Investigation of RNA-induced gene silencing in the fission yeast Schizosaccharomyces pombe

Abstract

RNA interference (RNAi) is a gene regulating system to which can silence genes at both transcriptional and post-transcriptional levels. In this thesis, a ura4-based RNAi selective assay was developed in the fission yeast Schizosaccharomyces pombe: antisense & sense constructs of mutually inverted ura4 DNA fragments were inserted under the regulation of a thiamine-repressible promoter; when induced by omission of thiamine, these transcripts triggered S. pombe ura4 gene silencing at the molecular level with the efficiency of -30%. Although dozens of proteins involved in RNAi pathways has been identified from different organisms, only a few are negative regulators of RNAi. The helF protein from the soil amoeba, Dictyostelium discoideum is one of the best characterized natural RNAi inhibitors. A homologue of Dictyostelium helF gene has also been identified in S. pombe. It is called. the mfhl gene. This thesis also addresses the question: is the mfhl gene a RNAi inhibitor in S. pombe. A haploid S. pombe mfhl deletion mutant was isolated, and the ura4-based RNAi selective assay was used to study RNAi in this mutant - there was no apparent effect on RNAi activity in this strain. Although no paralogue(s) of helF gene was found in Dictyostelium, a paralogue of S. pombe mfhl gene, mfh2 gene was identified -a mfhl & mf h2 double gene deletion mutant was also tested negative with respect to RNAi using the same assay. A phenotypic characterization of the S. pombe mfhl/mfh2 single/double gene deletion mutants compared to the wild type strain revealed that the mutants were more sensitive to ethanol, hydroxyurea and UV, which indicated that the mf hl and mf h2 genes appeared to protect S. pombe from chemical and UV induced DNA damage. The cellular localization of Dictyostelium helF and S. pombe mfhl genes was also attempted to be revealed by heterologous gene expressing studies using GFP-fusion expression plasmids in both Dictyostelium and S. pombe.