Cao Zubing, Zhang Ling, Hong Renyun, Li Yunsheng, Wang Yiqing, Qi Xin, Ning Wei, Gao Di, Xu Tengteng, Ma Yangyang, Yu Tong, Knott Jason G, Sathanawongs Anucha, Zhang Yunhai
Anhui Province Key Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, China.
Department of Reproductive Medicine, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, China.
Biol Reprod. 2021 May 7;104(5):1008-1021. doi: 10.1093/biolre/ioab022.
N6-methyladenosine (m6A) catalyzed by METTL3 regulates the maternal-to-zygotic transition in zebrafish and mice. However, the role and mechanism of METTL3-mediated m6A methylation in blastocyst development remains unclear. Here, we show that METTL3-mediated m6A methylation sustains porcine blastocyst development via negatively modulating autophagy. We found that reduced m6A levels triggered by METTL3 knockdown caused embryonic arrest during morula-blastocyst transition and developmental defects in trophectoderm cells. Intriguingly, overexpression of METTL3 in early embryos resulted in increased m6A levels and these embryos phenocopied METTL3 knockdown embryos. Mechanistically, METTL3 knockdown or overexpression resulted in a significant increase or decrease in expression of ATG5 (a key regulator of autophagy) and LC3 (an autophagy marker) in blastocysts, respectively. m6A modification of ATG5 mRNA mainly occurs at 3'UTR, and METTL3 knockdown enhanced ATG5 mRNA stability, suggesting that METTL3 negatively regulated autophagy in an m6A dependent manner. Furthermore, single-cell qPCR revealed that METTL3 knockdown only increased expression of LC3 and ATG5 in trophectoderm cells, indicating preferential inhibitory effects of METTL3 on autophagy activity in the trophectoderm lineage. Importantly, autophagy restoration by 3MA (an autophagy inhibitor) treatment partially rescued developmental defects of METTL3 knockdown blastocysts. Taken together, these results demonstrate that METTL3-mediated m6A methylation negatively modulates autophagy to support blastocyst development.
由METTL3催化的N6-甲基腺苷（m6A）调节斑马鱼和小鼠的母源-合子转变。然而，METTL3介导的m6A甲基化在囊胚发育中的作用和机制仍不清楚。在此，我们表明METTL3介导的m6A甲基化通过负向调节自噬来维持猪囊胚的发育。我们发现，METTL3敲低引发的m6A水平降低导致桑葚胚-囊胚转变期间胚胎停滞以及滋养外胚层细胞发育缺陷。有趣的是，早期胚胎中METTL3的过表达导致m6A水平升高，且这些胚胎表现出与METTL3敲低胚胎相似的表型。从机制上讲，METTL3敲低或过表达分别导致囊胚中自噬关键调节因子ATG5和自噬标志物LC3的表达显著增加或减少。ATG5 mRNA的m6A修饰主要发生在3'UTR，且METTL3敲低增强了ATG5 mRNA的稳定性，这表明METTL3以m6A依赖的方式负向调节自噬。此外，单细胞qPCR显示，METTL3敲低仅增加了滋养外胚层细胞中LC3和ATG5的表达，表明METTL3对滋养外胚层谱系中的自噬活性具有优先抑制作用。重要的是，用3MA（一种自噬抑制剂）处理恢复自噬可部分挽救METTL3敲低囊胚的发育缺陷。综上所述，这些结果表明METTL3介导的m6A甲基化通过负向调节自噬来支持囊胚发育。
