title: An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements.
abstract: A nonsense mutation c.4250T>A (p.Leu1417X) in the dystrophin gene of a patient with an intermediate phenotype of muscular dystrophy induces partial in-frame skipping of exon 31. On the basis of UV cross-linking assays and pull-down analysis, we present evidence that the skipping of this exon is because of the creation of an exonic splicing silencer, which acts as a highly specific binding site (UAGACA) for a known repressor protein, hnRNP A1. Recombinant hnRNP A1 represses exon inclusion both in vitro and in vivo upon transient transfection of C2C12 cells with Duchenne muscular dystrophy (DMD) minigenes carrying the c.4250T>A mutation. Furthermore, we identified a downstream splicing enhancer in the central region of exon 31. This region functions as a Tra2beta-dependent exonic splicing enhancer (ESE) in vitro when inserted into a heterologous splicing reporter, and deletion of the ESE showed that incorporation of exon 31 depends on the Tra2beta-dependent enhancer both in the wild-type and mutant context. We conclude that dystrophin exon 31 contains juxtaposed sequence motifs that collaborate to regulate exon usage. This is the first elucidation of the molecular mechanism leading to exon skipping in the dystrophin gene and allowing the occurrence of a milder phenotype than the expected DMD phenotype. The knowledge of which cis-acting sequence within an exon is important for its definition will be essential for the alternative gene therapy approaches based on modulation of splicing to bypass DMD-causing mutations in the endogenous dystrophin gene.
authors.affiliation: Laboratoire de Génétique Moléculaire, Institut Universitaire de Recherche Clinique (IURC), CHU Montpellier F34000, France.
authors.forename: A
authors.initials: A
authors.lastname: Disset
authors.forename: C F
authors.initials: CF
authors.lastname: Bourgeois
authors.forename: N
authors.initials: N
authors.lastname: Benmalek
authors.forename: M
authors.initials: M
authors.lastname: Claustres
authors.forename: J
authors.initials: J
authors.lastname: Stevenin
authors.forename: Sylvie
authors.initials: S
authors.lastname: Tuffery-Giraud
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D001483:Base Sequence; D018389:Codon, Nonsense; D015536:Down-Regulation / Q000235:genetics; D016189:Dystrophin / Q000096:biosynthesis / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D005786:Gene Expression Regulation / Q000502:physiology; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010641:Phenotype; D011485:Protein Binding / Q000235:genetics; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics
chemical_list: D018389:Codon, Nonsense; D016189:Dystrophin; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D012322:RNA Precursors; D012097:Repressor Proteins