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The pathway of hepatitis C virus mRNA recruitment to the human ribosome

Nature Structural & Molecular Biology volume 16, pages 397–404 (2009)Cite this article

Abstract

Eukaryotic protein synthesis begins with mRNA positioning in the ribosomal decoding channel in a process typically controlled by translation-initiation factors. Some viruses use an internal ribosome entry site (IRES) in their mRNA to harness ribosomes independently of initiation factors. We show here that a ribosome conformational change that is induced upon hepatitis C viral IRES binding is necessary but not sufficient for correct mRNA positioning. Using directed hydroxyl radical probing to monitor the assembly of IRES-containing translation-initiation complexes, we have defined a crucial step in which mRNA is stabilized upon initiator tRNA binding. Unexpectedly, however, this stabilization occurs independently of the AUG codon, underscoring the importance of initiation factor–mediated interactions that influence the configuration of the decoding channel. These results reveal how an IRES RNA supplants some, but not all, of the functions normally carried out by protein factors during initiation of protein synthesis.

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Figure 1: Directed hydroxyl radical probing of 18S rRNA from BABE-Fe–eIF3j–40S–HCV IRES complexes.
Figure 2: Toeprinting analysis of the 40S–HCV–eIF3j complexes.
Figure 3: Effects of eIF3 and eIF2–Met-tRNAi on directed hydroxyl radical probing of 18S rRNA with BABE-Fe–eIF3j.
Figure 4: Effects of eIF1, eIF1A, HCV and eIF3 on directed hydroxyl radical probing of 18S rRNA from BABE-Fe–eIF3j.
Figure 5: The effect of eIF2–Met-tRNAi on directed hydroxyl radical probing of 18S rRNA with BABE-Fe–eIF3j.
Figure 6: A model for HCV IRES association with the mRNA binding channel of the 40S subunit.

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References

  1. Pestova, T.V., Lorsch, J.R. & Hellen, C.U.T. The mechanism of translation initiation in eukaryotes. in Translational Control in Biology and Medicine (eds. Mathews, M.B., Sonenberg, N. & Hershey, J.W.B.) 87–128 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2007).

    Google Scholar 

  2. Fraser, C.S. & Doudna, J.A. Quantitative studies of ribosome conformational dynamics. Q. Rev. Biophys. 40, 163–189 (2007).

    Article  CAS  Google Scholar 

  3. Doudna, J.A. & Sarnow, P. Translation initiation by viral internal ribosome entry sites. in Translational Control in Biology and Medicine (eds. Mathews, M.B., Sonenberg, N. & Hershey, J.W.B.) 129–153 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2007).

    Google Scholar 

  4. Elroy-Stein, O. & Merrick, W.C. translation initiation via cellular internal ribosome entry sites. in Translational Control in Biology and Medicine (eds. Mathews, M.B., Sonenberg, N. & Hershey, J.W.B.) 155–172 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2007).

    Google Scholar 

  5. Pisarev, A.V., Shirokikh, N.E. & Hellen, C.U. Translation initiation by factor-independent binding of eukaryotic ribosomes to internal ribosomal entry sites. C. R. Biol. 328, 589–605 (2005).

    Article  CAS  Google Scholar 

  6. Fraser, C.S. & Doudna, J.A. Structural and mechanistic insights into hepatitis C viral translation initiation. Nat. Rev. Microbiol. 5, 29–38 (2007).

    Article  CAS  Google Scholar 

  7. 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, 67–83 (1998).

    Article  CAS  Google Scholar 

  8. Trachsel, H., Erni, B., Schreier, M.H. & Staehelin, T. Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors. J. Mol. Biol. 116, 755–767 (1977).

    Article  CAS  Google Scholar 

  9. Benne, R. & Hershey, J.W. The mechanism of action of protein synthesis initiation factors from rabbit reticulocytes. J. Biol. Chem. 253, 3078–3087 (1978).

    CAS  PubMed  Google Scholar 

  10. 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. USA 101, 16990–16995 (2004).

    Article  CAS  Google Scholar 

  11. Spahn, C.M. et al. Structure of the 80S ribosome from Saccharomyces cerevisiae–tRNA-ribosome and subunit-subunit interactions. Cell 107, 373–386 (2001).

    Article  CAS  Google Scholar 

  12. Taylor, D.J., Frank, J. & Kinzy, T.G. Structure and function of the eukaryotic ribosome and elongation fractors. in Translational Control in Biology and Medicine (eds. Mathews, M.B., Sonenberg, N. & Hershey, J.W.B.) pp. 59–85 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2007).

    Google Scholar 

  13. Spahn, C.M. et al. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40S ribosomal subunit. Science 291, 1959–1962 (2001).

    Article  CAS  Google Scholar 

  14. Passmore, L.A. et al. The eukaryotic translation initiation factors eIF1 and eIF1A induce an open conformation of the 40S ribosome. Mol. Cell 26, 41–50 (2007).

    Article  CAS  Google Scholar 

  15. Kolupaeva, V.G., Pestova, T.V. & Hellen, C.U. An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus. J. Virol. 74, 6242–6250 (2000).

