Dynamic modulation of N6-methyladenosine by ionising radiation in human cells
A cell's transcriptome is regulated through the integration of external and internal signals that activate intracellular signal pathways, epigenetic modifications and post-translational changes. Post-transcriptional regulation through RNA methylation has emerged as an important mechanism in cancer development, and informative for diagnosis and treatment.  The most abundant one, N6-methyladenosine (m6A), regulates gene expression in eukaryotes. In the present study m6A RNA modifications have been characterized in response to ionising radiation (IR) exposure in the HT1080 human cell line. Cells were exposed to a dose of 10Gy of X-rays and harvested 1, 2, 10 minutes, 1 and 24 hours after exposure. m6A sites were identified using long read nanopore direct RNA sequencing.  A pipeline was designed using m6Anet to estimate m6A stoichiometries transcriptome-wide, which were then analysed by a beta-binomial regression model with moderated dispersion estimates and independent filtering to detect differentially methylated (DM) sites between treated and control samples. We found that IR modifies m6A sites in a dynamic way, inducing site specific increase of methylation. Remarkably, it peaks within the first minute after exposure, followed by a sharp decrease at 1 hour without returning to baseline, increasing again after 24 hours.  Two transcripts of the nuclear encoded gene UQCR10, a subunit of the respiratory chain protein, sharing the same site presented a stable hypermethylation over time, confirmed by a modified quantitative PCR assay. Moreover, we generated Knockouts (KO) cell lines for 3 key enzymes involved in m6A methylation, a writer, a reader and an eraser namely METTl3, YTHDF2 and FTO, to better understand mechanistically IR driven m6A dynamics. Importantly, all three KOs presented a transcriptome wide decrease in RNA methylation following IR exposure. Last, m6A modifications were also confirmed in human skin biopsies exposed to IR, with the UQCR10 gene site also significantly hypermethylated 24h after a lower 2Gy X-rays dose, suggesting a universal mechanism. To summarise, we provided evidence that IR modulates m6A RNA patterns in a dynamic way. Representing a rapid cellular response to radiation; we identified DM sites on genes involved in bioenergetics, cell signalling/migration and apoptosis pathways. Considering the essential role of m6A in controlling gene expression and physiological activities, this study established the basis for further studies assessing IR driving m6A with potential role in radiation oncology and protection.