Enesis unknown/nuclear-transcribed 
mRNA catabolic process, nonsensemediated decayCycJ/CG10308 -/CG8086 bru/CG2478 -/CG8108 vnc/CG11989 Smg5/CGE E E E E EE E E E E ETable lists gene name (if applicable) and gene ID of all candidates identified to have a similar effect on polyQ- and Tau-induced REPs. Mode of modification is indicated (enhancement (E), suppression (S)). A brief summary of the molecular and biological functions assigned to the identified gene products is listed. doi:10.1371/SIS-3 site journal.pone.0047452.tDiscussionTo our knowledge, the present screen for modifiers of polyQ toxicity comprises the largest number of genes analyzed in such assays. Usage of the VDRC RNAi library allows large-scale, almost genome-wide screening. However, RNAi-mediated gene silencing approaches might cause off-target effects. Although the VDRC library was designed to limit off-target effects, we are aware that some of our candidates might result from off-target effects. Additionally, RNAi lines used in this screen were generated by random integrations of UAS-RNAi constructs into the fly genome. Consequently, we cannot exclude the possibility that the site of transgene insertion rather than the RNAi effect itself caused the observed modification on the polyQ-induced REP. In our screen, the plethora of individual RNAi lines and the high number of candidates prevented us 
to test for potential off-target and/or genetic background effects. Apart of these drawbacks, using RNAi libraries has certain advantages to screen for modifiers of polyQinduced induced toxicity. For example, previous screens on modifiers of polyQ-induced REPs utilized P-element gene disruption or EP-element-driven overexpression/silencing of genes [18,19,20]. Although these screens provided valuable insights inthe mechanisms of polyQ-induced toxicity, a drawback of P/EPelement-based screens is the limited amount of available elements and the unknown/low number of targeted genes. The expected low number of assayed genes might explain the small overlap of candidates identified by Bilen and (-)-Calyculin A biological activity Bonini [18] with our screen (Figure 4). In addition, we compared our data with selected RNAi screens for modifiers of polyQ aggregation performed in cultured insect cells [34] and in C. elegans [35]. Although the primary readout has been aggregation rather than toxicity, several common candidates were identified in comparison with our screen. To our surprise, the overlap of the two aggregation screens [34,35] was as high as with our screen (Figure 4). In a next step, we grouped overlapping candidate genes according to the reported function of their gene products. Almost all common candidates could be assigned to the following three categories: 1. Protein turnover/quality control (Trp2, DnaJ-1, Hop, Hsc70Cb, Hsc70-4, Pros?, etc); 2. Nuclear import/export (emb, Ntf-2 and CG5738) and 3. mRNA transport/editing/translation (orb, Nelf-E, Prp8, etc). These results suggest that impairment of these processes might contribute to disease. This is in line with previous reports showing a strong involvement of the UPS in polyQ toxicity [14,36,37,38,39,40]. In addition, network analysis of our candiModifiers of Polyglutamine ToxicityFigure 2. Analysis of polyQ aggregate load. (A) Exemplified filter retardation analysis to visualize polyQ aggregates. Decreasing amounts of loaded protein derived from fly heads of control (GMR-GAL4, top), GMR.polyQ (middle) or GMR.polyQ in combination with a candidate suppressor (bottom). (B).Enesis unknown/nuclear-transcribed mRNA catabolic process, nonsensemediated decayCycJ/CG10308 -/CG8086 bru/CG2478 -/CG8108 vnc/CG11989 Smg5/CGE E E E E EE E E E E ETable lists gene name (if applicable) and gene ID of all candidates identified to have a similar effect on polyQ- and Tau-induced REPs. Mode of modification is indicated (enhancement (E), suppression (S)). A brief summary of the molecular and biological functions assigned to the identified gene products is listed. doi:10.1371/journal.pone.0047452.tDiscussionTo our knowledge, the present screen for modifiers of polyQ toxicity comprises the largest number of genes analyzed in such assays. Usage of the VDRC RNAi library allows large-scale, almost genome-wide screening. However, RNAi-mediated gene silencing approaches might cause off-target effects. Although the VDRC library was designed to limit off-target effects, we are aware that some of our candidates might result from off-target effects. Additionally, RNAi lines used in this screen were generated by random integrations of UAS-RNAi constructs into the fly genome. Consequently, we cannot exclude the possibility that the site of transgene insertion rather than the RNAi effect itself caused the observed modification on the polyQ-induced REP. In our screen, the plethora of individual RNAi lines and the high number of candidates prevented us to test for potential off-target and/or genetic background effects. Apart of these drawbacks, using RNAi libraries has certain advantages to screen for modifiers of polyQinduced induced toxicity. For example, previous screens on modifiers of polyQ-induced REPs utilized P-element gene disruption or EP-element-driven overexpression/silencing of genes [18,19,20]. Although these screens provided valuable insights inthe mechanisms of polyQ-induced toxicity, a drawback of P/EPelement-based screens is the limited amount of available elements and the unknown/low number of targeted genes. The expected low number of assayed genes might explain the small overlap of candidates identified by Bilen and Bonini [18] with our screen (Figure 4). In addition, we compared our data with selected RNAi screens for modifiers of polyQ aggregation performed in cultured insect cells [34] and in C. elegans [35]. Although the primary readout has been aggregation rather than toxicity, several common candidates were identified in comparison with our screen. To our surprise, the overlap of the two aggregation screens [34,35] was as high as with our screen (Figure 4). In a next step, we grouped overlapping candidate genes according to the reported function of their gene products. Almost all common candidates could be assigned to the following three categories: 1. Protein turnover/quality control (Trp2, DnaJ-1, Hop, Hsc70Cb, Hsc70-4, Pros?, etc); 2. Nuclear import/export (emb, Ntf-2 and CG5738) and 3. mRNA transport/editing/translation (orb, Nelf-E, Prp8, etc). These results suggest that impairment of these processes might contribute to disease. This is in line with previous reports showing a strong involvement of the UPS in polyQ toxicity [14,36,37,38,39,40]. In addition, network analysis of our candiModifiers of Polyglutamine ToxicityFigure 2. Analysis of polyQ aggregate load. (A) Exemplified filter retardation analysis to visualize polyQ aggregates. Decreasing amounts of loaded protein derived from fly heads of control (GMR-GAL4, top), GMR.polyQ (middle) or GMR.polyQ in combination with a candidate suppressor (bottom). (B).
