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COMPARTMENTS WITHOUT BORDERS—DISSECTING THE ROLES FOR P-BODIES THROUGH HRR25 PROTEIN KINASE

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2017, Doctor of Philosophy, Ohio State University, Molecular Genetics.
RNA-protein (RNP) granules are non-membrane-bound organelles that function to compartmentalize the cytoplasm of eukaryotic cells. There are a number of distinct RNP granules, including the processing-body (P-body), stress granule and germ cell granule that form in response to specific stress conditions and developmental stimuli. These structures have been found to contain a number of signaling molecules important for the control of cell growth and survival and the determination of cell fate. However, relatively little is known about the mechanisms responsible for, and the ultimate consequences of, this signaling protein localization. The work here focuses on the essential CK1 protein kinase, Hrr25, that we showed was specifically associated with P-bodies in both yeast and mammalian cells. Hrr25 regulates a wide range of cellular activities including DNA damage repair, ribosome maturation, autophagy and meiosis. Our goal was to develop a better understanding of how this P-body localization influences the regulation of CK1 signaling in the model organism Saccharomyces cerevisiae. There are three major contributions to our understanding of P-body and CK1 biology. First, we determined the mechanisms responsible for the recruitment of Hrr25 to P-bodies. Specifically, we found that this localization of Hrr25 was dependent upon the presence of Dcp2, the catalytic subunit of the primary mRNA decapping enzyme. Hrr25 variants that could not interact with Dcp2 were not recruited to P-bodies. These variants include a kinase defective (KD) mutant and the PLS1* and PLS2* variants that possess mutations in two targeting motifs required for P-body localization. Altogether, these data suggested that Hrr25 is recruited to P-bodies in a kinase activity-dependent manner through an interaction with Dcp2. Second, we found that this association with the P-body is required for the maintenance of normal levels of Hrr25 protein in the cell. Hrr25 was rapidly degraded in mutants that failed to localize this protein kinase to P-bodies. This protein turnover occurs only under stress conditions that induce P-bodies formation. This degradation appears to occur within the proteasome as Hrr25 protein was stabilized by presence of proteasome inhibitor MG132. In all, this study has revealed a novel role for P-bodies in the protection of proteins from degradation, presumably by the ubiquitin-proteasome system during periods of stress. Finally, we uncovered a novel requirement for P-body localization of Hrr25 during meiotic transition. We found that P-bodies are present in diploid yeast cells undergoing meiosis and these structures contain Hrr25, an important regulator of meiosis. Meiosis was defective in cells expressing the Hrr25PLS1* variant that is not localized to P-bodies. Further analysis suggested that this deficiency might due to a reduced level of this variant as the over-expression of Hrr25PLS1* significantly increased meiotic efficiency. Consistent with this result, a heterozygotic diploid strain that has only one copy of the wild-type HRR25 gene was found to have reduced levels of the Hrr25 protein and to exhibit a corresponding drop in meiotic efficiency. Altogether, these analyses demonstrate that P-bodies are required for an efficient meiosis and that this requirement is at least partially due to the protective effects these RNP granules have on the Hrr25 protein. This work therefore suggests that RNP granules, like the P-body, can influence intracellular biology by affecting the levels of key signaling molecules in the cell.
Paul Herman (Advisor)
134 p.

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Citations

  • Zhang, B. (2017). COMPARTMENTS WITHOUT BORDERS—DISSECTING THE ROLES FOR P-BODIES THROUGH HRR25 PROTEIN KINASE [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1494291288298218

    APA Style (7th edition)

  • Zhang, Bo. COMPARTMENTS WITHOUT BORDERS—DISSECTING THE ROLES FOR P-BODIES THROUGH HRR25 PROTEIN KINASE. 2017. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1494291288298218.

    MLA Style (8th edition)

  • Zhang, Bo. "COMPARTMENTS WITHOUT BORDERS—DISSECTING THE ROLES FOR P-BODIES THROUGH HRR25 PROTEIN KINASE." Doctoral dissertation, Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1494291288298218

    Chicago Manual of Style (17th edition)