It is known that short-term exposures to major air pollutants increase acute risk of several pulmonary diseases and this remains mechanistically poorly understood. However, it has recently become clear that several environmental agents dynamically regulate chemical RNA modifications that influence numerous biological processes. Our goal in this two-year proposed work is to investigate the hypothesis that air pollution- generated ROS reprograms A-to-I RNA editing by affecting the activity of the ADAR1 protein and, through this mechanism, alters the cellular pool of m6A and A-to-I RNA modifications. This hypothesis is supported by our observations that the post-transcriptional modifications 8OG and m6A are influenced by environmentally relevant levels of oxidation-prone air pollution mixtures in model human bronchial epithelial cells. Importantly, one of the mRNA transcripts that is consistently most oxidized by these exposures (i.e. highly enriched with 8OG oxidations) encodes for ADAR1, a protein that induces A-to-I RNA modifications. ADAR1 has been identified as an oncogene in lung carcinoma; we have also observed decreased ADAR1 protein expression post exposure to environmentally-relevant air pollutants. Furthermore, our analysis of recently published CLIP-seq data suggests that ADAR1 associates with transcripts that encode for several methyltransferases, demethylase enzymes, and accessory proteins that are all known to regulate the overall m6A cellular pool. Overall, these preliminary data suggest a strong, but not yet examined, co-regulation between environmentally induced cellular oxidation, m6A methylations and Inosine (I) modifications that is relevant to cellular mechanisms underlying pulmonary distress. To address this, in Aim 1, we propose to determine the reprogramming of A-to-I edits by air pollution-induced ADAR1 misregulation in normal human epithelial bronchial (NHBE) primary cells using mass spectrometry-based approaches (LC-MS/MS) and next-generation sequencing (NGS) methods (ICE-seq). We propose to quantify the functional effect of 8OG accumulation on the ADAR1 transcript by analyzing the transcript stability using transcription inhibition-mediated mRNA half-life assays. To specifically test the role of ADAR1 oxidation, we propose experiments involving an antioxidant that has been shown to reduce levels of RNA oxidation.
In Aim 2, we propose to map changes in m6A RNA methylation patterns caused by air pollution-induced ADAR1 misregulation. To evaluate this, we will measure levels of proteins known to regulate m6A accumulation using Western blotting analysis, and map cellular patterns of m6A methylations using m6A immuno-detection coupled to NGS (i.e., miCLIP-seq) in wt and ADAR1 knockdown cell lines. Overall, our study paves the way for investigating a potential mechanistic role of environmentally-induced 8OG mRNA modifications in the regulation of m6A methylations, in the context of realistic concentrations and composition of PM 2.5 and submicron aerosol (PM1.0). This work is unique in proposing multi-epitranscriptomics analysis (e.g., 8OG-seq, m6A-seq and ICE- seq), that collectively will inform the dynamics of the most prevalent mRNA post-transcriptional modifications.
The association of human diseases to environmental exposures is becoming clearer. Reversible RNA methylations control gene expression, and as such affect cellular differentiation, cellular adaptation, cellular survival and death. We will test the concept that oxidative stress induced by environmental air pollution (i.e. airborne PM2.5), promotes changes in the cellular regulation of RNA methylation, perturbing important patterns of gene expression.
