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，如22:6n- 3,20:5 n-3）是美国人最常用的膳食补充剂之一。理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。脂肪酸去饱和酶基因簇（fads1 - 3,11q12 -13.1）编码的去饱和酶活性限制了LCPUFA的生物合成。FADS内含子和基因间snp在遗传研究中被不成比例地鉴定为重要的（例如GWAS）。近年来，我们发现所有的FADS基因都是保守的、高表达的和系统发育保守的替代转录本（AT）。一种新发现的FADS1 AT具有去饱和酶功能，新的siRNA数据表明，一种特殊的剪接因子（SF） PTB （hnRNP I）调节FADS AT的丰度。在2012年初，我们培育了第一只Fads3-/- (null)小鼠来研究这种广泛选择性剪接基因的未知功能。AT代表了非编码区介导的omega-3代谢控制的一种新颖的、必要的范式。目前关于忽略AT的调控机制的知识必然是不完整的，因为大约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来发现FADS AT的调控和功能及其部分遗传控制。假设1:FADS AT由膳食PUFA和激素通过剪接因子（SF）调节，其方式与增强催化（如FADS1AT1）或抑制（如FADS3 AT）去饱和一致。假设2:FADS AT调节通过剪接因子与snp和单倍型相关。具体目标定义FADS CS和AT的转录起始位点（TSS）和UTR。(a)利用人肝脏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PUFA的比例和人类细胞中的激素来确定FADS转录调节，从而确定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，并抑制其磷酸化，以研究SF对FADS转录物的调节，以及脂肪酸的去饱和和组成。具体目标2。FADS AT函数和调制的发现。(a)基于RNA-Seq和RNAi结果：(i)转染人细胞，测试特异性AT的激活或抑制作用。（ii）构建用于人类细胞功能研究的载体，使用全长ORF。(b)表征Fads3缺失小鼠的显性表型和分子/生化表型。(c)调查与多聚脂肪酸生物合成相关的人类snp和单倍型的流行情况。与人类健康有关。了解FADS AT对LCPUFA生物合成的调控、功能和影响，将有助于对LCPUFA生物合成的个体间变异性进行机制评估。人类基因变异和代谢条件对omega-3 LCPUFA补充的反应将被确定。