Defects of pre-mRNA splicing are increasingly recognized as an important mechanism through which gene mutations cause disease. In our group, the RNA-based strategy used to detect point mutations in the dystrophin gene from muscle samples of Duchenne (DMD) or Becker (BMD) muscular dystrophy patients has led to identify a large panel of mutations causing aberrant splicing. The dystrophin gene, the largest one, has a long coding sequence (11kb), a high number of exons (79), large introns (up to 180kb) so that sites for splicing mutations are numerous. The rules are still not clearly defined regarding exon definition and splicing regulation in eukaryotic genes. Data from naturally occurring mutations that cause splicing defects in patients provide valuable tools to identify auxiliary RNA cis-elements that participate in intron/exon recognition. We further investigate the mutations altering mRNA splicing in patients in view to identify which elements are essential for correct splicing in the dystrophin gene. A computational approach using the algorithms available in the Splice Site Finder software (http://www.umd.be/SSF) was first used to predict putative splicing signals in particular ESE and branch point sequences of domain of dystrophin. For functional validation of these sequences, we are developing an easy to use and reliable system based on fluorescent splicing reporter minigenes allowing flow cytometric analysis and determination of correct splicing of a tested exon. This approach is currently used to assess the pathogenic effect of the numerous missense mutations which have been identified in our laboratory in the genes responsible for the Usher syndrome. The systematic characterization of functional branch points and exons which are spliced as an exonic splicing enhancer (ESE) dependent manner contributes to the comprehensive knowledge of multiple splicing signals that could constitute therapeutic targets in the exon-skipping strategy to reverse the DMD phenotype into BMD.
Expertise :
Transcripts analtysis, minigenes constructs for splicing, PCR overlap, directed mutagenesis, computational analyses, in vitro RNA-proteins interactions studies (UV-cross linking), in vitro splicing, transient and stable transfections in eucaryotic cells, fluorescence microscopy, FACS, branch-point mapping, expression vector constructions (SR, hnRNPs proteins), RNAi.
Group members :
Sandie Le Guédard-Mereuze, PhD student
Mouna Messaoud Khelifi, PhD student