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Grant Abstract

Grant Number: 1R01AT007003-01A1
PI Name: Brenna
Project Title: Molecular mechanism of omega-3 response

Abstract: 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. PUBLIC HEALTH RELEVANCE: Relevance to public health. Omega-3 fatty acids are major over-the-counter supplements and are added to a vast array of food products. Results of randomized controlled trials (RCT) of omega-3 have shown many positive effects but also neutral and a few negative on cardiovascular risk factors and osteoarthritis in adults, visual and neurological development in infants, and psychiatric disorders in adults. These studies develop mechanistic background to evaluate who can benefit from omega-3 supplements and to assist in the interpretation of human RCT results to provide "reason-to-believe" bio- chemical support underpinning disease-related primary and secondary outcomes.

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