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Like Function in Myotonic Dystrophy

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Like Function in Myotonic Dystrophy Research Article TRANSPARENT OPEN Muscleblind deficiency in RNA‐mediated disease PROCESS ACCESS Compound loss of muscleblind‐like function in myotonic dystrophy Kuang-Yung Lee1,2†, Moyi Li1†, Mini Manchanda1, Ranjan Batra1, Konstantinos Charizanis1, Apoorva Mohan1, Sonisha A. Warren3, Christopher M. Chamberlain1, Dustin Finn1, Hannah Hong1, Hassan Ashraf3, Hideko Kasahara3, Laura P. W. Ranum1, Maurice S. Swanson1* Keywords: Mbnl1; Mbnl2; muscleblind- Myotonic dystrophy (DM) is a multi-systemic disease that impacts cardiac and like; myotonic dystrophy; RNA-mediated skeletal muscle as well as the central nervous system (CNS). DM is unusual disease because it is an RNA-mediated disorder due to the expression of toxic microsatellite expansion RNAs that alter the activities of RNA processing factors, including the muscleblind-like (MBNL) proteins. While these mutant RNAs inhibit DOI 10.1002/emmm.201303275 MBNL1 splicing activity in heart and skeletal muscles, Mbnl1 knockout mice fail Received July 10, 2013 to recapitulate the full-range of DM symptoms in these tissues. Here, we Revised August 30, 2013 generate mouse Mbnl compound knockouts to test the hypothesis that Mbnl2 Accepted September 06, 2013 functionally compensates for Mbnl1 loss. Although Mbnl1À/À; Mbnl2À/À double knockouts (DKOs) are embryonic lethal, Mbnl1À/À; Mbnl2þ/À mice are viable but develop cardinal features of DM muscle disease including reduced lifespan, heart conduction block, severe myotonia and progressive skeletal muscle weakness. Mbnl2 protein levels are elevated in Mbnl1À/À knockouts where Mbnl2 targets Mbnl1-regulated exons. These findings support the hypothesis that compound loss of MBNL function is a critical event in DM pathogenesis and provide novel mouse models to investigate additional pathways disrupted in this RNA- mediated disease. INTRODUCTION age‐of‐onset ranging from neonates to older adults with nearly all organ systems affected to different degrees. Myotonic dystrophy (DM) is an autosomal dominant neuromus- DM is caused by expansions of either CTG trinucleotide cular disease characterized by a multi‐systemic phenotype, repeats in the 30 untranslated region (30 UTR) of the DMPK gene including skeletal muscle myotonia and progressive weakness/ on chromosome 19 (DM type 1, DM1) or CCTG repeats in the wasting, cardiac arrhythmias, ocular ‘dust‐like’ cataracts, insulin first intron of the CNBP gene on chromosome 3 (DM type 2, insensitivity, hypogammaglobulinemia, hypersomnia and cere- DM2) (Ranum & Cooper, 2006; Udd & Krahe, 2012). These non‐ bral atrophy (Ranum & Cooper, 2006; Udd & Krahe, 2012). DM is coding C(C)TG expansion, or C(C)TGexp, mutations generate also one of the most variable human hereditary diseases with the pathogenic C(C)UGexp RNAs that are toxic because they perturb the normal cellular activities of several RNA binding factors, including the muscleblind‐like (MBNL), CUGBP1 and ETR3‐like (CELF), hnRNP H and STAU1 proteins (Ho et al, 2004; Kanadia (1) Department of Molecular Genetics and Microbiology, Center for et al, 2003; Miller et al, 2000; Paul et al, 2006; Philips et al, 1998; NeuroGenetics and the Genetics Institute, University of Florida, College Ravel‐Chapuis et al, 2012; Timchenko et al, 1996). The MBNL of Medicine, Gainesville, FL, USA (2) Department of Neurology, Chang Gung Memorial Hospital, Keelung, proteins appear to play a particularly prominent role in DM Taiwan pathogenesis since each of the three MBNL genes (MBNL1, (3) Department of Physiology and Functional Genomics, University of Florida, MBNL2, MBNL3) produce multiple isoforms with different, but College of Medicine, Gainesville, FL, USA relatively high, binding affinities for C(C)UGexp RNAs in vitro *Corresponding author: Tel: þ1 352 273 8076; Fax: þ1 352 273 8284; (Kino et al, 2004; Warf & Berglund, 2007; Yuan et al, 2007) and E-mail: mswanson@ufl.edu MBNL proteins are sequestered by mutant DM transcripts †These authors contributed equally to this work. in nuclear RNA foci in vivo (Jiang et al, 2004; Mankodi ß 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. EMBO Mol Med (2013) 5, 1887–1900 1887 Research Article www.embomolmed.org Muscleblind deficiency in RNA‐mediated disease D D D D et al, 2001; Miller et al, 2000). Mbnl1 E3/ E3, Mbnl2 E2/ E2 and (Charizanis et al, 2012). Interestingly, we noted that Mbnl2 D D Mbnl3 E2/ E2 isoform knockout (KO) mice recapitulate DM‐ protein levels increased strikingly in Mbnl1 KOs, particularly in relevant phenotypes with skeletal muscle myotonia, myopathy skeletal muscle (Fig 1A), in agreement with a recent study that D D and particulate cataracts in Mbnl1 E3/ E3 KOs, increased REM reported Mbnl2 protein upregulation following shRNA‐mediated D D sleep propensity and learning/memory deficits in Mbnl2 E2/ E2 knockdown of Mbnl1 in C2C12 myoblasts (Wang et al, 2012). KOs and an age‐associated decline in