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 (?KG)-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 ?KG, 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/?KG 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.

Public Health Relevance

Environmental exposures are associated with a host of human diseases. Reversible methylation of RNA controls gene expression, modulates physiologic processes and it is deregulated in benign and malignant disorders. We will test the concept that oxidative stress, by disrupting intermediary metabolism inhibits RNA demethylases, promotes RNA hypermethylation and perturbs gene expression and cell signaling.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES031522-02
Application #
10144440
Study Section
Special Emphasis Panel (ZES1)
Program Officer
Tyson, Frederick L
Project Start
2020-04-14
Project End
2025-01-31
Budget Start
2021-02-01
Budget End
2022-01-31
Support Year
2
Fiscal Year
2021
Total Cost
Indirect Cost
Name
University of Texas Health Science Center
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229