Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA

Abstract

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.

Pericentric and centromeric transcription: a perfect balance required

“…This enzyme produces doublestranded RNA from single-stranded PCTs that can be cleaved by the RNAse III cleavage enzyme Dicer to form short interfering RNAs (siRNAs) (Lippman and Martienssen 2004). The siRNA then associates with the RNA-induced initiation of transcriptional silencing (RITS) complex, which in turn binds another nascent pericentric transcript forming double-stranded RNA (Djupedal et al 2009). The process then cycles again, starting with Dicer cleaving the newly formed doublestranded RNA.…”

“…The process then cycles again, starting with Dicer cleaving the newly formed doublestranded RNA. The association of RITS with PCTs leads to recruitment of the histone methyltransferase containing Clr4 complex (CLRC) that methylates lysine 9 of histone H3, thus maintaining regional heterochromatin (Lippman and Martienssen 2004;Djupedal et al 2009). …”

“…Recent work in yeast has shown that an alternate RNAi pathway exists, in which certain PCTs form secondary stem loop structures that are recognized and cleaved by Dicer, thus bypassing the need for RDRP (Djupedal et al 2009). The resulting siRNAs associate with the argonaute containing complex RITS to assist in heterochromatin formation in a manner analogous to that produced from the RDRP pathway.…”

“…1a, b) and performs a variety of functions such as maintaining the boundary that separates the euchromatin from the centromere core (Chen et al 2008), providing sites for sister chromatid cohesion during mitosis (Lippman and Martienssen 2004), and repressing meiotic recombination around the centromere (Ellermeier et al 2010). Transcripts emanating from this region, known as pericentric transcripts, or PCTs, recruit heterochromatin factors that maintain the heterochromatic histone modifications, specifically H3K9me2, H3K9me3, H3K27me2, and H3K27me3 (Lippman and Martienssen 2004;Chen et al 2008;Djupedal et al 2009;Reyes-Turcu et al 2011).…”

“…Graph inset levels of CENP-A (red) and H3K4me2 (purple) within centrochromatin under normal (gray) and CT misregulated conditions. Gray nucleosomes represent unknown histone replacements and processing of the pericentric sequences into shorter fragments (Lippman and Martienssen 2004;Djupedal et al 2009;Reyes-Turcu et al 2011). The first mechanism of heterochromatin formation involves the RNA interference (RNAi) pathway through the action of RNAdependent RNA polymerase (RDRP) (Lippman and Martienssen 2004).…”