In eukaryotes, for a protein to be synthesized, the 40 S subunit has to first scan the 5'-UTR of the mRNA until it has encountered the AUG start codon. Several initiation factors that ensure high fidelity of AUG recognition were identified previously, including eIF1A, eIF1, eIF2, and eIF5. In addition, eIF3 was proposed to coordinate their functions in this process as well as to promote their initial binding to 40 S subunits. Here we subjected several previously identified segments of the N-terminal domain (NTD) of the eIF3c/Nip1 subunit, which mediates eIF3 binding to eIF1 and eIF5, to semirandom mutagenesis to investigate the molecular mechanism of eIF3 involvement in these reactions. Three major classes of mutant substitutions or internal deletions were isolated that affect either the assembly of preinitiation complexes (PICs), scanning for AUG, or both. We show that eIF5 binds to the extreme c/Nip1-NTD (residues 1-45) and that impairing this interaction predominantly affects the PIC formation. eIF1 interacts with the region (60-137) that immediately follows, and altering this contact deregulates AUG recognition. Together, our data indicate that binding of eIF1 to the c/Nip1-NTD is equally important for its initial recruitment to PICs and for its proper functioning in selecting the translational start site.

Laboratory of Regulation of Gene Expression Institute of Microbiology Academy of Sciences of the Czech Republic Videnska 1083 Prague 4 142 20 the Czech Republic

Zobrazit více v PubMed

Sonenberg N., Hinnebusch A. G. (2009) Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136, 731–745 PubMed PMC

Passmore L. A., Schmeing T. M., Maag D., Applefield D. J., Acker M. G., Algire M. A., Lorsch J. R., Ramakrishnan V. (2007) The eukaryotic translation initiation factors eIF1 and eIF1A induce an open conformation of the 40S ribosome. Mol. Cell 26, 41–50 PubMed

Hinnebusch A. G. (2011) Molecular mechanism of scanning and start codon selection in eukaryotes. Microbiol. Mol. Biol. Rev. 75, 434–467 PubMed PMC

Valášek L. S. (2012) 'Ribozoomin'—translation initiation from the perspective of the ribosome-bound eukaryotic initiation factors (eIFs). Curr. Protein Pept. Sci. 13, 305–330 PubMed PMC

Algire M. A., Maag D., Lorsch J. R. (2005) Pi release from eIF2, not GTP hydrolysis, is the step controlled by start-site selection during eukaryotic translation initiation. Mol. Cell 20, 251–262 PubMed

Maag D., Algire M. A., Lorsch J. R. (2006) Communication between eukaryotic translation initiation factors 5 and 1A within the ribosomal pre-initiation complex plays a role in start site selection. J. Mol. Biol. 356, 724–737 PubMed

Saini A. K., Nanda J. S., Lorsch J. R., Hinnebusch A. G. (2010) Regulatory elements in eIF1A control the fidelity of start codon selection by modulating tRNAiMet binding to the ribosome. Genes Dev. 24, 97–110 PubMed PMC

Cheung Y. N., Maag D., Mitchell S. F., Fekete C. A., Algire M. A., Takacs J. E., Shirokikh N., Pestova T., Lorsch J. R., Hinnebusch A. G. (2007) Dissociation of eIF1 from the 40S ribosomal subunit is a key step in start codon selection in vivo. Genes Dev. 21, 1217–1230 PubMed PMC

Elantak L., Wagner S., Herrmannová A., Karásková M., Rutkai E., Lukavsky P. J., Valásek L. (2010) The indispensable N-terminal half of eIF3j/HCR1 cooperates with its structurally conserved binding partner eIF3b/PRT1-RRM and with eIF1A in stringent AUG selection. J. Mol. Biol. 396, 1097–1116 PubMed PMC

Chiu W. L., Wagner S., Herrmannová A., Burela L., Zhang F., Saini A. K., Valásek L., Hinnebusch A. G. (2010) The C-terminal region of eukaryotic translation initiation factor 3a (eIF3a) promotes mRNA recruitment, scanning, and, together with eIF3j and the eIF3b RNA recognition motif, selection of AUG start codons. Mol. Cell. Biol. 30, 4415–4434 PubMed PMC

Valásek L., Nielsen K. H., Zhang F., Fekete C. A., Hinnebusch A. G. (2004) Interactions of eukaryotic translation initiation factor 3 (eIF3) subunit NIP1/c with eIF1 and eIF5 promote preinitiation complex assembly and regulate start codon selection. Mol. Cell. Biol. 24, 9437–9455 PubMed PMC

Nielsen K. H., Valásek L., Sykes C., Jivotovskaya A., Hinnebusch A. G. (2006) Interaction of the RNP1 motif in PRT1 with HCR1 promotes 40S binding of eukaryotic initiation factor 3 in yeast. Mol. Cell. Biol. 26, 2984–2998 PubMed PMC

