Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115, USA.
Hum Mol Genet. 2018 May 1;27(9):1654-1663. doi: 10.1093/hmg/ddy073.
Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by germline inactivating mutations of TSC1 or TSC2. In TSC-associated tumors of the brain, heart, skin, kidney and lung, inactivation of both alleles of TSC1 or TSC2 leads to hyperactivation of the mTORC1 pathway. The TSC/mTORC1 pathway is a key regulator of cellular processes related to growth, proliferation and autophagy. We and others have previously found that mTORC1 regulates microRNA biogenesis, but the mechanisms are not fully understood. Microprocessor, a multi-protein complex including the nuclease Drosha, processes the primary miR transcript. Using a dual-luciferase reporter, we found that inhibition of mTORC1 or downregulation of Raptor decreased Microprocessor activity, while loss of TSC2 led to a striking increase (∼5-fold) in Microprocessor activity. To determine the global impact of TSC2 on microRNAs we quantitatively analyzed 752 microRNAs in Tsc2-expressing and Tsc2-deficient cells. Out of 259 microRNAs expressed in both cell lines, 137 were significantly upregulated and 24 were significantly downregulated in Tsc2-deficient cells, consistent with the increased Microprocessor activity. Microprocessor activity is known to be regulated in part by GSK3β. We found that total GSK3β levels were higher in Tsc2-deficient cells, and the increase in Microprocessor activity associated with Tsc2 loss was reversed by three different GSK3β inhibitors. Furthermore, mTOR inhibition increased the levels of phospho-GSK3β (S9), which negatively affects Microprocessor activity. Taken together these data reveal that TSC2 regulates microRNA biogenesis and Microprocessor activity via GSK3β.
结节性硬化症复合征（TSC）是一种常染色体显性遗传疾病，由 TSC1 或 TSC2 的种系失活突变引起。在 TSC 相关的脑、心、皮肤、肾和肺肿瘤中，TSC1 或 TSC2 的两个等位基因失活导致 mTORC1 通路的过度激活。TSC/mTORC1 通路是与生长、增殖和自噬相关的细胞过程的关键调节剂。我们和其他人之前发现 mTORC1 调节 microRNA 的生物发生，但机制尚不完全清楚。Microprocessor 是一种包含核酸酶 Drosha 的多蛋白复合物，加工初级 miR 转录本。使用双荧光素酶报告基因，我们发现抑制 mTORC1 或下调 Raptor 降低了 Microprocessor 的活性，而 TSC2 的缺失导致 Microprocessor 的活性显著增加（约 5 倍）。为了确定 TSC2 对 microRNAs 的全局影响，我们定量分析了 Tsc2 表达和 Tsc2 缺失细胞中的 752 个 microRNAs。在这两种细胞系中表达的 259 个 microRNAs 中，有 137 个在 Tsc2 缺失细胞中显著上调，24 个显著下调，这与 Microprocessor 活性的增加一致。Microprocessor 活性的调节部分受 GSK3β 的影响。我们发现 Tsc2 缺失细胞中的总 GSK3β 水平升高，与 Tsc2 缺失相关的 Microprocessor 活性的增加可被三种不同的 GSK3β 抑制剂逆转。此外，mTOR 抑制增加了磷酸化 GSK3β（S9）的水平，这会负向影响 Microprocessor 的活性。总之，这些数据表明 TSC2 通过 GSK3β 调节 microRNA 的生物发生和 Microprocessor 的活性。
