Matthew S Alexander1,2,3,4, Rylie M Hightower1,2, Andrea L Reid1, Alexis H Bennett5, Lakshmanan Iyer6, Donna K Slonim7, Madhurima Saha8, Genri Kawahara9, Louis M Kunkel10,11,12,13, Alan S Kopin14, Vandana A Gupta5, Peter B Kang8,15,16,17,18,19, Isabelle Draper14. 1. Division of Neurology, Department of Pediatrics, University of Alabama at Birmingham and Children's of Alabama, Birmingham, Alabama, USA. 2. Center for Exercise Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA. 3. Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA. 4. Civitan International Research Center, University of Alabama at Birmingham, Birmingham, Alabama, USA. 5. Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA. 6. Department of Neuroscience, Tufts University, Boston, Massachusetts, USA. 7. Department of Computer Science, Tufts University, Medford, Massachusetts, USA. 8. Division of Pediatric Neurology, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida, USA. 9. Department of Pathophysiology, Tokyo Medical University, Tokyo, Japan. 10. Division of Genetics and Genomics, Boston Children's Hospital, Boston, Massachusetts, USA. 11. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. 12. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. 13. The Manton Center for Orphan Disease Research, Boston Children's Hospital, Boston, Massachusetts, USA. 14. Department of Medicine, Molecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts, USA. 15. Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida, USA. 16. Department of Neurology, University of Florida College of Medicine, Gainesville, Florida, USA. 17. Genetics Institute and Myology Institute, University of Florida, Gainesville, Florida, USA. 18. Paul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota Medical School, Minneapolis, Minnesota, USA. 19. Neurology Department, University of Minnesota Medical School, Minneapolis, Minnesota, USA.
Abstract
INTRODUCTION: RNA-binding proteins (RBPs) play an important role in skeletal muscle development and disease by regulating RNA splicing. In myotonic dystrophy type 1 (DM1), the RBP MBNL1 (muscleblind-like) is sequestered by toxic CUG repeats, leading to missplicing of MBNL1 targets. Mounting evidence from the literature has implicated other factors in the pathogenesis of DM1. Herein we sought to evaluate the functional role of the splicing factor hnRNP L in normal and DM1 muscle cells. METHODS: Co-immunoprecipitation assays using hnRNPL and MBNL1 expression constructs and splicing profiling in normal and DM1 muscle cell lines were performed. Zebrafish morpholinos targeting hnrpl and hnrnpl2 were injected into one-cell zebrafish for developmental and muscle analysis. In human myoblasts downregulation of hnRNP L was achieved with shRNAi. Ascochlorin administration to DM1 myoblasts was performed and expression of the CUG repeats, DM1 splicing biomarkers, and hnRNP L expression levels were evaluated. RESULTS: Using DM1 patient myoblast cell lines we observed the formation of abnormal hnRNP L nuclear foci within and outside the expanded CUG repeats, suggesting a role for this factor in DM1 pathology. We showed that the antiviral and antitumorigenic isoprenoid compound ascochlorin increased MBNL1 and hnRNP L expression levels. Drug treatment of DM1 muscle cells with ascochlorin partially rescued missplicing of established early biomarkers of DM1 and improved the defective myotube formation displayed by DM1 muscle cells. DISCUSSION: Together, these studies revealed that hnRNP L can modulate DM1 pathologies and is a potential therapeutic target.
INTRODUCTION: RNA-binding proteins (RBPs) play an important role in skeletal muscle development and disease by regulating RNA splicing. In myotonic dystrophy type 1 (DM1), the RBP MBNL1 (muscleblind-like) is sequestered by toxic CUG repeats, leading to missplicing of MBNL1 targets. Mounting evidence from the literature has implicated other factors in the pathogenesis of DM1. Herein we sought to evaluate the functional role of the splicing factor hnRNP L in normal and DM1 muscle cells. METHODS: Co-immunoprecipitation assays using hnRNPL and MBNL1 expression constructs and splicing profiling in normal and DM1 muscle cell lines were performed. Zebrafish morpholinos targeting hnrpl and hnrnpl2 were injected into one-cell zebrafish for developmental and muscle analysis. In human myoblasts downregulation of hnRNP L was achieved with shRNAi. Ascochlorin administration to DM1 myoblasts was performed and expression of the CUG repeats, DM1 splicing biomarkers, and hnRNP L expression levels were evaluated. RESULTS: Using DM1 patient myoblast cell lines we observed the formation of abnormal hnRNP L nuclear foci within and outside the expanded CUG repeats, suggesting a role for this factor in DM1 pathology. We showed that the antiviral and antitumorigenic isoprenoid compound ascochlorin increased MBNL1 and hnRNP L expression levels. Drug treatment of DM1 muscle cells with ascochlorin partially rescued missplicing of established early biomarkers of DM1 and improved the defective myotube formation displayed by DM1 muscle cells. DISCUSSION: Together, these studies revealed that hnRNP L can modulate DM1 pathologies and is a potential therapeutic target.
Authors: H Seznec; O Agbulut; N Sergeant; C Savouret; A Ghestem; N Tabti; J C Willer; L Ourth; C Duros; E Brisson; C Fouquet; G Butler-Browne; A Delacourte; C Junien; G Gourdon Journal: Hum Mol Genet Date: 2001-11-01 Impact factor: 6.150
Authors: F Rinaldi; C Terracciano; V Pisani; R Massa; E Loro; L Vergani; S Di Girolamo; C Angelini; G Gourdon; G Novelli; A Botta Journal: Neurobiol Dis Date: 2011-08-18 Impact factor: 5.996
Authors: Majid Fardaei; Mark T Rogers; Helena M Thorpe; Kenneth Larkin; Marion G Hamshere; Peter S Harper; J David Brook Journal: Hum Mol Genet Date: 2002-04-01 Impact factor: 6.150
Authors: Ramesh S Yadava; Qing Yu; Mahua Mandal; Frank Rigo; C Frank Bennett; Mani S Mahadevan Journal: Hum Mol Genet Date: 2020-06-03 Impact factor: 6.150
Authors: Thurman M Wheeler; Krzysztof Sobczak; John D Lueck; Robert J Osborne; Xiaoyan Lin; Robert T Dirksen; Charles A Thornton Journal: Science Date: 2009-07-17 Impact factor: 47.728
Authors: Maria de Haro; Ismael Al-Ramahi; Karlie R Jones; Jerrah K Holth; Lubov T Timchenko; Juan Botas Journal: PLoS Genet Date: 2013-04-18 Impact factor: 5.917