Date of Graduation


Document Type


Degree Type



School of Medicine



Committee Chair

Lisa M. Salati.


The expression of most lipogenic enzymes is regulated through the effects of dietary polyunsaturated fatty acids on transcription. However, we have observed that the regulation of Glucose-6-phosphate dehydrogenase (G6PD) by polyunsaturated fatty acids occurs through a novel posttranscriptional mechanism. RNA reporter assays were used to map the cis-acting element by measuring the regulation of each RNA reporter construct transfected into primary rat hepatocytes. All RNA reporter constructs were regulated by polyunsaturated fatty acids with the exception of RNA reporter constructs lacking exon 12. Therefore, polyunsaturated fatty acids regulate the constitutive splicing of G6PD mRNA through a cis-acting element located within exon 12. Likewise, G6PD expression is regulated by a similar mechanism in intact animals. Mice fed a high polyunsaturated fatty acid diet inhibit G6PD mRNA expression by 2-4 fold as compared to a low fat diet. Accordingly, starvation of mice induces a 12-15 fold inhibition of G6PD mRNA expression as compared to the refed mice.;The proteins that differentially bound to an exon 12 RNA element were observed and identified in nuclear extracts from starved and refed animals. The cis-acting element was identified as a region between 50 and 79 nucleotides of exon 12. Identification and purification of proteins differentially bound to the cis-acting element within exon 12 were identified by LC-MS/MS analysis, MALDI-TOF analysis and Western blot analysis. The proteins were identified as hnRNP K, L, and A2/B1. The 30 nucleotide binding site was further narrowed down to a region within nucleotides 65-79. HnRNP K, L and A2/B1 were shown to differentially bind in nuclear extracts of starved versus refed animals and this pattern of protein binding could potentially regulate G6PD mRNA splicing. All protein identifications were confirmed by Western blot analysis. Furthermore, a region within nucleotides 65-79 of exon 12 contained a strong C-rich patch located at nucleotides 65-67 and 69-71 of exon 12. C-rich patches found within mRNA sequences are thought to be potential binding sites for hnRNP K, L, A2/B1 proteins. The mutation of each of these C-rich patches disrupted protein binding to this region suggesting that the C-rich patch within 65-79 nucleotides is essential for hnRNP K, L, and A2/B1 protein binding. The binding of hnRNP K, L, and A2/B1 binding to the region of 65-79 nucleotides suggest the presence of an exon splicing silencer sequence within exon 12. The characterization of protein binding to this exon splicing silencer will be fundamental in providing new evidence on a novel lipogenic regulatory mechanism by polyunsaturated fatty acids.