Semester

Spring

Date of Graduation

2013

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Lisa Salati

Abstract

Splicing of nascent RNA transcripts is an essential step in gene expression and a target of nutrient regulation. Glucose-6-phosphate dehydrogenase (G6PD) is a lipogenic gene whose expression is regulated exclusively at this posttranscriptional stage. Expression of G6PD mRNA increases 15- to 17-fold during refeeding and is inhibited 80-90% by starvation and the addition of polyunsaturated fat to the diet. The large changes in the accumulation of G6PD mRNA are due to changes the rate of pre-mRNA splicing and not changes in its transcription. In this regard, dietary carbohydrate enhances intron removal, which increases the accumulation of G6PD mRNA. Starvation alters pre-mRNA splicing by decreasing the rate of intron removal, leading to intron retention and a decrease in the accumulation of mature mRNA. A regulatory element within exon 12 of the G6PD pre-mRNA that contains both an ESS and an ESE mediates these changes in splicing efficiency. SR proteins, like SRp20, along with hnRNP K, L and A2/B1 bind to this regulatory region. Starvation caused an increase in the expression of heterogeneous nuclear ribonucleoprotein (hnRNP) K protein and this increase coincided with the increase in the binding of hnRNP K to the regulatory element and a decrease in the expression of G6PD mRNA in vivo. HnRNP K bound to two C-rich motifs forming an ESS within exon 12. Overexpression of hnRNP K decreased the splicing and expression of G6PD mRNA, while siRNA-mediated depletion of hnRNP K caused an increase in the splicing and expression of G6PD mRNA. HnRNP K binding to the C- rich motifs blocked binding of serine-arginine rich, splicing factor 3 (SRSF3), a splicing enhancer. Thus hnRNP K is a nutrient regulated splicing factor responsible for the inhibition of the splicing of G6PD during starvation.

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