Investigation of factors involved in RNAi-mediated heterochromatic silencing in Schizosaccharomyces pombe

  • Francesca Taglini

    Student thesis: Doctoral ThesisDoctor of Science

    Abstract

    The genome of eukaryotic cells is organised into chromatin, a dynamic structure composed of DNA and specialised proteins that packages DNA to fit inside the nucleus, regulates gene expression and ensures genome stability. Heterochromatin is a repressive form of chromatin found at repetitive sequences such as centromeres, telomeres and transposons. Heterochromatin-mediated silencing of these loci prevents aberrant chromosomal rearrangements and constitutes a platform for recruiting factors responsible for faithful chromosome segregation. Studies in various organisms suggest that heterochromatin formation can be targeted by RNA interference (RNAi), a conserved regulatory mechanism mediated by small RNAs. RNAi-mediated heterochromatin assembly has been extensively studied in the fission yeast Schizosaccharomyces pombe, where RNAi is strictly required for the establishment and maintenance of heterochromatic silencing at centromeres. Indeed, the RNAi effector complex RITS recruits to chromatin the methyltransferase complex CLRC, which is responsible for methylation of histone H3 at lysine 9 (H3K9me), a repressive chromatin mark. In this work, I explored two fundamental aspects of the pathway: 1) the physical connection between the RNAi and chromatin-modifying enzymes, and 2) the requirements for de novo heterochromatin formation.

    1) The physical link between RITS and CLRC has recently been found to be mediated by the zinc finger protein Stc1. The zinc finger motif of Stc1 interacts with RITS, but nothing was known about the nature of the Stc1-CLRC interaction. By testing a series of Stc1 mutants for the ability to mediate silencing, either at centromeres or in an artificial RNAi-independent system, I discovered that the unstructured C-terminus of Stc1 is responsible for CLRC recruitment to chromatin, likely via electrostatic interactions.
    2) In order to identify novel factors involved in the initial phases of heterochromatin assembly, I analysed a set of candidate mutants previously identified in a sensitised genome-wide trans-silencing screen. My analyses identified a novel gene encoding a putative endonuclease, Mkt1, as the most promising candidate for further investigation. Employing genetic and molecular approaches, I discovered that Mkt1 is involved in de novo heterochromatin formation at centromeric repeats and at the mating-type locus. My findings suggest that Mkt1 could act in an RNA surveillance pathway, thus linking RNA metabolism with the establishment of heterochromatic silencing.
    Date of Award2015
    LanguageEnglish
    Awarding Institution
    • University of Dundee
    Add any sponsors of the thesis researchBiotechnology and Biological Sciences Research Council
    SupervisorElizabeth Bayne (Supervisor)

    Cite this

    Investigation of factors involved in RNAi-mediated heterochromatic silencing in Schizosaccharomyces pombe
    Taglini, F. (Author). 2015

    Student thesis: Doctoral ThesisDoctor of Science