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Free initiation factors eIF4A and eIF4B are dispensable for translation initiation on uncapped mRNAs

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Abstract

The formation of ribosomal 48S initiation complexes at the start AUG codon of uncapped mRNA leader sequences was studied using the methodology of primer extension inhibition (toe-printing). The experiments were performed in the system composed of purified individual components required for translation initiation. The formation of ribosomal 48S initiation complexes at the initiation codon was tested depending on the presence of the initiation factors eIF4F, eIF4A, and eIF4B. Several mRNAs containing short leader sequences lacking the extended secondary structure were studied. It was found that 48S ribosomal complexes at mRNAs with such leaders were not formed in the absence of eIF4F. In contrast, the removal of either eIF4A or eIF4B from the experimental system was found to be dispensable for the formation of the 48S complex.

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References

  1. Jackson, R. J., Hellen, C. U. T., and Pestova, T. V. (2010) The mechanism of eukaryotic translation initiation and principles of its regulation, Nat. Rev. Mol. Cell Biol., 11, 113–127.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kozak, M. (1978) How do eukaryotic ribosomes select initiation regions in messenger RNA? Cell, 15, 1109–1123.

    Article  CAS  PubMed  Google Scholar 

  3. Kozak, M. (1989) The scanning model for translation: an update, J. Cell. Biol., 108, 229–241.

    Article  CAS  PubMed  Google Scholar 

  4. Kozak, M. (1980) Role of ATP in binding and migration of 40S ribosomal subunits, Cell, 22, 459–457.

    Article  CAS  PubMed  Google Scholar 

  5. Vassilenko, K. S., Alekhina, O. M., Dmitriev, S. E., Shatsky, I. N., and Spirin, A. S. (2011) Unidirectional constant rate motion of the ribosomal scanning particle during eukaryotic translation initiation, Nucleic Acids Res., 39, 5555–5567.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Sonenberg, N. (1993) Remarks on the mechanism of ribosome binding to eukaryotic mRNAs, Gene Expr., 3, 317–323.

    CAS  PubMed  Google Scholar 

  7. Haghighat, A., and Sonenberg, N. (1997) eIF4G dramatically enhances the binding of eIF4E to the mRNA 5′-cap structure, J. Biol. Chem., 272, 21677–21680.

    Article  CAS  PubMed  Google Scholar 

  8. Pestova, T. V., and Kolupaeva, V. G. (2002) The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection, Genes. Dev., 16, 2906–2922.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Shirokikh, N. E., and Spirin, A. S. (2008) Poly(A) leader of eukaryotic mRNA bypasses the dependence of translation on initiation factors, Proc. Natl. Acad. Sci. USA, 2105, 10738–10743.

    Article  Google Scholar 

  10. Dmitriev, S. E., Terenin, I. M., Dunaevsky, Y. E., Merrick, W. C., and Shatsky, I. N. (2003) Assembly of 48S translation initiation complexes from purified components with mRNAs that have some base pairing within their 5′-untranslated regions, Mol. Cell. Biol., 23, 8925–8933.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Gudkov, A. T., Ozerova, M. V., Shiryaev, V. M., and Spirin, A. S. (2005) 5′-poly(A) sequence as an effective leader for translation in eukaryotic cell-free systems, Biotechnol. Bioeng., 91, 468–473.

    Article  CAS  PubMed  Google Scholar 

  12. Shaloiko, L. N., Granovsky, I. E., Ivashina, T. V., Ksenzenko, V. N., Shirokov, V. A., and Spirin, A. S. (2004) Effective non-viral leader for cap-independent translation in a eukaryotic cell-free system, Biotechnol. Bioeng., 88, 730–739.

    Article  CAS  PubMed  Google Scholar 

  13. Agalarov, S. Ch., Sakharov, P. A., Fattakhova, D. Kh., Sogorin, E. A., and Spirin, A. S. (2014) Internal translation initiation and eIF4F/ATP-independent scanning of mRNA by eukaryotic ribosomal particles, Sci. Rep., 4, doi: 10.1038/srep04438.

