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Mécanismes et fonctions de la voie d'ARN interférence induite par ARN double brin chez Paramecium tetraurelia

Abstract : The ciliate Paramecium tetraurelia is an interesting model to study the diversity and evolution of RNA interference (RNAi) pathways. One of the vegetative RNAi pathways is induced by feeding cells with bacteria producing double-stranded RNA (dsRNA) homologous to a given gene, which is then post-transcriptionally silenced through the production of 23-nt siRNAs. A forward genetic screen allowed us to obtain Mendelian mutants deficient in dsRNA-induced RNAi, and mutated genes were identified by whole-genome resequencing. 6 genes were identified: one Dicer, two RNA-dependent RNA polymerases (RDR1 et 2), one nucleotidyl-transferase (CID1) and two genes encoding novel poteins (PDS1 and 2). To study their roles in siRNA biosynthesis or action, we sequenced small RNAs from wild-type or mutants cells fed with a dsRNA homologous to a non-essential endogenous gene. Bioinformatic analyses showed that 'primary' siRNAs are produced from the bacterial dsRNA trigger, while 'secondary' siRNAs, predominantly of antisense polarity, are produced from the whole length of the targeted endogenous mRNA. While primary siRNA production requires all of the genes studied, the results only implicate RDR2 in the production of secondary siRNAs. Finally, I showed that some clusters of endogenous siRNAs depend on RDR1 and CID1, whereas others depend on RDR2. Paramecium was also shown to produce siRNAs that are antisense to bacterial ribosomal RNAs, suggesting new hypotheses about the possible natural functions of this pathway.
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Quentin Carradec. Mécanismes et fonctions de la voie d'ARN interférence induite par ARN double brin chez Paramecium tetraurelia. Génétique. Université Pierre et Marie Curie - Paris VI, 2014. Français. ⟨NNT : 2014PA066161⟩. ⟨tel-01077957⟩

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