Dysregulation of the Epitranscriptomic Mark mA in Ischemic Stroke.

Methylation of adenosine at N1 position yields N-methyladenosine (mA), which is an epitranscriptomic modification that regulates mRNA metabolism. Recent studies showed that altered mA methylation promotes acute and chronic neurological diseases. We currently evaluated the effect of focal ischemia on cerebral mA methylome and its machinery. Adult male C57BL/6J mice were subjected to transient middle cerebral artery occlusion, and the peri-infarct cortex was analyzed at 12 h and 24 h of reperfusion. The bulk abundance of mA was measured by mass spectrometry and dot blot, and transcriptome-wide mA alterations were profiled using antibody-independent mA-quant-seq. Expression of the mA writers and erasers was estimated by real-time PCR. Ischemia significantly decreased mA levels and concomitantly upregulated mA demethylase alkB homolog 3 at 24 h of reperfusion compared to sham. Transcriptome-wide profiling showed differential mA methylation at 14 sites (8 were hypo- and 6 were hypermethylated). Many of those are located in the 3'-UTRs of unannotated transcripts proximal to the genes involved in regulating protein complex assembly, circadian rhythms, chromatin remodeling, and chromosome organization. Using several different approaches, we show for the first time that mA epitranscriptomic modification in RNA is highly sensitive to cerebral ischemia.