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和进化上保守的稳态应答，其中编码肌钙蛋白T（一种影响肌肉力量产生的蛋白质）的前体mRNA的选择性剪接响应于体重变化而受到严格调控。该效应基于体重，而不是体内的质量或质量区室，因为外部负荷与天然体重的增加具有相同的效应。相反，反应受身体组成的影响，因为负荷诱导的肌钙蛋白T前mRNA选择性剪接的变化在肥胖但不瘦的Zucker大鼠中受损，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选择性剪接的变化在体重或成分发生可检测的变化之前就表现出来，这表明选择性剪接是对饮食宏量营养素的直接调节。在其他初步研究中，我们发现高脂饮食对前体mRNA剪接的影响并不是肌钙蛋白T所独有的，而是对编码一系列蛋白质的前体mRNA所观察到的。因此，本申请中提出的研究的目的是鉴定膳食脂肪酸调节前体mRNA的选择性剪接的机制，并描述这种改变的功能后果。我们假设高脂饮食导致骨骼肌前mRNA剪接模式改变，导致蛋白质亚型表达，表现出降低的力产生和/或钙敏感性，以及代谢改变。该假设将通过以下三个具体目标进行检验：（1）建立高脂饮食诱导的选择性剪接变化的最佳条件， 骨骼肌中肌钙蛋白T前体mRNA，（2）表征高脂饮食诱导的骨骼肌收缩特性变化，（3）表征和描述高脂饮食诱导的骨骼肌中前体mRNA在转录组中选择性剪接变化的分子机制，并鉴定改变的信号传导和代谢途径。从这些实验中，我们将获得前所未有的规模和深度的理解，了解选择性剪接的数量变化是如何控制的，以及饮食如何影响这种调节。此外，研究结果将为饮食如何改变参与体重稳态的途径打开一扇新的窗口。总的来说，这里提出的研究是非常原始的，并将解决我们对定量选择性剪接的可塑性的认识不足，以及大量营养素影响和在某些情况下破坏后生动物功能和代谢适应体重变化的机制。我们希望拟议的研究能够揭示由不良饮食引起的疾病前状态的生物标志物，以及用于药理学操作的候选分子和途径，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.