Abstract
(6)-methyladenosine (m(6)A) RNA methylation, regulated by writer, eraser, and reader proteins, modulates mRNA stability, splicing, and translation, thereby influencing key cellular processes. Environmental stressors, such as alcohol, may disrupt this epitranscriptomic machinery and contribute to disease vulnerability. In this study, we investigated how chronic exposure to ethanol, its toxic metabolite acetaldehyde, and subsequent withdrawal affect the expression of m(6)A regulatory genes. Neuron-like (SH-SY5Y) and non-neuronal (SW620) cells were exposed for 3 weeks to ethanol (40 mM) or acetaldehyde (30 μM) (concentrations comparable to blood levels after heavy drinking), followed by a 24-h withdrawal period. Gene expression of seven writers (KIAA1429, METTL3, METTL4, METTL14, RBM15, RBM15B, and WTAP), two erasers (ALKBH5, FTO), and nine readers (YTHDF1/2/3, YTHDC1/2, IGF2BP1/2/3, and HNRNPA2B1) was quantified by RT-qPCR. Concurrently, RNA-seq data from eight reward-related brain regions of 24 individuals of European ancestry (12 with alcohol use disorder [AUD] and 12 controls) were analyzed for AUD-associated expression changes in m(6)A regulatory genes. In cell models, ethanol broadly suppressed the expression of most m(6)A regulatory genes, whereas withdrawal largely restored their levels. Acetaldehyde induced subtler gene expression changes, likely reflecting its lower exposure concentration and rapid metabolism. Postmortem brain analysis revealed trends toward altered expression of m(6)A regulatory genes across multiple brain regions in individuals with AUD. Collectively, these findings suggest that chronic alcohol exposure dysregulates m(6)A regulatory gene expression and may impact downstream RNA regulatory pathways involved in AUD pathophysiology. Further studies are warranted to elucidate the mechanisms by which alcohol-induced dysregulation of m(6)A regulators influences AUD risk.