title: A genome-wide siRNA screen in mammalian cells for regulators of S6 phosphorylation.
abstract: mTOR complex1, the major regulator of mRNA translation in all eukaryotic cells, is strongly activated in most cancers. We performed a genome-wide RNAi screen in a human cancer cell line, seeking genes that regulate S6 phosphorylation, readout of mTORC1 activity. Applying a stringent selection, we retrieved nearly 600 genes wherein at least two RNAis gave significant reduction in S6-P. This cohort contains known regulators of mTOR complex 1 and is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. We next examined the effect of RNAi pools directed at 534 of these gene products on S6-P in TSC1 null mouse embryo fibroblasts. 76 RNAis reduced S6 phosphorylation significantly in 2 or 3 replicates. Surprisingly, among this cohort of genes the only elements previously associated with the maintenance of mTORC1 activity are two subunits of the vacuolar ATPase and the CUL4 subunit DDB1. RNAi against a second set of 84 targets reduced S6-P in only one of three replicates. However, an indication that this group also bears attention is the presence of rpS6KB1 itself, Rac1 and MAP4K3, a protein kinase that supports amino acid signaling to rpS6KB1. The finding that S6 phosphorylation requires a previously unidentified, functionally diverse cohort of genes that participate in fundamental cellular processes such as mRNA translation, RNA processing, DNA repair and metabolism suggests the operation of feedback pathways in the regulation of mTORC1 operating through novel mechanisms.
authors: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, 02114, United States of America; Diabetes Unit, Medical Services, Massachusetts General Hospital, Boston, MA, 02114, United States of America, 617-726-6909; Department of Medicine, Harvard Medical School, Boston, MA, 02115, United States of America.
authors: Angela
authors: A
authors: Papageorgiou
authors: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, 02114, United States of America; Diabetes Unit, Medical Services, Massachusetts General Hospital, Boston, MA, 02114, United States of America, 617-726-6909; Department of Medicine, Harvard Medical School, Boston, MA, 02115, United States of America.
authors: Joseph
authors: J
authors: Rapley
authors: Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts, 02142, United States of America.
authors: Jill P
authors: JP
authors: Mesirov
authors: Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts, 02142, United States of America.
authors: Pablo
authors: P
authors: Tamayo
authors: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, 02114, United States of America; Diabetes Unit, Medical Services, Massachusetts General Hospital, Boston, MA, 02114, United States of America, 617-726-6909; Department of Medicine, Harvard Medical School, Boston, MA, 02115, United States of America.
authors: Joseph
authors: J
authors: Avruch
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D016678:Genome; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D038601:Ribosomal Protein S6 / Q000378:metabolism*; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; D038601:Ribosomal Protein S6; D012313:RNA; D004247:DNA; C546842:MTOR protein, human; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases