Molecular mechanism of omega-3 response

omega-3反应的分子机制

• 批准号: 8992902
• 负责人: JAMES T. BRENNA
• 金额: $ 38.05万
• 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/8992902
• 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; Gender; Gene Cluster; Genes; Genetic; Genetic Variation; Genetic study; 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; Testing; Textbooks; Transcript; Transcription Initiation Site; Untranslated RNA; Untranslated Regions; Variant; Visual; Woman; Work; base; cardiovascular risk factor; desaturase; dietary supplements; fatty acid biosynthesis; fatty acid supplementation; genome wide association study; neglect; novel; pregnant; primary outcome; promoter; response; secondary outcome; transcriptome; transcriptome sequencing; vector; western diet

DESCRIPTION (provided by applicant): Omega-3 (ω3 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（ω3 或 n-3）长链多不饱和脂肪酸（LCPUFA，例如 22:6n-3、20:5n-3）是美国人最常用的膳食补充剂之一。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 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 SNP 在遗传学研究（例如 GWAS）中被不成比例地认为具有重要意义。近年来，我们发现所有 FADS 基因都具有保守、高表达且系统发育保守的替代转录本 (AT)。新描述的 FADS1 AT 具有去饱和酶功能，新的 siRNA 数据表明特定剪接因子 (SF)、PTB (hnRNP I) 调节 FADS AT 丰度。 2012 年初，我们培育了第一只 Fads3-/-（无效）小鼠，以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且重要的范例。目前关于忽略 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 FADS AT 的调控和功能及其一些遗传控制。假设 1：FADS AT 受膳食 PUFA 和激素通过剪接因子 (SF) 的调节，其调节方式与去饱和的增强催化（例如 FADS1AT1）或抑制（例如 FADS3 AT）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 1. 定义 FADS CS 和 AT 的转录起始位点 (TSS) 和 UTR。 (a) 使用人肝脏通过 RNA-Seq 确定 FADS CS 和 AT 的启动子和全长开放阅读框 (ORF)。 (b) 通过小鼠膳食 PUFA 的比例和人类细胞中的激素确定 FADS 转录调节，从而建立 AT 作为 LCPUFA 生物合成的中间生物标志物。 (c) 开发并实施剪接因子的 RNAi 及其磷酸化抑制，以研究 FADS 转录本的 SF 调节以及脂肪酸去饱和和组成。具体目标 2. 发现 FADS AT 功能和调制。 (a) 基于 RNA-Seq 和 RNAi 结果： (i) 转染人类细胞以测试特定 AT 的激活或抑制。 (ii) 使用全长 ORF 构建用于人类细胞功能研究的载体。 (b) 表征 Fads3 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 和单倍型的流行程度。与人类健康的相关性。了解 FADS AT 调节、功能和对 LCPUFA 生物合成的影响将能够对 LCPUFA 生物合成的个体间差异进行机械评估。将确定对 omega-3 LCPUFA 补充有反应的人类遗传变异和代谢状况。