METTL3 is a key regulator of milk synthesis in mammary epithelial cells

The enzyme m⁶ A methyltransferase-like 3 (METTL3) catalyzes N⁶ -methyladenosine (m⁶ A) modification in eukaryotic messenger RNAs (mRNAs). However, the physiological function and molecular mechanism of METTL3 in mammalian cells have not been fully understood. Here we showed that METTL3 was highly expressed in mouse mammary gland of the lactation period. METTL3 was located in the nucleus of bovine mammary epithelial cells (MECs), and methionine (Met) and β-estrodial (E2) upregulated METTL3 protein level. METTL3 knockdown decreased milk protein and fat synthesis, whereas its overexpression had the opposite effects. METTL3 overexpression stimulated mRNA expression and protein phosphorylation of the mechanistic target of rapamycin (mTOR) and mRNA and protein expression of sterol regulatory element binding protein 1 (SREBP1), whereas METTL3 knockdown blocked the stimulatory effects of Met and E2 on these processes. Furthermore, METTL3 overexpression led to increased mRNA m⁶ A methylation of mTOR and SREBP1, whereas METTL3 knockdown suppressed the stimulatory effects of Met and E2 on these processes. The interaction between METTL3 and glycyl-tRNA synthetase (GlyRS) was confirmed by Co-immunoprecipitation and fluorescence resonance energy transfer approaches, and colocalization observation further showed that Met and E2 treatment increased this interaction. GlyRS knockdown abolished METTL3 protein levels upregulated by Met and E2, and METTL3 knockdown markedly decreased the effects of GlyRS overexpression on mTOR expression and phosphorylation and SREBP1 expression. In summary, we demonstrate that METTL3 is a key positive regulator of Met and E2-stimulated and GlyRS-mediated mTOR and SREBP1 signaling pathways and milk protein and fat synthesis in mammary epithelial cells.