involved in the regulation of alternative splicing [27,28]. Seques- tration of MBNL-1 leads to mis-splicing of certain transcripts and subsequent disease [29]. Pathology is associated with the formation of nuclear RNA foci that co-localise with MBNL-1 and may therefore be sites at which sequestration occurs [24,30–32]. A number of other proteins have been identified that are bound or mis-regulated by repeat RNA, including CUG-binding protein (CUG-BP) [14,33], Pur-alpha [34] and some heterogeneous nuclear ribonucleoproteins (hnRNPs) [35,36], indicating a com- plex set of both common and repeat sequence specific interactions. The presence of RNA foci, co-localisation of MBNL-1 with foci and dysregulation of alternative splicing have also been observed in either human tissue or animal models of the untranslated repeat diseases, DM1, DM2 [29], FXTAS [37,38], SCA8 [39,40], HDL- 2 [41] and SCA10 [36]. Based on these hallmarks it is proposed that the sequestration of MBNL-1 and dysregulation of splicing may be a common contributor to pathology. Furthermore, RNA foci that co-localise with MBNL-1 have been observed in human HD cells [42], supporting a role for this pathway in the polyglutamine diseases, however evidence for perturbation of this pathway in human patients is limited. Recent studies have identified other mechanisms, distinct from RNA-binding protein sequestration, through which repeat RNA may contribute to common pathology. We and others have reported evidence supporting a pathogenic role for bi-directional transcription, producing complementary repeat transcripts that hybridise to form double-stranded RNA [19,20]. Pathology is modified by altering Dicer-2 levels and is associated with the formation of 21nt repeat RNAs, highlighting a role for small RNA processing pathways in disease [20]. Bi-directional transcription has been identified in a number of diseases and therefore this pathway may be a common contributor to dominant pathology [43–48]. Evidence for a repeat-mediated mechanism of non-ATG initiated translation, occurring in all three reading frames, highlights another alternative mechanism that may account for common pathology [49]. Combined with bi-directional repeat transcription this mechanism may produce multiple homopoly- meric protein tracts that can contribute to pathology [49,50]. The increasing number of pathogenic agents and pathways identified highlights the need for model systems in which to better define the contributors to pathology in each case. Expanded repeat disease involves late onset neurodegeneration and loss of affected tissue, making access to samples and the detection of early changes that underlie pathology difficult. Human studies are therefore limited and animal models have been essential in investigating pathology. Drosophila is well established as a model for expanded repeat disease and a number of key pathways are conserved and have been identified or characterised in this system [7,8,13–15,17,19,20,39,50,51]. Re- peats can be ectopically expressed in non-essential tissues such as the eye, allowing phenotypes involving cellular perturbation to be examined in an otherwise viable organism [52]. The Drosophila system is also amenable to the powerful approach of examining modifier genes, thus identifying genetic pathways required for pathology that will inform further studies in vertebrate models and human tissue. We recently reported phenotypes, due to co-expression in the Drosophila eye or neurons, of complementary repeat transcripts that form double-stranded repeat RNA [20]. However, no consistent phenotypes were observed in the eye or neurons when each repeat sequence was ectopically expressed in the absence of a comple- mentary transcript, thus acting only as a single-stranded ‘hairpin’ RNA [17,20]. Here we characterise a morphological phenotype CUG and CAG Repeat RNA Pathogenesis in Drosophila caused by ubiquitous expression of CUG or CAG repeat RNA- containing transgenes in Drosophila identifying a new system, independent of the Drosophila eye, in which to study common ‘hairpin’ repeat RNA-mediated pathology. Ubiquitous expression of either CUG or CAG repeat RNA disrupts the adult Drosophila dorsal abdominal tergites, possibly through an effect on the developing histoblast cells. Results indicate that the tergite phenotype is quantitative and dependent on repeat-transgene expression, providing a biological read-out of ‘hairpin’ RNA mediated pathology in this model system. Further experiments examine whether pathways known to contribute to repeat RNA-mediated pathology are rate limiting for tergite disruption. Reducing levels of Mbl, the Drosophila ortholog of MBNL-1, does not enhance pathology and only modifies CUG- mediated phenotypes. In contrast to recently reported double-stranded repeat RNA- mediated pathology [20], reducing Dicer-2 (Dcr-2) levels was not rate limiting for ‘hairpin’ RNA-mediated tergite disruption. However, reducing Dicer-1 (Dcr-1) levels gave opposing results for phenotypes caused by CAG compared to CUG repeat RNA, highlighting the possibility of a sequence-dependent role for this pathway. Finally, examining RNA localisation identified specific RNA foci formed due to CUG, but not CAG, RNA expression. Thus, in this system ‘hairpin’ RNA may contribute to pathology through multiple interactions. Results Ubiquitous CAG or CUG Repeat RNA Expression Perturbs Abdominal Developmental in Drosophila We previously reported a system to ectopically express ‘hairpin’- forming CUG or CAG repeat RNA transcripts under UAS control in Drosophila [8,17,20]. Co-expression of both CUG and CAG complementary repeat-containing transgenes gave Dcr-2 depen- dent eye phenotypes, behavioural phenotypes, and changes in the miRNA profile [20]. In contrast, expression of either one of CUG or CAG repeat-containing transgenes, in the absence of a complementary transcript, gave transcriptional changes indicative of cellular perturbation, but no observable phenotypes with ectopic expression in the neurons or eye [17,20]. The absence of an observable phenotype limits the ability to genetically examine the basis of cellular perturbation caused by CUG or CAG repeat ‘hairpin’ RNA. In this study we therefore set out to identify morphological phenotypes caused by ectopic expression of either CUG or CAG expanded repeat RNA in Drosophila. Ectopic expression of CUG or CAG expanded repeat RNA was compared to an empty vector (EV) negative control as well as ectopic expression of expanded CAA repeat RNA, which unlike CAG or CUG, is not predicted to form a stable secondary structure [53]. Experiments were undertaken using the system that we have previously reported, whereby different untranslated repeat RNA sequences are expressed within the 39 untranslated region of an RNA encoding a short, non-functional peptide [8,17]. Independent transgenic lines were examined for each repeat sequence, with each line carrying four independent insertions of the transgene to generate high steady state levels of RNA [20]. Multiple independent transgenic insertions were initially generated for each construct (rCUG,100, rCAG,100 or rCAA,100, named alphabetically, ‘A’ through to ‘K’) and used to create lines carrying sets of 4 independent transgene insertions (4xrCAG,100, 4xrCUG,100 or 4xrCAA,100, named numerically, ‘line 1’ onwards) [20]. We have previously shown that, in the case of CUG or CAG expression, these lines give comparable levels of transgene expression, so that PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e38516