Involvement of the Spliceosomal U4 Small Nuclear RNA in Heterochromatic Gene Silencing at Fission Yeast Centromeres

Abstract

prp13-1 is one of the mutants isolated in a screen for defective pre-mRNA splicing at a nonpermissive temperature in fission yeast Schizosaccharomyces pombe. We cloned the prp13 ؉ gene and found that it encodes U4 small nuclear RNA (snRNA) involved in the assembly of the spliceosome. The prp13-1 mutant produced elongated cells, a phenotype similar to cell division cycle mutants, and displays a high incidence of lagging chromosomes on anaphase spindles. The mutant is hypersensitive to the microtubule-destabilizing drug thiabendazole, supporting that prp13-1 has a defect in chromosomal segregation. We found that the prp13-1 mutation resulted in expression of the ura4 ؉ gene inserted in the pericentromeric heterochromatin region and reduced recruitment of the heterochromatin protein Swi6p to that region, indicating defects in the formation of pericentromeric heterochromatin, which is essential for the segregation of chromosomes, in prp13-1. The formation of centromeric heterochromatin is induced by the RNA interference (RNAi) system in S. pombe. In prp13-1, the processing of centromeric noncoding RNAs to siRNAs, which direct the heterochromatin formation, was impaired and unprocessed noncoding RNAs were accumulated. These results suggest that U4 snRNA is required for the RNAi-directed heterochromatic gene silencing at the centromeres. In relation to the linkage between the spliceosomal U4 snRNA and the RNAi-directed formation of heterochromatin, we identified a mRNA-type intron in the centromeric noncoding RNAs. We propose a model in which the assembly of the spliceosome or a sub-spliceosome complex on the intron-containing centromeric noncoding RNAs facilitates the RNAi-directed formation of heterochromatin at centromeres, through interaction with the RNA-directed RNA polymerase complex.The recognition and removal of introns from pre-mRNAs are essential for gene expression in eukaryotic cells. Pre-mRNA splicing takes place in a large complex, the spliceosome, which assembles through ordered interactions of four small nuclear ribonucleoprotein particles (snRNPs), 3 U1, U2, U4/U6, and U5 snRNPs, and numerous non-snRNP proteins (for a review, see Ref. 1,2). During assembly of the spliceosome and catalysis of the splicing reaction, the U snRNPs undergo ordered dynamic changes in composition and structure. The U1 and U2 snRNPs initially bind to the pre-mRNA and generate the pre-spliceosome, or complex A. The preformed U4/U6.U5 tri-snRNP is then recruited to complex A to form a pre-catalytic complex B containing all five snRNAs. Subsequently, a large conformational rearrangement occurs in the spliceosome in which U1 and U4 snRNPs are released, accompanied by base-pairing between U6 and U2, and generates complex B. After the first step of splicing, the spliceosome is converted into complex C. The second step generates the mature mRNA product and is followed by the release of the remaining snRNPs from the spliced-out intron (1, 2). In this way, pre-mRNA splicing is performed via RNA-RNA and RNA-protein interactions and ea...

RNA-mediated epigenetic regulation of gene expression

“…However, this Raf2–Rik1 complex seems to be distinct from the CLRC complex, and direct evidence of cooperation between the RNAi and DNA replication machineries is lacking. It has also been proposed that the splicing machinery contributes to heterochromatic silencing by directly interacting with the RDRC and the nascent transcript platform to promote siRNA biogenesis 43,44 . However, earlier biochemical purifications demonstrated that the RDRC subunit Cid12 forms RDRC-independent complexes with splicing factors 27 .…”

A systematic genetic screen identifies new factors influencing centromeric heterochromatin integrity in fission yeast

“…While we cannot exclude that defective splicing of some other contributory factor might be involved, we instead sought further evidence of a direct role of splicing components in processing of centromeric transcripts. Two previous studies have reported evidence of splicing of centromeric transcripts [44,45]. To confirm this and investigate the sensitivity of these splicing events to splicing mutants, we analysed centromeric transcripts by RT-PCR.…”

Post-transcriptional regulation of meiotic genes by a nuclear RNA silencing complex

“…The TRAMP complex regulates centromeric silencing by processing centromeric transcripts and promoting their degradation by the exosome (Buhler et al 2007;Reyes-Turcu et al 2011). Splicing has also been implicated in heterochromatin formation in S. pombe (Bayne et al 2008;Chinen et al 2010;Lee et al 2013). The NURS complex subunits Red1, Mtl1, Iss10, and Red5 are required for both meiotic mRNA degradation and H3K9 methylation at meiotic genes (Figs.…”