METTL3 Regulates Liver Homeostasis, Hepatocyte Ploidy, and Circadian Rhythm-Controlled Gene Expression in Mice

N⁶-methyladenosine (m⁶A), the most abundant internal modifier of mRNAs installed by the methyltransferase 13 (METTL3) at the (G/A)(m⁶A)C motif, plays a critical role in the regulation of gene expression. METTL3 is essential for embryonic development, and its dysregulation is linked to various diseases. However, the role of METTL3 in liver biology is largely unknown. In this study, METTL3 function was unraveled in mice depleted of Mettl3 in neonatal livers (Mettl3^fl/fl; Alb-Cre). Liver-specific Mettl3 knockout (M3LKO) mice exhibited global decrease in m⁶A on polyadenylated RNAs and pathologic features associated with nonalcoholic fatty liver disease (eg, hepatocyte ballooning, ductular reaction, microsteatosis, pleomorphic nuclei, DNA damage, foci of altered hepatocytes, focal lobular and portal inflammation, and elevated serum alanine transaminase/alkaline phosphatase levels). Mettl3-depleted hepatocytes were highly proliferative, with decreased numbers of binucleate hepatocytes and increased nuclear polyploidy. M3LKO livers were characterized by reduced m⁶A and expression of several key metabolic transcripts regulated by circadian rhythm and decreased nuclear protein levels of the core clock transcription factors BMAL1 and CLOCK. A significant decrease in total Bmal1 and Clock mRNAs but an increase in their nuclear levels were observed in M3LKO livers, suggesting impaired nuclear export. Consistent with the phenotype, methylated (m⁶A) RNA immunoprecipitation coupled with sequencing and RNA sequencing revealed transcriptome-wide loss of m⁶A markers and alterations in abundance of mRNAs involved in metabolism in M3LKO. Collectively, METTL3 and m⁶A modifications are critical regulators of liver homeostasis and function.