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 mRNA modification, N6-methyladenosine (m6A) in the regulation of alcohol drinking and alcohol mediated gene expression. The fat mass and obesity associated gene (FTO) plays a key role in erasing m6A RNA methylation marks. We generated brain m6A hypermethylated mice with selective deletion of FTO in neurons by crossing FTO-floxed mice (FTO-fl/fl) with the synapsin-1-CRE deleter line (Syn1-CRE). Neuronal FTO-deficient mice showed enhanced initial motivation for alcohol, achieved escalated (dependent) drinking more rapidly after repeated cycles of alcohol intoxication and were more susceptible to relapse to heavy drinking following a period of abstinence. Alcohol-naive neuronal FTO-deficient mice exhibited potentiated alcohol-induced anxiolytic and sedative effects and blunted anxiogenic responses, despite unaltered alcohol metabolism. We carried out RNA-Seq of enriched methylated RNA by immunoprecipitation (MeRIP) and RNA-Seq profiling of unenriched RNA to study alcohol's effect on the epitranscriptomic and transcriptional landscape. We observed that a history of alcohol intoxication induces robust RNA m6A hypermethylation in the hippocampus and that FTO-deficiency markedly alters m6A methylation and transcriptional dynamics. GSEA pathway analysis showed that neuronal FTO deficiency induces addiction-relevant pathways and alters monoaminergic and GABAergic neurotransmission, providing a molecular basis for the heightened vulnerability to alcohol abuse. Our findings demonstrate that neuronal m6A RNA methylation is a novel regulator of excessive alcohol drinking and alcohol-dependent gene expression and its dysregulation may contribute to the pathogenesis AUD.