CUG and CAG Repeat RNA Pathogenesis in Drosophila Figure 3. Reducing Dcr-2 levels is not rate limiting for the tergite phenotype. A-D, Proportion of progeny within each phenotype category when repeat expression is driven by da-GAL4 alone (left column), or in the presence of the Dcr-2L811fsX mutant allele such that Dicer-2 levels are reduced (right column). A, Wild-type control, B, 4xrCAA,100 [line 1], C, 4xrCUG,100 [line 1], D, 4xrCAG,100 [line 2]. E, F, Total proportion for each genotype that shows any phenotype (‘any phenotype’ - category 2, 3 and 4) and the proportion with a strong phenotype (‘strong phenotype’ – category 3 and 4). E, 4xrCUG,100 [line 1], F, 4xrCAG,100 [line 2]. None of the observed changes were statistically significant (Fisher’s exact test). doi:10.1371/journal.pone.0038516.g003 one of two lines expressing 4xrCUG,100 in the presence of Dcr- 1Q1147X the proportion of progeny with any phenotype, and those with a strong phenotype, was significantly reduced (Figure 4E, S5 and Table S7). A second 4xrCUG,100 line gave a reduction in the proportion of progeny with any phenotype, however this was not statistically significant (Figure S5, Table S7). In contrast, one of two 4xrCAG,100 lines showed a more severe phenotype in the presence of Dcr-1Q1147X than when the repeat was expressed alone, with both the proportion showing any phenotype, and a strong phenotype, significantly increased (Figure 4F and S5). It was not possible to confirm this finding in an independent line, as the second four transgene copy expression line already showed a strong phenotype and reduction in viability, giving insufficient progeny for analysis (Table S6). Results indicate that reducing Dcr-1 levels may have opposing effects on CUG or CAG mediated phenotypes in this system. A further reduction in Dcr-1 function may cause stronger effects, providing further evidence for this effect, however, flies homozygous for the Dcr-1Q1147X allele are not viable and the involvement of this protein in small RNA processing limits the feasibility of using RNAi methods. Nonethe- PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38516