Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay

Nature Structural & Molecular Biology volume 11, pages 346–351 (2004)Cite this article

Abstract

The exon junction complex (EJC), a set of proteins deposited on mRNAs as a consequence of pre-mRNA splicing, is a key effector of downstream mRNA metabolism. We have identified eIF4AIII, a member of the eukaryotic translation initiation factor 4A family of RNA helicases (also known as DExH/D box proteins), as a novel EJC core component. Crosslinking and antibody inhibition studies suggest that eIF4AIII constitutes at least part of the platform anchoring other EJC components to spliced mRNAs. A nucleocytoplasmic shuttling protein, eIF4AIII associates in vitro and in vivo with two other EJC core factors, Y14 and Magoh. In mammalian cells, eIF4AIII is essential for nonsense-mediated mRNA decay (NMD). Finally, a model is proposed by which eIF4AIII represents a new functional class of DExH/D box proteins that act as RNA clamps or 'place holders' for the sequence-independent attachment of additional factors to RNAs.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: eIF4AIII is associated with spliceosomes and binds directly to spliced mRNA.
Figure 2: eIF4AIII is a nucleocytoplasmic shuttling protein that associates with Y14 and Magoh.
Figure 3: Anti-eIF4AIII inhibits the second step of splicing and EJC deposition in vitro.
Figure 4: Depletion of eIF4AIII in vivo reverses NMD, but has no apparent effect on splicing.

Similar content being viewed by others

References

  1. Le Hir, H., Izaurralde, E., Maquat, L.E. & Moore, M.J. The spliceosome deposits multiple proteins 20–24 nucleotides upstream of mRNA exon-exon junctions. EMBO J. 19, 6860–6869 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Le Hir, H., Nott, A. & Moore, M.J. How introns influence and enhance eukaryotic gene expression. Trends Biochem. Sci. 28, 215–220 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Ishigaki, Y., Li, X., Serin, G. & Maquat, L.E. Evidence for a pioneer round of mRNA translation: mRNAs subject to nonsense-mediated decay in mammalian cells are bound by CBP80 and CBP20. Cell 106, 607–617 (2001).

    Article  CAS  PubMed  Google Scholar 

  4. Lejeune, F., Ishigaki, Y., Li, X. & Maquat, L.E. The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling. EMBO J. 21, 3536–3545 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Dostie, J. & Dreyfuss, G. Translation is required to remove Y14 from mRNAs in the cytoplasm. Curr. Biol. 12, 1060–1067 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Wagner, E. & Lykke-Andersen, J. mRNA surveillance: the perfect persist. J. Cell Sci. 115, 3033–3038 (2002).

    CAS  PubMed  Google Scholar 

  7. Culbertson, M.R. & Leeds, P.F. Looking at mRNA decay pathways through the window of molecular evolution. Curr. Opin. Genet. Dev. 13, 207–214 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Le Hir, H., Gatfield, D., Izaurralde, E. & Moore, M.J. The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay. EMBO J. 20, 4987–4997 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhou, Z. et al. The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans. Nature 407, 401–405 (2000).

    Article  CAS  PubMed  Google Scholar 

  10. Nott, A., Le Hir, H. & Moore, M.J. Splicing enhances translation in mammalian cells: an additional function of the exon junction complex. Genes Dev. 18, 210–222 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Palacios, I.M. RNA processing: splicing and the cytoplasmic localisation of mRNA. Curr. Biol. 12, R50–52 (2002).

    Article  CAS  PubMed  Google Scholar 

  12. Reichert, V.L., Le Hir, H., Jurica, M.S. & Moore, M.J. 5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly. Genes Dev. 16, 2778–2791 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Jurica, M.S. & Moore, M.J. Pre-mRNA splicing: awash in a sea of proteins. Mol. Cell 12, 5–14 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. Kataoka, N. et al. Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm. Mol. Cell 6, 673–682 (2000).

