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 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.
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
Robert A
RA
Lindquist
Kathleen A
KA
Ottina
Douglas B
DB
Wheeler
Peggy P
PP
Hsu
Carson C
CC
Thoreen
David A
DA
Guertin
Siraj M
SM
Ali
Shomit
S
Sengupta
Yoav D
YD
Shaul
Michael R
MR
Lamprecht
Katherine L
KL
Madden
Adam R
AR
Papallo
Thouis R
TR
Jones
David M
DM
Sabatini
Anne E
AE
Carpenter
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D053263:Gene Regulatory Networks; D016678:Genome; D023281:Genomics; D006801:Humans; D046228:Microarray Analysis; D058990:Molecular Targeted Therapy; D010766:Phosphorylation; D034622:RNA Interference; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D038601:Ribosomal Protein S6 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D029721:Drosophila Proteins; D011994:Recombinant Proteins; D038601:Ribosomal Protein S6; D014157:Transcription Factors
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