L’isomère de l’uridine (U), la pseudouridine (?), est le nucléoside le plus fréquemment rencontré dans les ARN. La réaction de pseudouridylation dans les ARN est catalysée par des ARN:?-synthases qui, soit fonctionnent comme des enzymes protéiques, soit sont portées chez les eucaryotes et les archaea par des particules ribonucléoprotéiques (RNP). Chaque RNP est composée d’un RNA guide dit à boîtes H/ACA (sno/sca/sRNA chez les eucaryotes et sRNA chez les archaea) et d’un ensemble invariable de protéines : l’enzyme ARN:?-synthase Dyskérine/aCBF5 et les 3 protéines NOP10/aNOP10, GAR1/aGAR1 et NHP2/L7Ae. L’ARN guide assure la reconnaissance de l’ARN cible à modifier par appariement de bases. Les sRNA H/ACA identifiés chez l’archae Pyrococcus abyssi ont servi de modèles pour des études de structure-fonction basées sur i) la mesure de l’activité de sRNP H/ACA reconstituées avec les composants protéiques et nucléiques purifiés, ii) l’analyse de la structure 2D des ARN au sein des RNP par des sondes chimiques et enzymatiques et par dichroïsme circulaire, iii) les diverses structures 3D disponibles. Les déterminants moléculaires présents sur les ARN guides ont été précisés, ainsi que le rôle fonctionnel des différentes protéines, de leurs domaines structuraux et de résidus conservés. Nous montrons que l’association entre aNOP10 et L7Ae est cruciale pour l’activité des RNP. Nous avons identifié que aCBF5 pouvait catalyser la formation des résidus ?55 dans les ARNt et ?2603 dans l’ARN 23S sans l’intervention de sRNA. Nous avons étudié le rôle de résidus conservés de aNOP10 ainsi que du site actif et de la boucle ß7/ß10 de aCBF5 dans les activités guidée et non guidée de aCBF5. / Pseudouridine (?), 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:?-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:?-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.

Ribosomal RNAs (rRNA) undergo several post-transcriptional modifications inside the cell. These modifications can be (1) RNA- independent (enzyme only) and (2) guide RNA-mediated. In the latter mechanism, a group of small, metabolically stable, non-coding RNAs, present as ribonucleoprotein (RNP) particles, modify ribosomal RNAs inside the cell. One of the highly abundant rRNA modifications is pseudouridine (Y) formation. In Archaea and Eukarya, pseudouridine synthases, with the help of small RNAs, form pseudouridines at functionally important regions in rRNA. Cbf5, the pseudouridine synthase, three other core proteins, and a box H/ACA RNA form the ribonucleoprotein complex in sRNP-mediated rRNA pseudouridylation. Certain Ys in rRNAs are evolutionarily conserved from Bacteria to human. Among those, two Ys are present in helix 69 of rRNA and one in helix 90. We successfully deleted Cbf5 in Haloferax volcanii, a haloarchaeon, and showed that the deleted strain was viable. It was the first report where Cbf5 deletion was achieved, because deletion or mutation of cbf5 or of its homologs is lethal in eukaryotes. We also found that the cbf5 deleted strain was unable to produce the three highly conserved Ys in rRNA of H. volcanii (position 1940, 1942 in helix 69, and 2605 in helix 90), whereas the tRNA Ys were intact. To identify the specific structural features of Cbf5 involved in rRNA Ψ formation, we used a cbf5 deleted strain which was complemented with a plasmid borne copy of the gene. Using the crystal structure of Pyrococcous furiosus Cbf5 as template, we created a homology model of H. volcanii Cbf5 (HvCbf5) and identified several residues and motifs/domains of HvCbf5 that might be important to the protein's enzymatic activity. By using an in vivo mutational approach, we confirmed some previously predicted and certain unidentified residues/motifs/domains that serve as positive determinants of rRNA Ys1940, 1942, and 2605 formation inside the cell. A box H/ACA RNA, sR-h45, was bioinformatically predicted before. We confirmed its presence as a double hairpin RNA inside the cell whose level goes down in the absence of Cbf5. We identified that sR-h45 is the guide RNA for sRNP-mediated Ys at the three above mentioned rRNA positions in H. volcanii. Each hairpin of this RNA can independently modify the substrate, both in vivo and in vitro. To characterize the structure of sR-h45, we have used a sR-h45 deleted strain where the function of sR-h45 was complemented with a plasmid-borne copy of the gene. By a combination of in vivo and in vitro mutagenic approaches, we determined specific nucleotides/structures of this RNA, involved in binding to the core proteins and also to the substrate RNA. We also identified that one hairpin of sR-h45 can modify two successive positions (1940 and 1942) in rRNA.

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