Herrmannová A., Daujotyte D., Yang J. C., Cuchalová L., Gorrec F., Wagner S., Dányi I., Lukavsky P. J., Valásek L. S. (2012) Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly. Nucleic Acids Res. 40, 2294–2311 PubMed PMC

Mitchell S. F., Walker S. E., Algire M. A., Park E. H., Hinnebusch A. G., Lorsch J. R. (2010) The 5′-7-methylguanosine cap on eukaryotic mRNAs serves both to stimulate canonical translation initiation and to block an alternative pathway. Mol. Cell 39, 950–962 PubMed PMC

Jivotovskaya A. V., Valásek L., Hinnebusch A. G., Nielsen K. H. (2006) Eukaryotic translation initiation factor 3 (eIF3) and eIF2 can promote mRNA binding to 40S subunits independently of eIF4G in yeast. Mol. Cell. Biol. 26, 1355–1372 PubMed PMC

Pisarev A. V., Kolupaeva V. G., Yusupov M. M., Hellen C. U., Pestova T. V. (2008) Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes. EMBO J. 27, 1609–1621 PubMed PMC

Szamecz B., Rutkai E., Cuchalová L., Munzarová V., Herrmannová A., Nielsen K. H., Burela L., Hinnebusch A. G., Valásek L. (2008) eIF3a cooperates with sequences 5′ of uORF1 to promote resumption of scanning by post-termination ribosomes for reinitiation on GCN4 mRNA. Genes Dev. 22, 2414–2425 PubMed PMC

Cuchalová L., Kouba T., Herrmannová A., Dányi I., Chiu W. L., Valásek L. (2010) The RNA recognition motif of eukaryotic translation initiation factor 3g (eIF3g) is required for resumption of scanning of posttermination ribosomes for reinitiation on GCN4 and together with eIF3i stimulates linear scanning. Mol. Cell. Biol. 30, 4671–4686 PubMed PMC

Asano K., Clayton J., Shalev A., Hinnebusch A. G. (2000) A multifactor complex of eukaryotic initiation factors, eIF1, eIF2, eIF3, eIF5, and initiator tRNAMet is an important translation initiation intermediate in vivo. Genes Dev. 14, 2534–2546 PubMed PMC

Valásek L., Nielsen K. H., Hinnebusch A. G. (2002) Direct eIF2-eIF3 contact in the multifactor complex is important for translation initiation in vivo. EMBO J. 21, 5886–5898 PubMed PMC

Nielsen K. H., Szamecz B., Valásek L., Jivotovskaya A., Shin B. S., Hinnebusch A. G. (2004) Functions of eIF3 downstream of 48S assembly impact AUG recognition and GCN4 translational control. EMBO J. 23, 1166–1177 PubMed PMC

Valásek L., Mathew A. A., Shin B. S., Nielsen K. H., Szamecz B., Hinnebusch A. G. (2003) The yeast eIF3 subunits TIF32/a, NIP1/c, and eIF5 make critical connections with the 40S ribosome in vivo. Genes Dev. 17, 786–799 PubMed PMC

Valásek L., Trachsel H., Hasek J., Ruis H. (1998) Rpg1, the Saccharomyces cerevisiae homologue of the largest subunit of mammalian translation initiation factor 3, is required for translational activity. J. Biol. Chem. 273, 21253–21260 PubMed

Nielsen K. H., Valásek L. (2007) In vivo deletion analysis of the architecture of a multiprotein complex of translation initiation factors. Methods Enzymol. 431, 15–32 PubMed

Grant C. M., Hinnebusch A. G. (1994) Effect of sequence context at stop codons on efficiency of reinitiation in GCN4 translational control. Mol. Cell. Biol. 14, 606–618 PubMed PMC

Donahue T. (2000) in Translational Control of Gene Expression (Sonenberg N., Hershey J. W. B., Mathews M. B., eds) pp. 487–502, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Hinnebusch A. G. (2005) Translational regulation of GCN4 and the general amino acid control of yeast. Annu. Rev. Microbiol. 59, 407–450 PubMed

Phan L., Schoenfeld L. W., Valásek L., Nielsen K. H., Hinnebusch A. G. (2001) A subcomplex of three eIF3 subunits binds eIF1 and eIF5 and stimulates ribosome binding of mRNA and tRNAiMet. EMBO J. 20, 2954–2965 PubMed PMC

Yamamoto Y., Singh C. R., Marintchev A., Hall N. S., Hannig E. M., Wagner G., Asano K. (2005) The eukaryotic initiation factor (eIF) 5 HEAT domain mediates multifactor assembly and scanning with distinct interfaces to eIF1, eIF2, eIF3, and eIF4G. Proc. Natl. Acad. Sci. U.S.A. 102, 16164–16169 PubMed PMC

