Analyse structurale et fonctionnelle des particules sRNP à boîtes H/ACA, catalysant la formation ciblée des pseudouridines dans les ARN d'archaea

Abstract : Pseudouridine ([Psi]), uridine's isomer, is the most abundant modified nucleoside found in structured RNAs. The reaction of pseudouridylation is either catalyzed by a limited number of standalone RNA:[Psi]-synthases, or in eukaryotes and archaea by multiple ribonucleoprotein particles (the so-called, box H/ACA sno/sca/sRNPs). Each RNP is composed of a specific box H/ACA RNA used as a guide to define the U residue for modification, and a common set of four proteins- the RNA:[Psi]-synthase Dyskerin/aCBF5, and the auxiliary proteins NOP10/aNOP10, GAR1/aGAR1, NHP2/L7Ae (respectively in human and archaea). Initially, 7 guide RNAs were identified in the archaeon P. abyssi, which were used as models for structure-function analyses. Activities of RNPs reconstituted with purified protein and RNA components were measured; RNA 2D structure within RNPs was investigated by chemical and enzymatic probing assays as well as by circular dichroism. By taking into consideration the various 3D structures recently resolved, we were able to pinpoint the RNA molecular determinants and to clarify the role played by each protein, their domains, and some of their conserved residues. Hence, interaction between aNOP10 and L7Ae was found to be crucial for RNP activity. Moreover, we show that the enzyme aCBF5 catalyzes pseudouridylation at the position 55 in tRNAs and the position 2603 in 23S rRNA without the use of any guide sRNA. The role of residues conserved in aNOP10, and in the active site and the ß7/ß10 loop of aCBF5 for the RNA and non-RNA guided activities were analyzed.
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Jean-Baptiste Fourmann. Analyse structurale et fonctionnelle des particules sRNP à boîtes H/ACA, catalysant la formation ciblée des pseudouridines dans les ARN d'archaea. Sciences agricoles. Université Henri Poincaré - Nancy 1, 2009. Français. ⟨NNT : 2009NAN10084⟩. ⟨tel-01747193⟩

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