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.

项目摘要 该R21提案的总体目标是确定环境化学化学的程度 暴露会影响最丰富的表面参考标记，N6-甲基 - 腺苷（M6A） 开发脊椎动物胚胎。像DNA一样，RNA进行可逆的化学修饰 潜在影响基因表达。到目前为止的研究表明，m6a修改mRNA和 NCRNA在许多生理过程中起着至关重要的作用，包括胚胎发育， 代谢，中枢神经系统功能和昼夜节律调节。 M6A修饰改变 还与包括癌症在内的许多疾病状态有关。像环境一样多 污染物改变基因表达谱，并对生理过程产生不利影响， 重要的是要了解暴露对基因调控的重要层的影响。我们的 初步结果表明，暴露于二恶英样多氯联苯（PCB）和A 发育过程中，芳基烃受体（AHR）改变了斑马鱼中的M6A模式。提议 研究有两个具体的目标。 AIM 1检验以下假设：多样化的AHR激动剂将 在一组独特的转录本中改变M6A RNA甲基化模式。使用三个不同 与环境相关的二恶英状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）。使用配对端 RNA的测序（RNASEQ），我们将确定M6A甲基化改变基因的影响 表达和mRNA剪接。在AIM 2中，我们将检验一个假设，即一个或多个参与者 M6A RNA甲基化（M6A作者（Mettl3），橡皮擦（FTO）和读取器（YTHDF2））将改变 对AHR激动剂的敏感性。我们将使斑马鱼胚胎从杂合突变体十字接触到 不同浓度的AHR激动剂，并比较响应之间的敏感性 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，阐明了M6A甲基化改变基因表达和 替代性（mRNA）剪接，并表征关键RNA甲基化蛋白在有毒物质中的作用 诱导M6A模式和基因调节的改变。这些结果将构成未来的基础 研究确定RNA甲基化在发育毒性以及 成人健康和疾病的发展基础。