Oxidative stress and RNA methylation

氧化应激和 RNA 甲基化

• 批准号: 10569629
• 负责人: Ricardo C Aguiar
• 金额: $ 38.75万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-14 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10569629
• 关键词: Abnormal Cell; Address; Agreement; Alcohol consumption; Antioxidants; Atmosphere; Autoimmunity; B-Cell Lymphomas; B-Lymphocytes; Benign; Biological Process; Chemicals; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Consumption; DNA; Data; Diabetes Mellitus; Dioxygenases; Disease; Embryonic Development; Environmental Exposure; Environmental Risk Factor; Enzymes; Ethanol; Event; Excision; Free Radicals; Gene Expression; Gene Expression Profile; Genetic Models; Growth; Homeostasis; Human; Hypermethylation; Impairment; In Vitro; Isocitrate Dehydrogenase; Knowledge; Link; Malignant - descriptor; Malignant Neoplasms; Mediating; Metabolism; Methyltransferase; Mitochondria; Mixed Function Oxygenases; Modeling; Modification; Nerve Degeneration; Non-Malignant; Obesity; Oncogenic; Oxidation-Reduction; Oxidative Stress; Oxidative Stress Induction; Oxidoreductase; Pathogenicity; Physiological; Physiological Processes; Play; Poison; Property; RNA; RNA methylation; Radiation; Reactive Oxygen Species; Recurrence; Reporting; Role; Secondary to; Signal Pathway; Signal Transduction; Supplementation; System; Testing; Transducers; Ultraviolet Rays; Untranslated RNA; Work; alpha ketoglutarate; cancer cell; cigarette smoking; cofactor; demethylation; epitranscriptome; exposed human population; histone demethylase; human disease; in vitro Model; in vivo; inhibitor; mitochondrial metabolism; mouse model; mutant; novel; pollutant; stem cell function; transcriptome

N6-methyladenosine (m6A) is a prevalent chemical modification of RNA that influences gene expression and cell signaling. The levels of m6A are dynamically regulated by a RNA methyltransferase complex and by the alpha-ketoglutarate (αKG)-dependent RNA demethylases, FTO and ALKBH5. Misregulated RNA methylation, and its attendant effects on the epitranscriptome, has been associated with a host of human diseases, including obesity/diabetes, auto-immunity, neurodegeneration and cancer. Notably, these conditions can all develop in association with environmental factors that influence oxidative stress, e.g., atmospheric pollutants, cigarette smoking, ultraviolet rays, radiation, toxic chemicals, etc., but the putative influence of redox homeostasis on RNA methylation is unknown. To start to address this knowledge gap, we first considered that the activity of the m6A “erasers” FTO and ALKBH5 rely on intact intermediary metabolism, a point that we illustrated with the discovery that accumulation of D-2-hydroxyglurate (D-2-HG) in IDH1/2 mutant cancers inhibits FTO/ALKBH5 and elevates m6A levels. We expanded on these data by showing that loss of D2- or L2- hydroxyglutarate dehydrogenase (D2HGDH, L2HGDH), which convert D- or L-2-HG into αKG, also suppress FTO/ALKBH5 activity and promotes RNA hypermethylation. Importantly, work from our group and others have uncovered a marked interplay between cellular accumulation of 2-HG, intermediary metabolism and redox homeostasis. These observations led us to speculate that high levels of reactive oxygen species (ROS) may broadly regulate the epitranscriptome. To start to test this concept, we exposed human B cells (normal and malignant) to physiologically relevant levels of H202 and ethanol and detected a marked increase in m6A levels. Using CRISPR KO models of FTO and ALKBH5, we preliminarily confirmed our hypothesis that ROS modify RNA methylation by inhibiting the activity of RNA demethylases. Notably, D2HGDH and L2HGDH are NAD+-dependent enzymes, and since ROS elevation consumes NAD+, it is possible that suppression of D2HGDH/L2HGDH play a part in the cross-talk between redox homeostasis and RNA methylation. Here, we will use genetic models in vitro an in vivo to test the overall hypothesis that oxidative stress-mediated disruption of intermediary metabolism modifies the epitranscriptome. More specifically, we postulate that NAD+ consumption secondary to oxidative stress impairs the activity of D2HGDH and L2HGDH, disrupts 2-HG/αKG homeostasis, thus inhibiting FTO/ALKBH5 activity and promoting RNA hypermethylation. In aim 1, we will mechanistically explain how ROS inhibits FTO/ALKBH5 activity and test if NAD+-modulating agents can correct the RNA hypermethylation associated with oxidative stress. In aim 2, using a novel compound mouse model of B-cell lymphoma, we will test the concept that suppression of RNA demethylases is integral to the oncogenic role of ROS. In aim 3, we will define the ROS-driven methylRNA/gene expression signatures and identify the signaling pathways that are deregulated at the intersection of redox imbalance and the epitranscriptome.

