Identification of Splicing Factors Involved in DMD Exon Skipping Events Using an In Vitro RNA Binding Assay

  • Julie Miro
  • Cyril F. Bourgeois
  • Mireille Claustres
  • Michel Koenig
  • Sylvie Tuffery-Giraud
Part of the Methods in Molecular Biology book series (MIMB, volume 1687)


Mutation-induced exon skipping in the DMD gene can modulate the severity of the phenotype in patients with Duchenne or Becker Muscular Dystrophy. These alternative splicing events are most likely the result of changes in recruitment of splicing factors at cis-acting elements in the mutated DMD pre-mRNA. The identification of proteins involved can be achieved by an affinity purification procedure. Here, we provide a detailed protocol for the in vitro RNA binding assay that we routinely apply to explore molecular mechanisms underlying splicing defects in the DMD gene. In vitro transcribed RNA probes containing either the wild type or mutated sequence are oxidized and bound to adipic acid dihydrazide–agarose beads. Incubation with a nuclear extract allows the binding of nuclear proteins to the RNA probes. The unbound proteins are washed off and then the specifically RNA-bound proteins are released from the beads by an RNase treatment. After separation by SDS-PAGE, proteins that display differential binding affinities for the wild type and mutant RNA probes are identified by mass spectrometry.

Key words

RNase-assisted RNA chromatography Binding affinity RNA pull-down RNA-binding protein Splicing factor Exon skipping ESS ESE DMD 


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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Julie Miro
    • 1
  • Cyril F. Bourgeois
    • 2
  • Mireille Claustres
    • 1
    • 3
  • Michel Koenig
    • 1
    • 3
  • Sylvie Tuffery-Giraud
    • 1
  1. 1.Laboratoire de Génétique de Maladies Rares (LGMR), EA7402Université de MontpellierMontpellierFrance
  2. 2.INSERM U1210, CNRS UMR 5239, Laboratory of Biology and Modelling of the Cell, Ecole Normale Supérieure de LyonUniversité de LyonLyonFrance
  3. 3.Laboratoire de Génétique Moléculaire, CHU MontpellierHôpital Arnaud de VilleneuveMontpellierFrance

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