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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
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1687)

Abstract

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 

References

  1. 1.
    Flanigan KM, Dunn DM, von Niederhausern A et al (2011) Nonsense mutation-associated Becker muscular dystrophy: interplay between exon definition and splicing regulatory elements within the DMD gene. Hum Mutat 32:299–308. doi: 10.1002/humu.21426 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Disset A, Bourgeois CF, Benmalek N et al (2006) An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements. Hum Mol Genet 15:999–1013. doi: 10.1093/hmg/ddl015 CrossRefPubMedGoogle Scholar
  3. 3.
    Miro J, Laaref AM, Rofidal V et al (2015) FUBP1: a new protagonist in splicing regulation of the DMD gene. Nucleic Acids Res 43:2378–2389. doi: 10.1093/nar/gkv086 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Guiraud S, Chen H, Burns DT, Davies KE (2015) Advances in genetic therapeutic strategies for Duchenne muscular dystrophy. Exp Physiol 100:1458–1467. doi: 10.1113/EP085308 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Hégarat N, François J-C, Praseuth D (2008) Modern tools for identification of nucleic acid-binding proteins. Biochimie 90:1265–1272. doi: 10.1016/j.biochi.2008.03.012 CrossRefPubMedGoogle Scholar
  6. 6.
    Goina E, Skoko N, Pagani F (2008) Binding of DAZAP1 and hnRNPA1/A2 to an exonic splicing silencer in a natural BRCA1 exon 18 mutant. Mol Cell Biol 28:3850–3860. doi: 10.1128/MCB.02253-07 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Dhir A, Buratti E, van Santen MA et al (2010) The intronic splicing code: multiple factors involved in ATM pseudoexon definition. EMBO J 29:749–760. doi: 10.1038/emboj.2009.397 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Pastor T, Pagani F (2011) Interaction of hnRNPA1/A2 and DAZAP1 with an Alu-derived intronic splicing enhancer regulates ATM aberrant splicing. PLoS One 6:e23349. doi: 10.1371/journal.pone.0023349 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Caputi M, Mayeda A, Krainer AR, Zahler AM (1999) hnRNP A/B proteins are required for inhibition of HIV-1 pre-mRNA splicing. EMBO J 18:4060–4067. doi: 10.1093/emboj/18.14.4060 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Buratti E, Dörk T, Zuccato E et al (2001) Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping. EMBO J 20:1774–1784. doi: 10.1093/emboj/20.7.1774 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Michlewski G, Cáceres JF (2010) RNase-assisted RNA chromatography. RNA 16:1673–1678. doi: 10.1261/rna.2136010 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Cavaloc Y, Bourgeois CF, Kister L, Stévenin J (1999) The splicing factors 9G8 and SRp20 transactivate splicing through different and specific enhancers. RNA 5:468–483CrossRefPubMedPubMedCentralGoogle Scholar

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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