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Analysis of Regulatory Mechanisms on RNA Interference by Molecular Chaperone Hsp90 and Protein Phosphorylation in Yeast

  • Author / Creator
    Wang, Yang
  • RNA interference (RNAi) is a conserved mechanism that eukaryotes employ small RNAs to regulate gene expression at transcriptional and post-trnascriptiona levels in a sequence-specific manner. However, current understanding on the regulatory mechanisms of RNAi via its core components is quite limited. In the RNAi-deficient budding yeast Saccharomyces cerevisiae (S. cerevisiae), I demonstrated that the integration of genes encoding Saccharomyces castellii (S. castellii) Dicer and S. castellii or human Argonaute restored RNAi-mediated reporter gene silencing. Conversely, the introduction of genes encoding human Dicer and human (or S. castellii) Argonaute, with or without Dicer co-factor TAR RNA-binding protein 2 (TRBP2), was unable to reconstitute RNAi. My studies also showed that S. castellii Dicer does not detectablly interact with either Argonuate protein, whereas human Dicer associates with human but not S. castellii Argonaute independently of TRBP2. Moreover, deletion of several genes proteins with one or more double-stranded RNA binding domains (dsRBDs) did not noticeably affect RNAi-mediated reporter gene silencing in S. cerevisiae. I hypothesizd Dicer proteins in budding yeast do not require dsRBP cofactor(s) to stabilize dsRNA substrtates as their counterparts in mammals. My study also revealed that the restored RNAi pathways in S. cerevisiae are dependent on the ATPase activity of the molecular chaperone Hsp90. One explanation is that Hsp90 facilitates a conformational change of Argonaute, which is required for the loading of small RNA duplexes. In the fission yeast Schizoaccharomyces pombe (S. pombe), I identified a number of kinases that are required for heterochromatin assembly at centromeres, a process that depends on the RNAi-mediated silencing of pericentromeric transcripts. Further research is needed to find out which kinase(s) catalyze the phosphorylation reaction targeting core RNAi components. Argonaute protein in the deletion strains of three kinase, Gsk3, Byr1, and Dsk1, strongly associated with Poly (A) binding protein (PABP) and non-selectively bound more RNAs. Moreover, the kinase Pka1 was found to be essential for the biogenesis and/or stability of Chp1, a binding partner of Argonaute and an essential component of RNA-induced transcriptional silencing complex (RITS). My results indicate that RNAi is subject to intricate and extensive regulation by the molecular chaperone Hsp90 and protein phosphorylation through RNAi core components.

  • Subjects / Keywords
  • Graduation date
    2016-06:Fall 2016
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3ZW19121
  • 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)
    • Hobman, Tom (Cell Biology)
    • LaPointe, Paul (Cell Biology)
  • Examining committee members and their departments
    • MacMillan, Andrew (Biochemistry)
    • Chua, Gordon (Biological Science, University of Calgary)
    • Simmonds, Andrew (Cell Biology)