REGULATION OF GENE EXPRESSION BY DIETARY FAT

这项工作的长期目标是了解饮食多不饱和脂肪抑制基因表达的分子机制。鼓励美国人少吃脂肪，提高多不饱和脂肪与饱和脂肪的比例。这一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导致的细胞内事件还没有得到很好的描述。使用葡萄糖-6-磷酸脱氢酶(G6PD)作为我们的模型基因，我们已经确定了一种由膳食多不饱和脂肪调控的非常新的表达形式。饮食改变G6PD的表达发生在核转录后步骤：调节前-mRNA的数量。G6PD前mRNA的数量在RNA加工途径的早期就被调节，而不是剪接或核质运输的调节。本申请中描述的研究计划将检验这样一个假设，即饮食多不饱和脂肪通过减少G6PD前体mRNA的处理来抑制G6PD的表达。这一机制涉及反式作用蛋白与前信使核糖核酸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加速降解。在具体目标1中，将确定参与抑制脂肪酸抑制G6PD前-mRNA积累的顺式作用元件。将嵌合构建体瞬时导入原代肝细胞，将在18kb的G6PD初级转录本的所有部分检测这些元件。在特定目的2的实验将表征G6PD前-mRNA的核定位。G6PD前体和成熟mRNA的数量将在核基质、可溶核部分和核膜上进行测量。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的调节表达发生在哪里。在特定的目标3中，将测量特定的降解途径对G6PD前mRNA积累的影响。改变G6PD Pre-mRNA的聚(A)尾的长度和/或增强3‘-5’降解可以减少摄入高多不饱和脂肪的小鼠G6PD Pre-mRNA在细胞核中的积聚。这些实验的结果将为饮食多不饱和脂肪调节基因表达的机制提供新的新信息。

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.