Abstract
We have developed an in vivo reporter of alternative splicing decisions that allows for the determination of FGF-R2 splicing patterns without the destruction of cells. This method has broad applications, including the study of other alternatively spliced genes in tissue culture and in whole animals, and may be useful in creating imaging markers for the study of tumor progression and metastasis. In this chapter, the authors present one example of this method using fluorescence reporters. As with any new assay, a series of experiments were performed to validate the method. This chapter documents some of these experiments.
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
Claverie, J. M. (2001) Gene number. What if there are only 30,000 human genes? Science 291, 1255–1257.
Venter, J. C., Adams, M. D., Myers, E. W., et al. (2001) The sequence of the human genome. Science 291, 1304–1351.
Black, D. L. (2000) Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology. Cell 103, 367–370.
Modrek, B. and Lee, C. (2002) A genomic view of alternative splicing. Nat. Genet. 30, 13–19.
Roberts, G. C. and Smith, C. W. (2002) Alternative splicing: combinatorial output from the genome. Curr. Opin. Chem. Biol. 6, 375–383.
Del Gatto, F. and Breathnach, R. (1995) Exon and intron sequences, respectively, repress and activate splicing of a fibroblast growth factor receptor 2 alternative exon. Mol. Cell. Biol. 15, 4825–4834.
Del Gatto, F., Plet, A., Gesnel, M. C., Fort, C., and Breathnach, R. (1997) Multiple interdependent sequence elements control splicing of a fibroblast growth factor receptor 2 alternative exon. Mol. Cell. Biol. 17, 5106–5116.
Del Gatto, F., Gesnel, M. C. and Breathnach, R. (1996) The exon sequence TAGG can inhibit splicing. Nucleic Acids Res. 24, 2017–2021.
Carstens, R. P., McKeehan, W. L. and Garcia-Blanco, M. A. (1998) An intronic sequence element mediates both activation and repression of rat fibroblast growth factor receptor 2 pre-mRNA splicing. Mol. Cell. Biol. 18, 2205–2217.
Carstens, R. P., Eaton, J. V., Krigman, H. R., Walther, P. J., and Garcia-Blanco, M. A. (1997) Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) in human prostate cancer. Oncogene 15, 3059–3065.
Wagner, E. J. and Garcia-Blanco, M. A. (2002) RNAi-mediated PTB depletion leads to enhanced exon definition. Mol. Cell 10, 943–949.
Wagner, E. J. and Garcia-Blanco, M. A. (2001) Polypyrimidine tract binding protein antagonizes exon definition. Mol. Cell. Biol. 21, 3281–3288.
Del Gatto-Konczak, F., Bourgeois, C. F., Le Guiner, C., et al. (2000) The RNA-binding protein TIA-1 is a novel mammalian splicing regulator acting through intron sequences adjacent to a 5′ splice site. Mol. Cell. Biol. 20, 6287–6299.
Gilbert, E., Del Gatto, F., Champion-Arnaud, P., Gesnel, M. C., and Breathnach, R. (1993) Control of BEK and K-SAM splice sites in alternative splicing of the fibroblast growth factor receptor 2 pre-mRNA. Mol. Cell. Biol. 13, 5461–5468.
Jensen, K. B., Dredge, B. K., Stefani, G., et al. (2000) Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability. Neuron 25, 359–371.
Carstens, R. P., Wagner, E. J. and Garcia-Blanco, M. A. (2000) An intronic splicing silencer causes skipping of the IIIb exon of fibroblast growth factor receptor 2 through involvement of polypyrimidine tract binding protein. Mol. Cell. Biol. 20, 7388–7400.
Jones, R. B., Wang, F., Luo, Y., et al. (2001) The nonsense-mediated decay pathway and mutually exclusive expression of alternatively spliced FGFR2IIIb and IIIc mRNAs. J. Biol. Chem. 276, 4158–4167.
Del Gatto-Konczak, F., Olive, M., Gesnel, M. C., and Breathnach, R. (1999) hnRNP A1 recruited to an exon in vivo can function as an exon splicing silencer. Mol. Cell. Biol. 19, 251–260.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Wagner, E.J., Baines, A., Albrecht, T., Brazas, R.M., Garcia-Blanco, M.A. (2004). Imaging Alternative Splicing in Living Cells. In: Schoenberg, D.R. (eds) mRNA Processing and Metabolism. Methods in Molecular Biology™, vol 257. Humana Press. https://doi.org/10.1385/1-59259-750-5:029
Download citation
DOI: https://doi.org/10.1385/1-59259-750-5:029
Publisher Name: Humana Press
Print ISBN: 978-1-58829-225-4
Online ISBN: 978-1-59259-750-5
eBook Packages: Springer Protocols