Macronutrient Regulation of Alternative Pre-mRNA Splicing

DESCRIPTION (provided by applicant): We have recently discovered a novel and evolutionarily conserved homeostatic response wherein alternative splicing of the pre-mRNA encoding troponin T, a gene that affects muscle force production, is tightly regulated in response to changes in body weight. The effect is based on weight rather than mass or compartmentation of mass within the body because an external load has the same effect as an increment in native body weight. However, this response occurs differently if the load comprises fat, as load-induced changes in troponin T pre-mRNA alternative splicing are impaired in obese, but not lean, Zucker rats, leading to inappropriate expression of particular troponin T isoforms. In preliminary studies, we found a similar dysregulation in rats fed a high-fat diet enriched in saturated fatty acids. Notably, high-fat diet-induced changes in troponin T mRNA alternative splicing manifest prior to detectable alterations in either body weight or body composition, suggesting that alternative splicing is directly modulated in response to the fat content of the diet. Hence, we seek to characterize and examine experimentally the mechanisms whereby a high-fat diet causes deviation from the normal weight response in troponin T alternative splicing. We hypothesize that the difference is due to the known inflammatory effects of a high-fat diet and so by studying this system we seek to answer important questions on a number of levels via the following three specific aims: (1) assess the effects of dietary fatty acids, e.g. saturated vs. unsaturated, on quantitative alternative splicing of pre-mRNA at the level of our focal gene and across the transcriptome, as revealed by an exon array, (2) assess the effects of a high-fat diet on alterations in the signaling pathways and regulatory mechanisms that control quantitative alternative splicing of pre-mRNA, and, (3) establish a cell culture experimental model based on cytokine and lipidomic analysis of plasma and tissue samples to define signaling pathways and molecular mechanisms through which selected nutrients act to control alternative splicing. From these experiments, we will obtain an unprecedented scale and depth of understanding of how quantitative variation in alternative splicing is controlled, and how diet affects that regulation. In addition, these results will open a new window into how diet changes homeostative pathways involved in body weight homeostasis. Overall, the studies proposed here are highly original and will address a deficit in our knowledge about the plasticity of quantitative alternative splicing in general, and mechanisms through which macronutrients affect and in some cases disrupt the way metazoans functionally and metabolically adapt to changes in their weight. We expect the proposed research to reveal biomarkers for pre-disease states caused by poor diet, and candidate molecules and pathways for pharmacological manipulation to provide new and innovative approaches for the prevention and treatment of metabolic disorders.