Investigation of the role of essential proteins in gene silencing at the centromere of Schizosaccharomyces pombe
The centromeres of eukaryotes have a region on which the kinetochore is assembled, flanked by heterochromatin which provides cohesion between the sister chromatids during cell division. When centromeric heterochromatin is lost chromosomes no longer segregate evenly into the daughter cells during cell division. In the fission yeast Schizosaccharomyces pombe (S. pombe) RNA interference (RNAi) is responsible for maintaining this heterochromatin. The pathway is part of a feedback loop whereby siRNAs generated from non-coding centromere transcripts are loaded into an Argonaute complex. The siRNAs guide the complex to the homologous centromere repeats in order to recruit Clr4 which modifies histone H3 with the heterochromatin mark H3K9me. A previous screen to find factors affecting centromere silencing isolated 13 loci termed centromere: suppressor of position-effect (csp) 1-13. Several csp mutants have been identified to be RNAi components. In this investigation the csp6 locus has been identified to be the Hsp70 gene ssa2+. It has been demonstrated that Argonaute proteins from plants and flies require Hsp70/90 chaperone activity for loading of siRNA. It therefore seems likely that Hsp70 may play a similar role in fission yeast. Genetic and biochemical techniques have been used in this study to investigate if the csp6 alleles are affecting siRNA loading in S. pombe. RNA Polymerase II (RNAPII) transcribes the pre-siRNA transcripts from the centromere repeats. csp3 was identified to be an allele of the RNAPII subunit rpb7+. rpb7-G150D was found to cause a silencing defect in the centromeric heterochromatin through a defect in transcription. Another RNAPII mutation, rpb2-m203, was found to have strong silencing defects caused by an unidentified non-transcriptional role in RNAi-mediated heterochromatin formation at the centromere. In order to gain more insight into the role of RNAPII in heterochromatin assembly I performed a screen in which the subunits rpb3 and rpb11 were subjected to random mutagenesis. Several mutants were isolated and characterisation of phenotypes regarding heterochromatin at the centromere has been carried out for nine of the mutants. As a result a novel phenomenon of RNAi-independent silencing at the centromere has been discovered.