mTORC1 regulates microRNA biogenesis in Hela cells

mTOR senses nutrient and energy status to regulate cell survival and metabolism in response to environmental changes. Surprisingly, targeted mutation of Tsc1, a negative regulator of mTORC1, caused a broad reduction in miRNAs due to Drosha degradation. Conversely, targeted mutation of Raptor, an essential component of mTORC 1, increased miRNA biogenesis. mTOR activation increased expression of Mdm2, which is hereby identified as the necessary and sufficient ubiquitin E3 ligase for Drosha. Drosha was induced by nutrient and energy deprivation and conferred resistance to glucose deprivation. Using a high throughput screen of a miRNA library, we identified 4 miRNAs that were necessary and sufficient to protect cells against glucose deprivation-induced apoptosis. These miRNA was regulated by glucose through the mTORC1-MDM2- Drosha axis. Taken together, our data reveal an mTOR-Mdm2-Drosha pathway in mammalian cells that broadly regulates miRNA biogenesis as a response to alteration in cellular environment. Knockdown of DROSHA or TSC1 caused a global decrease in both miRNA and pre-miRNA in Hela stable cell lines. To determine whether TSC1 regulates miRNA accumulation in human cells, we used shRNAs to silence TSC1 in HeLa cells and then compare the general profile of miRNA and pre-miRNA in Hela cells. We also used shRNAs against DROSHA as a positive control.

雷帕霉素靶蛋白（mTOR）可感知营养与能量状态，响应环境变化调控细胞存活与代谢过程。令人意外的是，作为雷帕霉素靶蛋白复合物1（mTORC1）负调控因子的Tsc1靶向突变，会因Drosha降解导致微小RNA（miRNAs）的广泛水平下降。与之相反，作为mTORC1必需组分的Raptor靶向突变，则可提升miRNA的生物合成效率。mTOR激活可上调鼠双微体2（Mdm2）的表达，本研究证实Mdm2是作用于Drosha的必需且充分的泛素E3连接酶。Drosha可被营养与能量剥夺诱导表达，并赋予细胞抵抗葡萄糖剥夺的能力。通过对miRNA文库进行高通量筛选，我们鉴定出4种可必需且充分地保护细胞免受葡萄糖剥夺诱导的细胞凋亡的miRNA。这些miRNA可通过mTORC1-MDM2-Drosha轴响应葡萄糖的调控。综上，本研究数据揭示了哺乳动物细胞内的mTOR-Mdm2-Drosha通路，该通路可广泛调控miRNA生物合成以响应细胞环境的改变。在稳定表达的海拉细胞（HeLa）系中，敲低DROSHA或TSC1会导致miRNA与前体微小RNA（pre-miRNA）的整体水平下降。为探究TSC1是否可调控人类细胞内的miRNA积累，我们通过短发夹RNA（shRNA）在HeLa细胞中沉默TSC1，随后比较HeLa细胞中miRNA与pre-miRNA的整体表达谱。我们同时以靶向DROSHA的shRNA作为阳性对照。