifically bind to eukaryotic translation initiation factor 4B: the role of the RNA remotif. RNA. 1996 Jan;2(1):38–50. [PMC free article] [PubMed]
  • Méthot N, Rom E, Olsen H, Sonenberg N. The human homologue of the yeast Prt1 protein is an integral part of the eukaryotic initiation factor 3 complex and interacts with p170. J Biol Chem. 1997 Jan 10;272(2):1110–1116. [PubMed]
  • Méthot N, Song MS, Sonenberg N. A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3. Mol Cell Biol. 1996 Oct;16(10):5328–5334. [PMC free article] [PubMed]
  • Meyer K, Petersen A, Niepmann M, Beck E. Interaction of eukaryotic initiation factor eIF-4B with a picornavirus internal translation initiation site. J Virol. 1995 May;69(5):2819–2824. [PMC free article] [PubMed]
  • Meyer LJ, Milburn SC, Hershey JW. Immunochemical characterization of mammalian protein synthesis initiation factors. Biochemistry. 1982 Aug 31;21(18):4206–4212. [PubMed]
  • Milburn SC, Duncan RF, Hershey JW. Immunoblot analysis of the structure of protein synthesis initiation factor eIF3 from HeLa cells. Arch Biochem Biophys. 1990 Jan;276(1):6–11. [PubMed]
  • Milburn SC, Hershey JW, Davies MV, Kelleher K, Kaufman RJ. Cloning and expression of eukaryotic initiation factor 4B cDNA: sequence determination identifies a common RNA recognition motif. EMBO J. 1990 Sep;9(9):2783–2790. [PMC free article] [PubMed]
  • Naranda T, Strong WB, Menaya J, Fabbri BJ, Hershey JW. Two structural domains of initiation factor eIF-4B are involved in binding to RNA. J Biol Chem. 1994 May 20;269(20):14465–14472. [PubMed]
  • Nygård O, Westermann P. Specific interaction of one subunit of eukaryotic initiation factor eIF-3 with 18S ribosomal RNA within the binary complex, eIF-3 small ribosomal subunit, as shown by cross-linking experiments. Nucleic Acids Res. 1982 Feb 25;10(4):1327–1334. [PMC free article] [PubMed]
  • Odreman-Macchioli FE, Tisminetzky SG, Zotti M, Baralle FE, Buratti E. Influence of correct secondary and tertiary RNA folding on the binding of cellular factors to the HCV IRES. Nucleic Acids Res. 2000 Feb 15;28(4):875–885. [PMC free article] [PubMed]
  • Pelletier J, Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature. 1988 Jul 28;334(6180):320–325. [PubMed]
  • Pestova TV, Borukhov SI, Hellen CU. Eukaryotic ribosomes require initiation factors 1 and 1A to locate initiation codons. Nature. 1998 Aug 27;394(6696):854–859. [PubMed]
  • Pestova TV, Hellen CU, Shatsky IN. Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry. Mol Cell Biol. 1996 Dec;16(12):6859–6869. [PMC free article] [PubMed]
  • Pestova TV, Shatsky IN, Fletcher SP, Jackson RJ, Hellen CU. 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. 1998 Jan 1;12(1):67–83. [PMC free article] [PubMed]
  • Pilipenko EV, Pestova TV, Kolupaeva VG, Khitrina EV, Poperechnaya AN, Agol VI, Hellen CU. A cell cycle-dependent protein serves as a template-specific translation initiation factor. Genes Dev. 2000 Aug 15;14(16):2028–2045. [PMC free article] [PubMed]
  • Preiss T, Hentze MW. From factors to mechanisms: translation and translational control in eukaryotes. Curr Opin Genet Dev. 1999 Oct;9(5):515–521. [PubMed]
  • Ramos R, Martínez-Salas E. Long-range RNA interactions between structural domains of the aphthovirus internal ribosome entry site (IRES). RNA. 1999 Oct;5(10):1374–1383. [PMC free article] [PubMed]
  • Rose JK, Buonocore L, Whitt MA. A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. Biotechniques. 1991 Apr;10(4):520–525. [PubMed]
  • Rozen F, Edery I, Meerovitch K, Dever TE, Merrick WC, Sonenberg N. Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F. Mol Cell Biol. 1990 Mar;10(3):1134–1144. [PMC free article] [PubMed]
  • Sizova DV, Kolupaeva VG, Pestova TV, Shatsky IN, Hellen CU. Specific interaction of eukaryotic translation initiation factor 3 with the 5' nontranslated regions of hepatitis C virus and classical swine fever virus RNAs. J Virol. 1998 Jun;72(6):4775–4782. [PMC free article] [PubMed]
  • Svitkin YV, Gradi A, Imataka H, Morino S, Sonenberg N. Eukaryotic initiation factor 4GII (eIF4GII), but not eIF4GI, cleavage correlates with inhibition of host cell protein synthesis after human rhinovirus infection. J Virol. 1999 Apr;73(4):3467–3472. [PMC free article] [PubMed]
  • Tarun SZ, Jr, Sachs AB. Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G. EMBO J. 1996 Dec 16;15(24):7168–7177. [PMC free article] [PubMed]
  • Wilson JE, Pestova TV, Hellen CU, Sarnow P. Initiation of protein synthesis from the A site of the ribosome. Cell. 2000 Aug 18;102(4):511–520. [PubMed]

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