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基因）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。然而，如果负荷包括脂肪，这种反应会发生不同，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，而在瘦的Zucker大鼠中没有，导致特定肌钙蛋白T亚型的不适当表达。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，高脂肪饮食诱导的肌钙蛋白T mRNA选择性剪接的变化在体重或身体组成的可检测变化之前就表现出来，这表明选择性剪接是直接调节的，以响应饮食中的脂肪含量。因此，我们试图通过实验来描述和检验高脂肪饮食导致肌钙蛋白T选择性剪接偏离正常体重反应的机制。我们假设这种差异是由于已知的高脂肪饮食的炎症作用，因此通过研究这个系统，我们试图通过以下三个具体目标在多个层面上回答重要问题：(1)评估膳食脂肪酸（如饱和脂肪酸和不饱和脂肪酸）对靶基因水平和转录组间pre-mRNA定量选择性剪接的影响，(2)评估高脂肪饮食对控制pre-mRNA定量选择性剪接的信号通路和调节机制改变的影响，以及(3)建立基于血浆和组织样本的细胞因子和脂质组学分析的细胞培养实验模型，明确选定的营养物质控制选择性剪接的信号通路和分子机制。从这些实验中，我们将获得一个前所未有的规模和深度的理解在选择性剪接的定量变化是如何控制的，以及饮食如何影响这种调节。此外，这些结果将为研究饮食如何改变参与体重平衡的稳态途径打开一扇新的窗口。总的来说，这里提出的研究是高度原创的，将解决我们在定量选择性剪接的可塑性方面的知识缺陷，以及宏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。我们希望这项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和创新的方法。

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 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.