A Network of Conserved Damage Survival Pathways Revealed by a Genomic RNAi Screen

Abstract

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.

DNA Repair inDrosophila: Mutagens, Models, and Missing Genes

“…Some studies have incorporated RNAi knockdown of DNA repair genes in vivo (e.g., Marek and Bale 2006), and in cells (e.g., Chiolo et al 2011). Ravi et al (2009) conducted an RNAi screen in Kc cells to identify genes required for resistance to MMS. Based on the 307 genes identified, and the 13 different pathways in which they function, these authors constructed an "MMS survival network.…”

Rates and Genomic Consequences of Spontaneous Mutational Events inDrosophila melanogaster

“…In particular, we looked for polymorphisms likely to affect the function of genes that could affect mutation rates. To do this, we examined mutations in genes identified as being important for DNA damage repair (Ravi et al 2009), particularly those mutations resulting in premature termination codons, frameshifts, and deletions of large portions of coding sequence. While we found many genes having different nonsynonymous alleles in the two inbred lines, we did not find any inactivating mutations in genes known to be involved in processes related to G:C/A:T transitions.…”

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

“…We performed a meta-analysis comparing hits between the present study and several other cell-growth-related Drosophila RNAi screens (Kiger et al 2003;Bettencourt-Dias et al 2004;Boutros et al 2004;Bjorklund et al 2006;Friedman and Perrimon 2006;Goshima et al 2007;Sepp et al 2008;Mummery-Widmer et al 2009;Ravi et al 2009;Sims et al 2009;Dekanty et al 2010;Kockel et al 2010). We also examined prior Drosophila genome-scale RNAi screens whose measured phenotypes were not explicitly growthrelated but whose hits included one or more canonical TORC1 pathway members (Agaisse et al 2005;Guo et al 2008;Zhou et al 2008;Zhang et al 2010).…”

“…A screen for regulators of cell survival following DNA damage by methane methylsulfonate (MMS) demonstrated that knockdown of Tor, raptor, S6k, or Pk61C/PDK1 promoted cell death in response to MMS, while knockdown of gig/TSC2 promoted survival (Ravi et al 2009). However, none of the canonical TORC1 pathway genes scored as hits in a prior Drosophila RNAi screen for regulators of cell count or viability under normal growth conditions (Boutros et al 2004).…”