    Google Scholar 

  14. Agalarov, S. C., Sogorin, E. A., Shirokikh, N. E., and Spirin, A. S. (2011) Insight into the structural organization of the omega leader of TMV RNA: the role of various regions of the sequence in the formation of a compact structure of the omega RNA, Biochem. Biophys. Res. Commun., 404, 250–253.

    Article  CAS  PubMed  Google Scholar 

  15. Kopeina, G. S., Afonina, Z. A., Gromova, K. V., Shirokov, V. A., Vasiliev, V. D., and Spirin, A. S. (2008) Step-wise formation of eukaryotic double-row polyribosomes and circular translation of polysomal mRNA, Nucleic Acids Res., 36, 2476–2478.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Alkalaeva, E. Z., Pisarev, A. V., Frolova, L. Y., Kisselev, L. L., and Pestova, T. V. (2006) In vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3, Cell, 125, 1125–1136.

    Article  CAS  PubMed  Google Scholar 

  17. Sakharov, P. A., Sokolov, A. S., and Agalarov, S. C. (2015) Nonhydrolyzable ATP analog 5′-adenylyl-imidodiphosphate (AMP-PNP) does not inhibit ATP-dependent scanning of leader sequence of mRNA, Biochemistry (Moscow), 80, 45–49.

    Article  CAS  Google Scholar 

  18. Hartz, D., McPheeters, D. S., Traut, R., and Gold, L. (1988) Extension inhibition analysis of translation initiation complexes, Methods Enzymol., 164, 419–425.

    Article  CAS  PubMed  Google Scholar 

  19. Gould, P. S., Bird, H., and Easton, A. J. (2005) Translation toeprinting assays using fluorescently labeled primers and capillary electrophoresis, Biotechniques, 38, 397–400.

    Article  CAS  PubMed  Google Scholar 

  20. Sleat, D. E., Gallie, D. R., Jefferson, R. A., Bevan, M. W., Turner, P. C., and Wilson, T. M. A. (1987) Characterization of the 50-leader sequence of tobacco mosaic virus RNA as general enhancer of translation in vitro, Gene, 60, 217–225.

    Article  CAS  PubMed  Google Scholar 

  21. Kovtun, A. A., Shirokikh, N. E., Gudkov, A. T., and Spirin, A. S. (2007) The leader sequence of tobacco mosaic virus RNA devoid of Watson–Crick secondary structure possesses a cooperatively melted, compact conformation, Biochem. Biophys. Res. Commun., 358, 368–372.

    Article  CAS  PubMed  Google Scholar 

  22. Shirokikh, N. E., Agalarov, S. C., and Spirin, A. S. (2010) Chemical and enzymatic probing of spatial structure of the omega leader of tobacco mosaic virus RNA, Biochemistry (Moscow), 75, 405–411.

    Article  CAS  Google Scholar 

  23. Gallie, D. R., and Walbot, V. (1992) Identification of the motifs within the tobacco virus 5′-leader responsible for enhancing translation, Nucleic Acids Res., 20, 4631–4638.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Sarabhai, A., and Brenner, S. (1967) A mutant which reinitiates the polypeptide chain after chain termination, J. Mol. Biol., 27, 145–162.

    Article  CAS  PubMed  Google Scholar 

  25. Svitkin, Y. V., Pause, A., Haghighat, A., Pyronnet, S., Witherell, G., Belsham, G. J., and Sonenberg, N. (2001) The requirement for eukaryotic initiation factor 4A (elF4A) in translation is in direct proportion to the degree of mRNA 5′-secondary structure, RNA, 7, 382–394.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Correspondence to S. Ch. Agalarov.

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Sakharov, P.A., Agalarov, S.C. Free initiation factors eIF4A and eIF4B are dispensable for translation initiation on uncapped mRNAs. Biochemistry Moscow 81, 1198–1204 (2016). https://doi.org/10.1134/S0006297916100175

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  • DOI: https://doi.org/10.1134/S0006297916100175

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