1 Scientific RepoRts | 7: 2843 | DOI:10.1038/s41598-017-02829-3 www.nature.com/scientificreports expanded CCUG repeat RNA expression in Drosophila heart and muscle trigger Myotonic Dystrophy type 1-like phenotypes and activate autophagocytosis genes estefania Cerro-Herreros 1,2,3 , Mouli Chakraborty 1,2,3 , Manuel pérez-Alonso 1,2,3 , Rubén Artero 1,2,3 & Beatriz Llamusí 1,2,3 Myotonic dystrophies (DM1–2) are neuromuscular genetic disorders caused by the pathological expansion of untranslated microsatellites. DM1 and DM2, are caused by expanded CTG repeats in the 3′UTR of the DMPK gene and CCTG repeats in the frst intron of the CNBP gene, respectively. Mutant RNAs containing expanded repeats are retained in the cell nucleus, where they sequester nuclear factors and cause alterations in RNA metabolism. However, for unknown reasons, DM1 is more severe than DM2. To study the diferences and similarities in the pathogenesis of DM1 and DM2, we generated model fies by expressing pure expanded CUG ([250]×) or CCUG ([1100]×) repeats, respectively, and compared them with control fies expressing either 20 repeat units or GFP. We observed surprisingly severe muscle reduction and cardiac dysfunction in CCUG-expressing model fies. The muscle and cardiac tissue of both DM1 and DM2 model fies showed DM1-like phenotypes including overexpression of autophagy-related genes, RNA mis-splicing and repeat RNA aggregation in ribonuclear foci along with the Muscleblind protein. These data reveal, for the frst time, that expanded non-coding CCUG repeat-RNA has similar in vivo toxicity potential as expanded CUG RNA in muscle and heart tissues and suggests that specifc, as yet unknown factors, quench CCUG-repeat toxicity in DM2 patients. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are dominantly-inherited multi-systemic genetic disorders. DM1 (OMIM: 160900) is caused by an unstable expansion of a CTG trinucleotide repeat motif located in the 3′ untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) gene 1 . Unafected individuals carry fewer than 37 triplet-repeats, whereas expansions ranging between 50 and 4000 CTG repeats have been found in afected individuals. DM2 (OMIM: 602668), initially named proximal myotonic myopathy due to the greater weakness of proximal compared to distal muscles 2 , is caused by a tetranucleotide (CCTG) expansion in intron 1 of the CCHC-type zinc fnger nucleic acid binding protein gene (CNBP, also known as ZNF9) 3 . Healthy individuals carry fewer than 30 tetra-nucleotide repeats, whereas repeat lengths found in afected patients are signifcantly longer than in DM1 (between 55 and 11000) 3 . In contrast to DM2, which does not have a congen- ital form, very large (>1,000 repeat) DMPK CTG mutations also cause congenital DM1 (CDM) characterized by neonatal hypotonia (foppy baby) and intellectual disability 4 . Te expansions are transcribed into (CUG)n and (CCUG)n-containing RNA, respectively, which form secondary structures and sequester RNA-binding pro- teins, such as the RNA processing factors Muscleblind-like proteins (MBNL1-3 in vertebrates, Muscleblind in Drosophila), forming nuclear aggregates known as foci 5–11 . Additional splicing factors, such as CUGBP Elav-like family member 1 (CELF1), are also disrupted, leading to the mis-splicing of a large number of downstream genes 12–14 . Among them, the alteration in the splicing pattern of CLCN1, INR, PKM, CACNA1S, and BIN1 1 translational Genomics Group, incliva Health Research institute, Valencia, Spain. 2 Department of Genetics and interdisciplinary Research Structure for Biotechnology and Biomedicine (eRi BiOtecMeD), University of Valencia, Valencia, Spain. 3 ciPf-incLiVA joint unit, Valencia, Spain. estefania cerro-Herreros and Mouli chakraborty contributed equally to this work. correspondence and requests for materials should be addressed to R.A. (email: ruben.artero@uv.es) Received: 31 October 2016 Accepted: 19 April 2017 Published: xx xx xxxx opeN