Asano K., Krishnamoorthy T., Phan L., Pavitt G. D., Hinnebusch A. G. (1999) Conserved bipartite motifs in yeast eIF5 and eIF2Bepsilon, GTPase-activating and GDP-GTP exchange factors in translation initiation, mediate binding to their common substrate eIF2. EMBO J. 18, 1673–1688 PubMed PMC

Kouba T., Rutkai E., Karásková M., Valášek L. S. (2012) The eIF3c/NIP1 PCI domain interacts with RNA and RACK1/ASC1 and promotes assembly of translation preinitiation complexes. Nucleic Acids Res. 40, 2683–2699 PubMed PMC

Rabl J., Leibundgut M., Ataide S. F., Haag A., Ban N. (2011) Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1. Science 331, 730–736 PubMed

Nanda J. S., Cheung Y. N., Takacs J. E., Martin-Marcos P., Saini A. K., Hinnebusch A. G., Lorsch J. R. (2009) eIF1 controls multiple steps in start codon recognition during eukaryotic translation initiation. J. Mol. Biol. 394, 268–285 PubMed PMC

Kouba T., Danyi I., Munzarová V., Vlčková V., Cuchalová L., Neueder A., Milkereit P., Valášek L. S. (2012) Small ribosomal protein RPS0 stimulates translation initiation by mediating 40S-binding of eIF3 via its direct contact with the eIF3a/TIF32 subunit. PLoS One, 7, e40464. PubMed PMC

Shin B. S., Kim J. R., Walker S. E., Dong J., Lorsch J. R., Dever T. E. (2011) Initiation factor eIF2γ promotes eIF2-GTP-Met-tRNAiMet ternary complex binding to the 40S ribosome. Nat. Struct. Mol. Biol. 18, 1227–1234 PubMed PMC

Lomakin I. B., Kolupaeva V. G., Marintchev A., Wagner G., Pestova T. V. (2003) Position of eukaryotic initiation factor eIF1 on the 40S ribosomal subunit determined by directed hydroxyl radical probing. Genes Dev. 17, 2786–2797 PubMed PMC

Reibarkh M., Yamamoto Y., Singh C. R., del Rio F., Fahmy A., Lee B., Luna R. E., Ii M., Wagner G., Asano K. (2008) Eukaryotic initiation factor (eIF) 1 carries two distinct eIF5-binding faces important for multifactor assembly and AUG selection. J. Biol. Chem. 283, 1094–1103 PubMed

Maag D., Fekete C. A., Gryczynski Z., Lorsch J. R. (2005) A conformational change in the eukaryotic translation preinitiation complex and release of eIF1 signal recognition of the start codon. Mol. Cell 17, 265–275 PubMed

Munzarová V., Pánek J., Gunišová S., Dányi I., Szamecz B., Valášek L. S. (2011) Translation reinitiation relies on the interaction between eIF3a/TIF32 and progressively folded cis-acting mRNA elements preceding short uORFs. PLoS Genet. 7, e1002137. PubMed PMC

Pöyry T. A., Kaminski A., Jackson R. J. (2004) What determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame? Genes Dev. 18, 62–75 PubMed PMC

Pisarev A. V., Hellen C. U., Pestova T. V. (2007) Recycling of eukaryotic posttermination ribosomal complexes. Cell 131, 286–299 PubMed PMC

Jennings M. D., Pavitt G. D. (2010) eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation. Nature 465, 378–381 PubMed PMC

Gietz R. D., Sugino A. (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74, 527–534 PubMed

Cigan A. M., Pabich E. K., Donahue T. F. (1988) Mutational analysis of the HIS4 translational initiator region in Saccharomyces cerevisiae. Mol. Cell. Biol. 8, 2964–2975 PubMed PMC

Mueller P. P., Hinnebusch A. G. (1986) Multiple upstream AUG codons mediate translational control of GCN4. Cell 45, 201–207 PubMed

Phan L., Zhang X., Asano K., Anderson J., Vornlocher H. P., Greenberg J. R., Qin J., Hinnebusch A. G. (1998) Identification of a translation initiation factor 3 (eIF3) core complex, conserved in yeast and mammals, that interacts with eIF5. Mol. Cell. Biol. 18, 4935–4946 PubMed PMC

Structural Differences in Translation Initiation between Pathogenic Trypanosomatids and Their Mammalian Hosts

uS3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3

Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes

Embraced by eIF3: structural and functional insights into the roles of eIF3 across the translation cycle

Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

Translation initiation factor eIF3 promotes programmed stop codon readthrough

Functional and biochemical characterization of human eukaryotic translation initiation factor 3 in living cells

Structural integrity of the PCI domain of eIF3a/TIF32 is required for mRNA recruitment to the 43S pre-initiation complexes