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:

Human CWC22 escorts the helicase eIF4AIII to spliceosomes and promotes exon junction complex assembly

Nature Structural & Molecular Biology volume 19, pages 983–990 (2012)Cite this article

Subjects

Abstract

The exon-junction complex (EJC) functionally links splicing to subsequent mRNA localization, translation and stability. Sequence-independent binding of the EJC core to RNA is ensured by the DEAD-box helicase eIF4AIII. Here, we identified the splicing factor CWC22 as a new eIF4AIII partner in flies and humans. CWC22 coexists with eIF4AIII in large protein complexes distinct from EJCs. Recombinant CWC22 directly contacts eIF4AIII and prevents it from binding RNA. In vitro splicing assays revealed that CWC22 introduces eIF4AIII to spliceosomes before remodeling to facilitate eIF4AIII incorporation into the EJC. Finally, using knockdowns in vivo, we showed that CWC22 is essential for EJC assembly. We elucidated the initial step of EJC assembly and the duality of CWC22 function that hinders eIF4AIII from nonspecifically binding RNA and escorts it to the splicing machinery to promote EJC assembly on mature mRNAs.

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: CWC22 is associated with eIF4AIII.
Figure 2: Recombinant CWC22 directly interacts with eIF4AIII.
Figure 3: CWC22 does not bind EJC core and inhibits eIF4AIII binding to RNA and ATPase activity.
Figure 4: CWC22 recruits eIF4AIII to active spliceosomes.
Figure 5: Knockdown of CWC22 prevents eIF4AIII binding to cellular mRNAs.

Similar content being viewed by others

References

  1. Moore, M.J. & Proudfoot, N.J. Pre-mRNA processing reaches back to transcription and ahead to translation. Cell 136, 688–700 (2009).

    Article  CAS  Google Scholar 

  2. 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  Google Scholar 

  3. Tange, T.Ø., Nott, A. & Moore, M.J. The ever-increasing complexities of the exon junction complex. Curr. Opin. Cell Biol. 16, 279–284 (2004).

    Article  CAS  Google Scholar 

  4. Le Hir, H. & Andersen, G.R. Structural insights into the exon junction complex. Curr. Opin. Struct. Biol. 18, 112–119 (2008a).

    Article  CAS  Google Scholar 

  5. Andersen, C.B. et al. Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA. Science 313, 1968–1972 (2006).

    Article  CAS  Google Scholar 

  6. Ballut, L. et al. The exon junction core complex is locked onto RNA by inhibition of eIF4AIII ATPase activity. Nat. Struct. Mol. Biol. 12, 861–869 (2005).

    Article  CAS  Google Scholar 

  7. Bono, F., Ebert, J., Lorentzen, E. & Conti, E. The crystal structure of the exon junction complex reveals how it maintains a stable grip on mRNA. Cell 126, 713–725 (2006).

    Article  CAS  Google Scholar 

  8. Fairman-Williams, M.E., Guenther, U.P. & Jankowsky, E. SF1 and SF2 helicases: family matters. Curr. Opin. Struct. Biol. 20, 313–324 (2010).

    Article  CAS  Google Scholar 

  9. Nielsen, K.H. et al. Mechanism of ATP turnover inhibition in the EJC. RNA 15, 67–75 (2009).

    Article  CAS  Google Scholar 

  10. Gehring, N.H., Lamprinaki, S., Kulozik, A.E. & Hentze, M.W. Disassembly of exon junction complexes by PYM. Cell 137, 536–548 (2009a).

    Article  CAS  Google Scholar 

  11. 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  Google Scholar 

  12. 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 (2001a).

    Article  CAS  Google Scholar 

  13. 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  Google Scholar 

  14. Ashton-Beaucage, D. et al. The exon junction complex controls the splicing of MAPK and other long intron-containing transcripts in Drosophila. Cell 143, 251–262 (2010).

    Article  CAS  Google Scholar 

  15. Michelle, L. et al. Proteins associated with the exon junction complex also control the alternative splicing of apoptotic regulators. Mol. Cell. Biol. 32, 954–967 (2012).

