College of Animal Science, Yangtze University, Jingzhou, China.
College of Life Science, Northeast Agricultural University, Harbin, China.
Cell Biol Int. 2022 Mar;46(3):359-369. doi: 10.1002/cbin.11733. Epub 2021 Dec 11.
The enzyme m A methyltransferase-like 3 (METTL3) catalyzes N -methyladenosine (m 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 m 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.
酶 m A 甲基转移酶样 3（METTL3）催化真核信使 RNA（mRNA）中的 N -甲基腺苷（m A）修饰。然而，METTL3 在哺乳动物细胞中的生理功能和分子机制尚未完全理解。在这里，我们发现 METTL3 在哺乳期的小鼠乳腺中高度表达。METTL3 位于牛乳腺上皮细胞（MECs）的细胞核中，蛋氨酸（Met）和β-雌二醇（E2）上调 METTL3 蛋白水平。METTL3 敲低降低了乳蛋白和脂肪的合成，而其过表达则有相反的效果。METTL3 过表达刺激了 mTOR 的机制靶标（mTOR）和固醇调节元件结合蛋白 1（SREBP1）的 mRNA 和蛋白磷酸化，而 METTL3 敲低则阻断了 Met 和 E2 对这些过程的刺激作用。此外，METTL3 过表达导致 mTOR 和 SREBP1 的 mRNA m A 甲基化增加，而 METTL3 敲低抑制了 Met 和 E2 对这些过程的刺激作用。通过免疫共沉淀和荧光共振能量转移方法证实了 METTL3 与甘氨酰-tRNA 合成酶（GlyRS）之间的相互作用，共定位观察进一步表明，Met 和 E2 处理增加了这种相互作用。GlyRS 敲低消除了 Met 和 E2 上调的 METTL3 蛋白水平，而 METTL3 敲低显著降低了 GlyRS 过表达对 mTOR 表达和磷酸化以及 SREBP1 表达的影响。综上所述，我们证明 METTL3 是 Met 和 E2 刺激以及 GlyRS 介导的 mTOR 和 SREBP1 信号通路以及乳腺上皮细胞中乳蛋白和脂肪合成的关键正调控因子。
