Role of m6A RNA modifications in AHR-mediated developmental toxicity

m6A RNA 修饰在 AHR 介导的发育毒性中的作用

• 批准号: 10647294
• 负责人: NEELAKANTESWAR Aluru
• 金额: $ 45.48万
• 依托单位: WOODS HOLE OCEANOGRAPHIC INSTITUTION
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-10 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10647294
• 关键词: 3' Untranslated Regions; Address; Adult; Affect; Agonist; Alternative Splicing; Aryl Hydrocarbon Receptor; Basic Science; Cell physiology; Central Nervous System; Chemical Exposure; Chemicals; Consensus Sequence; DNA; Dependence; Development; Developmental Process; Dioxins; Disease; Dose; Embryo; Embryonic Development; Environment; Environmental Exposure; Environmental Pollutants; Epigenetic Process; Exons; Exposure to; Future; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Genotype; Goals; Health; Immunoprecipitation; Individual; Life; Ligands; Link; Malignant Neoplasms; Measures; Mediating; Messenger RNA; Metabolism; Methylation; Modification; Molecular; Nervous System Physiology; Nucleotides; Pathway interactions; Pattern; Phenotype; Physiological Processes; Play; Protein Methylation; RNA; RNA Splicing; RNA methylation; Reader; Regulation; Research; Resolution; Role; Site; Specificity; Stimulus; Strategic Planning; Terminator Codon; Testing; Tetrachlorodibenzodioxin; Toxic effect; Transcript; Untranslated RNA; Vertebrates; Zebrafish; aryl hydrocarbon receptor ligand; circadian pacemaker; crosslink; developmental toxicity; diphenyl; environmental chemical; environmental chemical exposure; epigenetic regulation; epigenomics; epitranscriptome; epitranscriptomics; genome-wide; in vivo Model; loss of function; methylation pattern; mutant; response; screening; stressor; toxicant; transcriptome sequencing; vertebrate embryos; zebrafish development

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.

项目概要 该 R21 提案的总体目标是确定环境化学物质的影响程度 暴露影响最丰富的表观转录组标记，N6-甲基-腺苷（m6A） 正在发育的脊椎动物胚胎。 RNA 与 DNA 一样，会经历可逆的化学修饰， 潜在地影响基因表达。迄今为止的研究表明 mRNA 中的 m6A 修饰和 ncRNA 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。改变 m6A 修改 还与包括癌症在内的多种疾病有关。正如许多环保 污染物改变基因表达谱并对生理过程产生有害影响， 了解暴露对这一重要基因调控层的影响非常重要。我们的 初步结果表明，接触二恶英类多氯联苯 (PCB) 和 芳基碳氢化合物受体 (AHR) 在发育过程中会改变斑马鱼的 m6A 模式。拟议的 研究有两个具体目标。目标 1 检验以下假设：不同组的 AHR 激动剂将 改变一组独特转录本中的 m6A RNA 甲基化模式。使用三种不同的 环境相关的二恶英类 PCB（AHR 激动剂），我们将测量剂量依赖性和 AHR 在介导发育毒性和改变 m6A RNA 甲基化中的作用的配体特异性 模式。斑马鱼胚胎将暴露于有毒物质，以使用 m6A 评估 m6A 模式 个体核苷酸分辨率交联和免疫沉淀 (mi-CLIP)。使用双端 RNA 测序 (RNAseq)，我们将确定改变的 m6A 甲基化对基因的影响 表达和 mRNA 剪接。在目标 2 中，我们将检验以下假设： m6A RNA 甲基化（m6A writer (mettl3)、eraser (fto) 和 reader (ythdf2)）将发生改变 对 AHR 激动剂的敏感性。我们将把来自杂合突变体杂交的斑马鱼胚胎暴露于 不同浓度的 AHR 激动剂，并比较不同浓度的反应敏感性 基因型。拟议的研究将确定不同 AHR 配体对 m6A RNA 的影响 甲基化模式，阐明 m6A 甲基化改变对基因表达的影响， 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 m6A 模式和基因调控的改变。这些结果将成为未来的基础 研究确定 RNA 甲基化在发育毒性中的潜在作用以及 成人健康和疾病的发育基础。