Role of m6A RNA modifications in AHR-mediated developmental toxicity

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.

Grant Number: 1R21ES035153-01
NIH Institute/Center: NIH
Principal Investigator: NEELAKANTESWAR Aluru