Title: The mechanism of RNAi assembly and siRNA generation in fission yeast centromeres
Description: RNA interference, or RNAi, is a highly-conserved gene silencing mechanism mediated by base-pairing interactions of small, interfering RNAs, or siRNAs, to specifically target homologous gene products. This mechanism has been adapted to regulate diverse cellular processes through degradation or translational inhibition of mRNAs in a post-transcriptional manner or by recruitment of a transcriptionally-repressive heterochromatic structure. In fission yeast, centromere function requires RNAi components Ago1 (Argonaute), Dcr1 (Dicer), and Rdp1 (RNA-dependent RNA polymerase) that drive formation of heterochromatin structure at pericentric regions. The identification of Ago1 and the heterochromatic protein Chp1 as components of the RNA-induced Initiation of Transcriptional gene Silencing complex (RITS) provided the first physical evidence directly linking heterochromatin with RNAi. Recruitment of the RITS complex is a prerequisite for efficient dimethylation of histone H3 lysine 9 and localization of heterochromatic structural proteins like the HP1 homolog Swi6 that are absolutely required for heterochromatin formation in fission yeast. Initiation of heterochromatin assembly requires bi-directional transcription of centromeric domains that provide precursors for siRNA synthesis. However, the mechanisms of Rdp1 and Dcr1 function in this pathway and their coordination with RITS activity remain largely unknown. This study describes the purification of the RDRC, or RNA-dependent RNA polymerase complex, that contains Rdp1, a putative helicase Hrr1, and a poly-A polymerase member Cid12. RDRC, RITS, and Dcr1, are shown to interact whereas non-coding centromeric RNAs are implicated as assembly platforms for RNAi complexes.

Finally, siRNA generation by Dcr1 and double-stranded RNA synthesis by RDRC are reconstituted in vitro and shown to be coupled processes. The findings of this study define mechanisms of double-stranded RNA formation and siRNA generation central to RNAi in a transcriptional mode of gene silencing.