Antisense Methods to Modulate Pre-mRNA Splicing

  • Joonbae Seo
  • Eric W. Ottesen
  • Ravindra N. Singh
Part of the Methods in Molecular Biology book series (MIMB, volume 1126)


The dynamic process of pre-mRNA splicing is regulated by combinatorial control exerted by overlapping cis-elements that are unique to every exon and its flanking intronic sequences. Splicing cis-elements are usually 4–8-nucleotide-long linear motifs that furnish interaction sites for specific proteins. Secondary and higher-order RNA structures exert an additional layer of control by providing accessibility to cis-elements. Antisense oligonucleotides (ASOs) that block splicing cis-elements and/or affect RNA structure have been shown to modulate alternative splicing in vivo. Consistently, ASO-based strategies have emerged as a powerful tool for therapeutic manipulation of aberrant splicing in pathological conditions. Here we describe the application of an ASO-based approach for the enhanced production of the full-length mRNA of SMN2 in spinal muscular atrophy patient cells.

Key words

Antisense oligonucleotide (ASO) Survival motor neuron (SMN) Pre-mRNA splicing Multi-exon-skipping detection assay (MESDA) Intronic splicing silencer N1 (ISS-N1) GC-rich sequence GM03813 Spinal muscular atrophy (SMA) Phosphorothioate 2′-O-methyl modification Transfection 



This work was supported by grants from United States National Institutes of Health (R01 NS055925, R21 NS072259, and R21 NS080294) and Salsbury Endowment (Iowa State University, Ames, IA, USA) to RNS. Authors acknowledge Dr. Natalia Singh for providing critical comments on the manuscript.


  1. 1.
    Wang Z, Burge CB (2008) Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA 14:802–813PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Ke S, Shang S, Kalachikov SM et al (2011) Quantitative evaluation of all hexamers as exonic splicing elements. Genome Res 21:1360–1374PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Busch A, Hertel KJ (2012) HEXEvent: a database of Human EXon splicing Events. Nucleic Acids Res 41:D118–D124PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Singh NN, Singh RN, Androphy EJ (2007) Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes. Nucleic Acids Res 35:371–389PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Warf MB, Berglund JA (2010) Role of RNA structure in regulating pre mRNA splicing. Trends Biochem Sci 35:169–178PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    McManus CJ, Graveley BR (2011) RNA structure and the mechanisms of alternative splicing. Curr Opin Genet Dev 21:373–379PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Irimia M, Blencowe BJ (2012) Alternative splicing: decoding an expansive regulatory layer. Curr Opin Cell Biol 24:323–332PubMedCrossRefGoogle Scholar
  8. 8.
    Singh NK, Singh NN, Androphy EJ et al (2006) Splicing of a critical exon of human Survival Motor Neuron is regulated by a unique silencer element located in the last intron. Mol Cell Biol 26:1333–1346PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Singh NN, Shishimorova M, Cao LC et al (2009) A short antisense oligonucleotide masking a unique intronic motif prevents skipping of a critical exon in spinal muscular atrophy. RNA Biol 6:341–350PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Hammond SM, Wood MJ (2010) PRO-051, an antisense oligonucleotide for the potential treatment of Duchenne muscular dystrophy. Curr Opin Mol Ther 12:478–486PubMedGoogle Scholar
  11. 11.
    Singh NN, Seo J, Rahn S et al (2012) A multi-exon-skipping detection assay reveals surprising diversity of splice isoforms of spinal muscular atrophy genes. PLoS One 7:e49595PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Kole R, Krainer AR, Altman S (2012) RNA therapeutics: beyond RNA interference and antisense oligonucleotides. Nat Rev Drug Discov 11:125–140PubMedGoogle Scholar
  13. 13.
    Singh NN, Hollinger K, Bhattacharya D et al (2010) An antisense microwalk reveals critical role of an intronic position linked to a unique long-distance interaction in pre-mRNA splicing. RNA 16:1167–1181PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Markowitz JA, Singh P, Darras BT (2012) Spinal muscular atrophy: a clinical and research update. Pediatr Neurol 46:1–12PubMedCrossRefGoogle Scholar
  15. 15.
    Hua Y, Sahashi K, Rigo F et al (2011) Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model. Nature 478:123–126PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Osman EY, Yen PF, Lorson CL (2012) Bifunctional RNAs targeting the intronic splicing silencer N1 increase SMN levels and reduce disease severity in an animal model of spinal muscular atrophy. Mol Ther 20:119–126PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Porensky PN, Mitrpant C, McGovern VL et al (2012) A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse. Hum Mol Genet 21:1625–1638PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Joonbae Seo
    • 1
  • Eric W. Ottesen
    • 1
  • Ravindra N. Singh
    • 1
  1. 1.Department of Biomedical Sciences, College of Veterinary MedicineIowa State UniversityAmesUSA

Personalised recommendations