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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.

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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.

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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).

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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
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  2. Cheom-Gil Cheong
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  3. Traci M Tanaka Hall
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  4. Zefeng Wang
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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.

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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).

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

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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

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