Date of Graduation

2015

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Biochemistry

Committee Chair

Lisa M Salati

Committee Co-Chair

Yehenew Agazie

Committee Member

John Hollander

Committee Member

J Michael Ruppert

Committee Member

Maxim Sokolov

Abstract

Nutritional status is a powerful regulator of intracellular function. Dietary status can increase or decrease the rate of RNA splicing, thereby affecting gene expression; however, few molecular mechanisms have been identified that are responsible for this type of regulation.;The glucose-6-phosphate dehydrogenase (G6PD) gene has provided a useful tool for the study of nutrient-regulated splicing because accumulation of G6PD mRNA is dependent solely on changes in the rate of mRNA splicing in response to nutritional stimuli, consistent with its enzymatic role of converting dietary energy to fatty acids. Treatment of primary hepatocytes in culture with insulin or feeding a high-carbohydrate, low fat diet to rodents increases splicing by 7- and 15-fold, respectively, and increases the cellular content of G6PD. In contrast, starvation or treatment of primary hepatocytes in culture with polyunsaturated fatty acids reduces G6PD mRNA splicing by 70% or more. The unspliced RNA is degraded in the nucleus, thus reducing expression of the G6PD enzyme. Our laboratory identified a splicing regulatory element within exon 12 of the G6PD transcript is required for splicing-related changes in G6PD expression in response to nutrient stimuli. This element is bound by two splicing regulatory proteins, SRSF3 and hnRNP K.;To understand the mechanism of action of these proteins, we used a loss-of-function approach. SiRNA-mediated depletion of SRSF3 significantly decreased G6PD splicing and expression; in contrast, depletion of hnRNP K enhanced both splicing and mRNA accumulation. Consistent with their apparent roles as enhancers or silencers of splicing, respectively, the results of RNA immunoprecipitation (RIP) indicated binding of SRSF3 to exon 12 was enhanced 6-fold in the livers of refed mice and nearly undetectable in starved mice. Conversely, binding of hnRNP K to the regulatory element was increased 12.5-fold in the livers of starved mice and barely detectable in the livers of refed mice. Furthermore, RNA EMSA using purified SRSF3 and hnRNP K recombinant proteins, we demonstrated that SRSF3 and hnRNP K compete for binding to the same sequences within the regulatory element, which we hypothesize is a bifunctional ESE/ESS element. Thus, mutually exclusive binding of SRSF3 and hnRNP K to this regulatory element mediates the nutrient regulation of G6PD mRNA splicing and establishes a new intracellular mechanism for nutrient regulation of gene expression. We hypothesize that nutrient regulation of splicing is not unique to the G6PD mRNA, and thus the experiments described herein are focused identifying additional genes that are regulated by changes in alternative splicing in response to nutrient availability.

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