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Étude de l'effet d'un stress oxydant sur l'activité spliceosomale et sur les RNP à boîtes C/D et H/ACA

Abstract : During pre-RNA maturation process, alternative splicing (AS) selectively includes or excludes specific intronic or exonic sequences. This process leads from a given pre-RNA, to different isoforms. Some recent studies estimate that 95% of human genes are alternatively spliced. Then, it is not surprising that stress-induced modifications of splicing profils participate to the establishment of the cell response to stress or to the development of pathologies. Our global transcriptomic analysis has revealed significant splicing variations in response to an oxidative stress (500μM H2O2). That is why we were interested in such stress effects on the spliceosome, responsible for the dynamic catalysis of splicing. The functional spliceosome is composed by different ribonucleoproteins (RNPs), each being composed of a Uridine-rich small nuclear RNA (U snRNA) associated with specific proteins to form the mature UsnRNPs. To be functional, each UsnRNP should be maturated following different steps including the chemical modification of its RNA component. These post-transcriptional modifications are induced by another type of RNPs which are particularly enriched within nuclear Cajal bodies and are named scaRNPs (for small Cajal Bodies RNPs). Our results obtained by fluorescence microscopy techniques show that oxidative stress changes Cajal bodies structure by inducing the nucleoplasmic redistribution of its main structural and functional component, the coilin protein. Interestingly, we highlighted that coilin redistribution is accompanied with changes of the localization of others Cajal Body components: (i) factors involved in the formation of post-transcriptional modifications in UsnRNAs, (ii) factors involved in scaRNPs retention, (iii) UsnRNPs components. Oxidative stress also seems to induce the exclusion of SMN protein from the nucleus, knowing that it plays a central role in UsnRNP nuclear re-import during their maturation. This study has generated a large amount of fluorescence microscopy data, so that I have developed different bio-image analysis protocols in order to optimize the extraction and the processing of these data. In parallel, RNA-Immunoprecipitation (RIP) experiments coupled with high-throughput sequencing have revealed (i) a significant increase of U4, U5 and U6 snRNP fraction suggesting a potential effect of oxidative stress on the U4/U6-U5 tri-snRNP, (ii) oxidative stress-induced variations of the 2’-O-methylation level directly in mature U snRNPs.
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Géraud Heckler. Étude de l'effet d'un stress oxydant sur l'activité spliceosomale et sur les RNP à boîtes C/D et H/ACA. Biochimie, Biologie Moléculaire. Université de Lorraine, 2019. Français. ⟨NNT : 2019LORR0351⟩. ⟨tel-03133067⟩

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