Regulation of Gene Expression by Dietary Fat

描述（由申请人提供）：至少三十年来，美国公众一直被鼓励减少他们的总膳食脂肪摄入量，并增加饮食中多不饱和脂肪酸与饱和脂肪酸的比例。然而，脂肪酸在细胞内起作用的分子细节尚不清楚。长期目标是了解饮食中添加多不饱和脂肪是如何调节肝脏细胞内代谢的。本应用程序的目的是确定多不饱和脂肪酸在G6PD作为我们的模型基因表达过程中抑制转录后步骤的步骤。该应用程序的中心假设是G6PD转录物的有效剪接需要将特定剪接共激活子蛋白结合到前mrna外显子的剪接增强子序列上。在饲料中添加多不饱和脂肪会抑制剪接共激活子的活性，减少pre-mRNA剪接，从而靶向转录物进行降解。其基本原理是这种机制不是G6PD所独有的，而是与其他参与中间代谢的基因共享的，包括那些也在转录步骤中受到调节的基因。这种额外的调控水平将为细胞提供冗余机制，以确保这些蛋白质的调控表达，并对不断变化的环境条件做出更快速的反应。G6PD为研究这种新形式的转录后调控提供了一个理想的模型，因为缺乏转录变化使数据的解释更加容易。表征外显子剪接增强子元件序列的实验在具体目标1中进行了描述。特异性Aim 2测试剪接共激活蛋白是否与RNA元件结合，以及元件突变是否阻断该蛋白的结合。该项目的长期目标是确定膳食脂肪调节基因表达的机制。在Specific Aim 3中，将使用表达G6PD RNA报告子的转基因小鼠来测试外显子剪接增强子是否参与饮食对基因表达的调节。确定膳食脂肪改变细胞内代谢的分子途径对于理解肥胖、糖尿病和动脉粥样硬化等与脂肪过度消耗有关的疾病的分子基础至关重要，并且可以通过饮食或药物干预改善这些疾病的治疗策略。

DESCRIPTION (provided by applicant): For at least three decades, the American public has been encouraged to decrease their consumption of total dietary fat and to increase the ratio of polyunsaturated to saturated fatty acids in the diet. Yet the molecular details by which fatty acids act within cells is not well understood. The long-range goal is to understand how the addition of polyunsaturated fat in the diet regulates intracellular metabolism in the liver. The objective of this application is to define the steps by which polyunsaturated fatty acids inhibit posttranscriptional steps during gene expression using G6PD as our model gene. The central hypothesis of the application is that efficient splicing of the G6PD transcript requires the binding of a specific splicing co-activator protein to a splicing enhancer sequence in an exon of the pre-mRNA. The addition of polyunsaturated fat to the diet inhibits the activity of the splicing co-activator, decreasing pre-mRNA splicing and thereby targeting the transcript for degradation. The rationale is that this mechanism is not unique to G6PD but is shared by other genes involved in intermediary metabolism including those that are also regulated at transcriptional steps. This additional level of regulation would provide the cell with both redundant mechanisms to ensure regulated expression of these proteins and a more rapid response to changing environmental conditions. G6PD provides an ideal model to study this novel form of posttranscriptional regulation because the absence of transcriptional changes makes interpretation of the data much easier. Experiments to characterize the sequence of the exon splicing enhancer element are described in Specific Aims 1. Specific Aim 2 tests if a splicing co-activator protein binds the RNA element and if mutations in the element block binding of this protein. A long-term goal of this project has been to identify the mechanism by which dietary fat regulated gene expression. In Specific Aim 3, transgenic mice expressing G6PD RNA reporters will be used to test if the exon splicing enhancer is involved in dietary regulation of gene expression. Identification of the molecular pathway by which dietary fat alters intracellular metabolism is critical to understanding the molecular basis for diseases such as obesity, diabetes and atherosclerosis that are associated with over-consumption of fat, and can lead to improved strategies for treatment of these diseases via diet or drug intervention.