Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction
© 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...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Proceedings of the National Academy of Sciences
2021
|
| Online Access: | https://hdl.handle.net/1721.1/133370 |
| _version_ | 1826217936377872384 |
|---|---|
| author | 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 |
| author_facet | 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 |
| author_sort | Condon, Kendall J |
| collection | MIT |
| description | © 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. |
| first_indexed | 2024-09-23T17:11:24Z |
| format | Article |
| id | mit-1721.1/133370 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T17:11:24Z |
| publishDate | 2021 |
| publisher | Proceedings of the National Academy of Sciences |
| record_format | dspace |
| spelling | mit-1721.1/1333702021-10-28T03:01:11Z Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction 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 © 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. 2021-10-27T19:52:24Z 2021-10-27T19:52:24Z 2021 2021-07-23T17:50:18Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/133370 en 10.1073/pnas.2022120118 Proceedings of the National Academy of Sciences of the United States of America Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf Proceedings of the National Academy of Sciences PNAS |
| spellingShingle | 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 Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction |
| title | Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction |
| title_full | Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction |
| title_fullStr | Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction |
| title_full_unstemmed | Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction |
| title_short | Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction |
| title_sort | genome wide crispr screens reveal multitiered mechanisms through which mtorc1 senses mitochondrial dysfunction |
| url | https://hdl.handle.net/1721.1/133370 |
| work_keys_str_mv | AT condonkendallj genomewidecrisprscreensrevealmultitieredmechanismsthroughwhichmtorc1sensesmitochondrialdysfunction AT orozcojosem genomewidecrisprscreensrevealmultitieredmechanismsthroughwhichmtorc1sensesmitochondrialdysfunction AT adelmanncharlesh genomewidecrisprscreensrevealmultitieredmechanismsthroughwhichmtorc1sensesmitochondrialdysfunction AT spinellijessicab genomewidecrisprscreensrevealmultitieredmechanismsthroughwhichmtorc1sensesmitochondrialdysfunction AT vanderhelmpimw genomewidecrisprscreensrevealmultitieredmechanismsthroughwhichmtorc1sensesmitochondrialdysfunction AT robertsjustinm genomewidecrisprscreensrevealmultitieredmechanismsthroughwhichmtorc1sensesmitochondrialdysfunction AT kunchoktenzin genomewidecrisprscreensrevealmultitieredmechanismsthroughwhichmtorc1sensesmitochondrialdysfunction AT sabatinidavidm genomewidecrisprscreensrevealmultitieredmechanismsthroughwhichmtorc1sensesmitochondrialdysfunction |