Multiple binding of repressed mRNAs by the P-body protein Rck/p54 |
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Authors: | Michèle Ernoult-Lange Sonia Baconnais Maryannick Harper Nicola Minshall Sylvie Souquere Thomas Boudier Marianne Bénard Philippe Andrey Gérard Pierron Michel Kress Nancy Standart Eric le Cam Dominique Weil |
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Institution: | 1.UPMC Univ Paris 06, CNRS-FRE 3402, 75252 Paris cedex 5, France;2.CNRS UMR 8126, Institut Gustave Roussy, 94800 Villejuif, France;3.Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom;4.CNRS UMR 8122, Institut Gustave Roussy, 94800 Villejuif, France;5.UPMC Univ Paris 06, IFR83, 75252 Paris cedex 5, France;6.INRA, UMR1318, Institut Jean-Pierre Bourgin, RD10, 78000 Versailles, France;7.AgroParisTech, Institut Jean-Pierre Bourgin, RD10, 78000 Versailles, France |
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Abstract: | Translational repression is achieved by protein complexes that typically bind 3' UTR mRNA motifs and interfere with the formation of the cap-dependent initiation complex, resulting in mRNPs with a closed-loop conformation. We demonstrate here that the human DEAD-box protein Rck/p54, which is a component of such complexes and central to P-body assembly, is in considerable molecular excess with respect to cellular mRNAs and enriched to a concentration of 0.5 mM in P-bodies, where it is organized in clusters. Accordingly, multiple binding of p54 proteins along mRNA molecules was detected in vivo. Consistently, the purified protein bound RNA with no sequence specificity and high nanomolar affinity. Moreover, bound RNA molecules had a relaxed conformation. While RNA binding was ATP independent, relaxing of bound RNA was dependent on ATP, though not on its hydrolysis. We propose that Rck/p54 recruitment by sequence-specific translational repressors leads to further binding of Rck/p54 along mRNA molecules, resulting in their masking, unwinding, and ultimately recruitment to P-bodies. Rck/p54 proteins located at the 5' extremity of mRNA can then recruit the decapping complex, thus coupling translational repression and mRNA degradation. |
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Keywords: | DDX6 mRNA storage mRNA decay DEAD-box masking |
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