Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1–S6K pathway signaling

Abstract

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.

Targeting the Deregulated Spliceosome Core Machinery in Cancer Cells Triggers mTOR Blockade and Autophagy

“…Consistent with this observation, we have shown here that targeting SNRPE or SNRPD1 leads to cell death through autophagy. Interestingly, a recent report using a short hairpin RNA screening procedure in Drosophila melanogaster identified the spliceosome machinery as one of the main pathway regulating the mTORC1 signaling pathway (34). This observation, together with our study, highlights the importance of the spliceosome-mTOR axis and reinforces the interest of the core splicecosome as an interesting target in the control of eukaryotic cell growth and death.…”

SEACing the GAP that nEGOCiates TORC1 activation

“…For instance, compromised nuclear pore function or secretion may tie up or jam Seh1 and/or Sec13, thereby causing reduced SEAC assembly and, consequently, downregulation of TORC1. Interestingly, a genome-scale RNA interference screen by dsRNA reverse-transfection on living Drosophila cell microarrays identified nuclear pore components as TORC1 regulators 32 . In a similar vein, alterations in the yeast secretory pathway have also been found to converge on TORC1 regulation.…”

Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi

“…Second, to ensure that mutant cell lines do not revert to wild type, a method is required to grow cultures from individual cells, a historically difficult problem with Drosophila cells. Various methods for this problem have been proposed (32–34), but none have been widely used because of either difficulty in identifying single cell–derived cultures or very low efficiencies. To substitute for paracrine factors that promote the survival of individual Drosophila cells cultured in populations, we tested whether the use of culture media preconditioned with wild-type S2R+ cells would allow the efficient growth of individual S2R+ cells isolated by flow cytometry.…”