Role of m6A RNA modifications in AHR-mediated developmental toxicity

项目摘要 该R21提案的总体目标是确定环境化学物质 暴露影响最丰富的表位转录标记N6-甲基腺苷(M6A) 发育中的脊椎动物胚胎。RNA和DNA一样，经历了可逆的化学修饰，这种修饰可以 潜在地影响基因的表达。到目前为止的研究表明，mRNAs中的m6A修饰和 NcRNAs在许多生理过程中发挥着关键作用，包括胚胎发育， 新陈代谢、中枢神经系统功能和生物钟调节。更改的M6A修改 还与包括癌症在内的许多疾病状态有关。就像许多环境 污染物会改变基因表达谱，并对生理过程产生不利影响， 了解暴露对这一重要的基因调控层的影响是很重要的。我们的 初步结果表明，接触二恶英类多氯联苯(PCBs)和 芳香烃受体(AHR)在发育过程中改变斑马鱼的m6A模式。建议数 研究有两个具体目标。AIM 1测试了一组不同的AHR激动剂将 在一组独特的转录本中改变m6A RNA甲基化模式。使用三种不同的 与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关系和 AHR在调节发育毒性和改变m6A RNA甲基化中的配基特异性 模式。斑马鱼胚胎将暴露在毒物中，以使用m6A评估m6A模式 单核苷酸拆分、交联和免疫沉淀(MI-CLIP)。使用配对端 RNA测序(RNAseq)，我们将确定m6A甲基化改变对基因的影响 表达和信使核糖核酸剪接。在目标2中，我们将测试假设一个或多个玩家 M6A RNA甲基化(m6A写入器(METTL3)、擦除器(FTO)和读取器(Ythdf2))将发生更改 对AHR激动剂的敏感性。我们将把来自杂合突变杂交的斑马鱼胚胎暴露给 不同浓度的AHR激动剂，并比较不同浓度AHR激动剂 基因分型。拟议的研究将确定不同的AHR配体对m6A RNA的影响 甲基化模式，阐明改变的m6A甲基化对基因表达和 选择性(MRNA)剪接，并表征关键的RNA甲基化蛋白在毒物中的作用- 诱导M6A模式的改变和基因调控。这些结果将为今后的工作奠定基础 确定RNA甲基化在发育毒性中的潜在作用的研究以及 成人健康和疾病的发育基础。

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.