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NMD factors UPF2 and UPF3 bridge UPF1 to the exon junction complex and stimulate its RNA helicase activity

Nature Structural & Molecular Biology volume 15, pages 8593 (2008) | Download Citation

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Abstract

Nonsense-mediated mRNA decay (NMD) eliminates mRNAs containing a premature translation termination codon through the recruitment of the conserved NMD factors UPF1, UPF2 and UPF3. In humans, a dynamic assembly pathway allows UPF1 to join UPF2 and UPF3 recruited to the mRNA by the exon-junction complex (EJC). Here we show that the recombinant EJC core is sufficient to reconstitute, with the three UPF proteins, a stable heptameric complex on RNA. The EJC proteins MAGOH, Y14 and eIF4AIII provide a composite binding site for UPF3b that serves as a bridge to UPF2 and UPF1. In the UPF trimeric complex, UPF2 and UPF3b cooperatively stimulate both ATPase and RNA helicase activities of UPF1. This work demonstrates that the EJC core is sufficient to stably anchor the UPF proteins to mRNA and provides insights into the regulation of its central effector, UPF1.

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Acknowledgements

We thank E. Conti, (MPI of Biochemistry, Martinsried, Germany), S. Cusack (EMBL, Grenoble, France) and F. Lejeune (IGMM-CNRS, Montpellier, France) for UPF cDNAs. We are grateful to B. Marchadier, B. Séraphin and his laboratory for technical assistance, helpful advice and discussions. We acknowledge S. Camier, E. Conti, M. Dreyfus and A. Expert-Bezançon for carefully reading the manuscript. This work was supported in part by the Centre National de la Recherche Scientifique (CNRS), La Ligue Contre le Cancer, the Research Ministry (grant ACI-jeunes chercheurs) and the Agence Nationale de la Recherche (ANR). F.B. was supported by the Max Planck Institute of Biochemistry.

Author information

Author notes

    • Lionel Ballut

    Present addresses: IBCP, CNRS UMR5086, Université de Lyon 1, France (L.B.); Max Planck Institute of Biochemistry, Department of Structural Cell Biology, Am Klopferspitz 18, D-82152 Martinsried, Germany (F.B.).

    • Hala Chamieh
    •  & Lionel Ballut

    These authors contributed equally to this work.

Affiliations

  1. Equipe Labélisée La Ligue, Centre de Génétique Moléculaire, CNRS UPR 2167, Associée aux Universités Paris 6 et Paris 11, Avenue de la terrasse, Gif-sur-Yvette, F-91198, France.

    • Hala Chamieh
    • , Lionel Ballut
    • , Fabien Bonneau
    •  & Hervé Le Hir

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Contributions

L.B. initiated this work; H.C., L.B., F.B. and H.L.H. cloned cDNAs, and expressed and purified recombinant proteins; L.B., H.C. and F.B. performed protein binding assays; F.B. carried out RNA protection and ATPase assays; H.C. carried out duplex unwinding assays. H.L.H. provided resources, conceived and directed the project and wrote the article.

Corresponding author

Correspondence to Hervé Le Hir.

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https://doi.org/10.1038/nsmb1330

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