Molecular mechanism of omega-3 response

描述(申请人提供)：omega-3(？3或n-3)长链多不饱和脂肪酸(LCPUFA，例如22：6n-3，20：5n-3)是美国人使用的最受欢迎的膳食补充剂之一。要了解个体间的反应差异，需要阐明潜在的途径和基因类型的影响。LCPUFA的生物合成受到脂肪酸去饱和酶基因簇(FADS1-3，11q12-13.1)编码的去饱和酶活性的限制。FADS、内含子和基因间SNPs在遗传学研究中被不成比例地确定为重要的(例如，GWAs)。近年来，我们发现所有的FADS基因都具有保守性、高表达和系统发育保守的选择性转录本(AT)。新发现的FADS1AT具有去饱和酶功能，新的siRNA数据表明，一种特定的剪接因子PTB(HnRNP I)调节FADS AT的丰度。2012年初，我们培育了第一只Fads3-/-(空)小鼠，以研究这个广泛选择性剪接基因的未知功能。AT代表了在非编码区介导的omega-3代谢控制的一种新的、基本的范例。目前关于忽视AT的调节机制的知识必然是不完整的，因为人类95%的基因产生AT，包括FADS基因。总的目标是通过机制研究来发现FADS AT的调节和功能以及它们的一些遗传控制。假设1：FADS AT受膳食多不饱和脂肪酸和激素通过剪接因子(SF)调节，其方式与增强催化(例如FADS1AT1)或抑制去饱和(例如FADS3 AT)一致。假设2：FADS AT调控通过剪接因子与SNPs和单倍型相关。具体目的1.确定FADS、CS和AT的转录起始位点(TSS)和非编码区。(A)用人肝脏RNA-Seq测定FADS、CS和AT的启动子和全长开放阅读框(ORF)。(B)通过小鼠饮食中多不饱和脂肪酸的比例和人体细胞中激素的比例来确定FADS转录的调节，从而确立AT作为LCPUFA生物合成的中间生物标志物。(C)开发和实施剪接因子的RNAi及其磷酸化的抑制，以研究SF对FADS转录本的调节，以及脂肪酸的去饱和和组成。具体目标2.FADS AT功能的发现(S)与调制.(A)基于RNA-Seq和RNAi结果：(I)转染人细胞以检测特定AT的激活或抑制。(Ii)利用全长开放阅读框架构建人细胞功能研究载体。(B)鉴定Fads3基因缺失小鼠的外显性和分子/生化表型。(C)调查与多不饱和脂肪酸生物合成有关的人类SNPs和单倍型的流行情况。与人类健康相关。了解FADS AT的调节、功能和对LCPUFA生物合成的影响，将有助于从机制上评估LCPUFA生物合成的个体间变异性。将确定对补充omega-3 LCPUFA作出反应的人类遗传变异和代谢状况。

DESCRIPTION (provided by applicant): Omega-3 (?3 or n-3) long chain polyunsaturated fatty acids (LCPUFA, e.g., 22:6n-3, 20:5n-3) are among the most popular dietary supplements used by Americans. Understanding inter-individual response variation requires elucidation of the underlying pathways and the influence of genotypes. LCPUFA biosynthesis is limited by desaturase activity encoded by the fatty acid desaturase gene cluster (FADS1-3, 11q12-13.1). FADS intronic and intergenic SNPs are disproportionately identified as significant in genetic studies (e.g. GWAS). In recent years we showed that all the FADS genes have conserved, highly expressed, and phylogenetically conserved alternative transcripts (AT). A newly described FADS1 AT has desaturase function and new siRNA data show that a specific splicing factor (SF), PTB (hnRNP I) modulates FADS AT abundance. In early 2012, we generated the first Fads3-/- (null) mouse to investigate the as-yet-unknown function of this extensively alternatively spliced gene. AT represent a novel, essential paradigm for omega-3 metabolic control mediated in non-coding regions. Present knowledge of regulatory mechanisms neglecting AT are necessarily incomplete because >95% of human genes produce AT, including the FADS genes. The overall goal is to discover the regulation and function of FADS AT and some of their genetic control via mechanistic investigation. Hypothesis 1: FADS AT are modulated by dietary PUFA and by hormones via splicing factors (SF) in a manner consistent with enhanced catalysis (e.g. FADS1AT1) or inhibition (e.g. FADS3 AT) of desaturation. Hypothesis 2: FADS AT modulation is related to SNPs and haplotypes via splicing factors. Specific Aim 1. Define the transcription start sites (TSS) and UTR of FADS CS and AT. (a) Determine the promoters and full length open reading frames (ORFs) of FADS CS and AT with RNA-Seq using human liver. (b) Determine FADS transcript modulation by ratios of dietary PUFA in mice and by hormones in human cells, thereby establishing AT as intermediate biomarkers for LCPUFA biosynthesis. (c) Develop and implement RNAi of splicing factors and inhibition of their phosphorylation to investigate SF modulation of FADS transcripts, and fatty acid desaturation and composition. Specific Aim 2. Discovery of FADS AT function(s) and modulation. (a) Based on RNA-Seq and RNAi results: (i) Transfect human cells to test for activation or inhibition by specific AT. (ii) Construct vectors for functional studies in human cels, using full length ORF. (b) Characterize the overt and molecular/biochemical phenotype of Fads3 null mice. (c) Investigate the prevalence of human SNPs and haplotypes related to PUFA biosynthesis. Relevance to human health. Understanding of FADS AT regulation, function, and influence on LCPUFA bio- synthesis will enable mechanistic evaluation of interindividual variability in LCPUFA biosynthesis. Human genetic variation and metabolic conditions responsive to omega-3 LCPUFA supplementation will be identified.