The long-term objective of this work is to understand the molecular mechanism by which dietary polyunsaturated fats inhibit gene expression. Americans are encouraged to consume less fat and a higher polyunsaturated to saturated fat ratio. This recommended dietary goal is a preventative health measure because of the correlation between fat intake, serum lipids and the risk of heart disease. The intracellular events resulting from changing the type and quantity of fat in the diet have not been well characterized. Using glucose-6-phosphate dehydrogenase (G6PD) as our model gene, we have identified a very novel form of regulated expression by dietary polyunsaturated fat. Changes in the expression of G6PD by diet occur at a nuclear posttranscriptional step: regulation of the amount of pre-mRNA. The amount of G6PD pre-mRNA is regulated early in the RNA processing pathway, without regulation of splicing or nucleocytoplasmic transport. The research program described in this application will test the hypothesis that dietary polyunsaturated fats inhibit the expression of G6PD by decreasing processing of the G6PD pre-mRNA. This mechanism involves the binding of trans-acting proteins to cis-acting elements within the pre-mRNA. The consequence of this binding reaction is either a block in the entry of pre-mRNA into the processing pathway or enhanced degradation during processing.
In Specific Aim 1, the cis-acting elements involved in the inhibition of G6PD pre-mRNA accumulation by fatty acids will be identified. Transient transfection of chimeric constructs into primary hepatocytes will test for these elements in all parts of the 18 kb primary transcript of G6PD. Experiments in Specific Aim 2 will characterize the nuclear localization of G6PD pre-mRNA. The amount of G6PD pre- and mature mRNA will be measured on the nuclear matrix, in soluble nuclear fractions, and on the nuclear membrane. The relative amounts of precursor and mature mRNA and the kinetics by which these amounts change will indicate where during processing regulated expression of G6PD is occurring.
In Specific Aim 3, the effect of specific degradative pathways on the accumulation of G6PD pre-mRNA will be measured. Changes in the length of the poly(A) tail of G6PD pre-mRNA and/or enhanced 3' to 5' degradation could decrease G6PD pre-mRNA accumulation in the nucleus in mice consuming a diet high in polyunsaturated fat. The results of these experiments will provide novel new information regarding the mechanisms by which dietary polyunsaturated fats can regulate gene expression.
