Role of m6A RNA modifications in AHR-mediated developmental toxicity

项目摘要 本R21提案的总体目标是确定环境化学品 暴露会影响最丰富的表观转录组标志物，N6-甲基腺苷（m6 A）， 发育中的脊椎动物胚胎RNA，像DNA一样，经历可逆的化学修饰， 可能影响基因表达。迄今为止的研究表明，m6 A修饰的mRNA和 ncRNA在许多生理过程中起关键作用，包括胚胎发育， 代谢、中枢神经系统功能和生物钟调节。m6 A修饰 也与包括癌症在内的许多疾病有关。许多环境 污染物改变基因表达谱并对生理过程具有有害影响， 重要的是要了解暴露对基因调控这一重要层面的影响。我们 初步结果表明，接触二恶英类多氯联苯（PCB）和 芳烃受体（AHR）在发育过程中改变斑马鱼的m6 A模式。拟议 研究有两个具体目标。目的1检验一组不同的AHR激动剂将 在一组独特的转录本中改变m6 A RNA甲基化模式。使用三种不同 环境相关的二恶英类多氯联苯（AHR激动剂），我们将测量剂量依赖性， AHR在介导发育毒性和改变m6 A RNA甲基化中作用的配体特异性 模式.将斑马鱼胚胎暴露于毒物以使用m6 A评估m6 A模式 单个核苷酸分辨交联和免疫沉淀（mi-CLIP）。使用双端 通过RNA测序（RNAseq），我们将确定改变的m6 A甲基化对基因表达的影响。 表达和mRNA剪接。在目标2中，我们将检验一个假设，即一个或多个参与者 m6 A RNA甲基化（m6 A writer（mettl 3）、eraser（fto）和reader（ythdf 2））将改变 对AHR激动剂敏感。我们将把来自异基因突变杂交的斑马鱼胚胎暴露于 不同浓度的AHR激动剂，并比较 基因型这项研究将确定不同的AHR配体对m6 A RNA的影响， 甲基化模式，阐明改变m6 A甲基化对基因表达的影响， 选择性（mRNA）剪接，并表征关键RNA甲基化蛋白在毒物中的作用- 诱导m6 A模式和基因调控的改变。这些结果将为未来的研究奠定基础。 研究确定RNA甲基化在发育毒性中的潜在作用， 成人健康和疾病的发展基础。

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.