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.

Over one hundred types of chemical modifications have been characterized in cellular RNAs. Pseudouridines (Ψ) and 2’-O-methylation of ribose sugars are the two most widespread modifications present in rRNAs, tRNAs and snRNAs. These modifications can be either guide-RNA mediated or RNA-independent (enzyme only). The RNAs that guide pseudouridylations are called box H/ACA RNAs and the ones that carry out 2’-O-methylations modifications are called box C/D RNAs. Previously, we identified that sR-h45 is the box H/ACA guide RNA responsible for Ψ1940, 1942 and 2605 formation in 23S rRNA of Haloferax volcanii. This RNA has two stem loops – SL1 and SL2. SL1 acts as the guide for Ψ2605 formation and SL2 is responsible for guiding Ψ1940 and Ψ1942. We found that SL2 sequentially guides Ψ1940 and Ψ1942 formation in the unpaired "UNUN" target. Ψ1942 is produced after and only if Ψ1940 is produced. The requirement for conserved ACA box was determined by using variants of these two stem loops. We found that the ACA motif is not required either in vivo or in vitro for the activity of the typical variants of both SL1 and SL2 but required for the activity of the atypical variants of these guides. Cbf5 is the pseudouridine synthase involved in this box H/ACA RNA guided process. Mutants of Methanocaldococcus jannaschii Cbf5 were used with both typical and atypical guide variants in vitro and certain residues were found to be important only for the atypical reactions.We have also studied sR-h41, which is a unique single guide box C/D guide responsible for methylation of G1934 position of 23S rRNA of Haloferax volcanii. We have done in vitro assembly reactions using mutants of sR-h41 assembled with its cognate proteins from Methanocaldococcus jannaschii to study the structural determinants needed to convert it to a dual guide RNA. The assembly pattern of the core proteins on the conserved box C/D and box C’/D motifs steer the dual guide nature of these archaeal box C/D guide RNAs.Another aim of this study was to determine the role of pseudouridine synthases (Pus enzymes) for Ψ55 formation in mammalian tRNAs. We find that three Pus enzymes – TruB1 (in the nucleus), TruB2 (in the mitochondria) and Pus10 (in the cytoplasm) are responsible for this modification depending on the specific sub-cellular location in the cell. These enzymes exhibit different structural requirement for Ψ55 formation that are located on the TΨC loop of tRNAs. A subset of tRNAs like tRNAs for Trp and Gln are protected from the action of TRUB1 in the nucleus by binding to the nuclear version of Pus10 that lacks Ψ55 activity. Ψ55 in this subset of tRNAs is produced by the cytoplasmic version of Pus10.While studying pseudouridylation functions of Pus10, we also found that Pus10 regulates G1/S cell cycle progression in PC3 cells. It does so by directly repressing another protein c-Rel, that is a positive regulator of Cyclin D1 protein. Cyclin D1 is known to play a central role in transition of cell from G1 to S phase during cell cycle progression. c-Rel also regulates the levels of PUS10 by an unknown mechanism.

Ribonucleoprotein complexes are responsible for some of the post-transcriptional modifications of RNA that occur within the cell, including 2'-O-methylation and pseudouridylation. These modifications contribute, among other things, to RNA folding, inhibition of degradation, and general cellular viability. In this study, we identify residues within the proteins of these complexes that are important to the functioning of the Box C/D and Box H/ACA complexes. Candidates were selected based on previous work and mutant versions of the proteins were introduced in-vivo. Assays were done to determine the functionality of the mutant complex. This work is divided into three parts, focused on the three proteins investigated. The first part is concerned with Nop5, a protein in the Box C/D RNP complex. Nop5 is known to interact with all other proteins and RNAs in the complex, and is believed to serve a primarily structural role, aligning the other components. Mutagenesis study of suspected significant amino acids in this protein showed that it is difficult to disrupt the operation of Nop5 with single changes, but is possible with more extensive mutation. The second part concerns Fibrillarin, the catalytic protein of the Box C/D ribonucleoprotein complex. Previous mutagenesis work identified several important amino acids involved with AdoMet transfer and complex formation. The methylation ability of these mutant complexes were further examined in this work by confirming that the same modification, or lack thereof, occurred at a second rRNA position. The final part of this work is about Nop10, part of the Box H/ACA complex. This work is only preliminary, but begins the process of testing suspected essential amino acids in the structure.

La pseudouridylation, l'isomérisation du résidu urine (U) en pseudouridine ([PSI]) est la modification post-transcriptionnelle la plus fréquemment retrouvée dans les ARN. Elle est catalysée par une enzyme ARN:PSI-synthase. Chez les archées et les eucaryotes, cette activité est également portée par des particules ribonucléoprotéiques à boîtes H/ACA (RNP H/ACA). Chez les archées, le complexe comprend quatre protéines invariables dont l'ARN:PSI-synthase aCBF5 et trois protéines partenaires L7Ae, aNOP10 et aGAR1, ainsi qu'un ARN guide qui cible par appariement de bases la position de l'uridine à modifier de l'ARN substrat. Le rôle des partenaires a pu être identifié par des analyses structure-fonction basées sur des approches biochimiques, biophysiques et radiocristallographiques. Au cours de ce travail, nous avons démontré l'existence de disparités fonctionnelles entre les ARN guides d'un même organisme, et l'importance de l'interaction entre L7Ae et aNOP10 pour le positionnement correct de l'ARN substrat. Nous avons testé in vitro l'assemblage et l'activité de particules reconstituées en présence d'ARN guides non conventionnels. L'étude sur la dynamique de l'ARN substrat lors de la pseudouridylation a également été abordée et a permis de déterminer que aGAR1 n'était pas nécessaire pour le mécanisme de turnover de la particule, que la température jouait un rôle crucial pour cette activité, et que la nature du nucléotide cible ainsi que la longueur de l'ARN substrat étaient des éléments importants pour la sélection de cet ARN. Nous avons également mis au point une nouvelle technique basée sur le phénomène de FRET permettant de suivre l'association de l'ARN substrat à la RNP H/ACA / 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