mTORC1 regulates microRNA biogenesis in mouse embryonic fibroblast 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.In contrast, when the activated mTOR level is repressed by its inhibitor rapamycin, the repression of miRNA is released. 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. mTOR activation by Tsc1 knockout cause repression of both miRNA and pre-miRNA in mouse embryonic fibroblasts (MEFs) Tsc1-/- MEF cells were treated with vehicle or rapamycin (R, 5nM) for 4 days before harvesting, while Tsc1+/+ cells were only treated with vehicle for 4 days. Total RNA isolation was performed with TRIzol (Invitrogen) according to manufacturer’s instructions. RNA labeling and hybridization for miRNA expression profiling on miRNA microarray chips were performed as described (Liu, C.G., Calin, G.A., Volinia, S., and Croce, C.M. (2008). MicroRNA expression profiling using microarrays. Nat Protoc 3, 563-578). genotype: Tsc1+/+: Tsc1+/+_1, Tsc1+/+_2, Tsc1+/+_3, Tsc1+/+_4, Tsc1+/+_5 genotype: Tsc1-/-:Tsc1-/-_1, Tsc1-/-_2, Tsc1-/-_3, Tsc1-/-_4, Tsc1-/-_5, Tsc1-/-_R_1, Tsc1-/-_R_2, Tsc1-/-_R_3, Tsc1-/-_R-4, Tsc1-/-_R_5 biological replicate: Tsc1+/+_1, Tsc1+/+_2, Tsc1+/+_3, Tsc1+/+_4, Tsc1+/+_5 biological replicate: Tsc1-/-_1, Tsc1-/-_2, Tsc1-/-_3, Tsc1-/-_4, Tsc1-/-_5 biological replicate: Tsc1-/-_R_1, Tsc1-/-_R_2, Tsc1-/-_R_3, Tsc1-/-_R_4, Tsc1-/-_R_5