Ubiquitous Expression of CUG or CAG Trinucleotide Repeat RNA Causes Common Morphological Defects in a Drosophila Model of RNA-Mediated Pathology Kynan T. Lawlor, Louise V. O’Keefe, Saumya E. Samaraweera, Clare L. van Eyk, Robert I. Richards* Discipline of Genetics, School of Molecular and Biomedical Science and Australian Research Council Special Research Centre for the Molecular Genetics of Development, The University of Adelaide, Adelaide, South Australia, Australia Abstract Expanded DNA repeat sequences are known to cause over 20 diseases, including Huntington’s disease, several types of spinocerebellar ataxia and myotonic dystrophy type 1 and 2. A shared genetic basis, and overlapping clinical features for some of these diseases, indicate that common pathways may contribute to pathology. Multiple mechanisms, mediated by both expanded homopolymeric proteins and expanded repeat RNA, have been identified by the use of model systems, that may account for shared pathology. The use of such animal models enables identification of distinct pathways and their ‘molecular hallmarks’ that can be used to determine the contribution of each pathway in human pathology. Here we characterise a tergite disruption phenotype in adult flies, caused by ubiquitous expression of either untranslated CUG or CAG expanded repeat RNA. Using the tergite phenotype as a quantitative trait we define a new genetic system in which to examine ‘hairpin’ repeat RNA-mediated cellular perturbation. Further experiments use this system to examine whether pathways involving Muscleblind sequestration or Dicer processing, which have been shown to mediate repeat RNA- mediated pathology in other model systems, contribute to cellular perturbation in this model. Citation: Lawlor KT, O’Keefe LV, Samaraweera SE, van Eyk CL, Richards RI (2012) Ubiquitous Expression of CUG or CAG Trinucleotide Repeat RNA Causes Common Morphological Defects in a Drosophila Model of RNA-Mediated Pathology. PLoS ONE 7(6): e38516. doi:10.1371/journal.pone.0038516 Editor: Diego E. Rincon-Limas, University of Florida, United States of America Received February 9, 2012; Accepted May 8, 2012; Published June 8, 2012 Copyright: 2012 Lawlor et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Australian Postgraduate Awards (to KTL, SES and CLvE); a National Health and Medical Research Council Peter Doherty Fellowship (fellowship number 207830 to LVO); project grants from the National Health and Medical Research Council (grant numbers 453674, 627183 to RIR and LVO); and the Australian Research Council Special Research Centre for the Molecular Genetics of Development. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: robert.richards@adelaide.edu.au Introduction The expansion of polymorphic repeat sequences (dynamic mutation) is responsible for over 20 human diseases, including Huntington’s disease (HD), myotonic dystrophy and several types of spinocerebellar ataxia (SCA) [1–3]. In all cases affected individuals inherit a repeat number that exceeds a pathogenic threshold. Increased disease severity and earlier age-of-onset are correlated with repeat length expansion, which occurs within successive generations [1–3]. Expansion is associated with loss of repeat-containing gene function and recessive inheritance in a few cases [4,5], however the majority of diseases appear to be caused by dominant, repeat-mediated mechanisms. As well as a shared genetic basis, many diseases show similar clinical features, including late onset neurodegeneration and movement disorder, highlighting the possibility that common pathways contribute to pathology in most, if not all cases of dominantly inherited dynamic mutation [1–3,6]. In diseases including HD and some types of SCA, expanded CAG trinucleotide repeats are found within a coding region, producing an expanded polyglutamine tract within the final protein [2]. Polyglutamine is able to cause pathology through multiple possible pathways, including gain-of-function interactions inherent to the expanded protein tract and alterations to normal protein function [3,7,8]. Nevertheless, polyglutamine protein- mediated pathology is not able to account for diseases where repeats are unable to encode polyglutamine, due to the repeat sequence composition and/or presence of the repeat tract within non-coding regions of the associated gene. This class of ‘untranslated’ repeat diseases share similar repeat sequences, pathogenic thresholds and clinical features with the ‘polygluta- mine’ diseases and includes myotonic dystrophy type 1 (DM1) and 2 (DM2), Huntington’s disease-like-2 (HDL-2), fragile X tremor/ ataxia syndrome (FXTAS) and SCA8, 10 and 12. In these diseases, gain-of-function properties of the expanded repeat RNA transcript appear to be responsible for dominant pathology [9–11]. Since repeat-containing RNA is present whether repeats are coding or non-coding, it may also represent a common contributor to all the dominant expanded repeat diseases. Studies have reported the ability of repeat RNA to cause pathology in animal and cellular model systems, via multiple, and likely distinct pathways [12–21]. Pathology due to expanded repeat RNA was first attributed in DM1, caused by a non-coding CUG repeat within the 39UTR of the dystrophia myotonica-protein kinase (DMPK) gene, and DM2, caused by a non-coding CCUG repeat within an intron of ZNF9 (now called CNBP) [22,23]. Expanded repeat RNA transcripts form a ‘hairpin’ secondary structure, which is able to bind and sequester specific RNA-binding proteins [24–26]. The most extensively characterised of these is Muscleblind-like 1 (MBNL-1), which is PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e38516