REGULATION OF GENE EXPRESSION BY DIETARY FAT

这项工作的长期目标是了解膳食多不饱和脂肪抑制基因表达的分子机制。美国人被鼓励少吃脂肪，多不饱和脂肪与饱和脂肪的比例更高。这种推荐的饮食目标是一种预防性的健康措施，因为脂肪摄入量、血脂和心脏病风险之间存在相关性。改变饮食中脂肪的类型和数量所导致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（G6PD）作为我们的模式基因，我们已经确定了一种非常新颖的受膳食多不饱和脂肪调控的表达形式。饮食对G6PD表达的改变发生在细胞核转录后的一个步骤：pre-mRNA量的调节。G6PD前mrna的数量在RNA加工途径的早期受到调节，而不受剪接或核胞质运输的调节。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G6PD前mrna的加工来抑制G6PD表达的假设。该机制涉及反式作用蛋白与前体mrna中顺式作用元件的结合。这种结合反应的结果要么是阻止前mrna进入加工途径，要么是在加工过程中增强降解。在Specific Aim 1中，将鉴定参与脂肪酸抑制G6PD pre-mRNA积累的顺式作用元件。将嵌合构建物瞬时转染原代肝细胞，将测试G6PD原代转录物18kb片段中所有部分的这些元素。特异性靶2中的实验将表征G6PD前mrna的核定位。将在核基质、可溶核组分和核膜上测量G6PD预mRNA和成熟mRNA的量。前体和成熟mRNA的相对数量以及这些数量变化的动力学将表明在加工过程中G6PD的调节表达发生的位置。在Specific Aim 3中，我们将测量特定降解途径对G6PD pre-mRNA积累的影响。G6PD前体mrna聚(A)尾长度的变化和/或3‘至5’降解的增强可以减少G6PD前体mrna在高多元不饱和脂肪饮食小鼠细胞核中的积累。这些实验结果将为膳食多不饱和脂肪调节基因表达的机制提供新的信息。

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.