ARTICLE DOI: 10.1038/s41467-018-04370-x OPEN rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences Chantal Sellier1, Estefanía Cerro-Herreros2,3, Markus Blatter4, Fernande Freyermuth1, Angeline Gaucherot1, Frank Ruffenach1, Partha Sarkar5, Jack Puymirat6, Bjarne Udd7,8,9, John W. Day10, Giovanni Meola11,12, Guillaume Bassez13, Harutoshi Fujimura14, Masanori P. Takahashi15, Benedikt Schoser16, Denis Furling13, Ruben Artero 2,3, Frédéric H.T. Allain4, Beatriz Llamusi2,3 & Nicolas Charlet-Berguerand1,17,18,19 1234567890():,; Myotonic dystrophy type 1 and type 2 (DM1, DM2) are caused by expansions of CTG and CCTG repeats, respectively. RNAs containing expanded CUG or CCUG repeats interfere with the metabolism of other RNAs through titration of the Muscleblind-like (MBNL) RNA binding proteins. DM2 follows a more favorable clinical course than DM1, suggesting that specific modifiers may modulate DM severity. Here, we report that the rbFOX1 RNA binding protein binds to expanded CCUG RNA repeats, but not to expanded CUG RNA repeats. Interestingly, rbFOX1 competes with MBNL1 for binding to CCUG expanded repeats and overexpression of rbFOX1 partly releases MBNL1 from sequestration within CCUG RNA foci in DM2 muscle cells. Furthermore, expression of rbFOX1 corrects alternative splicing alterations and rescues muscle atrophy, climbing and flying defects caused by expression of expanded CCUG repeats in a Drosophila model of DM2. 1 IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, 67404 Illkirch, France. 2 Translational Genomics Group, Interdisciplinary Research Structure for Biotechnology and Biomedicine BIOTECMED, University of Valencia, 46010 Valencia, Spain. 3 INCLIVA Health Research Institute, 46010 Valencia, Spain. 4 Institute for Molecular Biology and Biophysics, Swiss Federal Institute of Technology (ETH) Zurich, 8092 Zurich, Switzerland. 5 Department of Neurology, University of Texas Medical Branch, Galveston, TX 77555, USA. 6 Human Genetics Research Unit, Laval University, CHUQ, Ste-Foy, Quebec QC G1V 4G2, Canada. 7 Neuromuscular Research Center, Tampere University Hospital, 33521 Tampere, Finland. 8 Department of Medical Genetics, Folkhälsan Institute of Genetics, Helsinki University, 00290 Helsinki, Finland. 9 Department of Neurology, Vasa Central Hospital, 65130 Vaasa, Finland. 10 Department of Neurology, Stanford University, San Francisco, CA 94305, USA. 11 Department of Biomedical Sciences for Health, University of Milan, 20097 Milan, Italy. 12 Neurology Unit, IRCCS Policlinico San Donato, San Donato Milanese, 20097 Milan, Italy. 13 Sorbonne Université, Inserm, Association Institut de Myologie, Center of Research in Myology, 75013 Paris, France. 14 Department of Neurology, Toneyama National Hospital, Toyonaka 560-0045, Japan. 15 Department of Neurology, Osaka University Graduate School of Medicine, Suita 565-0871, Japan. 16 Friedrich-Baur-Institute, Department of Neurology, Ludwig Maximilian University, 80539 Munich, Germany. 17 UMR7104, Centre National de la Recherche Scientifique, 67404 Illkirch, France. 18 Institut National de la Santé et de la Recherche Médicale, U964, 67404 Illkirch, France. 19 Université de Strasbourg, 67404 Illkirch, France. Correspondence and requests for materials should be addressed to B.L. (email: [email protected]) or to N.C.-B. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:2009 | DOI: 10.1038/s41467-018-04370-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04370-x yotonic dystrophy is the most common muscular dys- genomic localization as CCUG repeats are embedded in the first trophy in adults and comprises two genetically distinct intron of the CNBP pre-mRNA, which is presumably less stable M ′ forms. Myotonic dystrophy type 1 (DM1) and its severe compared to the 3 UTR of the DMPK mRNA that hosts the CUG congenital form (CDM1) are caused by an expansion of repeats. In favor of this hypothesis, transgenic Drosophila CTG repeats in the 3′-untranslated region (UTR) of the DMPK expressing either expanded CUG or CCUG repeats, embedded in – gene1 3. In contrast, myotonic dystrophy type 2 (DM2) is caused a comparable genomic context deprived of any DMPK or CNBP by an expansion of CCTG repeats within the first intron of the sequences, show similar DM-like phenotypes37,38. Nonetheless, it CNBP (also known as ZNF9) gene4. Expression of mutant RNAs is not excluded that other mechanisms may further contribute to containing hundreds to thousands of CUG or CCUG repeats the lesser toxicity of expanded CCUG repeats in DM2. interferes with the metabolism of other RNAs through dysfunc- Searching for novel RNA binding proteins that interact spe- tions of mainly two classes of RNA binding proteins. First, cifically with either