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The exon junction core complex is locked onto RNA by inhibition of eIF4AIII ATPase activity

Nature Structural & Molecular Biology volume 12, pages 861–869 (2005)Cite this article

Abstract

The multiprotein exon junction complex (EJC) is assembled on mRNAs as a consequence of splicing. EJC core components maintain a stable grip on mRNAs even as the overall EJC protein composition evolves while mRNAs travel to the cytoplasm. Here we show that recombinant EJC subunits MLN51, MAGOH and Y14, together with the DEAD-box protein eIF4AIII bound to ATP, are necessary and sufficient to form a highly stable complex on single-stranded RNA. Cross-linking and RNase protection studies indicate that this recombinant complex recapitulates the EJC core. The stable association of the recombinant EJC core with RNA is maintained by inhibition of eIF4AIII ATPase activity by MAGOH-Y14. We elucidate the modalities of EJC binding to RNA and provide the first example of how cellular machineries may use RNA helicases to clamp several proteins onto RNA in stable and sequence-independent manners.

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Figure 1: MLN51, eIF4AIII and MAGOH-Y14 form a stable complex.
Figure 2: The complex formed by eIF4AIII, MLN51-S and MAGOH-Y14ΔN strongly binds RNA.
Figure 3: Ability of various eIF4AIII and eIF4AI chimeric proteins to form the reconstituted complex.
Figure 4: Effect of site-specific MLN51-S mutations on complex formation.
Figure 5: Protein-RNA cross-linking analysis of the recombinant complex.
Figure 6: The reconstituted complex shows biochemical similarities to the EJC.
Figure 7: Effect of MLN51-S and MAGOH-Y14ΔN on eIF4AIII ATPase activity and complex stability.
Figure 8: Effect of MLN51-S mutations on its in vivo localization.

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Acknowledgements

We thank T. Tange, M.J. Moore, P. Linder and S. Degot for plasmids and J. Stévenin for antibodies to 9G8. We are grateful to A. Dziembowski for technical assistance. We acknowledge our laboratory and A. Expert-Bezancon for helpful advice and discussions and M. Jurica, D. Libri and A. Nott 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 and Human Frontier Science Program (B.S.) and the Research Ministry (grant ACI–Jeunes chercheurs to H.L.H.).

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  1. Equipe Labélisée La Ligue, Centre de Génétique Moléculaire, associé à l'Université Paris 6, CNRS UPR2167, Avenue de la Terrasse, Gif-sur-Yvette, 91198, France

    Lionel Ballut, Brice Marchadier, Bertrand Séraphin & Hervé Le Hir

  2. Département de Pathologie Moléculaire, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UPR 6520 / INSERM U596 / Université Louis Pasteur, BP 10142, Illkirch, 67404, C.U. de Strasbourg, France

    Aurélie Baguet & Catherine Tomasetto

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Correspondence to Hervé Le Hir.

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Supplementary Fig. 1

Stability of the complex in presence of the eIF4AIII mutant K92A (PDF 281 kb)

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Ballut, L., Marchadier, B., Baguet, A. 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). https://doi.org/10.1038/nsmb990

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