skeletal muscle regenera- Other factors implicated in DM pathogenesis (Mbnl3, Hnrnph1, D D tion in Mbnl3 E2/ E2 KO mice (Charizanis et al, 2012; Kanadia Stau1) did not show similar increases in protein level D D D D et al, 2003; Poulos et al, 2013). Mbnl1 E3/ E3 and Mbnl2 E2/ E2 (Supporting Information Fig S1A). The increase in Mbnl2 isoform knockout alleles do not express detectable Mbnl1 or protein was likely due to a corresponding increase in Mbnl2 Mbnl2 protein, respectively, so for simplicity they will be mRNA level (Fig 1B). To determine if combined loss of Mbnl1 À À subsequently referred to as Mbnl1 and Mbnl2 KOs or Mbnl1 / and Mbnl2 would recapitulate neonatal hypotonia and/or À À and Mbnl2 / . Additionally, Mbnl1 overexpression reverses progressive muscle wasting in adults, we attempted to generate myotonia in transgenic HSALR mice, which express a CUGexp Mbnl1; Mbnl2 DKO mice but loss of both proteins resulted in RNA in skeletal muscle, and transgenic MBNL1 overexpression embryonic lethality (Supporting Information Fig S1B). In À À þ À þ À À mice are viable with normal muscle structure and function contrast, Mbnl1 / ; Mbnl2 / , as well as Mbnl1 / ; Mbnl2 / À (Chamberlain & Ranum, 2012; Kanadia et al, 2006). , KOs were viable although the latter line showed reduced Sequestration and functional inhibition of all three MBNL embryonic viability so it was not characterized further. While paralogues depends on C(C)UGexp RNA repeat length and Mbnl2 is low in WT adult muscle, the upregulated Mbnl2 protein expression level, which are highly variable between tissues. in Mbnl1 KO muscle localized primarily to the nuclear Therefore, we propose that the full range of DM phenotypes compartment (Fig 1C). Elevated nuclear levels of Mbnl2 in cannot be adequately modelled in Mbnl single KO mice. Indeed, the Mbnl1 KO correlated with increased inclusion of Mbnl2 some prominent DM‐relevant manifestations, including skeletal exons 6 and 8 in skeletal muscle (Supporting Information Fig muscle weakness/wasting and cardiac arrhythmias, are not S2A, B) (Zhang et al, 2013). While the function of Mbnl2 exon 8 routinely observed in Mbnl1 and Mbnl2 single KOs. To test this is unknown, Mbnl2 exon 6 (54 nt) is homologous to Mbnl1 exon proposal, we investigated the effects of combined Mbnl1 and 7 that may encode a nuclear localization sequence (Lin Mbnl2 deficiency on cardiac and skeletal muscle structure and et al, 2006). function. While Mbnl1; Mbnl2 homozygous double KOs (DKOs) Despite the compensatory increase in Mbnl2 expression À À þ À were embryonic lethal, Mbnl1 / ; Mbnl2 / mice survived until in skeletal muscle, lifespan was severely compromised in À À þ À adulthood but developed skeletal and cardiac muscle defects, as Mbnl1 / ; Mbnl2 / KO animals with no mice surviving beyond well as alternative splicing changes, characteristic of DM. More 23 weeks of age (Fig 1D) and body weight was maintained at À À severe manifestations of DM skeletal muscle disease were 30% less than Mbnl1 / (Fig 1E). Since the MBNL loss‐of‐ À À þ À observed in Mbnl1 / ; Mbnl2c/c; Myo‐Cre / KO mice, with function model for DM proposes that both MBNL1 and MBNL2 complete absence of Mbnl2 protein expression restricted to are sequestered to varying levels depending on C(C)UGexp À À þ À muscle fibres. High‐throughput sequencing combined with RNA length and expression level, Mbnl1 / ; Mbnl2 / KO crosslinking/immunoprecipitation (HITS‐CLIP), a technique mice were further evaluated for DM‐relevant skeletal muscle that identifies binding sites for RNA‐binding proteins in vivo, defects to test the hypothesis that loss of both Mbnl1 and demonstrated that Mbnl2 upregulation following the loss of Mbnl2 proteins would increase the severity of skeletal muscle Mbnl1 expression resulted in an increase in Mbnl2 binding to deficits. Mbnl1 muscle RNA targets and partial correction of DM‐relevant mis‐splicing. Our results demonstrate that the major symptoms Progressive muscle weakness/wasting and NMJ defects in À À þ À of cardiac and skeletal muscle disease in DM can be recapitulated Mbnl1 / ; Mbnl2 / KOs in Mbnl compound knockout models and that sequestration of Histological hallmarks of DM patient muscle, which include both Mbnl1 and Mbnl2 by C(C)UGexp RNAs is an important centralized nuclei, fibre size heterogeneity and split fibres, have pathogenic feature of DM. been previously noted in older (>6 months of age) Mbnl1 KO quadriceps muscle (Kanadia et al, 2003; Kanadia et al, 2006). While these muscle abnormalities are considerably less severe in RESULTS younger Mbnl1 KO mice, more severe pathological changes (increased fibre size variation, atrophic and splitting fibres, Reduced lifespan in Mbnl1; Mbnl2 compound KO mice increased numbers of central nuclei indicative of muscle À À Mbnl1 KO adults develop skeletal muscle myotonia and DM‐ degeneration/regeneration) were observed in Mbnl1 / ; þ À associated muscle pathology, including centralized myonuclei Mbnl2 / muscles at 10–16 weeks of age (Fig 2A).
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