Dysregulation Of Cell Growth And Apoptosis Networks Through Altered Mrna Decay In Malignancy And Viral Infection

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Dysregulation Of Cell Growth And Apoptosis Networks Through Altered Mrna Decay In Malignancy And Viral Infection

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2018-08

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The gene expression in eukaryotic cells is tightly regulated at various levels, including transcription, RNA localization, RNA stability, translation, and post-translational modification. The coordinated regulation of messenger RNA (mRNA) stability enables the cell to control gene expression quickly and precisely in different cellular conditions. The work presented in this thesis focuses on investigating the regulation of gene expression at the level of mRNA decay in two disease status, viral infection and malignancy, and how would this coordinated regulation contribute to disease progression and malignant transformation. The first project presented in Chapter 2 was to elucidate the network of transcripts that are regulated by the RNA binding protein CUGBP1 and Etr3 Like Factor 1 (CELF1) in malignant T cells. CELF1 is an important RNA decay regulator which binds to GU-rich element (GRE) within its target transcripts and mediates rapid degradation of the transcripts. We performed immunoprecipitation using an anti- CELF1 antibody, followed by identification of copurified transcripts using microarrays. We found that CELF1 is bound to a distinct set of target transcripts in the H9 and Jurkat malignant T-cell lines, compared with primary human T cells. CELF1 was not phosphorylated in resting normal T cells, but in malignant T cells, phosphorylation of CELF1 correlated with its inability to bind to GRE-containing mRNAs that served as CELF1 targets in normal T cells. Lack of binding by CELF1 to these mRNAs in malignant T cells correlated with stabilization and increased expression of these transcripts. Several of these GRE-containing transcripts that encode regulators of cell growth were also stabilized and up-regulated in primary tumor cells from patients with T-cell acute lymphoblastic leukemia. Interestingly, transcripts encoding numerous suppressors of cell proliferation that served as targets of CELF1 in malignant T cells, but not normal T cells, exhibited accelerated degradation and reduced expression in malignant compared with normal T cells, consistent with the known function of CELF1 to mediate degradation of bound transcripts. Overall, CELF1 dysfunction in malignant T cells led to the up-regulation of a subset of GRE-containing transcripts that promote cell growth and down-regulation of another subset that suppress cell growth, producing a net effect that would drive a malignant phenotype. The next two projects presented in Chapter 3 and 4 were exploring the network of cellular transcripts with altered stability following viral infection, hepatitis C virus (HCV) and reovirus, respectively. We utilized RNA-immunoprecipitation followed by RNA sequencing on cell lysate of human hepatoma cells expressing a HCV subgenomic replicon, and found that the viral nonstructural protein 5A (NS5A), a protein known to bind to viral RNA, also bound specifically to human cellular transcripts that encode regulators of cell growth and apoptosis, and this binding correlated with transcript stabilization. An important subset of human NS5A-target transcripts contained GRE, sequences known to destabilize mRNA. We found that NS5A bound to GU-rich elements in vitro and in cells. Mutation of the NS5A zinc finger abrogated its GU-rich element-binding and mRNA stabilizing activities. Through this project, we identified a molecular mechanism whereby HCV manipulates host gene expression by stabilizing host transcripts in a manner that would promote growth and prevent death of virus-infected cells, allowing the virus to establish chronic infection and lead to the development of hepatocellular carcinoma. In the reovirus project, we utilized oligonucleotide microarrays to measure cellular mRNA decay rates in mock- or reovirus-infected murine L929 cells. Our analysis detected a subset of cellular transcripts that were coordinately induced and stabilized following infection with the reovirus isolates c87 and c8, which also induced inhibition of cellular translation. The induced and stabilized transcripts encoded multiple regulators of TGF- signaling, including components of the Smad signaling network and apoptosis/survival pathways. The coordinate induction through mRNA stabilization of multiple genes that encode components of TGF- signaling pathways represents a novel mechanism by which the host cell responds to viral infection.

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University of Minnesota Ph.D. dissertation. August 2018. Major: Veterinary Medicine. Advisors: Paul Bohjanen, Irina Vlasova-St. Louis. 1 computer file (PDF); xiv, 146 pages.

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Guo, Liang. (2018). Dysregulation Of Cell Growth And Apoptosis Networks Through Altered Mrna Decay In Malignancy And Viral Infection. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/201039.

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