(790d) Epitranscriptomics of RNA oxidation reveals mechanisms of lung toxicity and disease

Chemical modifications of proteins and nucleic acids play critical roles in the regulation of gene expression. New evidence suggests that environmental stresses may be involved in the misregulation of the functional activities of RNA modifications and pathways during lung distress and disease. Air pollution is the major source of environmental toxicity to the pulmonary cells. Components of air pollution can generate free radicals and perturb redox homeostasis in cells, leading to formation of the prominent RNA oxidative modification 8-oxo-7,8-dihydroguanine (8-oxoG). This mark is predominantly induced in respiratory cells, and it leads to genotoxicity as it preferentially mispairs with adenosine. Given the prevalence of RNA oxidation under environmental challenges, it is critical to identify novel molecular mechanisms involving RNA oxidation toxicity to cellular physiology and disease. To address this, we developed a high-throughput sequencing method to profile 8-oxoG in mRNAs of human bronchial epithelial BEAS-2B cells. Cells were exposed for 1.5 hours to realistic air pollution mixtures derived from the reaction of ozone (100 ppb) with acrolein (100 ppb), methacrolein (97ppb) and α-pinene (44ppb), common organic volatile compounds in the atmosphere. These compounds formed a multi-component gas-phase mixture and submicron secondary organic aerosols (PM 1.0) with concentrations ~ 40 μg/m³. Using this approach, we found 42 transcripts that are consistently oxidized by air pollution. These transcripts belong to key oxidative stress, signaling, and metabolic pathways. Importantly, one of the mRNA transcripts that is more susceptible to oxidation is FDFT1, a protein that catalyzes a regulatory step in cholesterol synthesis and free-radical scavenging. Moreover, we confirmed a ~2-fold (p-value < 0.05) decrease in expression of FDFT1 protein, and a ~1.7-fold (p-value < 0.001) decrease in cellular cholesterol levels. To further validate the involvement of misregulation of cholesterol on cellular toxicity of air pollution, we knocked down FDFT1 using anti-sense siRNA in BEAS-2B cells. We found that FDFT1 knockdown promotes distinct morphological phenotypes typically observed in studies of lung stress and inflammation. Overall, our findings indicate that air pollution influences the formation of 8-oxoG marks in transcripts of epithelial lung cells leading to alterations on cellular function and cell morphology.