mTORC1 regulates microRNA biogenesis in mouse bone marrow hematopoietic stem and progenitor 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. 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 causes a global decrease in both miRNA and pre-miRNA in mouse hematopoietic stem and progenitor cells(HSPCs). Tsc1(flox/flox) mice with conditional alleles for inactivating Raptor in C57BL/6 background were used for this study. Age (10 weeks old) and sex (all female)-matched Ctrl (Mx1-Cre-, 4 mice) and cKO (Mx1-Cre+, 4 mice) littermates were treated with 400 μg polyinosinic:polycytidylic acid (pIpC, 2mg/ml in 1xDPBS, Sigma-Aldrich) 7 times every other day by intraperitoneal injection (i.p.) to induce targeted gene deletion. At 10 days post pIpC treatment, bone marrow HSPCs (Lin-cKit+Sca1+) were purified with BD FACSAria. 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: Tsc1f/f, Mx1-Cre- (Cre negative Ctrl):WT LSK 1, WT LSK 2, WT LSK 3, WT LSK 4 genotype: Tsc1f/f, Mx1-Cre+ (Cre positive cKO): KO LSK 1, KO LSK 2, KO LSK 3, KO LSK 4 biological replicate: WT LSK 1, WT LSK 2, WT LSK 3, WT LSK 4 biological replicate: KO LSK 1, KO LSK 2, KO LSK 3, KO LSK 4