CUG and CAG Repeat RNA Pathogenesis in Drosophila Figure 4. Reduced Dicer-1 processing can have opposing effects on CUG or CAG RNA-mediated tergite phenotypes. A-D, Proportion of progeny within each phenotype category for expression with da-GAL4 alone (left column), or in the presence of the Dcr-1Q1147X mutant allele such that Dcr-1 levels are reduced (right column). A, wildtype 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] shows a suppression for both measures. F, 4xrCAG,100 [line 2] shows an enhancement for both measures. Comparisons were made between the populations using Fisher’s exact test, with significant results indicated above the proportion, where *p,0.05, **p,0.01 and ***p,0.001. doi:10.1371/journal.pone.0038516.g004 less, our results do not support a simple model whereby Dcr-1 processing is rate-limiting for the common pathway leading to tergite disruption. Ubiquitous CUG, but not CAG, Repeat RNA Expression Gives Nuclear RNA Foci in Specific Cells A hallmark of pathology associated with ‘hairpin’ repeat RNA is the formation of nuclear repeat RNA foci [61]. Thus RNA localisation was examined to determine whether each of the repeat-containing transgenes being expressed in our system is able to form nuclear RNA foci. Expanded repeat RNA was visualised in cryo-sections of whole larvae by in situ hybridisation with a fluorescently labelled probe complementary to the repeat sequence (Figure 5). With da-GAL4 driven 4xrCUG,100 expression, many nuclei contained up to four sites of RNA accumulation, possibly related to the sites of transcription for each transgene (data not PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e38516