REGULATION OF GENE EXPRESSION BY DIETARY FAT

膳食脂肪对基因表达的调节

• 批准号: 2859204
• 负责人: LISA M SALATI
• 金额: $ 19.03万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2859204
• 关键词: RNA binding protein; RNase protection assay; cell nucleus; dietary lipid; gene expression; genetic regulation; glucose 6 phosphate dehydrogenase; laboratory mouse; liver cells; messenger RNA; nuclear matrix; nuclear membrane; nucleic acid probes; nucleic acid sequence; nutrition related tag; polyadenylate; posttranscriptional RNA processing; precursor mRNA; tissue /cell culture; transcription factor; transcription termination; transfection; unsaturated fatty acids

The long-term objective of this work is to understand the molecular mechanism by which dietary polyunsaturated fats inhibit gene expression. Americans are encouraged to consume less fat and a higher polyunsaturated to saturated fat ratio. This recommended dietary goal is a preventative health measure because of the correlation between fat intake, serum lipids and the risk of heart disease. The intracellular events resulting from changing the type and quantity of fat in the diet have not been well characterized. Using glucose-6-phosphate dehydrogenase (G6PD) as our model gene, we have identified a very novel form of regulated expression by dietary polyunsaturated fat. Changes in the expression of G6PD by diet occur at a nuclear posttranscriptional step: regulation of the amount of pre-mRNA. The amount of G6PD pre-mRNA is regulated early in the RNA processing pathway, without regulation of splicing or nucleocytoplasmic transport. The research program described in this application will test the hypothesis that dietary polyunsaturated fats inhibit the expression of G6PD by decreasing processing of the G6PD pre-mRNA. This mechanism involves the binding of trans-acting proteins to cis-acting elements within the pre-mRNA. The consequence of this binding reaction is either a block in the entry of pre-mRNA into the processing pathway or enhanced degradation during processing. In Specific Aim 1, the cis-acting elements involved in the inhibition of G6PD pre-mRNA accumulation by fatty acids will be identified. Transient transfection of chimeric constructs into primary hepatocytes will test for these elements in all parts of the 18 kb primary transcript of G6PD. Experiments in Specific Aim 2 will characterize the nuclear localization of G6PD pre-mRNA. The amount of G6PD pre- and mature mRNA will be measured on the nuclear matrix, in soluble nuclear fractions, and on the nuclear membrane. The relative amounts of precursor and mature mRNA and the kinetics by which these amounts change will indicate where during processing regulated expression of G6PD is occurring. In Specific Aim 3, the effect of specific degradative pathways on the accumulation of G6PD pre-mRNA will be measured. Changes in the length of the poly(A) tail of G6PD pre-mRNA and/or enhanced 3' to 5' degradation could decrease G6PD pre-mRNA accumulation in the nucleus in mice consuming a diet high in polyunsaturated fat. The results of these experiments will provide novel new information regarding the mechanisms by which dietary polyunsaturated fats can regulate gene expression.

这项工作的长期目标是了解饮食中多不饱和脂肪抑制基因表达的分子机制。 美国人被鼓励消耗更少的脂肪和更高的多不饱和脂肪与饱和脂肪的比例。 由于脂肪摄入量、血脂和心脏病风险之间的相关性，这一推荐的饮食目标是一种预防性健康措施。 由于改变饮食中脂肪的类型和数量而引起的细胞内事件尚未得到很好的表征。 使用葡萄糖-6-磷酸脱氢酶（G6 PD）作为我们的模型基因，我们已经确定了一种非常新颖的形式，通过饮食多不饱和脂肪的调控表达。 饮食引起的G6 PD表达的变化发生在核转录后步骤：调节前mRNA的量。G6 PD前mRNA的量在RNA加工途径的早期受到调节，而不调节剪接或核质转运。 本申请中描述的研究计划将测试膳食多不饱和脂肪通过减少G6 PD前mRNA的加工来抑制G6 PD表达的假设。 该机制涉及反式作用蛋白质与前mRNA内的顺式作用元件的结合。 这种结合反应的结果是阻断前mRNA进入加工途径或在加工过程中增强降解。 在特定目的1中，将鉴定脂肪酸抑制G6 PD前mRNA蓄积的顺式作用元件。 将嵌合构建体瞬时转染到原代肝细胞中将测试G6 PD的18 kb初级转录物的所有部分中的这些元件。 特定目标2中的实验将表征G6 PD前mRNA的核定位。 将在核基质、可溶性核组分和核膜上测量G6 PD前mRNA和成熟mRNA的量。 前体和成熟mRNA的相对量以及这些量变化的动力学将指示在加工期间G6 PD的调节表达发生的位置。 在特定目的3中，将测定特定降解途径对G6 PD前mRNA蓄积的影响。 改变G6 PD前体mRNA的poly（A）尾的长度和/或增强的3'至5'降解可以减少进食高多不饱和脂肪饮食的小鼠中G6 PD前体mRNA在细胞核中的积累。这些实验的结果将提供有关膳食多不饱和脂肪调节基因表达的机制的新信息。