Alternative Splicing as a Therapeutic Target for Human Diseases

  • Kenneth J. Dery
  • Veronica Gusti
  • Shikha Gaur
  • John E. Shively
  • Yun Yen
  • Rajesh K. Gaur
Part of the Methods in Molecular Biology™ book series (MIMB, volume 555)


The majority of eukaryotic genes undergo alternative splicing, an evolutionarily conserved phenomenon, to generate functionally diverse protein isoforms from a single transcript. The fact that defective pre-mRNA splicing can generate non-functional and often toxic proteins with catastrophic effects, accurate removal of introns and joining of exons is vital for cell homeostasis. Thus, molecular tools that could either silence a disease-causing gene or regulate its expression in trans will find many therapeutic applications. Here we present two RNA-based approaches, namely RNAi and theophylline-responsive riboswitch that can regulate gene expression by loss-of-function and modulation of splicing, respectively. These strategies are likely to continue to play an integral role in studying gene function and drug discovery.

Key words

Alternative splicing spliceosome RNAi theophylline-responsive riboswitch 



We thank members of the Gaur laboratory for helpful discussions; Marieta Gencheva for valuable suggestions; and Faith Osep for administrative assistance. This work was supported in part by a Department of Defense (DOD; CDMRP) grant to RKG (BC023235), Beckman Research Institute excellence award to RKG, and NIH grant (CA 84202) to JES.


