PROJECT SUMMARY/ABSTRACT Alcohol use disorder (AUD) affects about 4.2% (or 14.1 million) of American adults each year and causes substantial morbidity and mortality. While genetic variation can influence an individual's vulnerability to AUD, chronic alcohol consumption can also lead to alcohol tolerance and dependence, but the underlying mechanism is unclear. There is evidence that that chronic alcohol use alters DNA methylation of specific genes, leading to gene expression changes and possibly an increased risk of AUD. RNA methylation is a common post- transcriptional modification, and it responds rapidly to a variety of stimuli and translates stimulatory signals into cellular activity. We hypothesize that alcohol use alters an individual's RNA methylome (or epitranscriptome), resulting in altered expression of genes involved in AUD-related pathways. The objective of this research is to explore messenger RNA (mRNA) methylomic changes in the brain of AUD subjects and analyze the effect of mRNA methylation on mRNA expression and neuronal activity, thus providing evidence for a new gene expression regulatory mechanism of AUD. To achieve this goal, we propose three specific aims. First, we will profile mRNA methylomic and transcriptomic changes in eight regions (amygdala, caudate, cerebellum, hippocampus, nucleus accumbens, prefrontal cortex, putamen, and ventral tegmental area) of postmortem brains of AUD subjects. Second, we will use chronic intermittent ethanol (CIE)-exposed mice as models to verify AUD-associated brain mRNA methylation and expression changes as well as the correlation of ethanol exposure-induced mRNA methylation and expression changes with the escalation of ethanol self-administration in mice. Third, we will employ a novel epitranscriptome editing approach to investigate the effect of AUD- associated mRNA methylation changes on mRNA expression and neuronal activity. We expect to (1) observe AUD-associated mRNA methylation and expression changes in specific (or across) brain regions; (2) verify AUD- associated mRNA methylation and expression changes by mouse modeling; and (3) confirm the functional role of AUD-associated mRNA methylation in regulating mRNA expression and neuronal activity. Our study design is innovative. AUD-associated mRNA methylation changes will be examined at single-base resolution. An integrative study approach (human postmortem brain studies, mouse modeling, and epitranscriptome editing) will be employed. The proposed research will provide evidence about the influence of brain epitranscriptomic changes on AUD risk. Given that mRNA methylation is dynamic and reversible, mRNAs with differential methylation in the brain of AUD subjects could be potential targets for AUD treatment by pharmacologically altering mRNA methylation status.

PROJECT NARRATIVE Chronic alcohol consumption may result in methyl-adenosine modification of brain RNAs, thus altering the stability and expression of brain RNAs involved in reward or addiction-related pathways. This proposed research will identify differentially methylated and expressed messenger RNAs (mRNAs) in postmortem brains of AUD subjects, validate AUD-associated brain mRNA methylation and expression changes by mouse modeling, and confirm the effect of AUD-associated brain mRNA methylation changes on mRNA expression and neuronal activity by an innovative epitranscriptome editing approach. The findings will provide insight into a novel epitranscriptomic mechanism of AUD and facilitate the design of a novel AUD therapeutic strategy through altering the methylation status of specific mRNAs.