N6-methyladenosine RNA methylation is a novel epitranscriptomic regulator of excessive alcohol drinking and vulnerability to relapse

While internal RNA modifications have been known for decades, the contribution of epitranscriptomics to alcohol use disorder (AUD) remains unexplored. Here we investigated, for the first time, the role of the most abundant RNA modification, N6-methyladenosine (m ⁶ A) in regulating alcohol drinking and alcohol-induced gene expression. Mice with selective neuronal deletion of the m ⁶ A demethylase fat mass and obesity associated gene ( Fto ) showed enhanced initial motivation for alcohol, achieved escalated drinking more rapidly, and displayed greater relapse-like drinking after abstinence. Alcohol-naive Fto -deficient mice also exhibited potentiated alcohol-induced anxiolysis and sedation and blunted anxiogenic responses, despite unaltered alcohol metabolism. We then performed RNA enrichment coupled with RNA sequencing to characterize alcohol-induced epitranscriptomic and transcriptional remodeling. We observed that a history of alcohol exposure induced robust m ⁶ A hypermethylation in the hippocampus and that Fto -deficiency markedly altered m ⁶ A methylation and transcriptional dynamics. Gene Set Enrichment Analysis (GSEA) indicated that neuronal Fto -deficiency engages addiction-relevant pathways and that transcriptional programs associated with neuronal Fto deletion overlapped with alcohol-induced gene expression signatures, consistent with increased alcohol vulnerability. Our findings demonstrate that neuronal m ⁶ A RNA methylation is a novel regulator of excessive alcohol drinking and alcohol-dependent gene expression, and suggest that dysregulated epitranscriptomic control may contribute to the pathogenesis of AUD.