Air pollution-induced Regulation of m6A methylations via ADAR oxidation

空气污染诱导通过 ADAR 氧化调节 m6A 甲基化

• 批准号: 10043757
• 负责人: Lydia Maria Contreras
• 金额: $ 24.04万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-11 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10043757
• 关键词: ADAR1; Acute; Address; Adenosine; Aerosols; Affect; Air; Air Pollutants; Air Pollution; Antioxidants; Autoimmunity; Basic Science; Biological; Biological Assay; Biological Process; Cell Line; Cell model; Cell physiology; Cells; Cessation of life; Chemicals; Complex; Coupled; Data; Detection; Diabetes Mellitus; Disease; Distress; Down-Regulation; Environmental Exposure; Environmental Impact; Environmental Pollution; Environmental Risk Factor; Enzymes; Epithelial; Epithelial Cells; Epithelium; Exposure to; Gene Expression; Gene Expression Profile; Genetic Transcription; Goals; Half-Life; Homeostasis; Human; Inosine; Knowledge; Lead; Life Cycle Stages; Lung; Lung diseases; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; Methods; Methylation; Methyltransferase; Modification; Nerve Degeneration; Obesity; Oncogenes; Oxidation-Reduction; Oxidative Stress; Oxides; Ozone; Particulate Matter; Pattern; Post-Transcriptional RNA Processing; Post-Transcriptional Regulation; Prevalence; Procedures; Proteins; Publishing; RNA; RNA Editing; RNA Processing; RNA methylation; Reactive Oxygen Species; Regulation; Reporting; Risk; Role; Site; Testing; Transcript; Viral; Western Blotting; Work; base; bronchial epithelium; cell growth regulation; crosslinking and immunoprecipitation sequencing; differential expression; environmental agent; environmental stressor; epitranscriptome; epitranscriptomics; experimental study; genome-wide; human disease; human model; interest; knock-down; lung Carcinoma; methylation pattern; next generation sequencing; oxidation; particle; protein expression; response; ribosome profiling; submicron; transcriptome; volatile organic compound

Abstract It is known that short-term exposures to major air pollutants increase acute risk of several pulmonary diseases and this remains mechanistically poorly understood. However, it has recently become clear that several environmental agents dynamically regulate chemical RNA modifications that influence numerous biological processes. Our goal in this two-year proposed work is to investigate the hypothesis that air pollution- generated ROS reprograms A-to-I RNA editing by affecting the activity of the ADAR1 protein and, through this mechanism, alters the cellular pool of m6A and A-to-I RNA modifications. This hypothesis is supported by our observations that the post-transcriptional modifications 8OG and m6A are influenced by environmentally relevant levels of oxidation-prone air pollution mixtures in model human bronchial epithelial cells. Importantly, one of the mRNA transcripts that is consistently most oxidized by these exposures (i.e. highly enriched with 8OG oxidations) encodes for ADAR1, a protein that induces A-to-I RNA modifications. ADAR1 has been identified as an oncogene in lung carcinoma; we have also observed decreased ADAR1 protein expression post exposure to environmentally-relevant air pollutants. Furthermore, our analysis of recently published CLIP-seq data suggests that ADAR1 associates with transcripts that encode for several methyltransferases, demethylase enzymes, and accessory proteins that are all known to regulate the overall m6A cellular pool. Overall, these preliminary data suggest a strong, but not yet examined, co-regulation between environmentally induced cellular oxidation, m6A methylations and Inosine (I) modifications that is relevant to cellular mechanisms underlying pulmonary distress. To address this, in Aim 1, we propose to determine the reprogramming of A-to-I edits by air pollution-induced ADAR1 misregulation in normal human epithelial bronchial (NHBE) primary cells using mass spectrometry-based approaches (LC-MS/MS) and next-generation sequencing (NGS) methods (ICE-seq). We propose to quantify the functional effect of 8OG accumulation on the ADAR1 transcript by analyzing the transcript stability using transcription inhibition-mediated mRNA half-life assays. To specifically test the role of ADAR1 oxidation, we propose experiments involving an antioxidant that has been shown to reduce levels of RNA oxidation. In Aim 2, we propose to map changes in m6A RNA methylation patterns caused by air pollution-induced ADAR1 misregulation. To evaluate this, we will measure levels of proteins known to regulate m6A accumulation using Western blotting analysis, and map cellular patterns of m6A methylations using m6A immuno-detection coupled to NGS (i.e., miCLIP-seq) in wt and ADAR1 knockdown cell lines. Overall, our study paves the way for investigating a potential mechanistic role of environmentally-induced 8OG mRNA modifications in the regulation of m6A methylations, in the context of realistic concentrations and composition of PM 2.5 and submicron aerosol (PM1.0). This work is unique in proposing multi-epitranscriptomics analysis (e.g., 8OG-seq, m6A-seq and ICE- seq), that collectively will inform the dynamics of the most prevalent mRNA post-transcriptional modifications.

摘要 众所周知，短期暴露于主要空气污染物会增加几种肺部疾病的急性风险。 疾病，这仍然是机制知之甚少。然而，最近很明显， 环境因子动态调节影响许多生物学特性的化学RNA修饰， 流程.在这项为期两年的拟议工作中，我们的目标是调查空气污染的假设- 产生的ROS通过影响ADAR 1蛋白的活性来重新编程A-to-I RNA编辑， 机制，改变m6 A和A至I RNA修饰的细胞池。这一假设得到了我们的支持。 观察到转录后修饰8 OG和m6 A受到环境相关的影响， 模型人支气管上皮细胞中易氧化空气污染混合物的水平。重要的是， 在这些暴露条件下始终最易氧化的mRNA转录物（即高度富集8 OG氧化） 编码ADAR 1，一种诱导A到I RNA修饰的蛋白质。ADAR 1已被鉴定为癌基因， 在肺癌中，我们也观察到暴露于 与环境有关的空气污染物。此外，我们对最近发表的CLIP-seq数据的分析表明， ADAR 1与编码几种甲基转移酶、脱甲基酶和 辅助蛋白，这些都是已知的调节整个m6 A细胞池。总的来说，这些初步数据 表明环境诱导的细胞氧化，m6 A， 甲基化和肌苷（I）修饰，其与肺窘迫的细胞机制相关。 为了解决这一问题，在目标1中，我们提出通过空气污染诱导的细胞毒性来确定A-to-I编辑的重编程。 基于质谱的正常人上皮支气管（NHBE）原代细胞中ADAR 1的失调 方法（LC-MS/MS）和下一代测序（NGS）方法（ICE-seq）。我们建议量化 通过分析转录物稳定性，使用 转录抑制介导的mRNA半衰期测定。为了具体测试ADAR 1氧化的作用，我们 提出了涉及抗氧化剂的实验，这种抗氧化剂已被证明可以降低RNA氧化水平。在目标2中， 我们建议绘制由空气污染诱导的ADAR 1引起的m6 A RNA甲基化模式的变化， 监管不当为了评估这一点，我们将测量已知调节m6 A积累的蛋白质水平， Western印迹分析，并使用m6 A免疫检测偶联的方法绘制m6 A甲基化的细胞模式 到NGS（即，miCLIP-seq）在wt和ADAR 1敲除细胞系中的作用。总的来说，我们的研究为 研究环境诱导的8 OG mRNA修饰在调节中的潜在机制作用， 在PM 2.5和亚微米气溶胶的实际浓度和组成的背景下， （PM1.0）。这项工作在提出多表位转录组学分析方面是独特的（例如，8OG-seq、m6A-seq和ICE- seq），其共同地将告知最普遍的mRNA转录后修饰的动态。