Fig. 2

A screen of translation factors reveals those important for expression of GR from (G4C2)_EXP. a. To identify canonical translation factors that may be involved in RAN translation of G4C2 in the GR reading frame, a loss-of-function (LOF) based screen was designed utilizing previously developed RNAi [57, 60] or LOF mutant fly lines [6, 7, 80, 81] targeting 48 of 56 (86%) known translation factors [50]. Individual translation factors were downregulated in animals expressing LDS-(G4C2)_EXP and any that altered the external eye phenotype and/or GR-GFP levels were defined (Step 1). These 28 LOF lines were further tested in (GR)₃₆ expressing animals, defining 6 that acted similarly on GR-associated toxicity (Step 2). Additional quality control experiments excluded LOF lines that altered expression from a control (LacZ) transgene by western immunoblot and/or altered a WT eye morphology when expressed alone, as described [13, 26, 41] (Step 3). b. Summary of screen results. Overall, 11 translation factors were identified as candidate RAN translation factors as their depletion reduced GR-GFP levels. Overall, excluded LOF lines either: altered toxicity of G4C2 flies but did not alter GR-GFP levels, similarly altered toxicity in a non-G4C2, GR fly model arguing that these acted downstream of GR production, had no effect on G4C2 toxicity or GR-GFP levels, or were “unspecific” modifiers identified by quality control experiments. Shown: representative images while all RNAi were tested 2+ times for effects under each condition. Details on LOF lines used and complete results with each line can be found in Additional file 2: Table S2. For full genotypes and RNAi lines see Additional file 7: Table S1