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Polypyrimidine tract binding protein controls the transition from exon definition to an intron defined spliceosome

Nature Structural & Molecular Biology volume 15, pages 183–191 (2008)Cite this article

Abstract

The polypyrimidine tract binding protein (PTB) binds pre-mRNAs to alter splice-site choice. We characterized a series of spliceosomal complexes that assemble on a pre-mRNA under conditions of either PTB-mediated splicing repression or its absence. In the absence of repression, exon definition complexes that were assembled downstream of the regulated exon could progress to pre-spliceosomal A complexes and functional spliceosomes. Under PTB-mediated repression, assembly was arrested at an A-like complex that was unable to transition to spliceosomal complexes. Trans-splicing experiments indicated that, even when the U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) are properly bound to the upstream and downstream exons, the presence of PTB prevents the interaction of the two exon complexes. Proteomic analyses of these complexes provide a new description of exon definition complexes, and indicate that splicing regulators can act on the transition between the exon definition complex and an intron-defined spliceosome.

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Figure 1: An exon definition complex forms in both HeLa and WERI extracts.
Figure 2: The U2 snRNA is base-paired to the pre-mRNA in the EDA complex.
Figure 3: U2 small ribonucleoprotein particle assembly via exon definition does not overcome splicing repression.
Figure 4: Splicing repression after exon definition in HeLa extract is dependent on polypyrimidine tract binding protein (PTB).
Figure 5: ATP-independent E-like (E′) complex assembles in both HeLa and WERI extracts.
Figure 6: ATP-dependent splicing complex assembly in HeLa extract is stalled at the A-like (A′) complex.
Figure 7: Trans-splicing occurs in WERI but not HeLa extract.
Figure 8: Model for polypyrimidine tract binding protein (PTB)-mediated splicing repression.

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Acknowledgements

We thank K. Lynch, T. Nilsen and members of the Black laboratory for helpful discussion and comments. We thank B. Jokusch (Technical University of Braunschweig, Braunschweig, Germany) for the gift of anti–Raver-1 antibody and C. Lutz (University of Medicine and Dentistry of New Jersey, Newark, USA) for the anti-U1A antibody. This work was supported by US National Institutes of Health grants RO1:GM49662 to D.L.B., and RO1:GM61987 and 1S10RR017780-01 to D.C.R. D.L.B. is an investigator of the Howard Hughes Medical Institute, Los Angeles, California, USA.

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Authors and Affiliations

  1. Howard Hughes Medical Institute, University of California, Los Angeles, MRL5-748, Charles E. Young Drive South, Los Angeles, 90095, California, USA

    Shalini Sharma, Andrey Damianov & Douglas L Black

  2. Cancer Research Laboratory, Mass Spectrometry Facility, University of California, Berkeley, 525 Life Science Addition.,

    Lori A Kohlstaedt

  3. Department of Molecular and Cell Biology, University of California, Berkeley, 16 Barker Hall, Berkeley, 94720, California, USA

    Donald C Rio

  4. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, 1602 Molecular Sciences Building, 405 Hilgard Avenue, Los Angeles, 90095, California, USA

    Douglas L Black

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Contributions

S.S. and D.L.B. designed the experiments and analyzed the data. S.S. performed the experiments. L.A.K. and D.C.R. performed and analyzed the MS experiments. A.D. contributed to Figure 2. S.S. and D.L.B. wrote the paper.

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Correspondence to Douglas L Black.

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Sharma, S., Kohlstaedt, L., Damianov, A. et al. Polypyrimidine tract binding protein controls the transition from exon definition to an intron defined spliceosome. Nat Struct Mol Biol 15, 183–191 (2008). https://doi.org/10.1038/nsmb.1375

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