Macronutrient Regulation of Alternative Pre-mRNA Splicing

描述(申请人提供)：我们最近发现了一种新颖的、进化上保守的动态平衡反应，其中编码肌钙蛋白T的前mRNA的选择性剪接受到严格调控，以响应体重的变化。肌钙蛋白T是一种影响肌肉力量产生的蛋白质。这种影响是基于体重而不是质量或身体内质量的划分，因为外部负荷与自然体重的增加具有相同的效果。相反，这种反应受身体成分的影响，因为在肥胖的Zucker大鼠中，负荷诱导的肌钙蛋白T前体mRNA选择性剪接的变化受到损害，但不是瘦的，导致肌钙蛋白T亚型的不适当表达。此外，在初步研究中，我们在喂食富含饱和脂肪酸的高脂肪饮食的大鼠中发现了类似的失调。值得注意的是，高脂饮食诱导的肌钙蛋白T前体mRNA选择性剪接的变化先于体重或成分的可检测到的变化，这表明选择性剪接直接受到饮食中大量营养素的调节。在其他的初步研究中，我们发现高脂饮食对前mRNA剪接的影响并不是肌钙蛋白T独有的，而是在编码一系列蛋白质的前mRNA中观察到的。因此，本申请中提出的研究的目的是确定膳食脂肪酸调节Pre-mRNAs选择性剪接的机制(S)，并描述这种改变的功能后果。我们假设，摄入高脂肪饮食会导致骨骼肌中Pre-mRNA剪接模式的改变，导致蛋白质异构体的表达，表现出力量产生和/或钙敏感性的减少，以及代谢的改变。这一假说将通过以下三个具体目标进行检验：(1)为高脂饮食诱导的选择性剪接改变建立最佳条件 (2)描述高脂饮食诱导的骨骼肌收缩特性的变化，以及(3)表征和描绘高脂饮食诱导的骨骼肌前-mRNA跨转录组选择性剪接改变的分子机制(S)，并确定改变的信号和代谢途径。从这些实验中，我们将获得前所未有的规模和深度的理解，如何控制选择性剪接的数量变异，以及饮食如何影响这一调节。此外，这一结果将为饮食如何改变与体重动态平衡有关的途径打开一扇新的窗口。总体而言，这里提出的研究具有很高的原创性，将解决我们对定量交替剪接一般可塑性的认识不足，以及大量营养素影响并在某些情况下扰乱后生动物在功能和代谢上适应其重量变化的机制。我们期望这项拟议的研究将揭示饮食不良引起的疾病前状态的生物标志物，以及药物操纵的候选分子和途径，为预防和治疗代谢紊乱提供新的创新方法。

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.