Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Black, D. L. (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu. Rev. Biochem. 72, 291–336.
House, A. E. and Lynch, K. W. (2008) Regulation of alternative splicing: more than just the ABCs. J. Biol. Chem. 283, 1217–1221.
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.
Mironov, A. A., Fickett, J. W. and Gelfand, M. S. (1999) Frequent alternative splicing of human genes. Genome Res. 9, 1288–1293.
Johnson, J. M., et al. (2003) Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science 302, 2141–2144.
Kan, Z., et al. (2001) Gene structure prediction and alternative splicing analysis using genomically aligned ESTs. Genome Res. 11, 889–900.
Garcia-Blanco, M. A. (2006) Alternative splicing: therapeutic target and tool. Prog. Mol. Subcell. Biol. 44, 47–64.
Faustino, N. A. and Cooper, T. A. (2003) Pre-mRNA splicing and human disease. Genes Dev. 17, 419–437.
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.
Kurreck, J. (2006) siRNA Efficiency: Structure or sequence – that is the question. J. Biomed. Biotechnol. 2006, 83757.
Leuschner, P. J., et al. (2006) Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep. 7, 314–320.
Gaur, R. K. (2006) RNA interference: a potential therapeutic tool for silencing splice isoforms linked to human diseases. Biotechniques Suppl, 15–22.
Kim, D. S., et al. (2008) Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing. BMC Mol. Biol. 9, 23.
Kim, D. S., et al. (2005) An artificial riboswitch for controlling pre-mRNA splicing. RNA 11, 1667–1677.
Kole, R., Vacek, M. and Williams, T. (2004) Modification of alternative splicing by antisense therapeutics. Oligonucleotides 14, 65–74.
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.
Tucker, B. J. and Breaker, R. R. (2005) Riboswitches as versatile gene control elements. Curr. Opin. Struct. Bio. 15, 342–348.
Nudler, E. and Mironov, A. S. (2004) The riboswitch control of bacterial metabolism. Trends Biochem. Sci. 29, 11–17.
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.
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.
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.
Fischer, D. C., et al. (2004) Expression of splicing factors in human ovarian cancer. Oncol. Rep. 11, 1085–1090.
Ghigna, C., et al. (2005) Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene. Mol. Cell 20, 881–890.
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.
Karni, R., et al. (2007) The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat. Struct. Mol. Biol. 14, 185–193.
Zhu, H., et al. (2005) Enhancing TRAIL-induced apoptosis by Bcl-X(L) siRNA. Cancer Biol. Ther. 4, 393–397.
Chevinsky, A. H. (1991) CEA in tumors of other than colorectal origin. Semin. Surg. Oncol. 7, 162–166.
Hammarstrom, S. (1999) The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin. Cancer Biol. 9, 67–81.
Li, W. and Cha, L. (2007) Predicting siRNA efficiency. Cell. Mol. Life Sci. 64, 1785–1792.
Yiu, S.M., et al. (2005) Filtering of ineffective siRNAs and improved siRNA design tool. Bioinformatics 21, 144–151.
Patzel, V., et al. (2005) Design of siRNAs producing unstructured guide-RNAs results in improved RNA interference efficiency. Nat. Biotechnol. 23, 1440–1444.
Huesken, D., et al. (2005) Design of a genome-wide siRNA library using an artificial neural network. Nat. Biotechnol. 23, 995–1001.
Tuschl, T. (2004) Targeting genes expressed in mammalian cells using siRNAs. Nat. Methods X, 13–17.
Reynolds, A., et al. (2004) Rational siRNA design for RNA interference. Nat. Biotechnol. 22, 326–330.
Daoud, R., et al. (1999) Activity-dependent regulation of alternative splicing patterns in the rat brain. Eur. J. Neurosci. 11, 788–802.
Venables, J. P. (2004) Aberrant and alternative splicing in cancer. Cancer Res. 64, 7647–7654.
Tazi, J., Durand, S. and Jeanteur, P. (2005) The spliceosome: a novel multi-faceted target for therapy. Trends Biochem. Sci. 30, 469–478.
Jenison, R. D., et al. (1994) High-resolution molecular discrimination by RNA. Science 263, 1425–1429.
Mayeda, A. and Krainer, A. R. (1999) Mammalian in vitro splicing assays. Methods Mol. Biol. 118, 315–321.
Ge, L. and Rudolph, P. (1997) Simultaneous introduction of multiple mutations using overlap extension PCR. Biotechniques 22, 28–30.
Visitsunthorn, N., Udomittipong, K. and Punnakan, L. (2001) Theophylline toxicity in Thai children. Asian Pac. J. Allergy Immunol. 19, 177–182.
Acknowledgments
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Dery, K.J., Gusti, V., Gaur, S., Shively, J.E., Yen, Y., Gaur, R.K. (2009). Alternative Splicing as a Therapeutic Target for Human Diseases. In: Rondinone, C., Reidhaar-Olson, J. (eds) Therapeutic Applications of RNAi. Methods in Molecular Biology™, vol 555. Humana Press. https://doi.org/10.1007/978-1-60327-295-7_10
Download citation
DOI: https://doi.org/10.1007/978-1-60327-295-7_10
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-294-0
Online ISBN: 978-1-60327-295-7
eBook Packages: Springer Protocols