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Étude biochimique et biophysique de l’ARN hélicase UPF1 : un moteur moléculaire hautement régulé

Abstract : UPF1 (Up-Frameshift 1) is a multifunctional helicase that unwinds nucleic acids and is conserved throughout the eukaryote kingdom. UPF1 is required for the Nonsense Mediated mRNA Decay (NMD) surveillance pathway, which degrades mRNAs carrying premature termination codons, among other substrates. UPF1 is the archetype of a family of 11 helicases sharing similar cores but involved in various cellular pathways. However, the structure-function relationship and intrinsic biophysical properties of these molecular engines remain poorly described. In vitro, the UPF1 helicase core is highly processive, it travels along thousands of RNA or DNA bases and unwinds double-strands. In this work, we looked for key factors governing this remarkable processivity. We combined biochemical and biophysical techniques. In particular, we used magnetic tweezers to study helicases in real time at a single molecule scale. In contrast to UPF1, the related IGHMBP2 is not processive, thus processivity is not a shared family trait. Based on the 3D structures of both proteins, we designed various mutants and used them to identify structural elements that modulate processivity. Our approach reveals that UPF1 has a very firm grip on nucleic acids, guaranteeing long binding lifetimes and action times that dictate its high processivity. Thanks to the variety in mutant behaviors, we built a novel mechanistic model linking binding energy to processivity. Furthermore, we show that UPF1 processivity is required for an efficient NMD in vivo. In addition, we used the same biochemical and biophysical tools to investigate a natural human UPF1 isoform moving faster than the major isoform, and to compare the regulation of human andyeast UPF1 by their flanking domains. We also characterized the interaction of yeast UPF1 with new NMD partners. Our work shows how a combination of biochemical, biophysical, structural and in vivo tools can offer unexpected insights into the operating mode of molecular motors.
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Joanne Kanaan. Étude biochimique et biophysique de l’ARN hélicase UPF1 : un moteur moléculaire hautement régulé. Biochimie, Biologie Moléculaire. Université Paris sciences et lettres, 2018. Français. ⟨NNT : 2018PSLEE008⟩. ⟨tel-01878209⟩

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