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Analyses of trans-acting factors that regulate RNA interference in Schizosaccharomyces pombe

  • Author / Creator
    Park, Jungsook
  • RNA interference (RNAi) is a phenomenon in which small RNAs induce an efficient, sequence-specific silencing of gene expression at transcriptional (TGS) and post-transcriptional (PTGS) levels. In the fission yeast Schizosaccharomyces pombe (S. pombe), the basic roles of Argonaute protein (Ago1), Dicer (Dcr1), and RNA-dependent RNA polymerase (Rdp1) in TGS and PTGS are relatively well understood. The core RNAi effector proteins are differentially localized in this organism. Specifically, most Ago1 and Dcr1 reside in the cytoplasm, whereas the bulk of Rdp1 is found in the nucleus. However, it is not known how RNAi effector protein functions are regulated in different cellular compartments. Emerging evidence suggests that various cis- and trans-acting factors are involved in the regulation of RNAi pathways. In this thesis, I characterized trans-acting factors that modulate small RNA-mediated gene silencing mechanisms in S. pombe. I demonstrated that the β-karyopherin Sal3 is required for the nuclear import of Rdp1 in S. pombe. Loss of nuclear Rdp1 was associated with substantially reduced transcriptional gene-silencing. Surprisingly, post-transcriptional gene-silencing, which occurs in the cytoplasm of other eukaryotes, was also affected. In addition to identifying Sal3 as a modulator of RNAi-dependent transcriptional gene-silencing, a potential link between nuclear import and post-transcriptional gene silencing was discovered. As well, I identified a previously unknown modulator of the Argonaute protein in S. pombe. In the cytoplasm, Argonaute proteins are incorporated into large, mobile puncta known as processing bodies (PBs) that travel along microtubules. Using a candidate gene approach, I discovered that a microtubule-associated motor protein, Cut7, is important for the homeostasis of Ago1-containing PBs in the cytoplasm. The observation that Cut7 activity enhances post-transcriptional gene silencing provides direct evidence that microtubule motor proteins are part of the gene silencing machinery in the cytoplasm. Finally, preliminary data derived from the study of Ago1 mutants as well as inhibitor experiments suggest that kinases and phosphates control the localization and, by extension, the function of Argonaute proteins in fission yeast.

  • Subjects / Keywords
  • Graduation date
    2013-06
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R36T4H
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Cell Biology
  • Supervisor / co-supervisor and their department(s)
    • Dr. Hobman, Tom (Department of Cell Biology)
  • Examining committee members and their departments
    • Dr. MacMillan, Andrew (Biochemistry, U of Alberta)
    • Dr. Hobman, Tom (Cell Biology, U of Alberta)
    • Dr. Wozniack, Richard (Cell Biology, U of Alberta)
    • Dr. Provost, Patrick (CHUQ Research Center, Department of Microbiology-Infectiology and Immunology, Universite´ Laval)
    • Dr. Simmonds, Andrew (Cell Biology, U of Alberta)