Abstract
Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that degrades aberrant mRNAs containing premature termination codons (PTCs). NMD is triggered upon the assembly of the UPF surveillance complex near a PTC. In humans, UPF assembly is prompted by the exon junction complex (EJC). We investigated the molecular architecture of the human UPF complex bound to the EJC by cryo-EM and using positional restraints from additional EM, MS and biochemical interaction data. The heptameric assembly is built around UPF2, a scaffold protein with a ring structure that closes around the CH domain of UPF1, keeping the helicase region in an accessible and unwinding-competent state. UPF2 also positions UPF3 to interact with the EJC. The geometry is such that this transient complex poises UPF1 to elicit helicase activity toward the 3′ end of the mRNP.
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Acknowledgements
We thank E. Arias-Palomo, E. Torreira and U. Jayachandran for help in the initial stages of this project, and C. Boulegue and S. Uebel (at the Max Planck Institute Martinsried Core Facility) and members of our labs for critical reading of the manuscript. This work was funded by the Spanish Government (SAF2008-00451 and SAF2011-22988 to O.L.) and the Red Temática de Investigación Cooperativa en Cáncer from the Instituto de Salud Carlos III (RD06/0020/1001 to O.L. and contract to R.M.). O.L. is additionally supported by the Human Frontiers Science Program (RGP39/2008 to O.L.), the Fundación Ramón Areces and the Government from the Autonomous Region of Madrid (S2010-BMD-2316). This work was also supported by the Max Planck Gesellschaft, the Sonderforschungsbereich SFB646, the Gottfried Wilhelm Leibniz Program of the Deutsche Forschungsgemeinschaft and the Center for Integrated Protein Science Munich (E.C.).
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R.M., R.C., D.G. and M.L. carried out EM. R.M. did all the image processing. R.C. purified and analyzed UPF2–UPF3 complexes. M.R. and H.U. did MS experiments. G.B. purified all proteins and complexes used, cross-linked complexes and analyzed data. H.U., E.C. and O.L. designed experiments, analyzed results and wrote the manuscript.
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Melero, R., Buchwald, G., Castaño, R. et al. The cryo-EM structure of the UPF–EJC complex shows UPF1 poised toward the RNA 3′ end. Nat Struct Mol Biol 19, 498–505 (2012). https://doi.org/10.1038/nsmb.2287
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DOI: https://doi.org/10.1038/nsmb.2287
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