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Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB

Nature Structural & Molecular Biology volume 17, pages 1114–1123 (2010)Cite this article

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Abstract

To gain global insights into the role of the well-known repressive splicing regulator PTB, we analyzed the consequences of PTB knockdown in HeLa cells using high-density oligonucleotide splice-sensitive microarrays. The major class of identified PTB-regulated splicing event was PTB-repressed cassette exons, but there was also a substantial number of PTB-activated splicing events. PTB-repressed and PTB-activated exons showed a distinct arrangement of motifs with pyrimidine-rich motif enrichment within and upstream of repressed exons but downstream of activated exons. The N-terminal half of PTB was sufficient to activate splicing when recruited downstream of a PTB-activated exon. Moreover, insertion of an upstream pyrimidine tract was sufficient to convert a PTB-activated exon to a PTB-repressed exon. Our results show that PTB, an archetypal splicing repressor, has variable splicing activity that predictably depends upon its binding location with respect to target exons.

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Figure 1: Microarray analysis of PTB/nPTB targets in Hela cells.
Figure 2: PTB regulation of mutually exclusive exons.
Figure 3: Tissue specificity of PTB-regulated exons.
Figure 4: Motifs associated with PTB-repressed exons.
Figure 5: Motifs associated with PTB-activated exons.
Figure 6: PTB acts via an ESS.
Figure 7: PTB promotes exon inclusion via an ISE.
Figure 8: MS2 recruitment to downstream intron reveals PTB minimal activation domain.
Figure 9: Position-dependent PTB mode of action.

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Acknowledgements

We thank M. Hallegger, N. McGlincy and J. Ule for comments on the manuscript. This work was supported by a Wellcome Trust programme grant to C.W.J.S. (077877) and European Commission grant EURASNET-LSHG-CT-2005-518238 (C.W.J.S. and G.A.). G.A. is supported by grants from the Israel Science Foundation (ISF 61/09), Joint Germany-Israeli Research Program (ca-139), Deutsche-Israel Project (DIP MI-1317), the Israel Cancer Association and the Israel Cancer Research Foundation (ICRF). P.G. was supported by the Association Française contre les Myopathies and European Commission grant EURASNET-LSHG-CT-2005-518238. S.S. and D.H. are fellows of the Edmond J. Safra bioinformatics program at Tel Aviv University. This work was performed in partial fulfillment of the requirements for a PhD degree of S.S. and D.H., Sackler Faculty of Medicine, Tel Aviv University, Israel.

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  1. Rachel Spellman

    Present address: Present address: Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, UK.,

  2. Miriam Llorian and Schraga Schwartz: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, University of Cambridge, Cambridge, UK

    Miriam Llorian, Lit-Yeen Tan, Rachel Spellman, Adele Gordon & Christopher W J Smith

  2. Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv, Israel

    Schraga Schwartz, Dror Hollander & Gil Ast

  3. Affymetrix, Inc. Santa Clara, California, USA

    Tyson A Clark & Anthony C Schweitzer

  4. GenoSplice Technology, Centre Hayem, Hôpital Saint-Louis, Paris, France

    Rachel Spellman & Pierre de la Grange

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Contributions

M.L. and R.S. prepared materials for array analyses; T.A.C. and A.C.S. carried out the array analyses; M.L., L.-Y.T. and A.G. validated array predictions; S.S., D.H., P.G. and G.A. carried out bioinformatic analyses; M.L. carried out all other wet experimental work; M.L., S.S. and C.W.J.S. wrote the manuscript with input from other authors; C.W.J.S. conceived the project and coordinated the collaborations with important input from G.A., M.L., S.S. and T.A.C.

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Correspondence to Christopher W J Smith.

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Llorian, M., Schwartz, S., Clark, T. et al. Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB. Nat Struct Mol Biol 17, 1114–1123 (2010). https://doi.org/10.1038/nsmb.1881

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