CUG and CAG Repeat RNA Pathogenesis in Drosophila foci as in C. I, J, Expression of 4xrCAG,100-GFP or 4xrCAA,100-GFP leads to a similar pattern as in D and E. K, Expression of a single copy of GFP, not containing a repeat, leads to the formation of a single similar site of RNA concentration. doi:10.1371/journal.pone.0038516.g005 reducing Dcr-2. In contrast to double-stranded repeat RNA mediated pathology, reduced Dcr-2 levels were not rate limiting for either CUG or CAG-mediated tergite phenotypes [20]. This result suggests that mechanisms responsible for ‘hairpin’ RNA- mediated tergite pathology are not identical to those involved in double-stranded repeat RNA mediate pathology. For technical reasons it was not possible to examine progeny homozygous for the Dcr-2 loss of function allele, and the participation of this enzyme in RNAi processing limits the feasability of reducing Dcr-2 function via this targeted RNAi approach. Thus, we cannot exclude the possibility that some level of Dcr-2 processing, of CUG or CAG ‘hairpin’ RNA, may be necessary for the phenotype. However, our results suggest that even if Dcr-2 is required in both double-stranded and ‘hairpin’-mediated pathology, different dy- namics must be involved. Further studies will be necessary to dissect the mechanisms involved in each case. We further examined whether Dcr-1 processing is rate limiting for the tergite phenotype. Reduced Dcr-1 levels gave opposing effects, with a suppression observed with CUG transgene expression and an enhancement with CAG transgene expression. If pathology occurs through a mechanism dependent on the processing of ‘hairpin’ repeats by Dcr-1, it would be expected that a reduction in processing would give a similar effect in each case. In contrast, the ability to induce opposing effects is suggestive of a more complex interaction. It is unclear how this may occur, however these results highlight the need to further investigate Dcr- 1 processing as a candidate pathogenic pathway. Future studies examining other small RNA processing components, such as the Argonaut proteins, may be necessary to understand how this pathway may differentially interact with CUG or CAG repeat- mediated pathology. We have previously reported that double- stranded repeat RNA may cause pathology through the alteration of endogenous miRNA levels [20]. Thus, a possible interaction of ‘hairpin’ RNA with Dcr-1 processing pathways, known to regulate miRNA processing, may provide a candidate pathway that is a common downstream target of ‘hairpin’ and double-stranded RNA-mediated pathology. Finally, we examined the localisation of ‘hairpin’ repeat RNA in our system, as the formation of nuclear foci has previously been shown to be a hallmark of ‘hairpin’ RNA pathology [61]. Results indicate, that in our model system, CUG expression is sufficient to produce certain, specific RNA foci, while CAG expression is not. Expression of non-hairpin-forming CAA repeat RNA led to identical nuclear staining as with CAG expression. Thus, the ability to form a ‘hairpin’ structure is not sufficient to enable interactions required for the formation of cell specific foci observed with CUG RNA expression. CAG RNA foci have been previously observed in Drosophila, mouse and human cells, indicating that CAG expression may induce specific foci under some conditions [15,18,42]. These different observations may be due to charac- teristics of the transgenic expression system. Examining RNA localisation in histoblast cells was not possible for technical reasons, and thus we could not determine whether foci are formed specifically in cells that are perturbed by repeat RNA expression. Nonetheless, CAG transcripts in our system, that did not form specific foci, gave a strong tergite phenotype, and thus are able to undergo as yet undefined interactions necessary to induce pathology. These results, together with our observations of sequence specific modification with Mbl and Dcr-1, are consistent with the possibily that ‘hairpin’ repeat RNA may undergo multiple interactions that contribute to pathology. The tergite phenotype should be considered in the context of recent findings that non-coding repeat RNA sequences may undergo translation through a repeat-mediated mechanism known as repeat-associated non-ATG (RAN) translation, producing transcripts in all possible reading frames [49]. This process is yet to be observed in Drosophila, however, it is possible that homopolymeric peptides are produced at some frequency from our repeat RNA expression constructs including polyglutamine and polyalanine proteins that are known to be pathogenic [8,49,50]. Indeed, we previously reported a tergite phenotype due to the expression of a single copy of a transgene encoding a translated GCA repeat, producing a polyalanine peptide [50]. This phenotype may potentially be caused by either the repeat- containing GCA RNA, which is structurally identical to CAG repeat RNA, the presence of polyalanine protein, or a combina- tion of the two. Ubiquitous expression of a polyglutamine encoding CAG repeat is lethal in this system, preventing the analysis of adult tergites [50]. However, expression of a single translated CUG repeat-encoding transgene did not lead to a tergite phenotype [50], perhaps consistent with the weaker CUG phenotypes observed in this current study. As positively confirming the presence of homopolymeric peptides in multiple reading frames is technically complex, further analysis will be required to determine the relative contribution of RNA and homopolymeric protein to the tergite phenotype. Recent findings suggest that multiple pathways, mediated by ‘hairpin’ and double-stranded repeat RNA, as well as homopol- ymeric protein tracts, are able to independently cause pathology in Drosophila models of expanded repeat disease. Here we characterise a Drosophila tergite disruption phenotype caused by CUG or CAG ‘hairpin’ repeat RNA, providing a new, independent system in which to study common pathways that lead to pathology. Drosophila models such as this will enable the genetic pathways contributing to each type of repeat-mediated pathology to be defined. Identifying conserved pathways will provide candidates for future studies in vertebrate models, as well as defining ‘molecular hallmarks’ that may be used to verify the contribution of specific pathways modelled in Drosophila to human disease. Materials and Methods Drosophila Stocks Stocks were obtained from the Bloomington Drosophila Stock Centre (Bloomington, IN, USA) unless otherwise noted. Nomen- clature used for Drosophila genotypes is as used on Flybase (www. flybase.org). da-GAL4 (Bloomington stock #8641) was originally described in [54]. Act5c-GAL4 (Bloomington stock #4414) was originally described in [64]. T155-GAL4 (Bloomington stock #5076) was originally described by [57]. Dcr-1Q1147X and Dcr- 2L811fsX were obtained from Professor Richard Carthew and are described in [60]. mblE27 (Bloomington stock #7318) is caused by imprecise excision of a P-element, removing exon 1 and 2 [27]. Repeat Expression Lines Generation of repeat constructs has been previously described [8,17]. Generation of rCAG,100 and rCUG,100 four transgene copy lines, and nomenclature is described in [20]. Identical methods were used to generate 4xrCAA,100. To generate GFP- PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e38516