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.

抽象的 众所周知，对主要空气污染物的短期暴露会增加几种肺部的急性风险 疾病和这种疾病在机械上仍然了解不足。但是，最近很明显有几个 环境药物动态调节化学RNA修饰，影响众多生物学 过程。我们在这项为期两年的工作中的目标是调查空气污染的假设 - 通过影响ADAR1蛋白的活性并通过此而产生的ROS重编程A-to-I RNA编辑 机理，改变了M6a的细胞池和A-to-I RNA修饰。这个假设得到了我们的支持 观察转录后修改8OG和M6A受环境相关的影响 模型人支气管上皮细胞中容易氧化的空气污染混合物的水平。重要的是，之一 通过这些暴露量最大程度地氧化的mRNA转录本（即高度富集8og氧化） 编码ADAR1，ADAR1是一种诱导A到I RNA修饰的蛋白质。 ADAR1已被确定为癌基因 在肺癌中；我们还观察到暴露于 与环境相关的空气污染物。此外，我们对最近发表的剪辑数据的分析提出了 该ADAR1与编码几种甲基转移酶，脱甲基酶和的转录本相关联 众所周知的辅助蛋白可以调节整体M6A细胞池。总体而言，这些初步数据 建议在环境诱导的细胞氧化之间进行强但尚未检查的共同调节M6A 甲基化和肌苷（i）修饰与肺部遇到的细胞机制相关。 为了解决这个问题，在AIM 1中，我们建议确定通过空气污染引起的A到I编辑的重编程 使用基于质谱的正常人上皮支气管（NHBE）原代细胞中的ADAR1正上调 方法（LC-MS/MS）和下一代测序（NGS）方法（ICE-SEQ）。我们建议量化 8og积累对ADAR1转录本的功能效应，通过使用使用 转录抑制介导的mRNA半衰期测定。为了特别测试ADAR1氧化的作用，我们 提出涉及抗氧化剂的实验，该实验已被证明降低了RNA氧化水平。在AIM 2中， 我们建议绘制由空气污染引起的ADAR1引起的M6A RNA甲基化模式的变化 正直。为了评估这一点，我们将测量已知的蛋白质水平，以调节M6A的积累 蛋白质印迹分析和使用M6A免疫检测的M6A甲基化的细胞模式 在WT和ADAR1敲低细胞系中的NGS（即Miclip-Seq）。总体而言，我们的研究为 研究环境诱导的8og mRNA修饰在调节中的潜在机械作用 M6a甲基化，在PM 2.5和亚微米气溶胶的逼真浓度和组成的背景下 （PM1.0）。这项工作在提出多本文组合分析方面是独一无二的（例如，8og-seq，m6a-seq和Ice- Seq），这将共同告知转录后最普遍的mRNA的动态。