Thesis advisor: Clare O'Connor / As organisms get older, the proteins in their cells also age, and as this happens, the amino acids that make up these proteins may become chemically modified and begin to lose their integrity. One example of an age-related modification occurs when the amino acid residue methionine is oxidized by a reactive oxygen species to methionine sulfoxide. Methionine sulfoxide reductase is an enzyme that repairs this damage to the protein by catalyzing a reaction that reduces methionine sulfoxide back to methionine. The fission yeast Schizosachharomyces pombe was used as the experimental model to study methionine sulfoxide reductase in vivo, taking advantage of the variety of tools available with which to study the organism. In S. pombe there are two genes encoding methionine reductase activities, msrA and msrB. The first goal of this project was to construct yeast strains in which the endogenous msrA and msrB genes had been inactivated. This was accomplished via homologous recombination reactions in which the msr genes were replaced with a selectable marker for biosynthesis of uracil (ura4+). After the construction and verification of the two knockout strains, the sensitivities of the strains to reactive oxygen species were tested. Both strains showed reduced resistance to oxidative stress. Future experiments will include more detailed analyses of the abilities of the strains to survive oxidative stress. Finally, the two knockout strains of yeast will be mated with one another in order to produce a double msr knockout, in order to examine the effects of a complete lack of methionine sulfoxide reductase activity on the organism. / Thesis (BS) — Boston College, 2005. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Les petites particules ribonucléoprotéiques nucléaires (snRNPs) sont les composants majeurs du splicéosome, la machinerie responsable de l'épissage des pré-messagers. La biogenèse des snRNPs est un processus complexe qui fait intervenir de nombreux facteurs comme les protéines SMN et ICln. Au cours de ma thèse, je me suis intéressé à l'étude du rôle de ces deux protéines dans la maturation et la production des snRNPs du splicéosome.Dans la première partie de mon travail, les modifications internes des snRNAs ont été caractérisées dans des cellules dont les corpuscules de Cajal sont dispersés à cause d'une déficience de la protéine SMN. En effet, en plus de son rôle dans les étapes précoces de formation des snRNPs, la protéine SMN est également requise pour la formation des corpuscules de Cajal, structures nucléaires qui concentrent les scaRNAs impliqués dans le processus de modification post-transcriptionnelle des ARNs. Nous avons pu ainsi montrer que la protéine SMN et les corps de Cajal ne sont pas essentiels à la production des résidus 2'-O-methyl et pseudouridine dans les snRNAs majeurs et mineurs.La deuxième partie de mon travail a porté sur l'étude des relations fonctionnelles entre les protéines ICln et SMN in vivo en utilisant l'organisme modèle S. pombe. Après avoir caractérisé un homologue de la protéine humaine dans la levure fissipare, nous avons montré que la protéine ICln n'est pas essentielle mais est importante pour une croissance optimale des cellules de levure. Notre étude montre aussi que la modulation de l'activité de la protéine ICln ne permet pas de compenser les défauts dans la production de snRNPs observés dans les cellules portant un allèle muté de SMN. Finalement, l'utilisation d'une approche génomique montre que la délétion du gène ICln entraine des défauts différentiels d'épissage, indiquant que le choix des sites et la cinétique d'épissage sont fortement dépendants de la concentration des composants de base du splicéosome. / Small nuclear ribonucleoproteins (snRNPs) are the major components of the spliceosome, the machinery responsible for the splicing of pre-messenger RNAs. The biogenesis of snRNPs is a complex process that involves many factors such as the SMN and ICln proteins. During my thesis, I studied the role of these two proteins in the maturation and the production of the spliceosome snRNPs.The goal of the first part of my work was to characterize the internal modifications of snRNAs in SMN-deficient cells carrying disrupted Cajal bodies. Indeed, in addition to its role in the early stages of snRNPs assembly, the SMN protein is also required for the formation of Cajal bodies which are nuclear structures carrying the scaRNAs involved in the post-transcriptional modification process of RNAs. We could show that the SMN protein and Cajal bodies are not essential for the formation of 2'-O-methyl and pseudouridine residues in the major and minor snRNAs.In the second part of my work, the functional relationships between the ICln and SMN proteins were examined in vivo using the S. pombe model organism. We first identified a fission yeast homologue of the human ICln protein and found that the ICln protein is not essential but important for optimal growth of yeast cells. Our study also showed that the modulation of the activity of the ICln protein does not compensate for defects in the production of snRNPs observed in yeast cells carrying a SMN mutated allele. Finally, the use of a genome-wide approach allowed us to show that deletion of the ICln gene resulted in differential splicing defects, indicating that the choice of splice sites and the kinetics of splicing are strongly dependent on the concentration of the basic components of the spliceosome.

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