Genome-scale RNAi on living-cell microarrays identifies novel regulators ofDrosophila melanogasterTORC1–S6K pathway signaling
Copyright policy )Genome-scale RNAi on living-cell microarrays identifies novel regulators ofDrosophila melanogasterTORC1–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 ofDrosophila melanogastercells 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 inDrosophila, revealing regulators that may represent drug targets for cancers and other diseases of deregulated TORC1 signaling.
- Massachusetts Institute of Technology United States
- Broad Institute United States
- Whitehead Institute for Biomedical Research United States
Ribosomal Protein S6, Genome, Blotting, Western, Fluorescent Antibody Technique, Genomics, Microarray Analysis, Recombinant Proteins, Drosophila melanogaster, Animals, Drosophila Proteins, Humans, Gene Regulatory Networks, RNA Interference, Molecular Targeted Therapy, Phosphorylation, Cells, Cultured, Signal Transduction, Transcription Factors
Ribosomal Protein S6, Genome, Blotting, Western, Fluorescent Antibody Technique, Genomics, Microarray Analysis, Recombinant Proteins, Drosophila melanogaster, Animals, Drosophila Proteins, Humans, Gene Regulatory Networks, RNA Interference, Molecular Targeted Therapy, Phosphorylation, Cells, Cultured, Signal Transduction, Transcription Factors
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