Oxidative stress and RNA methylation

N6-甲基腺苷（m6 A）是一种普遍的RNA化学修饰，影响基因表达， 细胞信号m6 A的水平受RNA甲基转移酶复合物和DNA甲基转移酶的动态调节。 α-酮戊二酸（αKG）依赖性RNA脱甲基酶、FTO和ALKBH 5。RNA甲基化失调， 以及其对表转录组的伴随作用，与许多人类疾病有关， 包括肥胖/糖尿病、自身免疫、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有关，例如，大气污染物， 吸烟、紫外线、辐射、有毒化学物质等，但氧化还原的影响 RNA甲基化的稳态是未知的。为了开始解决这一知识差距，我们首先考虑， m6 A“擦除器”FTO和ALKBH 5的活性依赖于完整的中间代谢，这一点我们认为， 发现IDH 1/2突变型癌症中D-2-羟基谷氨酸（D-2-HG）的积累 抑制FTO/ALKBH 5并升高m6 A水平。我们扩展了这些数据，显示D2-或L2- 将D-或L-2-HG转化为αKG的羟基戊二酸脱氢酶（D2 HGDH，L2 HGDH）也抑制 FTO/ALKBH 5活性并促进RNA超甲基化。重要的是，我们小组和其他人的工作 揭示了2-HG的细胞积累，中间代谢和氧化还原之间的显着相互作用 体内平衡这些观察结果使我们推测，高水平的活性氧簇（ROS）可能 广泛地调节着表转录组。为了开始测试这一概念，我们将人类B细胞（正常和 恶性）的H2 O2和乙醇的生理相关水平，并检测到m6 A 程度.使用FTO和ALKBH 5的CRISPR KO模型，我们初步证实了我们的假设，ROS 通过抑制RNA去甲基化酶的活性来修饰RNA甲基化。值得注意的是，D2 HGDH和L2 HGDH是 NAD+依赖性酶，并且由于ROS升高消耗NAD+，因此抑制NAD+是可能的。 D2 HGDH/L2 HGDH在氧化还原稳态和RNA甲基化之间的相互作用中发挥作用。这里我们 将在体外和体内使用遗传模型来测试氧化应激介导的 中间代谢的破坏改变了表转录组。更具体地说，我们假设NAD+ 继发于氧化应激的消耗损害D2 HGDH和L2 HGDH的活性，破坏2-HG/αKG 体内平衡，从而抑制FTO/ALKBH 5活性并促进RNA超甲基化。在目标1中， 机械地解释ROS如何抑制FTO/ALKBH 5活性，并测试NAD+调节剂是否可以纠正 与氧化应激相关的RNA超甲基化。在aim 2中，使用一种新的复合小鼠模型， B细胞淋巴瘤，我们将测试的概念，抑制RNA去甲基化酶是不可或缺的致癌基因， ROS的作用。在目标3中，我们将定义ROS驱动的甲基RNA/基因表达特征，并识别 在氧化还原不平衡和表转录组的交叉点被解除调节的信号传导途径。

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.