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CBP80 promotes interaction of Upf1 with Upf2 during nonsense-mediated mRNA decay in mammalian cells

Nature Structural & Molecular Biology volume 12, pages 893–901 (2005)Cite this article

Abstract

In mammalian cells, nonsense-mediated messenger RNA decay (NMD) targets newly synthesized nonsense-containing mRNA bound by the cap-binding-protein heterodimer CBP80-CBP20 and at least one exon-junction complex (EJC). An EJC includes the NMD factors Upf3 or Upf3X and Upf2, and Upf2 recruits Upf1. Once this pioneer translation initiation complex is remodeled so that CBP80-CBP20 is replaced by eukaryotic initiation factor 4E, the mRNA is no longer detectably targeted for NMD. Here, we provide evidence that CBP80 augments the efficiency of NMD but not of Staufen1 (Stau1)-mediated mRNA decay (SMD). SMD depends on the recruitment of Upf1 by the RNA-binding protein Stau1 but does not depend on the other Upf proteins. We find that CBP80 interacts with Upf1 and promotes the interaction of Upf1 with Upf2 but not with Stau1.

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Figure 1: Downregulating CBP80 abrogates the reduction in mRNA abundance that is elicited by tethering Upf2 or Upf3, but not Upf1 or Stau1, downstream of a termination codon.
Figure 2: siRNA-mediated downregulation of CBP80 inhibits NMD but not SMD, and the inhibition of NMD is reversed by expressing siRNA-resistant CBP80.
Figure 3: 4E-BP1 inhibits the reduction in FLuc-MS2bs mRNA abundance mediated by the tethering of MS2-Upf1 or MS2-HA-Stau1 but not MS2-Upf2 or MS2-Upf3.
Figure 4: 4E-BP1 inhibits the reduction of FLuc-MS2bs mRNA abundance mediated by the tethering of MS2-Upf1 or MS2-HA-Stau1 by affecting eIF4E-bound but not CBP80-bound mRNA.
Figure 5: CBP80 interacts with Upf1.
Figure 6: CBP80 promotes interaction of Upf1 with Upf2 but does not detectably affect interaction of Upf1 with Stau1.
Figure 7: CBP80 promotes interaction of Upf1 with Upf2 in vitro.
Figure 8: Model proposing how CBP80 promotes NMD but not SMD.

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Acknowledgements

We thank E. Wolcott for technical assistance; members of the Maquat lab, especially H. Kuzmiak, for comments on the manuscript; E. Izaurralde (European Molecular Biology Laboratory, Heidelberg, Germany) for anti-CBP80; L. DesGroseillers (Université de Montreal, Montreal) for anti-Stau1; H. Baumann and B. Held (Roswell Park Cancer Institute, Buffalo, New York, USA) for anti-MUP; J. Lykke-Andersen (University of Colorado, Boulder, Colorado, USA) for anti-Upf1 and pcNMS2-UPF plasmids; N. Sonenberg (McGill Cancer Center, Montreal) for pACTAG2-HA3, pACTAG2-HA3-4E-BP1, pACTAG2-heIF4E and anti-4E-BP1; S. Morley (University of Sussex, Falmer, Brighton, UK) for pBS-Hs-CBP80 and pRSET-Hs-CBP80; J. Lewis (Anadys Pharmaceuticals, Inc., UK) for pRSET-Hs-CBP20; and F. LeRoy and S. Peltz (University of Medicine and Dentistry of New Jersey, Pistcataway, New Jersey, USA) for the FLAG-Upf1 baculovirus expression vector. This work was supported by US National Institutes of Health grant GM059614 to L.E.M.

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Author notes

  1. Yoon Ki Kim

    Present address: School of Life Sciences and Biotechnology, Korea University, Seoul, 136-701, Korea

  2. Fabrice Lejeune

    Present address: Institut de Génétique Moléculaire de Montpellier, Centre National de la Recherche Scientifique, 34290, Montpellier, France

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, 601 Elmwood Avenue, Rochester, Box 712, 14642, New York, USA

    Nao Hosoda, Yoon Ki Kim, Fabrice Lejeune & Lynne E Maquat

  2. School of Life Sciences and Biotechnology, Korea University, Seoul, 136-701, Korea

    Fabrice Lejeune

  3. Institut de Génétique Moléculaire de Montpellier, Centre National de la Recherche Scientifique, 34290, Montpellier, France

    Yoon Ki Kim

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Supplementary information

Supplementary Fig. 1

SMD targets eIF4E-bound mRNA. (PDF 123 kb)

Supplementary Fig. 2

4E-BP1 inhibits the reduction in FLuc-MS2bs mRNA abundance that is mediated by tethering either MS2-Upf1 or MS2-HA-Stau1. (PDF 129 kb)

Supplementary Fig. 3

Tethering CBP80 or CBP20 downstream of a termination codon triggers NMD. (PDF 5272 kb)

Supplementary Methods (PDF 56 kb)

Supplementry Data (PDF 94 kb)

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Hosoda, N., Kim, Y., Lejeune, F. et al. CBP80 promotes interaction of Upf1 with Upf2 during nonsense-mediated mRNA decay in mammalian cells. Nat Struct Mol Biol 12, 893–901 (2005). https://doi.org/10.1038/nsmb995

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