    Article  CAS  Google Scholar 

  16. Otto, G.A. & Puglisi, J.D. The pathway of HCV IRES-mediated translation initiation. Cell 119, 369–380 (2004).

    Article  CAS  Google Scholar 

  17. Spahn, C.M. et al. Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes: the IRES functions as an RNA-based translation factor. Cell 118, 465–475 (2004).

    Article  CAS  Google Scholar 

  18. Unbehaun, A., Borukhov, S.I., Hellen, C.U. & Pestova, T.V. Release of initiation factors from 48S complexes during ribosomal subunit joining and the link between establishment of codon-anticodon base-pairing and hydrolysis of eIF2-bound GTP. Genes Dev. 18, 3078–3093 (2004).

    Article  CAS  Google Scholar 

  19. Fraser, C.S., Berry, K.E., Hershey, J.W. & Doudna, J.A. eIF3j is located in the decoding center of the human 40S ribosomal subunit. Mol. Cell 26, 811–819 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. Anthony, D.D. & Merrick, W.C. Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition. J. Biol. Chem. 267, 1554–1562 (1992).

    CAS  PubMed  Google Scholar 

  23. Kozak, M. Primer extension analysis of eukaryotic ribosome-mRNA complexes. Nucleic Acids Res. 26, 4853–4859 (1998).

    Article  CAS  Google Scholar 

  24. Kieft, J.S., Zhou, K., Jubin, R. & Doudna, J.A. Mechanism of ribosome recruitment by hepatitis C IRES RNA. RNA 7, 194–206 (2001).

    Article  CAS  Google Scholar 

  25. ElAntak, L., Tzakos, A.G., Locker, N. & Lukavsky, P.J. Structure of eIF3b RNA recognition motif and its interaction with eIF3j: structural insights into the recruitment of eIF3b to the 40 S ribosomal subunit. J. Biol. Chem. 282, 8165–8174 (2007).

    Article  CAS  Google Scholar 

  26. Joseph, S. & Noller, H.F. Directed hydroxyl radical probing using iron(II) tethered to RNA. Methods Enzymol. 318, 175–190 (2000).

    Article  CAS  Google Scholar 

  27. Reynolds, J.E. et al. Unique features of internal initiation of hepatitis C virus RNA translation. EMBO J. 14, 6010–6020 (1995).

    Article  CAS  Google Scholar 

  28. Jivotovskaya, A.V., Valasek, L., Hinnebusch, A.G. & Nielsen, K.H. 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 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  31. Fraser, C.S. et al. The j-subunit of human translation initiation factor eIF3 is required for the stable binding of eIF3 and its subcomplexes to 40 S ribosomal subunits in vitro. J. Biol. Chem. 279, 8946–8956 (2004).

    Article  CAS  Google Scholar 

  32. Pestova, T.V. & Hellen, C.U. Preparation and activity of synthetic unmodified mammalian tRNAi(Met) in initiation of translation in vitro. RNA 7, 1496–1505 (2001).

    Article  CAS  Google Scholar 

  33. Spanggord, R.J., Siu, F., Ke, A. & Doudna, J.A. RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle. Nat. Struct. Mol. Biol. 12, 1116–1122 (2005).

    Article  CAS  Google Scholar 

  34. Kieft, J.S. et al. The hepatitis C virus internal ribosome entry site adopts an ion-dependent tertiary fold. J. Mol. Biol. 292, 513–529 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  36. Culver, G.M. & Noller, H.F. Directed hydroxyl radical probing of RNA from iron(II) tethered to proteins in ribonucleoprotein complexes. Methods Enzymol. 318, 461–475 (2000).

    Article  CAS  Google Scholar 

  37. Pisarev, A.V., Unbehaun, A., Hellen, C.U. & Pestova, T.V. Assembly and analysis of eukaryotic translation initiation complexes. Methods Enzymol. 430, 147–177 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank D. King at the University of California, Berkeley, for expert MS analysis of modified proteins. We gratefully acknowledge members of the Doudna laboratory for discussions and comments on the manuscript. In particular, we would like to thank R. Spanggord for advice on hydroxyl radical probing and F. Siu for advice on RNA transcription protocols. This work was supported in part by a grant from the US National Institutes of Health to J.A.D. and J.W.B.H.

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Author notes

  1. Christopher S Fraser

    Present address: Present address: Section of Molecular and Cellular Biology, College of Biological Sciences, University of California, Davis, California 95616, USA.,

Authors and Affiliations

  1. Howard Hughes Medical Institute, University of California, Berkeley, 94720, California, USA

    Christopher S Fraser & Jennifer A Doudna

  2. Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, 95616, California, USA

    John W B Hershey

  3. Department of Molecular and Cell Biology, University of California, Berkeley, 94720, California, USA

    Jennifer A Doudna

  4. Department of Chemistry, University of California, Berkeley, 94720, California, USA

    Jennifer A Doudna

  5. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Jennifer A Doudna

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  1. Christopher S Fraser
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Contributions

C.S.F. performed the experiments; C.S.F., J.W.B.H. and J.A.D. designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Jennifer A Doudna.

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Fraser, C., Hershey, J. & Doudna, J. The pathway of hepatitis C virus mRNA recruitment to the human ribosome. Nat Struct Mol Biol 16, 397–404 (2009). https://doi.org/10.1038/nsmb.1572

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