Splicing factors facilitate RNAi-directed silencing in fission yeast

Abstract

Heterochromatin formation at fission yeast centromeres is directed by RNA interference (RNAi). Noncoding transcripts derived from centromeric repeats are processed into small interfering RNAs (siRNAs) that direct the RNA-induced transcriptional silencing (RITS) effector complex to engage centromere transcripts, resulting in recruitment of the histone H3 lysine 9 methyltransferase Clr4, and hence silencing. We have found that defects in specific splicing factors, but not splicing itself, affect the generation of centromeric siRNAs and consequently centromeric heterochromatin integrity. Moreover, splicing factors physically associate with Cid12, a component of the RNAi machinery, and with centromeric chromatin, consistent with a direct role in RNAi. We propose that spliceosomal complexes provide a platform for siRNA generation and hence facilitate effective centromere repeat silencing.

Fig. 1

Specific splicing mutants affect RNAi-directed silencing at centromeres (A) Schematic of fission yeast centromere 1, indicating site of integration of cen1:ade6⁺ marker, outer repeat (otr) dg and dh elements, inner repeats (imr), and central core (cnt). (B) Silencing assay on strains bearing cen1:ade6⁺ (red, silent; pink/white, alleviated). (C) RT-PCR and quantitative RT-PCR (qRT-PCR) analysis of transcripts from ade6, cen-otr (dg and dh), and act1 control (-RT, no reverse transcriptase). Histograms show transcript levels relative to act1, normalized to the wild type. (D and E) Northern analysis of siRNAs corresponding to cen-dh or dg/dh. snoRNA58 (snR58) is a loading control.

Fig. 2

Defective splicing does not cause defects in centromere silencing. (A and B) RT-PCR analysis of transcripts from tbp1, ade6, cen-dh, and act1. Spliced, mature (m), and unspliced, pre- (p) tbp1⁺ mRNA are indicated. Strains were grown at permissive (25°C) or semipermissive (30°C) temperatures or, for analysis at restrictive temperature, grown at 25°C and then shifted to 36°C for 6 hours. The histogram shows qRT-PCR analysis of tbp1⁺ mature transcript levels relative to act1, normalized to the wild type, at 25°C. (C) Silencing assay on strains bearing cen1:ade6⁺, with endogenous ago1⁺ or hrr1⁺ replaced by cDNAs.

Fig. 3

Splicing mutants disrupt RNAi-dependent heterochromatin. (A and B) ChIP analysis of H3K9me2 and Swi6 at cen-dg or cen1:ura4⁺ relative to a euchromatic control locus (fbp1 or ura4 DS/E, respectively). Representative gels are shown. Relative enrichments were calculated as the ratio of product of interest to control product in immunoprecipitate (IP) relative to input (in). The histogram represents qPCR analysis of four independent experiments; relative enrichments (cen-dg/fbp1 and cen1:ura/fbp1) are shown as a percentage of the wild type. See also fig. S2. (C) ChIP analysis of H3K9me2 at cen-dg sequences on endogenous centromeres, or on plasmid pH-cc2 [see schematic and (20, ​27)]. Plasmid was introduced by transformation and maintained under selection. Primers spanning the dg-plasmid backbone junction were used to specifically analyze H3K9me2 on the plasmid.

Fig. 4

Splicing factors are specifically required for RNAi-dependent silencing and physically associate with the RNAi machinery and centromere repeats. (A and B) Assays for silencing of ura4⁺ at mating-type locus [mat3-M::ura4⁺ (21) (A)] or with tethered Tas3 [ura4-5BoxB/tas3-lN (22) (B)]. Plates are nonselective (N/S), lacking uracil (-ura), or supplemented with FOA (+FOA). (C) Proteins specifically associated with Cid12-FLAG, analyzed by LC-MS/MS, as described (​27). For splicing factors, human (H.s.) and budding yeast (S.c.) homologs are shown. (D) Coimmunoprecipitation of Cwf10-HA with Cid12-FLAG. (E and F) ChIP analysis of Cwf10 enrichment at cen-dh or cen-dg relative to a euchromatic, unspliced control locus act1⁺, normalized to an untagged control strain.