Abstract
Non-coding CUG repeat expansions interfere with the activity of human Muscleblind-like (MBNL) proteins contributing to myotonic dystrophy 1 (DM1). To understand this toxic RNA gain-of-function mechanism we developed a Drosophila model expressing 60 pure and 480 interrupted CUG repeats in the context of a non-translatable RNA. These flies reproduced aspects of the DM1 pathology, most notably nuclear accumulation of CUG transcripts, muscle degeneration, splicing misregulation, and diminished Muscleblind function in vivo. Reduced Muscleblind activity was evident from the sensitivity of CUG-induced phenotypes to a decrease in muscleblind genetic dosage and rescue by MBNL1 expression, and further supported by the co-localization of Muscleblind and CUG repeat RNA in ribonuclear foci. Targeted expression of CUG repeats to the developing eye and brain mushroom bodies was toxic leading to rough eyes and semilethality, respectively. These phenotypes were utilized to identify genetic and chemical modifiers of the CUG-induced toxicity. 15 genetic modifiers of the rough eye phenotype were isolated. These genes identify putative cellular processes unknown to be altered by CUG repeat RNA, and they include mRNA export factor Aly, apoptosis inhibitor Thread, chromatin remodelling factor Nurf-38, and extracellular matrix structural component Viking. Ten chemical compounds suppressed the semilethal phenotype. These compounds significantly improved viability of CUG expressing flies and included non-steroidal anti-inflammatory agents (ketoprofen), muscarinic, cholinergic and histamine receptor inhibitors (orphenadrine), and drugs that can affect sodium and calcium metabolism such as clenbuterol and spironolactone. These findings provide new insights into the DM1 phenotype, and suggest novel candidates for DM1 treatments.
Highlights
Myotonic dystrophy 1 (DM1) is an autosomal dominant neuromuscular disease involving the expansion of unstable CTG repeats in the 39 untranslated region (UTR) of the DM protein kinase (DMPK) gene
To understand the molecular and cellular mechanisms underlying the DM1 pathology we generated transgenic Drosophila lines that express 60 uninterrupted or 480 interrupted CUG repeats as a noncoding transcript under the control of the Gal4/UAS system. 480 repeats consisted of synthetic CTG repeats interrupted every 20 units by the CTCGA sequence (hereafter referred to as i(CTG)480).The effect of expressing CUG repeat RNA in the Drosophila muscles or ubiquitously in the fly was studied with Myosin heavy chain (Mhc)-Gal4 and daughterless-Gal4 lines, respectively
Drosophila flies expressing 162 pure CTG repeats in the context of a 39UTR reporter gene show no detectable pathological phenotype despite forming discrete ribonuclear foci in muscle cells [4]. This suggests that ribonuclear foci are not directly pathogenic and that Drosophila might be refractory to CUG-induced toxicity since 162 pure CTG repeats are well within the pathogenic range in humans
Summary
Myotonic dystrophy 1 (DM1) is an autosomal dominant neuromuscular disease involving the expansion of unstable CTG repeats in the 39 untranslated region (UTR) of the DM protein kinase (DMPK) gene. Expression of 250 CUG repeats within a heterologous RNA gives rise to DM1-like phenotypes demonstrating that expanded CUG repeat transcripts are themselves toxic to cells [3]. Expression of 162 pure CTG repeats in the context of the 39UTR of a Green Fluorescent Protein (GFP) reporter gene has been reported not to cause signs of pathology [4] whereas larger, interrupted, CTG repeats induced muscle degeneration [5]. Several RNA binding proteins, most notably human Muscleblind-like proteins MBNL1, MBNL2 and MBNL3, are sequestered by mutant DMPK transcripts
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