mRNA Processing and Metabolism pp 29-46

Part of the Methods in Molecular Biology™ book series (MIMB, volume 257) | Cite as

Imaging Alternative Splicing in Living Cells

  • Eric J. Wagner
  • Andrea Baines
  • Todd Albrecht
  • Robert M. Brazas
  • Mariano A. Garcia-Blanco

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.

Key Words

Alternative splicing in vivo imaging fluorescence intronic splicing silencers green fluorescent protein fibroblast growth factor receptor 

References

  1. 1.
    Claverie, J. M. (2001) Gene number. What if there are only 30,000 human genes? Science 291, 1255–1257.PubMedCrossRefGoogle Scholar
  2. 2.
    Venter, J. C., Adams, M. D., Myers, E. W., et al. (2001) The sequence of the human genome. Science 291, 1304–1351.PubMedCrossRefGoogle Scholar
  3. 3.
    Black, D. L. (2000) Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology. Cell 103, 367–370.PubMedCrossRefGoogle Scholar
  4. 4.
    Modrek, B. and Lee, C. (2002) A genomic view of alternative splicing. Nat. Genet. 30, 13–19.PubMedCrossRefGoogle Scholar
  5. 5.
    Roberts, G. C. and Smith, C. W. (2002) Alternative splicing: combinatorial output from the genome. Curr. Opin. Chem. Biol. 6, 375–383.PubMedCrossRefGoogle Scholar
  6. 6.
    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.PubMedGoogle Scholar
  7. 7.
    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.PubMedGoogle Scholar
  8. 8.
    Del Gatto, F., Gesnel, M. C. and Breathnach, R. (1996) The exon sequence TAGG can inhibit splicing. Nucleic Acids Res. 24, 2017–2021.PubMedCrossRefGoogle Scholar
  9. 9.
    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.PubMedGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    Wagner, E. J. and Garcia-Blanco, M. A. (2002) RNAi-mediated PTB depletion leads to enhanced exon definition. Mol. Cell 10, 943–949.PubMedCrossRefGoogle Scholar
  12. 12.
    Wagner, E. J. and Garcia-Blanco, M. A. (2001) Polypyrimidine tract binding protein antagonizes exon definition. Mol. Cell. Biol. 21, 3281–3288.PubMedCrossRefGoogle Scholar
  13. 13.
    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.PubMedCrossRefGoogle Scholar
  14. 14.
    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.PubMedGoogle Scholar
  15. 15.
    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.PubMedCrossRefGoogle Scholar
  16. 16.
    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.PubMedCrossRefGoogle Scholar
  17. 17.
    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.PubMedCrossRefGoogle Scholar
  18. 18.
    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.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Eric J. Wagner
    • 1
  • Andrea Baines
    • 2
  • Todd Albrecht
    • 2
  • Robert M. Brazas
    • 2
  • Mariano A. Garcia-Blanco
    • 2
  1. 1.Program in Molecular Biology and Biotechnology, Department of Biochemistry and BiophysicsUniversity of North CarolinaChapel Hill
  2. 2.Department of Molecular Genetics and MicrobiologyDuke University Medical CenterDurham

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