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 new 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 upregulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to make mTORC1 largely resistant to mitochondrial dysfunction. 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.

中文翻译：

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全基因组CRISPR筛选揭示了mTORC1感知线粒体功能障碍的多层机制

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发出信号，从而调节mTORC1的生长控制途径。因为尚未完全鉴定出这些蛋白质的全部互补物，所以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。连同mTORC1途径的几乎所有已知阳性成分一起，我们鉴定了许多影响mTORC1活性的新基因，包括DCAF7，CSNK2B，SRSF2，IRS4，CCDC43和HSD17B10。使用全基因组范围的筛选数据，我们生成了一个包含针对数百个基因的单向导RNA（sgRNA）的集中子库，并对缺乏营养和胁迫响应性mTORC1调节剂的细胞（包括GATOR1，AMPK，GCN2和ATF4）进行了上位筛选。根据这些数据，我们将线粒体功能定位为mTORC1信号的特别重要输入。尽管线粒体向mTORC1发出信号已广为人知，但其机制尚不完全清楚。我们发现激酶AMPK和HRI信号具有变化的动力学，线粒体对mTORC1的困扰，并且HRI通过Sestrin2和Redd1的ATF4依赖性上调起作用。AMPK和HRI的损失都足以使mTORC1对线粒体功能障碍产生很大的抵抗力。综上所述，我们的数据揭示了影响mTORC1途径的基因目录，并阐明了mTORC1感知线粒体功能障碍的多种方式。我们发现激酶AMPK和HRI信号具有变化的动力学，线粒体对mTORC1的困扰，并且HRI通过Sestrin2和Redd1的ATF4依赖性上调起作用。AMPK和HRI的损失都足以使mTORC1对线粒体功能障碍产生很大的抵抗力。综上所述，我们的数据揭示了影响mTORC1途径的基因目录，并阐明了mTORC1感知线粒体功能障碍的多种方式。我们发现激酶AMPK和HRI信号具有变化的动力学，线粒体对mTORC1的困扰，并且HRI通过Sestrin2和Redd1的ATF4依赖性上调起作用。AMPK和HRI的损失都足以使mTORC1对线粒体功能障碍产生很大的抵抗力。综上所述，我们的数据揭示了影响mTORC1途径的基因目录，并阐明了mTORC1感知线粒体功能障碍的多种方式。