Human Molecular Genetics Advance Access published online on February 6, 2006
Human Molecular Genetics, doi:10.1093/hmg/ddl015
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1 Laboratoire de Génétique Moléculaire, Institut Universitaire de Recherche Clinique (IURC), CHU Montpellier, F34000, France; CNRS UPR1142, Montpellier, F 34000, France
* To whom correspondence should be addressed. 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. Based on UV cross-linking assays and pull-down analysis, we present evidence that the skipping of this exon is due to the creation of an exonic splicing silencer (ESS), 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 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 Tra2ß-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 Tra2ß-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 Duchenne muscular dystrophy 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.
Received November 28, 2005
Revised February 1, 2006
Accepted February 1, 2006
Article
An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements
A. Disset 1,
C. F. Bourgeois 2,
N. Benmalek 1,
M. Claustres 1,
J. Stevenin 2,
and
S. Tuffery-Giraud 3 *
2 IGBMC, Illkirch, F-67400 France; INSERM U596, Illkirch, F-67400 France; CNRS UMR7104, Illkirch, F-67400 France; Université Louis Pasteur, Strasbourg, F-67000 France
3 Laboratoire de Génétique Moléculaire, Institut Universitaire de Recherche Clinique (IURC), CHU Montpellier, F34000, France; CNRS UPR1142, Montpellier, F 34000, France; Laboratoire de Génétique Moléculaire, Institut Universitaire de Recherche Clinique (IURC), 641 Avenue du Doyen G. Giraud, 34093 Montpellier cedex 5, France
S. Tuffery-Giraud, E-mail: tuffery{at}igh.cnrs.fr
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