Molecular mechanism of omega-3 response

omega-3反应的分子机制

• 批准号: 8433842
• 负责人: JAMES T. BRENNA
• 金额: $ 37.83万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8433842
• 关键词: 11q12; 5' Untranslated Regions; Adult; American; Anabolism; Arachidonic Acids; Biochemical; Biological Markers; Cardiovascular system; Catalysis; Cells; Chemicals; Complex; DNA; Data; Degenerative polyarthritis; Development; Diabetes Mellitus; Diet; Disease; Docosahexaenoic Acids; Dominant-Negative Mutation; Eicosanoid Production; Eicosapentaenoic Acid; Euphausiacea; Evaluation; Fatty Acid Desaturases; Fatty Acids; Fishes; Food; Functional RNA; Gender; Gene Cluster; Gene Expression Profile; Genes; Genetic; Genetic Variation; Genotype; Goals; Growth and Development function; Haplotypes; Health; Heart; Heterogeneous-Nuclear Ribonucleoproteins; Hormonal; Hormones; Human; Human Genetics; Individual; Industry; Infant; Intake; Intercistronic Region; Investigation; Knockout Mice; Knowledge; Lactation; Learning; Length; Link; Linolenic Acids; Liver; MCF7 cell; Mediating; Mental disorders; Metabolic; Metabolic Control; Metabolic syndrome; Molecular; Mus; Neurologic; Nutrient; Oils; Omega-3 Fatty Acids; Open Reading Frames; Papio; Pathway interactions; Phenotype; Phosphorylation; Polypyrimidine Tract-Binding Protein; Polyunsaturated Fatty Acids; Positioning Attribute; Pregnancy; Prevalence; Process; Protein Isoforms; Public Health; RNA; RNA Interference; RNA Splicing; Randomized Controlled Trials; Regulation; Reporting; Role; Shotgun Sequencing; Small Interfering RNA; Spliced Genes; Supplementation; Testing; Textbooks; Transcript; Transcription Initiation Site; Untranslated Regions; Variant; Visual; Woman; Work; base; cardiovascular risk factor; desaturase; dietary supplements; fatty acid biosynthesis; genome wide association study; neglect; novel; pregnant; primary outcome; promoter; public health relevance; response; secondary outcome; transcriptome sequencing; vector

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.

描述（由申请人提供）：Omega-3（？3或n-3）长链多不饱和脂肪酸（LCPUFA，例如，22：6 n-3，20：5 n-3）是美国人使用的最受欢迎的膳食补充剂之一。了解个体间的反应差异需要阐明潜在的途径和基因型的影响。LCPUFA的生物合成受到脂肪酸去饱和酶基因簇（FADS 1 -3，11 q12 -13.1）编码的去饱和酶活性的限制。FADS内含子和基因间SNP在遗传研究中被不成比例地鉴定为显著的（例如GWAS）。近年来，我们发现所有的FADS基因都具有保守的、高表达的和遗传上保守的替代转录物（AT）。新描述的FADS 1 AT具有去饱和酶功能，新的siRNA数据显示特异性剪接因子（SF）PTB（hnRNP I）调节FADS 1 AT丰度。在2012年初，我们产生了第一只Fads 3-/-（null）小鼠，以研究这种广泛可变剪接基因的未知功能。AT代表非编码区介导的ω-3代谢控制的一种新的、必要的范例。目前忽视AT的调控机制的知识必然是不完整的，因为>95%的人类基因产生AT，包括FADS基因。总体目标是通过机制研究发现FADSAT的调节和功能以及它们的一些遗传控制。假设1：FADS AT由膳食PUFA和激素通过剪接因子（SF）以与去饱和的增强催化（例如FADS 1AT 1）或抑制（例如FADS 3AT）一致的方式调节。假设2：FADSAT调节通过剪接因子与SNP和单倍型相关。具体目标1.定义FADS CS和AT的转录起始位点（TSS）和UTR。(a)利用RNA-Seq技术对人肝FADS CS和AT的启动子和全长开放阅读框（ORF）进行了检测。(b)通过小鼠中饮食PUFA的比例和人类细胞中激素的比例确定FADS转录调节，从而将AT确定为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及其磷酸化抑制，以研究FADS转录物的SF调节，以及脂肪酸去饱和和组成。具体目标2。发现FADS AT功能和调节。(a)基于RNA-Seq和RNAi结果：（i）转染人细胞以测试特异性AT的激活或抑制。(ii)利用全长ORF构建载体用于人类乳腺癌的功能研究。(b)表征Fads 3缺失小鼠的明显和分子/生化表型。(c)调查与PUFA生物合成相关的人类SNP和单倍型的流行率。与人类健康的相关性。了解FADS AT的调节、功能和对LCPUFA生物合成的影响将有助于对LCPUFA生物合成中的个体间差异进行机制评估。将确定对omega-3 LCPUFA补充剂有反应的人类遗传变异和代谢状况。