Études des aspects structuraux et dynamiques liés à l'activité des particules ribonucléoprotéiques sRNP à boîtes H/ACA catalysant chez les archées l'isomérisation de résidus uridines en pseudouridines

Abstract : Pseudouridylation reaction that consists in the isomerization of uridines (U) into pseudouridines (PSI) is the most frequent post-transcriptional modification found in RNAs. It is catalyzed by enzymes with RNA:PSI-synthase activity. In Archaea and Eukarya, ribonucleoprotein particles, the so-called box H/ACA RNPs, possess such activity. In Archaea, the box H/ACA complex comprises four invariable proteins namely the RNA:PSI-synthase aCBF5 and three protein partners L7Ae, aNOP10 and aGAR1, and specific to each RNP, an RNA acting as a guide to secure by base pairing the RNA substrate and define the position to be modify. During these last years, several crystal structures of components of archaeal H/ACA RNP and fully assembled RNP have been resolved. Complementary biochemical and biophysical studies allowed detailed structure-function analyses to identify the role of the different components. During this work we identified functional differences between two RNA guides expressed in the same archaea, and demonstrated that the interaction between L7Ae and aNOP10 is important for a correct positioning of substrate RNA. We also tested in vitro the assembly and activity of RNP reconstituted on H/ACA-like guide RNAs. We investigated dynamics of substrate RNA during the pseudouridylation. We found that aGAR1 was not necessary for the turnover of the particle, that the temperature was crucial for such activity, and that the chemical structure of the targeted residue and length of the substrate RNA were important determinants for substrate selectivity. Finally, we have also developed a new technic based on FRET adapted to monitor binding of the susbtrate RNA to the box H/ACA RNP enzyme
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Anne-Sophie Tillault. Études des aspects structuraux et dynamiques liés à l'activité des particules ribonucléoprotéiques sRNP à boîtes H/ACA catalysant chez les archées l'isomérisation de résidus uridines en pseudouridines. Biochimie, Biologie Moléculaire. Université de Lorraine, 2013. Français. ⟨NNT : 2013LORR0150⟩. ⟨tel-01750220⟩

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