CUG or CCUG expanded repeats, we iden- expression and phosphorylation of the CUG-binding protein 1 tified that the rbFOX RNA binding proteins bind to expanded (CUGBP1, encoded by the CELF1 gene) are increased in DM1 CCUG repeats, but not to expanded CUG repeats. The rbFOX heart samples, especially in the most severely affected indivi- family comprises three members, rbFOX1 (also named Fox-1, duals5. Second, Muscleblind-like proteins (MBNL1, MBNL2 and A2BP1 or HRNBP1), rbFOX2 (also known as Fox-2, RBM9, Fxh MBNL3), which are RNA binding proteins specifically recogniz- or HRNBP2) and rbFOX3 (also called Fox-3, NeuN or HRNBP3), ing YGC RNA motifs, are titrated away from their normal mRNA which are involved in the regulation of various aspects of mRNA – targets as a result of their binding to expanded CUG and CCUG metabolism39 47. While rbFOX2 is widely expressed, rbFOX1 is RNA repeats. MBNL proteins titration is illustrated by their enriched in skeletal muscle, heart and brain, and rbFOX3 mislocalization within nuclear RNA foci formed by expanded expression is restricted to neuronal cells48. All three rbFOX CUG and CCUG repeats in cell and animal models of myotonic proteins possess a near-identical RNA-recognition motif (RRM) – – dystrophy6 8. MBNL and CUGBP1 are RNA binding proteins that recognizes the UGCAUGY RNA sequence39 41,49. – that regulate pre-mRNA alternative splicing9 14. Thus, alterations Here, we find that rbFOX1 also binds to expanded CCUG of MBNL and CUGBP1 functional levels result in reversion to repeats, albeit with a lesser affinity compared to its favorite embryonic splicing patterns for various mRNAs, which are UGCAUGY sequence. Interestingly, rbFOX1 co-localizes with – associated with several symptoms of myotonic dystrophy15 22. CCUG RNA foci in muscle cells and skeletal muscle tissues of Importantly, knockout of Mbnl1 and/ or Mbnl2 in mouse individuals with DM2. In contrast, rbFOX1 does not bind to reproduces splicing alterations and keys features of myotonic expanded CUG repeats and does not co-localize with CUG RNA dystrophy10,13,14,20,23. Conversely, overexpression of MBNL1 foci in DM1 samples. Furthermore, we find that the binding of corrects splicing alterations and myotonia in mice expressing MBNL1 and rbFOX1 to expanded CCUG repeats are mutually expanded CUG repeats24. Finally, a higher number of pathogenic exclusive. Addition of rbFOX1 competes with MBNL1 binding to CUG repeats leads to a greater titration of MBNL proteins, CCUG repeats and partially releases MBNL1 from CCUG RNA resulting in increased RNA metabolism alterations that correlate foci in DM2 muscle cells. Importantly, expression of rbFOX1 with increased disease severity in DM1 individuals23,25. These corrects splicing alterations and reduces muscle atrophy, as well results highlight the importance of MBNL titration in myotonic as climbing and flying defects caused by expression of expanded dystrophy type 1. CCUG repeats in Drosophila models of DM2. Thus, we propose Myotonic dystrophy type 2 resembles adult-onset DM1 with that the rbFOX proteins, by competing with and reducing the autosomal dominant inheritance pattern and similar clinical titration of MBNL1 within CCUG RNA foci, may participate to multi-organ features, including progressive skeletal muscle atro- the lesser toxicity of the CCTG repeat expansion in myotonic phy and weakness, myotonia, cardiac arrhythmia and conduction dystrophy type 2. defects, posterior subcapsular iridescent cataract, insulin resis- tance, hypogammaglobulinemia, as well as cognitive and per- – sonality changes26 28. Despite these similarities, there are Results significant differences between DM1 and DM2, including a Identification of proteins associated with expanded CCUG usually more favorable clinical course in DM2 compared to DM1. repeats. To identify novel RNA binding proteins involved in Indeed, symptoms are generally milder in DM2 compared to myotonic dystrophy, we incubated radioactively labeled CUG or DM1 and include slower and less severe progression of the dis- CCUG RNA repeats with nuclear extract of differentiated mouse ease, reduced severity of the cardiac involvement, later and less C2C12 muscle cells and analyzed the RNA-bound proteins by prominent weakness of the respiratory, facial and bulbar muscles, UV-light crosslink followed by SDS page gel electrophoresis less evocable myotonia and preserved social and cognitive abil- (Fig. 1a). Consistent with the pioneering work of the Swanson – ities29 31. A milder involvement in DM2 compared to DM1 is group6, a UV-crosslink signal around 42 to 45 kDa, which cor- also found at the cellular level as in vitro cultures of muscle cells responds to Mbnl1 molecular weight, was evident for