    Article  CAS  Google Scholar 

  16. Roignant, J.Y. & Treisman, J.E. Exon junction complex subunits are required to splice Drosophila MAP kinase, a large heterochromatic gene. Cell 143, 238–250 (2010).

    Article  CAS  Google Scholar 

  17. Hachet, O. & Ephrussi, A. Splicing of oskar RNA in the nucleus is coupled to its cytoplasmic localization. Nature 428, 959–963 (2004).

    Article  CAS  Google Scholar 

  18. Le Hir, H. & Seraphin, B. EJCs at the heart of translational control. Cell 133, 213–216 (2008b).

    Article  CAS  Google Scholar 

  19. Rebbapragada, I. & Lykke-Andersen, J. Execution of nonsense-mediated mRNA decay: what defines a substrate? Curr. Opin. Cell Biol. 21, 394–402 (2009).

    Article  CAS  Google Scholar 

  20. Hwang, J. & Maquat, L.E. Nonsense-mediated mRNA decay (NMD) in animal embryogenesis: to die or not to die, that is the question. Curr. Opin. Genet. Dev. 21, 422–430 (2011).

    Article  CAS  Google Scholar 

  21. Mühlemann, O. & Lykke-Andersen, J. How and where are nonsense mRNAs degraded in mammalian cells? RNA Biol. 7, 28–32 (2010).

    Article  Google Scholar 

  22. Giorgi, C. et al. The EJC factor eIF4AIII modulates synaptic strength and neuronal protein expression. Cell 130, 179–191 (2007).

    Article  CAS  Google Scholar 

  23. Saulière, J. et al. The exon junction complex differentially marks spliced junctions. Nat. Struct. Mol. Biol. 17, 1269–1271 (2010).

    Article  Google Scholar 

  24. Silver, D.L. et al. The exon junction complex component Magoh controls brain size by regulating neural stem cell division. Nat. Neurosci. 13, 551–558 (2010).

    Article  CAS  Google Scholar 

  25. Wahl, M.C., Will, C.L. & Luhrmann, R. The spliceosome: design principles of a dynamic RNP machine. Cell 136, 701–718 (2009).

    Article  CAS  Google Scholar 

  26. Bessonov, S., Anokhina, M., Will, C.L., Urlaub, H. & Luhrmann, R. Isolation of an active step I spliceosome and composition of its RNP core. Nature 452, 846–850 (2008).

    Article  CAS  Google Scholar 

  27. 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  Google Scholar 

  28. Makarov, E.M. et al. Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome. Science 298, 2205–2208 (2002).

    Article  CAS  Google Scholar 

  29. Merz, C., Urlaub, H., Will, C.L. & Luhrmann, R. Protein composition of human mRNPs spliced in vitro and differential requirements for mRNP protein recruitment. RNA 13, 116–128 (2007).

    Article  CAS  Google Scholar 

  30. 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  Google Scholar 

  31. Zhang, Z. & Krainer, A.R. Splicing remodels messenger ribonucleoprotein architecture via eIF4A3-dependent and -independent recruitment of exon junction complex components. Proc. Natl. Acad. Sci. USA 104, 11574–11579 (2007).

    Article  CAS  Google Scholar 

  32. Degot, S. et al. Association of the breast cancer protein MLN51 with the exon junction complex via its speckle localizer and RNA binding module. J. Biol. Chem. 279, 33702–33715 (2004).

    Article  CAS  Google Scholar 

  33. Gehring, N.H., Lamprinaki, S., Hentze, M.W. & Kulozik, A.E. The hierarchy of exon-junction complex assembly by the spliceosome explains key features of mammalian nonsense-mediated mRNA decay. PLoS Biol. 7, e1000120 (2009b).

    Article  Google Scholar 

  34. Tange, T.Ø., Shibuya, T., Jurica, M.S. & Moore, M.J. Biochemical analysis of the EJC reveals two new factors and a stable tetrameric protein core. RNA 11, 1869–1883 (2005).

    Article  CAS  Google Scholar 

  35. Ohi, M.D. & Gould, K.L. Characterization of interactions among the Cef1p-Prp19p-associated splicing complex. RNA 8, 798–815 (2002).

