A network of conserved damage survival pathways revealed by a genomic RNAi screen

Damage initiates a pleiotropic cellular response aimed at cellular survival when appropriate. To identify genes required for damage survival, we used a cell-based RNAi screen against the Drosophila genome and the alkylating agent methyl methanesulphonate (MMS). Similar studies performed in other model organisms report that damage response may involve pleiotropic cellular processes other than the central DNA repair components, yet an intuitive systems level view of the cellular components required for damage survival, their interrelationship, and contextual importance has been lacking. Further, by comparing data from different model organisms, identification of conserved and presumably core survival components should be forthcoming. We identified 307 genes, representing 13 signaling, metabolic, or enzymatic pathways, affecting cellular survival of MMS–induced damage. As expected, the majority of these pathways are involved in DNA repair; however, several pathways with more diverse biological functions were also identified, including the TOR pathway, transcription, translation, proteasome, glutathione synthesis, ATP synthesis, and Notch signaling, and these were equally important in damage survival. Comparison with genomic screen data from Saccharomyces cerevisiae revealed no overlap enrichment of individual genes between the species, but a conservation of the pathways. To demonstrate the functional conservation of pathways, five were tested in Drosophila and mouse cells, with each pathway responding to alkylation damage in both species. Using the protein interactome, a significant level of connectivity was observed between Drosophila MMS survival proteins, suggesting a higher order relationship. This connectivity was dramatically improved by incorporating the components of the 13 identified pathways within the network. Grouping proteins into “pathway nodes” qualitatively improved the interactome organization, revealing a highly organized “MMS survival network.” We conclude that identification of pathways can facilitate comparative biology analysis when direct gene/orthologue comparisons fail. A biologically intuitive, highly interconnected MMS survival network was revealed after we incorporated pathway data in our interactome analysis.
Author Summary Cellular damage is known to elicit a pleiotropic response, but the relative importance of the constituent components in cell survival is poorly understood. To provide an unbiased identification of the proteins utilized in damage survival, we performed an RNAi survival screen in fly cells with methyl methanesulfonate (MMS). The genes identified are involved in 13 biologically diverse pathways. Comparison with analogous yeast data demonstrated a lack of conservation of the individual MMS survival genes but a conservation of pathways. We went on to demonstrate the MMS responsiveness for five representative pathways in both fly and mouse cells. We conclude that identification of pathways can facilitate comparative biology analysis when direct gene/orthologue comparisons fail. Incorporation of pathway data in interactome analysis also improved connectivity and, more importantly, revealed a biologically intuitive, highly inter-connected “MMS survival network.” This pathway conservation and inter-connectivity implies extensive interaction between pathways; for diseases such as cancer, such crosstalk may dictate disparate cellular responses not necessarily expected and confound treatments that are not tailored to the individual molecular context.