In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10. Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.

在哺乳动物细胞中，营养物质和生长因子通过一系列上游蛋白质发出信号来调节 mTORC1 生长控制途径。由于这些蛋白质的完整互补体尚未得到系统鉴定，我们开发了一种基于 FACS 的 CRISPR-Cas9 遗传筛选策略，以查明调节 mTORC1 活性的基因。除了 mTORC1 通路中几乎所有已知的阳性成分之外，我们还鉴定了许多影响 mTORC1 活性的基因，包括DCAF7 、 CSNK2B 、 SRSF2 、 IRS4 、 CCDC43和HSD17B10 。利用全基因组筛选数据，我们生成了一个包含针对数百个基因的单向导 RNA (sgRNA) 的重点子文库，并在缺乏营养和应激响应性 mTORC1 调节剂（包括 GATOR1、AMPK、GCN2 和 ATF4）的细胞中进行了上位筛选。 。从这些数据中，我们确定线粒体功能是 mTORC1 信号传导的一个特别重要的输入。虽然线粒体向 mTORC1 发出信号已得到广泛认可，但其机制尚不完全清楚。我们发现激酶 AMPK 和 HRI 信号以不同的动力学、mTORC1 的线粒体窘迫以及 HRI 通过 ATF4 依赖性上调 Sestrin2 和 Redd1 发挥作用。 AMPK 和 HRI 的缺失足以使 mTORC1 信号传导在很大程度上抵抗 ATP 合酶抑制剂寡霉素以及电子传递链抑制剂哌粉菌素和抗霉素诱导的线粒体功能障碍。总而言之，我们的数据揭示了影响 mTORC1 通路的基因目录，并阐明了 mTORC1 感知线粒体功能障碍的多方面方式。