    Article  CAS  Google Scholar 

  36. Kelly, W.G., Xu, S., Montgomery, M.K. & Fire, A. Distinct requirements for somatic and germline expression of a generally expressed Caernorhabditis elegans gene. Genetics 146, 227–238 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Yeh, T.C. et al. Splicing factor Cwc22 is required for the function of Prp2 and for the spliceosome to escape from a futile pathway. Mol. Cell. Biol. 31, 43–53 (2011).

    Article  CAS  Google Scholar 

  38. Daguenet, E. et al. Perispeckles are major assembly sites for the exon junction core complex. Mol. Biol. Cell 23, 1765–1782 (2012).

    Article  CAS  Google Scholar 

  39. Custódio, N. et al. In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei. RNA 10, 622–633 (2004).

    Article  Google Scholar 

  40. 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  Google Scholar 

  41. Schmidt, U., Richter, K., Berger, A.B. & Lichter, P. In vivo BiFC analysis of Y14 and NXF1 mRNA export complexes: preferential localization within and around SC35 domains. J. Cell Biol. 172, 373–381 (2006).

    Article  CAS  Google Scholar 

  42. Hall, L.L., Smith, K.P., Byron, M. & Lawrence, J.B. Molecular anatomy of a speckle. Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 288, 664–675 (2006).

    Article  Google Scholar 

  43. 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  Google Scholar 

  44. Shibuya, T., Tange, T.O., Stroupe, M.E. & Moore, M.J. Mutational analysis of human eIF4AIII identifies regions necessary for exon junction complex formation and nonsense-mediated mRNA decay. RNA 12, 360–374 (2006).

    Article  CAS  Google Scholar 

  45. Chamieh, H., Ballut, L., Bonneau, F. & Le Hir, H. NMD factors UPF2 and UPF3 bridge UPF1 to the exon junction complex and stimulate its RNA helicase activity. Nat. Struct. Mol. Biol. 15, 85–93 (2008).

    Article  CAS  Google Scholar 

  46. Henn, A., Bradley, M.J. & De La Cruz, E.M. ATP utilization and RNA conformational rearrangement by DEAD-box proteins. Annu Rev Biophys 41, 247–267 (2012).

    Article  CAS  Google Scholar 

  47. Gozani, O., Patton, J.G. & Reed, R. A novel set of spliceosome-associated proteins and the essential splicing factor PSF bind stably to pre-mRNA prior to catalytic step II of the splicing reaction. EMBO J. 13, 3356–3367 (1994).

    Article  CAS  Google Scholar 

  48. Jankowsky, E. RNA helicases at work: binding and rearranging. Trends Biochem. Sci. 36, 19–29 (2011).

    Article  CAS  Google Scholar 

  49. Alexandrov, A., Colognori, D. & Steitz, J.A. Human eIF4AIII interacts with an eIF4G-like partner, NOM1, revealing an evolutionarily conserved function outside the exon junction complex. Genes Dev. 25, 1078–1090 (2011).

    Article  CAS  Google Scholar 

  50. Budiman, M.E. et al. Eukaryotic initiation factor 4a3 is a selenium-regulated RNA-binding protein that selectively inhibits selenocysteine incorporation. Mol. Cell 35, 479–489 (2009).

    Article  CAS  Google Scholar 

  51. Linder, P. & Jankowsky, E. From unwinding to clamping—the DEAD box RNA helicase family. Nat. Rev. Mol. Cell Biol. 12, 505–516 (2011).

    Article  CAS  Google Scholar 

  52. Hilbert, M., Kebbel, F., Gubaev, A. & Klostermeier, D. eIF4G stimulates the activity of the DEAD box protein eIF4A by a conformational guidance mechanism. Nucleic Acids Res. 39, 2260–2270 (2011).

    Article  CAS  Google Scholar 

  53. Marintchev, A. et al. Topology and regulation of the human eIF4A/4G/4H helicase complex in translation initiation. Cell 136, 447–460 (2009).

