Dysregulation of the Epitranscriptomic Mark m¹A in Ischemic Stroke

Methylation of adenosine at N1 position yields N¹-methyladenosine (m¹A), which is an epitranscriptomic modification that regulates mRNA metabolism. Recent studies showed that altered m¹A methylation promotes acute and chronic neurological diseases. We currently evaluated the effect of focal ischemia on cerebral m¹A 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 m¹A was measured by mass spectrometry and dot blot, and transcriptome-wide m¹A alterations were profiled using antibody-independent m¹A-quant-seq. Expression of the m¹A writers and erasers was estimated by real-time PCR. Ischemia significantly decreased m¹A levels and concomitantly upregulated m¹A demethylase alkB homolog 3 at 24 h of reperfusion compared to sham. Transcriptome-wide profiling showed differential m¹A 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 m¹A epitranscriptomic modification in RNA is highly sensitive to cerebral ischemia.