Investigating the Role of RNA Polymerase II in RNAi-dependent Heterochromatin Assembly at Centromeric Repeats

In Schizosaccharomyces pombe, a fission yeast, large domains of heterochromatin are found at telomeres, silent mating-type loci, and centromeric repeat regions of DNA (Bühler and Moazed, 2007). Much of the work done with S. pombe has shown that the assembly of heterochromatin around centromeric repeats depends on the coordination of two pathways: RNAi and histone modification. Current models suggest that initiation of heterochromatin formation begins with transcription of centromeric RNA by RNA polymerase II. These RNA transcripts are converted to dsRNA by RNA-dependent polymerase, cleaved to siRNA’s by the Dicer protein, and that it is these siRNA’s which are responsible for directing an RNA-induced transcriptional silencing (RITS) complex back to nascent centromeric RNA transcripts, from which it can recruit histone modification machinery (Grewal and Jia, 2007). It is not completely understood how targeting via siRNA is mediated, though mutations in several subunits of RNA polymerase II (RNAPII) result in phenotypes that mimic complete loss of the RNAi pathway and lead to a loss of heterochromatin formation. Our intention is to investigate RNAPII’s interaction with the RNAi proteins, and the status of siRNA binding of the RITS complex in RNAPII mutants, to generate a more complete understanding of RNAPII’s role in the RNAi silencing pathway. Introduction Chromatin states are an important part of gene regulation. In order to be packaged on chromosomes, DNA is wound about histones and this DNA/histone structure is called chromatin, of which there are two kinds. In eukaryotes, euchromatin is associated with transcriptionally