Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
Genome Res. 2011 Mar;21(3):433-46. doi: 10.1101/gr.111492.110. Epub 2011 Jan 14.
The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls cell growth in response to nutrient availability and growth factors. TORC1 signaling is hyperactive in cancer, and regulators of TORC1 signaling may represent therapeutic targets for human diseases. To identify novel regulators of TORC1 signaling, we performed a genome-scale RNA interference screen on microarrays of Drosophila melanogaster cells expressing human RPS6, a TORC1 effector whose phosphorylated form we detected by immunofluorescence. Our screen revealed that the TORC1-S6K-RPS6 signaling axis is regulated by many subcellular components, including the Class I vesicle coat (COPI), the spliceosome, the proteasome, the nuclear pore, and the translation initiation machinery. Using additional RNAi reagents, we confirmed 70 novel genes as significant on-target regulators of RPS6 phosphorylation, and we characterized them with extensive secondary assays probing various arms of the TORC1 pathways, identifying functional relationships among those genes. We conclude that cell-based microarrays are a useful platform for genome-scale and secondary screening in Drosophila, revealing regulators that may represent drug targets for cancers and other diseases of deregulated TORC1 signaling.
雷帕霉素复合物 1（TORC1）的进化保守靶标控制着细胞对营养物质可用性和生长因子的生长反应。TORC1 信号在癌症中过度活跃，TORC1 信号的调节剂可能代表人类疾病的治疗靶点。为了鉴定 TORC1 信号的新调节剂，我们在表达人 RPS6 的黑腹果蝇细胞的微阵列上进行了全基因组 RNAi 筛选，RPS6 是 TORC1 效应物，我们通过免疫荧光检测其磷酸化形式。我们的筛选揭示了 TORC1-S6K-RPS6 信号轴受到许多亚细胞成分的调节，包括 I 类囊泡外衣（COPI）、剪接体、蛋白酶体、核孔和翻译起始机制。使用额外的 RNAi 试剂，我们确认了 70 个新基因作为 RPS6 磷酸化的重要靶标调节剂，并通过广泛的二次检测对它们进行了表征，这些检测探测了 TORC1 途径的各个分支，确定了这些基因之间的功能关系。我们得出结论，基于细胞的微阵列是在果蝇中进行全基因组和二次筛选的有用平台，揭示了可能代表 TORC1 信号失调的癌症和其他疾病的药物靶点的调节剂。
