By contrast, in the presence of expanded (CCUG)106 repeats (transgenic flies display a spliceopathy phenotype similar to that seen in human DM2 patients and thus validate it as a suitable DM2 model. Expression of (CCUG)106 in IFM does not cause muscle atrophy To study the extent of (CCUG)DM2 toxicity in our DM2 model, we analyzed IFM samples expressing control or expanded (CCUG)n (or flies appeared to be able to fly normally and even aged flies (40?days) did not display any obvious flight defects. 2010). For example, aberrant splicing of the muscle-specific chloride channel and the insulin receptor (flies recapitulate many features observed in the human disease condition. They form RNA foci in muscles and retinal cells and affect RNA splicing of splicing reporter genes. APG-115 Although we did not observe muscle atrophy in flies, they displayed strong disruption in the external morphology of the eye and underlying retina. Expression of MBNL1, but not CUGBP1, was able to rescue the eye phenotype of flies. Furthermore, flies exhibited a strong apoptotic response in developing retinae, and inhibition of apoptosis rescued the retinal disruption. Finally, we tested two chemical compounds with therapeutic potential in DM1. Whereas treatment of flies with pentamidine had no effect, treatment with a PKR inhibitor blocked both the formation of RNA foci and apoptosis in retinae of flies. These data suggest that the DM2 model described here may provide a suitable tool for drug screening. RESULTS Transcripts with expanded (CCUG)n repeats form RNA foci The smallest reported DM2 expansions associated with clinically detectable manifestations are between 55 and 100 CCTG repeats (Liquori et al., 2001; Lucchiari et al., 2008; Bachinski et al., 2009). To generate a DM2 model in allele had a (TG)20(TCTG)12(CCTG)16 motif, while the allele had a (TG)22(TCTG)2(CCTG)106 motif (Fig.?1A). These transgenes are under the control of a UAS promoter (Brand and Perrimon, 1993) and expression can be induced using convenient Gal4 drivers, such as muscle-specific and eye-specific DM2 model forms nuclear CCUG foci. (A) Schematic (not to scale) of the noncoding CCTG repeat constructs used in this study. The control contains (CCTG)16 repeats (hybridization using a locked nucleic acid (LNA) probe was performed on 15 m cryosections of thoracic muscles of flies expressing and control repeats using the myosin driver. expression is associated with the presence of ribonuclear foci (red) in DAPI-stained nuclei (blue), APG-115 whereas no foci are detected in controls using the same driver. Two representative foci are indicated (arrows). (C) Quantification of nuclei with ribonuclear foci in control and muscle cells using and analyzed the morphology of the indirect flight muscle (IFM). As nuclear retention of RNA-protein aggregates (foci) is a hallmark of DM2 (Mankodi et al., 2003; Jones et al., 2011; Udd and Krahe, 2012; Meola et al., 2013), we first determined that flies mirror this disease-linked trait and performed FISH analysis to detect foci in the nucleus of IFM cells of flies. No foci were detected in control IFM, whereas more than 50% of the cells analyzed had nuclear foci in flies (Fig.?1B,C), demonstrating that 106 APG-115 CCUG repeats are sufficient to cause biochemical changes. The average fraction of nuclei with ribonuclear APG-115 foci in muscle cells is similar to that observed in a DM1 fly model expressing 480 CTG repeats (Garca-Alcover et al., 2014). Expression of in muscles causes mis-splicing In order to evaluate flies as a suitable DM2 model, we examined mis-splicing events in transgenic flies APG-115 expressing the 106 CCUG Rabbit polyclonal to AFP repeats in IFM. We studied alternative splicing of the endogenous gene (Fig.?2A), which showed aberrant splicing regulation in DM1 flies expressing a (CTG)480 tract (Garcia-Lopez et al., 2008) (see also Fig.?2B). For this analysis, we used two different transgenes for control and constructs, located on chromosomes 2 and 3. Expression of both transgenes increased the frequency at which exon 24 was aberrantly included (Fig.?2B): quantification revealed an increase from 30% in control flies to >70% in flies (Fig.?2C), similar to DM1. Open in a separate window Fig. 2. expression in muscle causes mis-splicing of MBNL1-dependent transcripts. (A) Outline of the intron/exon structure of (expression in IFM led to aberrant inclusion of exon 24 (dotted lines). Arrows indicate primers used for semi-quantitative PCR analysis. (B,C) Agarose gel and quantification of RT-PCR products from IFM expressing control (transgenes located on chromosomes 2 and 3. These transgenes were driven by driver without a UAS transgene show an average frequency of exon 24 inclusion of 30%. Compared with this control, expression of normal repeat length (CCUG)16 does not significantly alter splicing, whereas in the (CCUG)106 repeat-expressing cells exon 24 is retained at 70%, levels similar to those of DM1 flies expressing an interrupted 480 CUG repeat sequence (iCUG)480. (D,E) Agarose gel and quantification of RT-PCR products from flies expressing the indicated transgenes with the driver. Simultaneous expression of human and induces exon 24 exclusion, restoring wild-type levels.