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siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells
Published on: May 24, 2014
A genome-wide siRNA screen in mammalian cells for regulators of S6 phosphorylation
Angela Papageorgiou1, Joseph Rapley1, Jill P Mesirov2
1Department 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.
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.
Insights
A genome-wide screen identified nearly 600 genes regulating mTORC1 activity, crucial for cancer cell growth. Many of these genes are involved in fundamental cellular processes, suggesting novel feedback mechanisms controlling mTORC1 signaling.
Area of Science:
- Molecular Biology
- Cancer Research
- Cellular Signaling
Background:
- Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of mRNA translation, frequently hyperactivated in cancers.
- S6 phosphorylation serves as a reliable readout for mTORC1 activity.
Purpose of the Study:
- To identify novel genes regulating mTORC1 activity through a genome-wide RNA interference (RNAi) screen.
- To investigate the functional diversity of genes involved in mTORC1 pathway regulation.
Main Methods:
- Genome-wide RNAi screen in a human cancer cell line to identify genes affecting S6 phosphorylation.
- Validation of candidate genes in TSC1-deficient mouse embryo fibroblasts.
- Analysis of gene enrichment in cancer cell line proliferation/viability datasets.
Main Results:
- Nearly 600 genes were identified that significantly reduce S6 phosphorylation upon depletion.
- 76 genes consistently reduced S6 phosphorylation in validation experiments.
- Previously unrecognized genes involved in mRNA translation, RNA processing, DNA repair, and metabolism were identified as regulators.
Conclusions:
- The study reveals a large, functionally diverse set of genes critical for maintaining mTORC1 activity.
- These findings suggest the existence of novel feedback pathways regulating mTORC1 through previously uncharacterized mechanisms.
- The identified genes represent potential therapeutic targets for cancer treatment.
