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Marasovic, Mirela (2015): Toward unraveling biogenesis of Dicer-independent priRNAs and siRNAs in Schizosaccharomyces pombe. Dissertation, LMU München: Faculty of Chemistry and Pharmacy



RNA interference (RNAi) is a highly conserved process of gene silencing in which Argonaute family proteins are guided by small RNA molecules to complementary targets. In the fission yeast Schizosaccharomyces pombe, RNAi is required for heterochromatin formation at centromeres. Although it seems counterintuitive, pericentromeric heterochromatin in fission yeast is transcribed. The transcripts are processed by RNAi machinery, which is in turn guided back to the pericentromeric repeats by sequence complementarity of the Argonaute-bound small interfering RNA (siRNA) and the nascent transcript. This generates a positive-feedback loop of siRNA amplification that recruits factors required for the assembly of heterochromatin. Previously, it was suggested that a fission yeast class of Dicer-independent small RNAs called primal small RNAs (priRNAs) initiates the positive-feedback loop of siRNA generation and heterochromatin assembly. However, the biogenesis of priRNAs as well as of Dicer-independent small RNAs from other organisms was not well understood. The results presented here identify Triman, a novel 3’-5’ exonuclease that is involved in the final step of biogenesis of both priRNAs and siRNAs in fission yeast. It was observed that Argonaute binds longer priRNA and siRNA precursors from the total RNA fraction. This is followed by the recruitment of Triman to trim 3’ ends of Argonaute-bound small RNAs to the mature size. The final trimming of priRNAs and siRNAs is required for de novo heterochromatin formation at centromeres and the mating-type locus as well as for the maintenance of facultative heterochromatin islands. Furthermore, it was shown that in cells lacking Rrp6, a nuclease subunit of the exosome, RNAi targets various genes across the yeast genome. This demonstrated that the exosome protects the genome against aberrant RNAi. Spurious RNAi targeting in rrp6∆ cells at majority of loci occurs via accumulation of antisense transcripts that are processed into priRNAs in a Triman-dependent manner. These results suggest that Argonaute association with cellular degradation products which are processed into priRNAs might serve as a surveillance mechanism to guard the genome against invading genomic elements (Marasovic et al. 2013).