Rbfox1 downregulation and altered calpain 3 splicing by FRG1 in a mouse model of Facioscapulohumeral muscular dystrophy (FSHD)

PLoS Genet. 2013;9(1):e1003186. doi: 10.1371/journal.pgen.1003186. Epub 2013 Jan 3.

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1) in mice, frogs, and worms perturbs muscle development and causes FSHD-like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6-) is increased. Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing / genetics
  • Animals
  • Calpain* / genetics
  • Calpain* / metabolism
  • Cells, Cultured
  • Disease Models, Animal
  • Exons
  • Gene Expression Regulation, Developmental
  • HEK293 Cells
  • Humans
  • Mice
  • Microfilament Proteins
  • Muscle Development / genetics
  • Muscle Proteins* / genetics
  • Muscle Proteins* / metabolism
  • Muscular Dystrophy, Facioscapulohumeral* / genetics
  • Muscular Dystrophy, Facioscapulohumeral* / metabolism
  • Muscular Dystrophy, Facioscapulohumeral* / pathology
  • Myoblasts / cytology
  • Myoblasts / metabolism
  • Proteins* / genetics
  • Proteins* / metabolism
  • RNA Splicing Factors
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism

Substances

  • Frg1 protein, mouse
  • Microfilament Proteins
  • Muscle Proteins
  • Proteins
  • RNA Splicing Factors
  • RNA-Binding Proteins
  • Rbfox1 protein, mouse
  • Calpain
  • Capn3 protein, mouse