Muscleblind-Like 1 Knockout Mice Reveal Novel Splicing Defects in the Myotonic Dystrophy Brain Koichi Suenaga 1. , Kuang-Yung Lee 2,3. , Masayuki Nakamori 4¤a , Yoshiki Tatsumi 1 , Masanori P. Takahashi 4 , Harutoshi Fujimura 5 , Kenji Jinnai 6 , Hiroo Yoshikawa 1 , Hongqing Du 7¤b , Manuel Ares Jr. 7 , Maurice S. Swanson 2 , Takashi Kimura 1 * 1 Division of Neurology, Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan, 2 Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, United States of America, 3 Department of Neurology, Chang Gung Memorial Hospital, Keelung, Taiwan, 4 Department of Neurology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan, 5 Department of Neurology, National Hospital Organization Toneyama Hospital, Toyonaka, Osaka, Japan, 6 Department of Neurology, National Hospital Organization Hyogo-Chuo Hospital, Ohara, Hyogo, Japan, 7 Department of Molecular, Cell and Developmental Biology, RNA Center, Sinsheimer Labs, University of California Santa Cruz, Santa Cruz, California, United States of America Abstract Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTG exp ) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSA LR transgenic mouse model which expresses a pathogenic range CTG exp . In the present study, we addressed the possibility that MBNL1 sequestration by CUG exp RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1 DE3/DE3 knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1 DE3/DE3 knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1 DE3/DE3 brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUG exp RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain. Citation: Suenaga K, Lee K-Y, Nakamori M, Tatsumi Y, Takahashi MP, et al. (2012) Muscleblind-Like 1 Knockout Mice Reveal Novel Splicing Defects in the Myotonic Dystrophy Brain. PLoS ONE 7(3): e33218. doi:10.1371/journal.pone.0033218 Editor: Thomas H. Gillingwater, University of Edinburgh, United Kingdom Received September 20, 2011; Accepted February 9, 2012; Published March 13, 2012 Copyright: ß 2012 Suenaga et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: TK was supported by Research Grant for Intractable Disease from the Ministry of Health Labour and Welfare 22-118 and Grants-in-aid for Scientific Research from JSPS 23591258. MSS was supported by the National Institutes of Health (NIH) grant NS058901. MA was supported by the NIH grant GM084317. KJ was supported by the Intramural Research Grant for Neurological and Psychiatric Disorders of NCNP 22-7. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kimura@hyo-med.ac.jp . These authors contributed equally to this work. ¤a Current address: Department of Neurology, University of Rochester Medical Center, Rochester, New York, United States of America ¤b Current address: Department of Pediatrics, Stanford School of Medicine, Stanford, California, United States of America Introduction Myotonic dystrophy type1 (DM1) is a multi-systemic disorder affecting skeletal muscle, heart, ocular lens, testis, and the central nervous system (CNS). CNS involvement in adult-onset DM1 includes visual spatial and attention deficits, dysexecutive syndrome, apathy, avoidant behavior and excessive daytime sleepiness (review [1]). Although neuropathological studies have revealed several morpholog- ical changes [2,3,4], whether these changes contribute to the clinical symptoms observed in DM1 remains to be determined. DM1 is caused by the unstable expansion of CTG trinucleotide repeats in the 39 untranslated region of the DM protein kinase (DMPK) gene [5]. Recent evidence suggests that transcripts containing expanded CUG repeats (CUG exp ) accumulate in nuclear RNA foci and exert toxic effects on a variety of cellular regulatory pathways including splicing and transcription [6]. One disease model that is supported by considerable experimental evidence proposes that CUG exp RNAs cause sequestration and inhibition of the RNA-binding protein MBNL1 [7]. In support of this model, Mbnl1 knockout mice develop the muscle, eye, and RNA splicing abnormalities that are characteristic of DM1 disease [8]. In addition, we have reported a strong correlation in skeletal muscle splicing changes between two mouse models of DM1, the HSA LR transgenic and the Mbnl1 knockout [9]. In the CNS of DM1 patients, mutant DMPK transcripts accumulate in neuronal nuclei and sequester MBNL1 and MBNL2 [10]. Although abnormal regulation of several alterna- tively spliced exons has also been documented in the DM1 brain [10,11,12,13], it is not clear if MBNL1 sequestration contributes to aberrant splicing. Here we used splicing-sensitive microarrays to detect mis-splicing in the Mbnl1 DE3/DE3 knockout brain. PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e33218