GSE173837
RNA-seq and small RNA-seq in fission yeast CPF mutants that enable small-RNA-mediated epigenetic gene silencing
Small RNAs are the common denominator of various RNA silencing pathways that regulate gene expression and protect the genome against mobile repetitive DNA sequences, retroelements, and transposons. In Schizosaccharomyces pombe, small interfering RNAs (siRNAs) are required for the faithful propagation of heterochromatin that is found at peri-centromeric repeats. In contrast to repetitive DNA, protein-coding genes are refractory to siRNA-mediated heterochromatin formation, unless siRNAs are expressed in mutant cells.  Different studies have shown that siRNAs become potent mediators of RNAi-mediated epigenetic gene silencing in S. pombe cells that are mutant for mlo3+, dss1+, mst2+, or genes encoding subunits of the Paf1 complex (Paf1C). In this study, we have combined chemical mutagenesis with whole-genome sequencing in a sensitized S. pombe reporter strain to obtain a more comprehensive list of putative suppressors of small-RNA-mediated epigenetic gene silencing. This revealed more than 20 novel silencing-enabling mutations in genes that are associated with RNA processing, regulation of transcription, or post-translational protein modification. Focusing on factors involved in pre-mRNA cleavage and polyadenylation, we for example show that single amino acid substitutions in Yth1, which is responsible for polyadenylation signal recognition, lead to nearly 100% effective de novo formation of silent heterochromatin.   In S. pombe, small RNA-mediated silencing relies on the continuous amplification of the siRNA pool through a positive feedback loop. The deposited data shows the generation of secondary small RNAs over the reporter gene exclusively in the presence of RNAi-enabling mutations in the pre-mRNA cleavage and polyadenylation machinery. Furthermore, the RNA-seq data show that overall transcriptome levels are not markedly changing in the mutations themselves.   Altogether, our work shows that epigenetic gene silencing can be enabled by the acquisition of a plethora of mutant alleles in fission yeast. Overall design: In the course of this project, we have identified mutations in the pre-mRNA cleavage and polyadenylation machinery that enable RNAi-dependent heterochromatin formation. To show the direct effect of the mutants on siRNA generation and subsequent reporter gene silencing, we performed small RNA-seq and total RNA-seq experiments. To this end, total RNA from exponentially growing cultures was extracted with the MasterPure Yeast RNA Purification Kit (Epicenter). Small RNA libraries were prepared with the QIAseq miRNA Library Kit (QIAGEN) according to the manufacturer's instructions and sequenced with an Illumina NextSeq500 (75bp single-end). Total RNA libraries were prepared with TruSeq Stranded Total RNA kit (Illumina) according to the manufacturer's instructions and sequenced with an Illumina HiSeq2500 (50bp single-end).