Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1–S6K pathway signaling
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 regulat...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Cold Spring Harbor Laboratory Press
2012
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| Online Access: | http://hdl.handle.net/1721.1/74542 https://orcid.org/0000-0002-1446-7256 |
| _version_ | 1811077971270696960 |
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| author | Lindquist, Robert A. Ottina, Kathleen Wheeler, Douglas B. Hsu, Peggy P. Thoreen, Carson C. Guertin, David A. Ali, Siraj M. Sengupta, Shomit Shaul, Yoav D. Lamprecht, Michael R. Madden, Katherine L. Papallo, Adam R. Jones, Thouis R. Sabatini, David M. Carpenter, Anne E. |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Lindquist, Robert A. Ottina, Kathleen Wheeler, Douglas B. Hsu, Peggy P. Thoreen, Carson C. Guertin, David A. Ali, Siraj M. Sengupta, Shomit Shaul, Yoav D. Lamprecht, Michael R. Madden, Katherine L. Papallo, Adam R. Jones, Thouis R. Sabatini, David M. Carpenter, Anne E. |
| author_sort | Lindquist, Robert A. |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T10:51:21Z |
| format | Article |
| id | mit-1721.1/74542 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T10:51:21Z |
| publishDate | 2012 |
| publisher | Cold Spring Harbor Laboratory Press |
| record_format | dspace |
| spelling | mit-1721.1/745422022-09-30T23:30:13Z Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1–S6K pathway signaling Lindquist, Robert A. Ottina, Kathleen Wheeler, Douglas B. Hsu, Peggy P. Thoreen, Carson C. Guertin, David A. Ali, Siraj M. Sengupta, Shomit Shaul, Yoav D. Lamprecht, Michael R. Madden, Katherine L. Papallo, Adam R. Jones, Thouis R. Sabatini, David M. Carpenter, Anne E. Massachusetts Institute of Technology. Department of Biology Whitehead Institute for Biomedical Research Lindquist, Robert A. Ottina, Kathleen Wheeler, Douglas B. Hsu, Peggy P. Thoreen, Carson C. Guertin, David A. Ali, Siraj M. Sengupta, Shomit Shaul, Yoav D. Lamprecht, Michael R. Papallo, Adam R. Jones, Thouis R. Sabatini, David M. Carpenter, Anne E. 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. National Institutes of Health (U.S.) (R01 GM072555) National Institutes of Health (U.S.) (CA103866) National Institutes of Health (U.S.) (R01 GM089652) United States. Dept. of Defense (DOD TSC Research Program (W81XWH-05-1-0318-DS)) LAM Foundation Society for Biomolecular Sciences Society for Biomolecular Sciences Whitehead Institute for Biomedical Research Broad Institute 2012-11-01T16:36:53Z 2012-11-01T16:36:53Z 2011-01 2010-06 Article http://purl.org/eprint/type/JournalArticle 1088-9051 http://hdl.handle.net/1721.1/74542 Lindquist, R. A. et al. “Genome-scale RNAi on Living-cell Microarrays Identifies Novel Regulators of Drosophila Melanogaster TORC1-S6K Pathway Signaling.” Genome Research 21.3 (2011): 433–446. Web. https://orcid.org/0000-0002-1446-7256 en_US http://dx.doi.org/10.1101/gr.111492.110 Genome Research Creative Commons Attribution Non-Commercial http://creativecommons.org/licenses/by-nc/3.0/ application/pdf Cold Spring Harbor Laboratory Press Cold Spring Harbor Laboratory Press |
| spellingShingle | Lindquist, Robert A. Ottina, Kathleen Wheeler, Douglas B. Hsu, Peggy P. Thoreen, Carson C. Guertin, David A. Ali, Siraj M. Sengupta, Shomit Shaul, Yoav D. Lamprecht, Michael R. Madden, Katherine L. Papallo, Adam R. Jones, Thouis R. Sabatini, David M. Carpenter, Anne E. Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1–S6K pathway signaling |
| title | Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1–S6K pathway signaling |
| title_full | Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1–S6K pathway signaling |
| title_fullStr | Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1–S6K pathway signaling |
| title_full_unstemmed | Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1–S6K pathway signaling |
| title_short | Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1–S6K pathway signaling |
| title_sort | genome scale rnai on living cell microarrays identifies novel regulators of drosophila melanogaster torc1 s6k pathway signaling |
| url | http://hdl.handle.net/1721.1/74542 https://orcid.org/0000-0002-1446-7256 |
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