N6-甲基腺苷（M6A）是RNA的普遍化学修饰，影响基因表达和 细胞信号传导。 M6a的水平由RNA甲基转移酶复合酶的动态调节，并由 α-酮戊二酸（αkg）依赖性RNA脱甲基酶FTO和ALKBH5。 RNA甲基化不正常， 它的随之而来的对同意组的影响与许多人类疾病有关， 包括肥胖/糖尿病，自身免疫性，神经变性和癌症。值得注意的是，这些条件都可以 与影响氧化应激的环境因素相关的发展，例如大气污染物， 吸烟，紫外线，辐射，有毒化学物质等，但氧化还原的推定影响 RNA甲基化上的稳态尚不清楚。为了开始解决这个知识差距，我们首先考虑 M6A“橡皮” FTO和ALKBH5的活性依赖于完整的中介代谢，这一点是我们 用IDH1/2突变体癌症中D-2-羟基氟化物（D-2-HG）的积累进行了说明 抑制FTO/ALKBH5并提高M6A水平。我们通过显示D2-或L2-的损失来扩展这些数据 将D-或L-2-HG转换为αkg的羟基谷酸脱氢酶（D2HGDH，L2HGDH），也抑制 FTO/ALKBH5活性并促进RNA高甲基化。重要的是，我们小组和其他人的工作有 发现了2-Hg，中间代谢和氧化还原的细胞积累之间的明显相互作用 稳态。这些观察结果使我们推测高水平的活性氧（ROS）可能 广泛调节同源体。为了开始测试这个概念，我们暴露了人类B细胞（正常和 恶性）与H202和乙醇的物理相关水平，并检测到M6A的显着增加 水平。使用FTO和ALKBH5的CRISPR KO模型，我们先确认了ROS的假设 通过抑制RNA脱甲基酶的活性来修饰RNA甲基化。值得注意的是，D2HGDH和L2HGDH是 NAD+依赖性酶，并且由于ROS升高会消耗NAD+，因此有可能抑制 D2HGDH/L2HGDH在氧化还原稳态和RNA甲基化之间的串扰中发挥了作用。在这里，我们 将在体内使用遗传模型来检验氧化应激介导的总体假设 中间新陈代谢的破坏修饰了上映组。更具体地说，我们假设NAD+ 氧化应激继发的消费会损害D2HGDH和L2HGDH的活性，破坏2-Hg/αkg 稳态，从而抑制FTO/ALKBH5活性并促进RNA高甲基化。在AIM 1中，我们将 机械解释ROS如何抑制FTO/ALKBH5活性并测试NAD+调节剂是否可以纠正 与氧化应激相关的RNA高甲基化。在AIM 2中，使用新型的复合鼠标模型 B细胞淋巴瘤，我们将测试以下概念，即RNA脱甲基酶的抑制是致癌性不可或缺的 ROS的角色。在AIM 3中，我们将定义由ROS驱动的甲基NRA/基因表达特征，并确定 在氧化还原不平衡和表面转录组的交点上进行管制的信号通路。