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Développement et application de méthodes bioinformatiques pour la recherche de gènes de sRNA à boîtes H/ACA dans les génomes d'archaea et étude fonctionnelle des ARN et sRNP H/ACA correspondants

Abstract : The conversion of U into Pseudouridine (Y) residue is the most prevalent RNA modification. This reaction is catalysed either by stand-alone RNA:Y-synthases or by H/ACA snoRNPs composed of 4 proteins and a snoRNA. In snoRNPs the recognition of the U residue is carried out by base-pair interactions between the snoRNA and the RNA target. SnoRNPs are involved in eukaryal rRNA modifications whereas stand-alone RNA:Y-synthase are involved in tRNA and bacterial rRNA modifications. Three counterparts of the H/ACA snoRNAs had been discovered in Archaea when I started my PhD work, but their contribution to rRNA modification was not demonstrated. We developed a computational approach to identify the H/ACA sRNA genes and their rRNA targets. Its application to the Pyrococcus abyssi genome revealed 7 candidate genes. We verified their expression and experimentally identified 14 Y in the P. abyssi rRNAs. We succeeded in the in vitro reconstitution of active H/ACA sRNPs and thereby studied the sRNP assembly mechanism, identified important H/ACA sRNA structural features and assigned 12 of the identified Y residues to the 7 sRNAs. The application of the computational approach to the other archaeal genomes revealed 90 sRNA genes. This repertoire shed new light on the function and the evolution of this ncRNA class. In addition, we experimentally demonstrated for the first time that a few H/ACA sRNAs are involved in tRNA modification. We also showed that the enzyme of the H/ACA sRNP (aCBF5) is involved in Y55 formation in all archaeal tRNAs without the use of an H/ACA sRNA. We compared the guided and non-guided CBF5 activities and identified aminoacids which are important for these activities.
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https://hal.univ-lorraine.fr/tel-01748244
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Submitted on : Thursday, March 29, 2018 - 11:29:38 AM
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Sébastien Muller. Développement et application de méthodes bioinformatiques pour la recherche de gènes de sRNA à boîtes H/ACA dans les génomes d'archaea et étude fonctionnelle des ARN et sRNP H/ACA correspondants. Biologie moléculaire. Université Henri Poincaré - Nancy 1, 2007. Français. ⟨NNT : 2007NAN10110⟩. ⟨tel-01748244⟩

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