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Permanent link (DOI): https://doi.org/10.7939/R36T4H

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

Descriptions

Other title
Subject/Keyword
Fission yeast
TGS, PTGS, Rdp1, Cut7
RAN interference
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Park, Jungsook
Supervisor and department
Dr. Hobman, Tom (Department of Cell Biology)
Examining committee member and department
Dr. Wozniack, Richard (Cell Biology, U of Alberta)
Dr. Simmonds, Andrew (Cell Biology, U of Alberta)
Dr. Hobman, Tom (Cell Biology, U of Alberta)
Dr. MacMillan, Andrew (Biochemistry, U of Alberta)
Dr. Provost, Patrick (CHUQ Research Center, Department of Microbiology-Infectiology and Immunology, Universite´ Laval)
Department
Department of Cell Biology
Specialization

Date accepted
2013-01-25T16:14:53Z
Graduation date
2013-06
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
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.
Language
English
DOI
doi:10.7939/R36T4H
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
“Park J, Freitag SI, Young PG, Hobman TC. (2012). The karyopherin Sal3 is required for nuclear import of the core RNA interference pathway protein Rdp1. Traffic. 13(4):520-531.”“C. Stoica (†), J. Park (†), J. Pare, S. Willows, T. Hobman (2010), The Kinesin Motor Protein Cut7 Regulates Biogenesis and Function of Ago1-Complexes, Traffic. 11(1):25-36.”

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