Bishop1,3* Abstract A Network of Conserved Damage Survival Pathways Revealed by a Genomic RNAi Screen Dashnamoorthy Ravi1, Amy M. Wiles1, Selvaraj Bhavani1, Jianhua Ruan 2 , Philip Leder3, Alexander J. R. 1 Greehey Children’s Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America, 2 Department of Computer Science, University of Texas at San Antonio, San Antonio, Texas, United States of America, 3 Harvard Medical School, Department of Genetics, Harvard University, Boston, Massachusetts, United States of America 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. Citation: Ravi D, Wiles AM, Bhavani S, Ruan J, Leder P, et al. (2009) A Network of Conserved Damage Survival Pathways Revealed by a Genomic RNAi Screen. PLoS Genet 5(6): e1000527. doi:10.1371/journal.pgen.1000527 Editor: Amy Kiger, University of California San Diego, United States of America Received January 5, 2009; Accepted May 19, 2009; Published June 19, 2009 Copyright: 2009 Ravi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the NIEHS (K22-ES12264; AJRB). JR was supported in part by a NIH grant 1SC3GM086305-01. We thank the Drosophila RNAi Screening Center (http://flyrnai.org/), which is supported by NIGMS (2R01-GM067761-05). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: bishopa@uthscsa.edu Introduction Cellular damage is a normal component of life, with constant damage exposure from both endogenous and exogenous sources. Damage to DNA is considered to be the most biologically relevant lesion with the potential of mutagenic results, though most exogenous agents have the potential to damage many components of the cell. Responding appropriately to such insults, either mitigating cellular toxicity or initiating an appropriate cell death response, is critical, particularly in multi-cellular organisms. Inappropriate responses may facilitate deleterious effects, such as a destabilized genome and diseases such as cancer [1]. As such, DNA damage response (DDR) components are critical suppressors of deleterious effects of genotoxic agents by controlling cell cycle progression, DNA repair, and apoptosis [2]. For this reason, there have been many investigations using a variety of model organisms to identify components of DDR and subsequently to determine how they function and the consequences of their dysfunction. Recent reports suggest that DDR may involve pleiotropic cellular processes other than the central DDR components [3,4], yet an intuitive systems level view of the cellular components required for damage survival, their interrelationship, and their contextual importance has been lacking. The most comprehensive attempt at understanding the interrelationship of damage response and survival components in yeast at a systems level has been mapping identified genes to a network by integrating the general biological processes as distinct modules [5]. It has been suggested that the inclusion of well-defined biological pathways in protein networks might provide a better understanding of biological interactions therein [6,7]. In order to identify genes involved in damage response in an unbiased manner and to put them in a functional context, we used an RNAi library [8] to knock-down every predicted protein in the Drosophila melanogaster genome and assessed whether knock-down of PLoS Genetics | www.plosgenetics.org 1 June 2009 | Volume 5 | Issue 6 | e1000527