Fission yeast centromeric repeats are transcribed into small interfering RNA (siRNA) precursors (pre-siRNAs), which are processed by Dicer to direct heterochromatin formation. Recently, Rpb1 and Rpb2 subunits of RNA polymerase II (RNA Pol II) were shown to mediate RNA interference (RNAi)-directed chromatin modification but did not affect pre-siRNA levels. Here we show that another Pol II subunit, Rpb7 has a specific role in presiRNA transcription. We define a centromeric presiRNA promoter from which initiation is exquisitely sensitive to the rpb7-G150D mutation. In contrast to other Pol II subunits, Rpb7 promotes pre-siRNA transcription required for RNAi-directed chromatin silencing. In RNA interference (RNAi), Dicer processes doublestranded RNA to produce short small interfering RNAs (siRNAs) that act to silence gene expression post-transcriptionally (Fire et al. 1998;Zamore et al. 2000;Bernstein et al. 2001). RNAi also mediates transcriptional silencing via formation of heterochromatin. Centromeres in many organisms are composed of repetitive DNAs, which act as a substrate for the formation of kinetochores, flanked by domains of centromeric heterochromatin (Cleveland et al. 2003). In fission yeast (Schizosaccharomyces pombe) genes inserted within this heterochromatin are transcriptionally silenced (Allshire et al. 1995). Paradoxically, the centromeric repeats themselves are transcribed to form long siRNA precursors (pre-siRNAs), which are required for the RNAi-mediated chromatin modifications resulting in transcriptional silencing (Provost et al. 2002;Volpe et al. 2002). Defective RNAi leads to loss of silencing and defective heterochromatin formation in several organisms (Mochizuki et al. 2002;Pal-Bhadra et al. 2002;Zilberman et al. 2003) and transcripts homologous to centromere repeats have been detected in chicken cells as well as mice (Fukagawa et al. 2004;Kanellopoulou et al. 2005). Interestingly, in plants, specialized RNA polymerase IV (RNA Pol IV) subunits have been implicated in RNAidirected silencing (Herr et al. 2005;Onodera et al. 2005). Animal and fungal genomes lack such Pol IV subunits. Very recently, however, two subunits of RNA pol II (Rpb1 and Rpb2) were shown to have specific roles in chromatin modification and siRNA production, respectively, but did not affect pre-siRNA levels (Kato et al. 2005;Schramke et al. 2005).Here, we have characterized the temperature sensitive csp3 (centromere: suppressor of position effect) mutant isolated in a screen for trans-acting mutants that are required for centromeric silencing (Ekwall et al. 1999). csp3 is a G150D missense mutation in the rpb7 gene, a subunit of Pol II. We show that Rpb7 has a specific defect in centromeric pre-siRNA transcription. We define a centromeric pre-siRNA promoter from which initiation is exquisitely sensitive to rpb7-G150D. Thus, in contrast to the previously characterized silencing defective Pol II subunits, which affect siRNA production and/or downstream events, Rpb7 has a distinct role in generating centromeric pre-siRNAs ne...

RNA interference is a form of gene silencing in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. Here we report a role for Dicer in chromosome segregation of fission yeast. Deletion of the Dicer (dcr1 ؉ ) gene caused slow growth, sensitivity to thiabendazole, lagging chromosomes during anaphase, and abrogated silencing of centromeric repeats. As Dicer in other species, Dcr1p degraded double-stranded RNA into Ϸ23 nucleotide fragments in vitro, and dcr1⌬ cells were partially rescued by expression of human Dicer, indicating evolutionarily conserved functions. Expression profiling demonstrated that dcr1 ؉ was required for silencing of two genes containing a conserved motif.

The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 5 0 -monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably wellconserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1D cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity.