| RNA interference (RNAi) and related RNA silencing mechanisms regulate gene expression at both the transcriptional and posttranscriptional levels. Small RNA molecules, called micro RNAs (miRNA) or small interfering RNAs (siRNAs), play a central role in RNAi as guides that recognize complementary sequences in other RNAs and target them for inactivation. During post-transcriptional gene silencing, siRNAs or miRNAs are loaded onto the RNA-Induced Silencing Complex (RISC), containing a conserved Argonaute protein, which binds to siRNAs and also directly cleaves target mRNA sequences. In the fission yeast Schizosaccharomyces pombe, repetitive DNA elements that surround centromeres give rise to siRNAs, and the formation of heterochromatin at these repeats requires components of the RNAi pathway. Centromeric siRNAs are found in the RNA-Induced Transcriptional gene Silencing (RITS) complex, which also contains the fission yeast Argonaute protein, Ago1, the chromodomain protein Chp1, and Tas3, a GW-repeat protein of unknown function. The identification of RITS represented the first biochemical link between the RNAi pathway and heterochromatin. Here, we identify a second fission yeast Argonaute complex (Argonaute siRNA chaperone, ARC), which contains, in addition to Ago1, two previously uncharacterized proteins, Arb1 and Arb2, both of which are required for histone H3 Lys9 (H3-K9) methylation, heterochromatin assembly and siRNA generation. Furthermore, whereas siRNAs in the RITS complex are mostly single-stranded, siRNAs associated with ARC are mostly double-stranded, indicating that Arb1 and Arb2 inhibit the release of the siRNA passenger strand from Ago1. Consistent with this observation, purified Arb1 inhibits the slicer activity of Ago1 in vitro, and purified catalytically inactive Ago1 contains only double-stranded siRNA. Finally, we show that slicer activity is required for the siRNA-dependent association of Ago1 with chromatin and for the spreading of histone H3-K9 methylation. The findings of this study describe a role for previously uncharacterized proteins in the maturation of heterochromatic siRNAs that is essential for RNAi-mediated transcriptional gene silencing. |
