The Proper Splicing of RNAi Factors Is Critical for Pericentric Heterochromatin Assembly in Fission Yeast

摘要

Heterochromatin preferentially assembles at repetitive DNA elements, playing roles in transcriptional silencing, recombination suppression, and chromosome segregation. The RNAi machinery is required for heterochromatin assembly in a diverse range of organisms. In fission yeast, RNA splicing factors are also required for pericentric heterochromatin assembly, and a prevailing model is that splicing factors provide a platform for siRNA generation independently of their splicing activity. Here, by screening the fission yeast deletion library, we discovered four novel splicing factors that are required for pericentric heterochromatin assembly. Sequencing total cellular RNAs from the strongest of these mutants, cwf14Δ , showed intron retention in mRNAs of several RNAi factors. Moreover, introducing cDNA versions of RNAi factors significantly restored pericentric heterochromatin in splicing mutants. We also found that mutations of splicing factors resulted in defective telomeric heterochromatin assembly and mis-splicing the mRNA of shelterin component Tpz1, and that replacement of tpz1~(+) with its cDNA partially rescued heterochromatin defects at telomeres in splicing mutants. Thus, proper splicing of RNAi and shelterin factors contributes to heterochromatin assembly at pericentric regions and telomeres. Author Summary Heterochromatin formation at specific genomic regions is critical for processes as diverse as gene expression and chromosome segregation. The formation of silent heterochromatin at repetitive DNA elements requires processing of transcripts by the RNA interference machinery. Curiously, factors involved in proper RNA splicing are required for heterochromatin assembly, and it was proposed that splicing factors provide a platform for the recruitment of RNAi complexes independently of their role in regulating splicing. In this study, we found several novel splicing factors involved in heterochromatin assembly. Our analysis of genome-wide splicing patterns by RNA sequencing showed that the mRNAs of RNAi factors are very sensitive to perturbations of RNA splicing machinery. Moreover, we found that splicing factors are critical for the production of a telomere shelterin component and proper telomeric heterochromatin assembly. Most importantly, we showed that introducing the cDNA versions of RNAi and shelterin components alleviates heterochromatin defects associated with splicing factor mutations. Thus splicing factors are required for heterochromatin assembly mainly by regulating the proper splicing of heterochromatin assembly factors.