Role of m6A RNA modifications in AHR-mediated developmental toxicity

点击翻译按钮获取中文摘要

Project Summary The overall objective of this R21 proposal is to determine the extent to which environmental chemical exposures affect the most abundant epitranscriptomic mark, N6-methyl-adenosine (m6A) in developing vertebrate embryos. RNA, like DNA, undergo reversible chemical modifications that can potentially influence gene expression. Research so far indicates that m6A modification in mRNAs and ncRNAs plays a critical role in a number of physiological processes including embryonic development, metabolism, central nervous system function and circadian clock regulation. Altered m6A modification has also been linked to a number of disease states including cancer. As many environmental contaminants alter gene expression profiles and have detrimental effects on physiological processes, it is important to understand the effects of exposure on this important layer of gene regulation. Our preliminary results demonstrated that exposure to a dioxin-like polychlorinated biphenyl (PCB) and an aryl hydrocarbon receptor (AHR) during development alter m6A patterns in zebrafish. The proposed research has two specific aims. Aim 1 tests the hypothesis that a diverse group of AHR agonists will alter m6A RNA methylation patterns in a unique set of transcripts. Using three different environmentally relevant dioxin-like PCBs (AHR agonists), we will measure the dose-dependence and ligand-specificity of AHR's role in mediating developmental toxicity and altered m6A RNA methylation patterns. Zebrafish embryos will be exposed to toxicants to evaluate the m6A patterns using m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (mi-CLIP). Using paired-end sequencing of RNA (RNAseq), we will determine the impact of altered m6A methylation on gene expression and mRNA splicing. In Aim 2, we will test the hypothesis that one or more of the players in m6A RNA methylation (m6A writer (mettl3), eraser (fto) and reader (ythdf2)) will have altered sensitivity to AHR agonists. We will expose zebrafish embryos from heterozgyous mutant crosses to different concentrations of an AHR agonist, and compare the sensitivity in responses between genotypes. The proposed research will establish the effects of diverse AHR ligands on m6A RNA methylation patterns, elucidate the impact of altered m6A methylation on gene expression and alternative (mRNA) splicing, and characterize the role of key RNA methylation proteins in toxicant- induced alteration of m6A patterns and gene regulation. These results will form the basis for future studies determining the potential roles of RNA methylation in developmental toxicity as well as developmental basis of adult health and disease.