title: Rbfox1 downregulation and altered calpain 3 splicing by FRG1 in a mouse model of Facioscapulohumeral muscular dystrophy (FSHD).
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.
authors.affiliation: Dulbecco Telethon Institute and Division of Regenerative Medicine, San Raffaele Scientific Institute, Milano, Italy.
authors.forename: Mariaelena
authors.initials: M
authors.lastname: Pistoni
authors.forename: Lily
authors.initials: L
authors.lastname: Shiue
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Cline
authors.forename: Sergia
authors.initials: S
authors.lastname: Bortolanza
authors.forename: Maria Victoria
authors.initials: MV
authors.lastname: Neguembor
authors.forename: Alexandros
authors.initials: A
authors.lastname: Xynos
authors.forename: Manuel
authors.initials: M
authors.lastname: Ares
authors.forename: Davide
authors.initials: D
authors.lastname: Gabellini
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D002154:Calpain / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005091:Exons; D018507:Gene Expression Regulation, Developmental; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins; D024510:Muscle Development / Q000235:genetics; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D020391:Muscular Dystrophy, Facioscapulohumeral / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: C484440:Frg1 protein, mouse; D008840:Microfilament Proteins; D009124:Muscle Proteins; D011506:Proteins; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; C465036:Rbfox1 protein, mouse; D002154:Calpain; C491373:Capn3 protein, mouse