    Article  CAS  Google Scholar 

  54. Montpetit, B. et al. A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP6 in mRNA export. Nature 472, 238–242 (2011).

    Article  CAS  Google Scholar 

  55. Ideue, T., Sasaki, Y.T., Hagiwara, M. & Hirose, T. Introns play an essential role in splicing-dependent formation of the exon junction complex. Genes Dev. 21, 1993–1998 (2007).

    Article  CAS  Google Scholar 

  56. Schwer, B. A conformational rearrangement in the spliceosome sets the stage for Prp22-dependent mRNA release. Mol. Cell 30, 743–754 (2008).

    Article  CAS  Google Scholar 

  57. Dignam, J.D., Lebovitz, R.M. & Roeder, R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983).

    Article  CAS  Google Scholar 

  58. Lee, K.K., Florens, L., Swanson, S.K., Washburn, M.P. & Workman, J.L. The deubiquitylation activity of Ubp8 is dependent upon Sgf11 and its association with the SAGA complex. Mol. Cell. Biol. 25, 1173–1182 (2005).

    Article  CAS  Google Scholar 

  59. Chakrabarti, S. et al. Molecular mechanisms for the RNA-dependent ATPase activity of Upf1 and its regulation by Upf2. Mol. Cell 41, 693–703 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank E. Izaurralde (Max Planck Institute for Developmental Biology, Tuebingen, Germany) for anti-MAGOH antibodies and E. Conti (Max Planck Institute for Biochemistry, Martinsried, Germany) for purified EJC core. We thank the Institut de Génétique et de Biologie Moléculaire et Cellulaire peptide-synthesis and antibody facilities. We thank M. Noizet and X. Darzacq for technical assistance for cellular imagery. We are grateful to the members of the lab for technical assistance, helpful advice and discussions. This work was supported in part by the Centre National de la Recherche Scientifique (ATIP programme blanc 2008, to H.L.H.), the Agence Nationale de la Recherche (2008-BLAN-0323, 2011-BLAN-01801, to H.L.H.), the Fondation pour la Recherche Médicale (H.L.H.) and the Stowers Institute for Medical Research (M.B.).

Author information

Authors and Affiliations

  1. Institut de Biologie de l'Ecole Normale Supérieure, Centre National de la Recherche Scientifique UMR8197, Paris, France

    Isabelle Barbosa, Francesca Fiorini, Charlotte Barrandon & Hervé Le Hir

  2. Institut de Biologie de l'Ecole Normale Supérieure, Institut National de la Santé et de la Recherche Médicale U1024, Paris, France

    Isabelle Barbosa, Francesca Fiorini, Charlotte Barrandon & Hervé Le Hir

  3. Stowers Institute for Medical Research, Kansas City, Missouri, USA

    Nazmul Haque & Marco Blanchette

  4. Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique UMR7104, Illkirch, France

    Catherine Tomasetto

Authors
  1. Isabelle Barbosa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nazmul Haque
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Francesca Fiorini
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Charlotte Barrandon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Catherine Tomasetto
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marco Blanchette
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hervé Le Hir
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.H. established Drosophila cell lines, purified and identified Drosophila CWC22 and performed all experiments with Drosophila cells. I.B. and F.F. cloned reporter constructs for human protein expression. I.B. performed cellular imagery, produced recombinant proteins and performed the interaction assays. F.F. performed ATPase assays. C.T. produced anti-eIF4AIII and anti-CWC22 rabbit polyclonal antibodies. H.L.H. performed in vitro splicing reactions and coprecipitations. I.B. and C.B. performed immunoprecipitations and knockdowns. M.B. and H.L.H. provided resources, conceived and directed the project. H.L.H. wrote the paper.

Corresponding author

Correspondence to Hervé Le Hir.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1 and 2, and Supplementary Note (PDF 6095 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Barbosa, I., Haque, N., Fiorini, F. et al. Human CWC22 escorts the helicase eIF4AIII to spliceosomes and promotes exon junction complex assembly. Nat Struct Mol Biol 19, 983–990 (2012). https://doi.org/10.1038/nsmb.2380

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2380

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