Oxidative stress and RNA methylation

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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.