Air pollution-induced Regulation of m6A methylations via ADAR oxidation

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，这一点从机械上讲仍然知之甚少。然而，最近变得明显的是，有几个 环境因子动态调节影响众多生物的化学RNA修饰 流程。我们在这项为期两年的拟议工作中的目标是调查空气污染的假设- 生成的ROS通过影响ADAR1蛋白的活性重新编程A-to-I RNA编辑，并通过这一点 机制，改变m6A和A-to-I RNA修饰的细胞池。这一假设得到了我们的 观察到转录后修饰80G和M6A受环境相关的影响 模型人支气管上皮细胞中易氧化的空气污染混合物的水平。重要的是，其中一个 持续被这些暴露氧化最多的mRNA转录本(即高度富含80G氧化) 编码ADAR1，这是一种诱导A-to-I RNA修饰的蛋白质。ADAR1已被确定为癌基因 在肺癌中，我们也观察到ADAR1蛋白在暴露于 与环境有关的空气污染物。此外，我们对最近发布的CLIP-SEQ数据的分析表明 ADAR1与编码几种甲基转移酶、去甲基酶和 所有已知的调节整个M6A细胞池的辅助蛋白。总的来说，这些初步数据 提示环境诱导的细胞氧化，m6A之间存在强烈的，但尚未被研究的共同调节 甲基化和肌苷(I)修饰，这与肺窘迫的细胞机制有关。 为了解决这个问题，在目标1中，我们建议确定由空气污染引起的A-to-I编辑的重新编程 基于质谱学的人上皮支气管(NHBE)原代细胞ADAR1错配研究 方法(LC-MS/MS)和下一代测序(NGS)方法(ICE-seq)。我们建议量化 8OG的积累对ADAR1转录本的功能影响 转录抑制介导的信使核糖核酸半衰期分析。为了具体测试ADAR1氧化的作用，我们 建议使用一种抗氧化剂进行实验，这种抗氧化剂已被证明可以降低RNA的氧化水平。在目标2中， 我们建议定位空气污染诱导的ADAR1引起的m6A RNA甲基化模式的变化 监管不善。为了评估这一点，我们将测量已知的调节m6A积累的蛋白质水平。 Western blotting分析，并利用m6A免疫检测联用绘制m6A甲基化的细胞模式 在WT和ADAR1基因敲除细胞系中转化为NGS(即miCLIP-seq)。总体而言，我们的研究为 研究环境诱导的80G基因修饰在调节中的潜在机制作用 在PM2.5和亚微米气溶胶的实际浓度和组成的背景下，m6A甲基化 (PM1.0)。这项工作在提出多表位转录分析(例如，80G-seq、m6A-seq和ICE-seq)方面是独一无二的。 SEQ)，这将共同告知最普遍的mRNA转录后修饰的动态。

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.