  1. 1.
    Black, D. L. (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu. Rev. Biochem. 72, 291–336.PubMedCrossRefGoogle Scholar
  2. 2.
    House, A. E. and Lynch, K. W. (2008) Regulation of alternative splicing: more than just the ABCs. J. Biol. Chem. 283, 1217–1221.PubMedCrossRefGoogle Scholar
  3. 3.
    Wang, Z. and Burge, C. B. (2008) Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA 14, 802–813.PubMedCrossRefGoogle Scholar
  4. 4.
    Mironov, A. A., Fickett, J. W. and Gelfand, M. S. (1999) Frequent alternative splicing of human genes. Genome Res. 9, 1288–1293.PubMedCrossRefGoogle Scholar
  5. 5.
    Johnson, J. M., et al. (2003) Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science 302, 2141–2144.PubMedCrossRefGoogle Scholar
  6. 6.
    Kan, Z., et al. (2001) Gene structure prediction and alternative splicing analysis using genomically aligned ESTs. Genome Res. 11, 889–900.PubMedCrossRefGoogle Scholar
  7. 7.
    Garcia-Blanco, M. A. (2006) Alternative splicing: therapeutic target and tool. Prog. Mol. Subcell. Biol. 44, 47–64.PubMedCrossRefGoogle Scholar
  8. 8.
    Faustino, N. A. and Cooper, T. A. (2003) Pre-mRNA splicing and human disease. Genes Dev. 17, 419–437.PubMedCrossRefGoogle Scholar
  9. 9.
    Benz, E. J., Jr. and Huang, S. C. (1997) Role of tissue specific alternative pre-mRNA splicing in the differentiation of the erythrocyte membrane. Trans. Am. Clin. Climatol. Assoc. 108, 78–95.PubMedGoogle Scholar
  10. 10.
    Kurreck, J. (2006) siRNA Efficiency: Structure or sequence – that is the question. J. Biomed. Biotechnol. 2006, 83757.PubMedCrossRefGoogle Scholar
  11. 11.
    Leuschner, P. J., et al. (2006) Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep. 7, 314–320.PubMedCrossRefGoogle Scholar
  12. 12.
    Gaur, R. K. (2006) RNA interference: a potential therapeutic tool for silencing splice isoforms linked to human diseases. Biotechniques Suppl, 15–22.PubMedCrossRefGoogle Scholar
  13. 13.
    Kim, D. S., et al. (2008) Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing. BMC Mol. Biol. 9, 23.PubMedCrossRefGoogle Scholar
  14. 14.
    Kim, D. S., et al. (2005) An artificial riboswitch for controlling pre-mRNA splicing. RNA 11, 1667–1677.PubMedCrossRefGoogle Scholar
  15. 15.
    Kole, R., Vacek, M. and Williams, T. (2004) Modification of alternative splicing by antisense therapeutics. Oligonucleotides 14, 65–74.PubMedCrossRefGoogle Scholar
  16. 16.
    Dominski, Z. and Kole, R. (1993) Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides. Proc. Natl. Acad. Sci. USA 90, 8673–8677.PubMedCrossRefGoogle Scholar
  17. 17.
    Tucker, B. J. and Breaker, R. R. (2005) Riboswitches as versatile gene control elements. Curr. Opin. Struct. Bio. 15, 342–348.CrossRefGoogle Scholar
  18. 18.
    Nudler, E. and Mironov, A. S. (2004) The riboswitch control of bacterial metabolism. Trends Biochem. Sci. 29, 11–17.PubMedCrossRefGoogle Scholar
  19. 19.
    Goguel, V., Wang, Y. and Rosbash, M. (1993) Short artificial hairpins sequester splicing signals and inhibit yeast pre-mRNA splicing. Mol. Cell. Biol. 13, 6841–6848.PubMedGoogle Scholar
  20. 20.
    Gusti, V., Kim, D. S. and Gaur, R. K. (2008) Sequestering of the 3' splice site in a theophylline-responsive riboswitch allows ligand-dependent control of alternative splicing. Oligonucleotides 18, 93–99.PubMedCrossRefGoogle Scholar
  21. 21.
    Dignam, J. D., Lebovitz, R. M. and Roeder, R. G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489.PubMedCrossRefGoogle Scholar
  22. 22.
    Fischer, D. C., et al. (2004) Expression of splicing factors in human ovarian cancer. Oncol. Rep. 11, 1085–1090.PubMedGoogle Scholar
  23. 23.
    Ghigna, C., et al. (2005) Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene. Mol. Cell 20, 881–890.PubMedCrossRefGoogle Scholar
  24. 24.
    He, X., et al. (2004) Alternative splicing of the multidrug resistance protein 1/ATP binding cassette transporter subfamily gene in ovarian cancer creates functional splice variants and is associated with increased expression of the splicing factors PTB and SRp20. Clin. Cancer Res. 10, 4652–4660.PubMedCrossRefGoogle Scholar
  25. 25.
    Karni, R., et al. (2007) The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat. Struct. Mol. Biol. 14, 185–193.PubMedCrossRefGoogle Scholar
  26. 26.
    Zhu, H., et al. (2005) Enhancing TRAIL-induced apoptosis by Bcl-X(L) siRNA. Cancer Biol. Ther. 4, 393–397.PubMedCrossRefGoogle Scholar
  27. 27.
    Chevinsky, A. H. (1991) CEA in tumors of other than colorectal origin. Semin. Surg. Oncol. 7, 162–166.PubMedCrossRefGoogle Scholar
  28. 28.
    Hammarstrom, S. (1999) The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin. Cancer Biol. 9, 67–81.PubMedCrossRefGoogle Scholar
  29. 29.
    Li, W. and Cha, L. (2007) Predicting siRNA efficiency. Cell. Mol. Life Sci. 64, 1785–1792.PubMedCrossRefGoogle Scholar
  30. 30.
    Yiu, S.M., et al. (2005) Filtering of ineffective siRNAs and improved siRNA design tool. Bioinformatics 21, 144–151.PubMedCrossRefGoogle Scholar
  31. 31.
    Patzel, V., et al. (2005) Design of siRNAs producing unstructured guide-RNAs results in improved RNA interference efficiency. Nat. Biotechnol. 23, 1440–1444.PubMedCrossRefGoogle Scholar
  32. 32.
    Huesken, D., et al. (2005) Design of a genome-wide siRNA library using an artificial neural network. Nat. Biotechnol. 23, 995–1001.PubMedCrossRefGoogle Scholar
  33. 33.
    Tuschl, T. (2004) Targeting genes expressed in mammalian cells using siRNAs. Nat. Methods X, 13–17.Google Scholar
  34. 34.
    Reynolds, A., et al. (2004) Rational siRNA design for RNA interference. Nat. Biotechnol. 22, 326–330.PubMedCrossRefGoogle Scholar
  35. 35.
    Daoud, R., et al. (1999) Activity-dependent regulation of alternative splicing patterns in the rat brain. Eur. J. Neurosci. 11, 788–802.PubMedCrossRefGoogle Scholar
  36. 36.
    Venables, J. P. (2004) Aberrant and alternative splicing in cancer. Cancer Res. 64, 7647–7654.PubMedCrossRefGoogle Scholar
  37. 37.
    Tazi, J., Durand, S. and Jeanteur, P. (2005) The spliceosome: a novel multi-faceted target for therapy. Trends Biochem. Sci. 30, 469–478.PubMedCrossRefGoogle Scholar
  38. 38.
    Jenison, R. D., et al. (1994) High-resolution molecular discrimination by RNA. Science 263, 1425–1429.PubMedCrossRefGoogle Scholar
  39. 39.
    Mayeda, A. and Krainer, A. R. (1999) Mammalian in vitro splicing assays. Methods Mol. Biol. 118, 315–321.PubMedGoogle Scholar
  40. 40.
    Ge, L. and Rudolph, P. (1997) Simultaneous introduction of multiple mutations using overlap extension PCR. Biotechniques 22, 28–30.PubMedGoogle Scholar
  41. 41.
    Visitsunthorn, N., Udomittipong, K. and Punnakan, L. (2001) Theophylline toxicity in Thai children. Asian Pac. J. Allergy Immunol. 19, 177–182.PubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Kenneth J. Dery
    • 1
  • Veronica Gusti
    • 2
  • Shikha Gaur
    • 3
  • John E. Shively
    • 4
  • Yun Yen
    • 5
  • Rajesh K. Gaur
    • 6
  1. 1.Divisions of Molecular Biology and ImmunologyBeckman Research Institute of the City of HopeDuarteUSA
  2. 2.Division of Molecular BiologyBeckman Research Institute of the City of HopeDuarteUSA
  3. 3.Department of Clinical and Molecular PharmacologyBeckman Research Institute of the City of HopeDuarteUSA
  4. 4.Division of ImmunologyBeckman Research Institute of the City of HopeDuarteUSA
  5. 5.Department of Clinical and Molecular PharmacologyBeckman Research Institute of the City of HopeDuarteUSA
  6. 6.Division of Molecular Biology and Graduate School of Biological SciencesBeckman Research Institute of the City of HopeDuarteUSA

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