    Article  CAS  PubMed  Google Scholar 

  15. Kataoka, N., Diem, M.D., Kim, V.N., Yong, J. & Dreyfuss, G. Magoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing-dependent exon-exon junction complex. EMBO J. 20, 6424–6433 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Le Hir, H., Gatfield, D., Braun, I.C., Forler, D. & Izaurralde, E. The protein Mago provides a link between splicing and mRNA localization. EMBO Rep. 2, 1119–1124 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kim, V.N. et al. The Y14 protein communicates to the cytoplasm the position of exon-exon junctions. EMBO J. 20, 2062–2068 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Fribourg, S., Gatfield, D., Izaurralde, E. & Conti, E. A novel mode of RBD-protein recognition in the Y14-Mago complex. Nat. Struct. Biol. 10, 433–439 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Shi, H. & Xu, R.M. Crystal structure of the Drosophila Mago nashi-Y14 complex. Genes Dev. 17, 971–976 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lau, C.K., Diem, M.D., Dreyfuss, G. & Van Duyne, G.D. Structure of the y14-magoh core of the exon junction complex. Curr. Biol. 13, 933–941 (2003).

    Article  CAS  PubMed  Google Scholar 

  21. Jurica, M.S., Licklider, L.J., Gygi, S.R., Grigorieff, N. & Moore, M.J. Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis. RNA 8, 426–439 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Li, Q. et al. Eukaryotic translation initiation factor 4AIII (eIF4AIII) is functionally distinct from eIF4AI and eIF4AII. Mol. Cell. Biol. 19, 7336–7346 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Holzmann, K. et al. A human common nuclear matrix protein homologous to eukaryotic translation initiation factor 4A. Biochem. Biophys. Res. Commun. 267, 339–344 (2000).

    Article  CAS  PubMed  Google Scholar 

  24. Weinstein, D.C., Honoré, E. & Hemmati-Brivanlou, A. Epidermal induction and inhibition of neural fate by translation initiation factor 4AIII. Development 124, 4235–4242 (1997).

    CAS  PubMed  Google Scholar 

  25. Gingras, A.C., Raught, B. & Sonenberg, N. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem. 68, 913–963 (1999).

    Article  CAS  PubMed  Google Scholar 

  26. Rogers, G.W., Jr., Komar, A.A. & Merrick, W.C. eIF4A: the godfather of the DEAD box helicases. Prog. Nucleic Acid Res. Mol. Biol. 72, 307–331 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Zhou, Z., Licklider, L.J., Gygi, S.P. & Reed, R. Comprehensive proteomic analysis of the human spliceosome. Nature 419, 182–185 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Le Hir, H., Moore, M.J. & Maquat, L.E. Pre-mRNA splicing alters mRNP composition: evidence for stable association of proteins at exon-exon junctions. Genes Dev. 14, 1098–1108 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Lykke-Andersen, J., Shu, M.D. & Steitz, J.A. Communication of the position of exon-exon junctions to the mRNA surveillance machinery by the protein RNPS1. Science 293, 1836–1839 (2001).

    Article  CAS  PubMed  Google Scholar 

  30. Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Mendell, J.T., ap Rhys, C.M. & Dietz, H.C. Separable roles for rent1/hUpf1 in altered splicing and decay of nonsense transcripts. Science 298, 419–422 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Zhou, Z. & Reed, R. Human homologs of yeast prp16 and prp17 reveal conservation of the mechanism for catalytic step II of pre-mRNA splicing. EMBO J. 17, 2095–2106 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Perlick, H.A., Medghalchi, S.M., Spencer, F.A., Kendzior, R.J. Jr. & Dietz, H.C. Mammalian orthologues of a yeast regulator of nonsense transcript stability. Proc. Natl. Acad. Sci. USA 93, 10928–10932 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wilusz, C.J., Wang, W. & Peltz, S.W. Curbing the nonsense: the activation and regulation of mRNA surveillance. Genes Dev. 15, 2781–2785 (2001).

