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RNA interference, Hsp90, Protein phosphorylation, S. cerevisiae, S. pombe

Wang, Yang

Supervisor and department: Hobman, Tom Cell Biology LaPointe, Paul Cell Biology

Examining committee member and department: Simmonds, Andrew Cell Biology MacMillan, Andrew Biochemistry Chua, Gordon Biological Science, University of Calgary

Department: Department of Cell Biology

Specialization:

Date accepted: 2016-09-22T13:47:35Z

Graduation date: 2016-06:Fall 2016

Degree: Doctor of Philosophy

Degree level: Doctoral

Abstract: 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 cofactors 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 kinases 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.

Language: English

DOI: doi:10.7939-R3ZW19121

Rights: This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. 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.





Autor: Wang, Yang

Fuente: https://era.library.ualberta.ca/


Introducción



Analysis of Regulatory Mechanisms on RNA Interference by Molecular Chaperone Hsp90 and Protein Phosphorylation in Yeast by Yang Wang A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Cell Biology University of Alberta © Yang Wang, 2016 ABSTRACT RNA interference (RNAi) is a conserved mechanism that eukaryotes employ small RNAs to regulate gene expression at transcriptional and post-transcriptional 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 detectably interact with either Argonaute 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 hypothesized Dicer proteins in budding yeast do not require dsRBP cofactor(s) to stabilize dsRNA substrates 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 Schizosaccharomyces pombe (S.
pombe), I identified a number of kinases that are re...





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