For at least three decades, the American public has been encouraged to decrease their consumption of total dietary fat and to increase the ratio of polyunsaturated to saturated fatty acids in the diet. Yet the molecular details by which fatty acids act within cells is not well understood. The long-range goal is to understand how the addition of polyunsaturated fat in the diet regulates intracellular metabolism in the liver. The objective of this application is to define the steps by which polyunsaturated fatty acids inhibit posttranscriptional steps during gene expression using G6PD as our model gene. The central hypothesis of the application is that efficient splicing of the G6PD transcript requires the binding of a specific splicing co-activator protein to a splicing enhancer sequence in an exon of the pre-mRNA. The addition of polyunsaturated fat to the diet inhibits the activity of the splicing co-activator, decreasing pre-mRNA splicing and thereby targeting the transcript for degradation. The rationale is that this mechanism is not unique to G6PD but is shared by other genes involved in intermediary metabolism including those that are also regulated at transcriptional steps. This additional level of regulation would provide the cell with both redundant mechanisms to ensure regulated expression of these proteins and a more rapid response to changing environmental conditions. G6PD provides an ideal model to study this novel form of posttranscriptional regulation because the absence of transcriptional changes makes interpretation of the data much easier. Experiments to characterize the sequence of the exon splicing enhancer element are described in Specific Aims 1.
Specific Aim 2 tests if a splicing co-activator protein binds the RNA element and if mutations in the element block binding of this protein. A long-term goal of this project has been to identify the mechanism by which dietary fat regulated gene expression.
In Specific Aim 3, transgenic mice expressing G6PD RNA reporters will be used to test if the exon splicing enhancer is involved in dietary regulation of gene expression. Identification of the molecular pathway by which dietary fat alters intracellular metabolism is critical to understanding the molecular basis for diseases such as obesity, diabetes and atherosclerosis that are associated with over-consumption of fat, and can lead to improved strategies for treatment of these diseases via diet or drug intervention. ? ?
