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Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction
Name
e2022120118.full.pdf
Description
Published version
Size
1.32 MB
Format
Adobe PDF
Checksum (MD5)
5b1175bee905db04a1f2ab3c95f96612
Author(s)
Condon, Kendall J
Orozco, Jose M
Adelmann, Charles H
Spinelli, Jessica B
van der Helm, Pim W
Roberts, Justin M
Kunchok, Tenzin
Sabatini, David M
Date Issued
2021
Journal
Proceedings of the National Academy of Sciences of the United States of America
Publisher
Proceedings of the National Academy of Sciences
Version
Final published version
Abstract
© 2021 National Academy of Sciences. All rights reserved. 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.
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DOI of Published Version
10.1073/pnas.2022120118