    CAS  PubMed  Google Scholar 

  35. Mühlemann, O. et al. Precursor RNAs harboring nonsense codons accumulate near the site of transcription. Mol. Cell 8, 33–43 (2001).

    Article  PubMed  Google Scholar 

  36. Tanner, N.K. & Linder, P. DExD/H box RNA helicases: from generic motors to specific dissociation functions. Mol. Cell 8, 251–262 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Kim, J.L. et al. Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure 6, 89–100 (1998).

    Article  CAS  PubMed  Google Scholar 

  38. de la Cruz, J., Kressler, D. & Linder, P. Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families. Trends Biochem. Sci. 24, 192–198 (1999).

    Article  CAS  PubMed  Google Scholar 

  39. Linder, P., Tanner, N.K. & Banroques, J. From RNA helicases to RNPases. Trends Biochem. Sci. 26, 339–341 (2001).

    Article  CAS  PubMed  Google Scholar 

  40. Schwer, B. A new twist on RNA helicases: DExH/D box proteins as RNPases. Nat. Struct. Biol. 8, 113–116 (2001).

    Article  CAS  PubMed  Google Scholar 

  41. Jankowsky, E., Gross, C.H., Shuman, S. & Pyle, A.M. Active disruption of an RNA-protein interaction by a DExH/D RNA helicase. Science 291, 121–125 (2001).

    Article  CAS  PubMed  Google Scholar 

  42. Query, C.C., Strobel, S.A. & Sharp, P.A. Three recognition events at the branch-site adenine. EMBO J. 15, 1392–1402 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Maroney, P.A., Romfo, C.M. & Nilsen, T.W. Functional recognition of 5′ splice site by U4/U6. U5 tri-snRNP defines a novel ATP-dependent step in early spliceosome assembly. Mol. Cell 6, 317–328 (2000).

    Article  CAS  PubMed  Google Scholar 

  44. Gozani, O., Feld, R. & Reed, R. Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A. Genes Dev. 10, 233–243 (1996).

    Article  CAS  PubMed  Google Scholar 

  45. Palacios, I.M., Gatfield, D., St Johnston, D. & Izaurralde, E. An eIF4AIII-containing complex required for mRNA localization and nonsense-mediated mRNA decay. Nature 427, 753–757 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Fan, X.C. & Steitz, J.A. Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs. EMBO J. 17, 3448–3460 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Chan, C.C. et al. eIF4A3 is a novel component of the exon junction complex. RNA 10, 200–209 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ferraiuolo, M.A. et al. A nuclear translation-like factor eIF4AIII is recruited to the mRNA during splicing and functions in nonsense-mediated decay. Proc. Natl. Acad. Sci. USA. (in the press).

Download references

Acknowledgements

We thank E. Izaurralde, R. Reed and J. Steitz for plasmids and antibodies against Y14 and Magoh. We are grateful to G. Dreyfuss, E. Izaurralde and R. Reed for communicating results prior to publication. We acknowledge our laboratory members, especially M. Jurica, A. Nott, F. LaRiviere and C. Mock-Casagrande for helpful advice and discussions. We are grateful to N. Levin for synthesizing siRNA oligos. M.J.M. is a Howard Hughes Medical Institute associate investigator. This work was supported in part by US National Institutes of Health grant GM53007 (M.J.M.) and a research stipend from the Alfred Benzon Foundation (T.Ø.T.).

Author information

Author notes

  1. Toshiharu Shibuya and Thomas Ø Tange: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, 02454, Massachusetts, USA

    Toshiharu Shibuya, Thomas Ø Tange & Melissa J Moore

  2. Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, H3G 1Y6, Quebec, Canada

    Nahum Sonenberg

Authors
  1. Toshiharu Shibuya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas Ø Tange
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nahum Sonenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Melissa J Moore
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Melissa J Moore.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shibuya, T., Tange, T., Sonenberg, N. et al. eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay. Nat Struct Mol Biol 11, 346–351 (2004). https://doi.org/10.1038/nsmb750

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb750

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing