Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Engineering splicing factors with designed specificities

Nature Methods volume 6, pages 825–830 (2009)Cite this article

Abstract

Alternative splicing is generally regulated by trans-acting factors that specifically bind pre-mRNA to activate or inhibit the splicing reaction. This regulation is critical for normal gene expression, and dysregulation of splicing is closely associated with human diseases. Here we engineered artificial splicing factors by combining sequence-specific RNA-binding domains of human Pumilio1 with functional domains that regulate splicing. We applied these factors to modulate different types of alternative splicing in selected targets, to examine the activity of effector domains from natural splicing factors and to modulate splicing of an endogenous human gene, Bcl-X, an anticancer target. The designer factor targeted to Bcl-X increased the amount of pro-apoptotic Bcl-xS splice isoform, thus promoting apoptosis and increasing chemosensitivity of cancer cells to common antitumor drugs. Our approach permitted the creation of artificial factors to target virtually any pre-mRNA, providing a strategy to study splicing regulation and to manipulate disease-associated splicing events.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Design of ESFs to modulate exon skipping.
Figure 2: Effect of ESFs on alternative splice site usage.
Figure 3: Various arginine/serine–rich domains or glycine-rich domains can function as the effector module of ESFs.
Figure 4: Design of an ESF to modulate splicing of endogenous Bcl-X pre-mRNA.
Figure 5: ESFs affect Bcl-X splicing in multiple cancer cells.

Similar content being viewed by others

References

  1. Wang, E.T. et al. Alternative isoform regulation in human tissue transcriptomes. Nature 456, 470–476 (2008).

    Article  CAS  Google Scholar 

  2. Cooper, T.A., Wan, L. & Dreyfuss, G. RNA and disease. Cell 136, 777–793 (2009).

    Article  CAS  Google Scholar 

  3. Graveley, B.R. & Maniatis, T. Arginine/serine-rich domains of SR proteins can function as activators of pre-mRNA splicing. Mol. Cell 1, 765–771 (1998).

    Article  CAS  Google Scholar 

  4. Del Gatto-Konczak, F., Olive, M., Gesnel, M.C. & Breathnach, R. hnRNP A1 recruited to an exon in vivo can function as an exon splicing silencer. Mol. Cell. Biol. 19, 251–260 (1999).

    Article  CAS  Google Scholar 

  5. Wickens, M., Bernstein, D.S., Kimble, J. & Parker, R.A. PUF family portrait: 3′UTR regulation as a way of life. Trends Genet. 18, 150–157 (2002).

    Article  CAS  Google Scholar 

  6. Wang, X., McLachlan, J., Zamore, P.D. & Hall, T.M. Modular recognition of RNA by a human pumilio-homology domain. Cell 110, 501–512 (2002).

    Article  CAS  Google Scholar 

  7. Cheong, C.G. & Hall, T.M. Engineering RNA sequence specificity of Pumilio repeats. Proc. Natl. Acad. Sci. USA 103, 13635–13639 (2006).

    Article  CAS  Google Scholar 

  8. Black, D.L. Mechanisms of alternative pre-messenger RNA splicing. Annu. Rev. Biochem. 72, 291–336 (2003).

    Article  CAS  Google Scholar 

  9. Wang, Z. & Burge, C.B. Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA 14, 802–813 (2008).

    Article  CAS  Google Scholar 

  10. Bai, Y., Lee, D., Yu, T. & Chasin, L.A. Control of 3′ splice site choice in vivo by ASF/SF2 and hnRNP A1. Nucleic Acids Res. 27, 1126–1134 (1999).

    Article  CAS  Google Scholar 

  11. Wang, Z., Xiao, X., Van Nostrand, E. & Burge, C.B. General and specific functions of exonic splicing silencers in splicing control. Mol. Cell 23, 61–70 (2006).

    Article  CAS  Google Scholar 

  12. Eperon, I.C. et al. Selection of alternative 5′ splice sites: role of U1 snRNP and models for the antagonistic effects of SF2/ASF and hnRNP A1. Mol. Cell. Biol. 20, 8303–8318 (2000).

    Article  CAS  Google Scholar 

  13. Long, J.C. & Caceres, J.F. The SR protein family of splicing factors: master regulators of gene expression. Biochem. J. 417, 15–27 (2009).

    Article  CAS  Google Scholar 

  14. Martinez-Contreras, R. et al. hnRNP proteins and splicing control. Adv. Exp. Med. Biol. 623, 123–147 (2007).

    Article  Google Scholar 

  15. Keryer-Bibens, C., Barreau, C. & Osborne, H.B. Tethering of proteins to RNAs by bacteriophage proteins. Biol. Cell 100, 125–138 (2008).

    Article  CAS  Google Scholar 

  16. Philipps, D., Celotto, A.M., Wang, Q.Q., Tarng, R.S. & Graveley, B.R. Arginine/serine repeats are sufficient to constitute a splicing activation domain. Nucleic Acids Res. 31, 6502–6508 (2003).

    Article  CAS  Google Scholar 

  17. Boise, L.H. et al. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74, 597–608 (1993).

    Article  CAS  Google Scholar 

  18. Adams, J.M. & Cory, S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene 26, 1324–1337 (2007).

    Article  CAS  Google Scholar 

  19. Taylor, J.K., Zhang, Q.Q., Wyatt, J.R. & Dean, N.M. Induction of endogenous Bcl-xS through the control of Bcl-x pre-mRNA splicing by antisense oligonucleotides. Nat. Biotechnol. 17, 1097–1100 (1999).

    Article  CAS  Google Scholar 

  20. Mercatante, D.R., Bortner, C.D., Cidlowski, J.A. & Kole, R. Modification of alternative splicing of Bcl-x pre-mRNA in prostate and breast cancer cells. analysis of apoptosis and cell death. J. Biol. Chem. 276, 16411–16417 (2001).

    Article  CAS  Google Scholar 

  21. Wilusz, J.E., Devanney, S.C. & Caputi, M. Chimeric peptide nucleic acid compounds modulate splicing of the bcl-x gene in vitro and in vivo. Nucleic Acids Res. 33, 6547–6554 (2005).

    Article  CAS  Google Scholar 

  22. Gendron, D. et al. Modulation of 5′ splice site selection using tailed oligonucleotides carrying splicing signals. BMC Biotechnol. 6, 5 (2006).

    Article  Google Scholar 

  23. Zhou, A., Ou, A.C., Cho, A., Benz, E.J. Jr. & Huang, S.C. Novel splicing factor RBM25 modulates Bcl-x pre-mRNA 5′ splice site selection. Mol. Cell. Biol. 28, 5924–5936 (2008).

    Article  CAS  Google Scholar 

  24. Oltersdorf, T. et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435, 677–681 (2005).

    Article  CAS  Google Scholar 

  25. Shoemaker, A.R. et al. A small-molecule inhibitor of Bcl-XL potentiates the activity of cytotoxic drugs in vitro and in vivo. Cancer Res. 66, 8731–8739 (2006).

    Article  CAS  Google Scholar 

  26. Hua, Y., Vickers, T.A., Baker, B.F., Bennett, C.F. & Krainer, A.R. Enhancement of SMN2 exon 7 Inclusion by antisense oligonucleotides targeting the exon. PLoS Biol. 5, e73 (2007).

    Article  Google Scholar 

  27. Cartegni, L. & Krainer, A.R. Correction of disease-associated exon skipping by synthetic exon-specific activators. Nat. Struct. Biol. 10, 120–125 (2003).

    Article  CAS  Google Scholar 

  28. Skordis, L.A., Dunckley, M.G., Yue, B., Eperon, I.C. & Muntoni, F. Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts. Proc. Natl. Acad. Sci. USA 100, 4114–4119 (2003).

    Article  CAS  Google Scholar 

  29. Jumaa, H. & Nielsen, P.J. The splicing factor SRp20 modifies splicing of its own mRNA and ASF/SF2 antagonizes this regulation. EMBO J. 16, 5077–5085 (1997).

    Article  CAS  Google Scholar 

  30. Hutchison, S., LeBel, C., Blanchette, M. & Chabot, B. Distinct sets of adjacent heterogeneous nuclear ribonucleoprotein (hnRNP) A1/A2 binding sites control 5′ splice site selection in the hnRNP A1 mRNA precursor. J. Biol. Chem. 277, 29745–29752 (2002).

    Article  CAS  Google Scholar 

  31. Del Gatto-Konczak, F., Olive, M., Gesnel, M.C. & Breathnach, R. hnRNP A1 recruited to an exon in vivo can function as an exon splicing silencer. Mol. Cell. Biol. 19, 251–260 (1999).

    Article  CAS  Google Scholar 

  32. Cheong, C.G. & Hall, T.M. Engineering RNA sequence specificity of Pumilio repeats. Proc. Natl. Acad. Sci. USA 103, 13635–13639 (2006).

    Article  CAS  Google Scholar 

  33. Xiao, X., Wang, Z., Jang, M. & Burge, C.B. Coevolutionary networks of splicing cis-regulatory elements. Proc. Natl. Acad. Sci. USA 104, 18583–18588 (2007).

    Article  CAS  Google Scholar 

  34. Wang, Z., Xiao, X., Van Nostrand, E. & Burge, C.B. General and specific functions of exonic splicing silencers in splicing control. Mol. Cell 23, 61–70 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. Burge and our colleagues for critical reading of our manuscript. This work was supported by a grant from the Beckman Foundation (Z.W.) and the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences (T.M.T.H).

Author information

Authors and Affiliations

  1. Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Yang Wang & Zefeng Wang

  2. Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

    Cheom-Gil Cheong & Traci M Tanaka Hall

Authors
  1. Yang Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Cheom-Gil Cheong
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Traci M Tanaka Hall
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Zefeng Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

Z.W. and T.M.T.H. conceived the ideas. Z.W. and Y.W. designed and conducted the splicing experiments. C.-G.C. modified the PUF domains. Z.W. and T.M.T.H. wrote the paper.

Corresponding author

Correspondence to Zefeng Wang.

Ethics declarations

Competing interests

Y.W., Z.W. and T.M.T. H. are authors on a US patent application that has been filed related to this project (US application 61/140,326).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 (PDF 1270 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, Y., Cheong, CG., Tanaka Hall, T. et al. Engineering splicing factors with designed specificities. Nat Methods 6, 825–830 (2009). https://doi.org/10.1038/nmeth.1379

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1379

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing