Chronic nitric oxide mediates dual-layer gene regulation through mRNA m⁶A positional remodeling and parallel...

Nitric oxide (NO) is a pleiotropic free radical that functions as a master regulator of gene expression, and its sustained production within the tumor microenvironment reshapes the epitranscriptomic state of cancer cells. We previously demonstrated that NO inhibits the m⁶A mRNA demethylases FTO and ALKBH5 through dinitrosyliron complex formation while leaving the methyltransferase METTL3 intact, a demethylase-specific perturbation that increases global m⁶A on mRNA. Here, integrating m⁶A-RIP-seq and RNA-seq from triple-negative breast cancer cells, we show that chronic NO does not produce the uniform hypermethylation anticipated from demethylase inhibition. Instead, it redistributes m⁶A on mRNA, enriching the 5'UTR and coding sequence while depleting the 3'UTR and departing from the canonical stop-codon and 3'UTR topology. We found that the position of m⁶A, rather than its intensity or mere presence, shapes the outcome, in part by determining which reader protein is predicted to recognize it. In parallel, NO drives a canonical NF-κB and inflammatory transcriptional program. The transcriptional program is independent of the m⁶A methylome in both which genes respond and how strongly they respond, ruling out a linear methylome-to-transcriptome cascade; even so, m⁶A position remains associated with the direction of change among responding transcripts. The 3'UTR is the primary site of m⁶A loss and shows a suggestive computational link to miRNA-mediated regulation. Sense-antisense coordination reinforces the transcriptional response without bridging the two programs. These findings demonstrate that NO not only increases m⁶A abundance, but it also rewrites the m⁶A positional code, establishing spatial reprogramming of the epitranscriptome as a previously unrecognized mode of gene regulation.