Macronutrient Regulation of Alternative Pre-mRNA Splicing

描述（由申请人提供）：我们最近发现了一种新的进化保守的稳态反应，其中编码肌钙蛋白T（一种影响肌肉力量产生的蛋白质）的前mrna的选择性剪接在体重变化的反应中受到严格调节。这种影响是基于体重，而不是基于质量或体内质量的区隔，因为外部负荷与自然体重的增加具有相同的效果。相反，这种反应受到身体成分的影响，因为负荷引起的肌钙蛋白T前mrna选择性剪接的变化在肥胖的Zucker大鼠中受损，而在瘦弱的Zucker大鼠中没有，从而导致肌钙蛋白T亚型的不适当表达。此外，在初步研究中，我们发现在喂食富含饱和脂肪酸的高脂肪饮食的大鼠中也存在类似的失调。值得注意的是，高脂肪饮食诱导的肌钙蛋白T前mrna选择性剪接的变化在体重或组成的可检测变化之前就表现出来，这表明选择性剪接是对饮食常量营养素的直接调节。在其他初步研究中，我们发现高脂肪饮食对前mrna剪接的影响并非肌钙蛋白T所独有，而是对编码一系列蛋白质的前mrna的影响。因此，本应用中提出的研究目的是确定膳食脂肪酸调节前mrna选择性剪接的机制，并描述这种改变的功能后果。我们假设，高脂肪饮食的消耗导致骨骼肌前mrna剪接模式的改变，导致蛋白质异构体的表达，表现出力产生和/或钙敏感性的降低，以及代谢的改变。该假设将通过以下三个具体目标进行验证：(1)建立高脂肪饮食诱导的选择性剪接变化的最佳条件

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 protein 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. In contrast, the response is affected by body composition, 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 troponin T isoforms. Moreover, in preliminary studies, we have 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 pre-mRNA alternative splicing manifest prior to detectable alterations in either body weight or composition, suggesting that alternative splicing is directly modulated in response to dietary macronutrients. In other preliminary studies we have found that the effect of a high-fat diet on pre-mRNA splicing is not unique to troponin T, but instead is observed for pre-mRNAs encoding an array of proteins. Hence, the objective of the studies proposed in the present application is to identify the mechanism(s) through which dietary fatty acids regulate alternative splicing of pre-mRNA, and delineate the functional consequences of such alterations. We hypothesize that consumption of a high-fat diet results in an altered pattern of pre-mRNA splicing in skeletal muscle, leading to expression of protein isoforms that exhibit reduced force production and/or calcium sensitivity, as well as altered metabolism. The hypothesis will be tested via the following three specific aims: (1) Establish optimal conditions for high-fat diet-induced changes in alternative splicing of the troponin T pre- mRNA in skeletal muscle, (2) Characterize high-fat diet-induced changes in the contractile properties of skeletal muscle, and, (3) Characterize and delineate the molecular mechanism(s) involved in high-fat diet- induced changes in alternative splicing of pre-mRNA in skeletal muscle across the transcriptome, and identify altered signaling and metabolic pathways. 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, the results will open a new window into how diet changes 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 alternatie 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.