Dynamic modulation of N6-methyladenosine by ionizing 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 ionizing radiation (IR) exposure in the HT1080 human cell line. Cells were exposed to a dose of 10 Gy of X-rays and harvested 1, 2, 10 min, 1 and 24 h 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 h without returning to baseline, increasing again after 24 h. 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. Lastly, m⁶A modifications were also confirmed in human skin biopsies exposed to IR, with the UQCR10 gene site also hypermethylated 24 h after a lower 2 Gy X-rays dose. To summarise, we provide evidence that IR modulates RNA m⁶A levels in a site-specific and dynamic way, with DM sites enriched in genes involved in bioenergetics, cell signalling/migration and apoptosis pathways, thus representing a rapid cellular response to radiation. 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 a potential role in radiation oncology and protection.