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A role for ubiquitin in the spliceosome assembly pathway

Nature Structural & Molecular Biology volume 15, pages 444–451 (2008)Cite this article

Abstract

The spliceosome uses numerous strategies to regulate its function in mRNA maturation. Ubiquitin regulates many cellular processes, but its potential roles during splicing are unknown. We have developed a new strategy that reveals a direct role for ubiquitin in the dynamics of splicing complexes. A ubiquitin mutant (I44A) that can enter the conjugation pathway but is compromised in downstream functions diminishes splicing activity by reducing the levels of the U4/U6-U5 small nuclear ribonucleoprotein (snRNP). Similarly, an inhibitor of ubiquitin's protein-protein interactions, ubistatin A, reduces U4/U6-U5 triple snRNP levels in vitro. When ubiquitin interactions are blocked, ATP-dependent disassembly of purified U4/U6-U5 particles is accelerated, indicating a direct role for ubiquitin in repressing U4/U6 unwinding. Finally, we show that the conserved splicing factor Prp8 is ubiquitinated within purified triple snRNPs. These results reveal a previously unknown ubiquitin-dependent mechanism for controlling the pre-mRNA splicing pathway.

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Figure 1: A mutant form of ubiquitin (Ub) inhibits pre-mRNA splicing activity in vitro.
Figure 2: The U2 snRNP-containing pre-spliceosome accumulates in the presence of I44A ubiquitin.
Figure 3: I44A ubiquitin blocks spliceosome assembly by interfering with U4/U6-U5 triple snRNP accumulation.
Figure 4: The ubiquitin (Ub) binding small molecule ubistatin A recapitulates the inhibitory effects of I44A ubiquitin.
Figure 5: Conjugated mutant I44A ubiquitin or the deubiquitinating cysteine protease USP2 derepresses U4/U6 unwinding in purified U4/U6-U5 snRNPs.
Figure 6: Affinity-purified U4/U6-U5 triple snRNPs contain Prp8-ubiquitin conjugates.
Figure 7: A model for ubiquitin's involvement in U4/U6-U5 triple snRNP accumulation and pre-mRNA splicing.

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Acknowledgements

We thank L. Hicke and D. Finley for strains and plasmids, J. Beggs for anti-Prp8 antibodies and the Drug Synthesis & Chemistry Branch of the National Cancer Institute for ubistatin A. We are grateful to L. Hicke, S. Prakash and members of the Sontheimer and Staley laboratories for advice and discussions, to S. Stevens (University of Texas, Austin) for communicating unpublished results and to M. French, D. Golden, J. Pellino, S. Prakash and J. Preall for critical reading of this manuscript. This work was supported by a US National Institutes of Health grant (GM62264) to J.P.S. and a US National Science Foundation CAREER Award (MCB-0093003) and a US National Institutes of Health grant (GM072830) to E.J.S.

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Authors and Affiliations

  1. Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2205 Tech Drive, Evanston, 60208, Illinois, USA

    Priya Bellare & Erik J Sontheimer

  2. Department of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, 60637, Illinois, USA

    Eliza C Small

  3. Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, 615 Charles E. Young Drive South, Los Angeles, 90095-1737, California, USA

    Xinhua Huang & James A Wohlschlegel

  4. Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, 60637, Illinois, USA

    Jonathan P Staley

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Contributions

P.B., E.C.S., X.H., J.A.W., J.P.S. and E.J.S. designed the experiments; P.B., E.C.S., X.H. and J.A.W. performed the experiments; P.B., E.C.S., X.H., J.A.W., J.P.S. and E.J.S. analyzed the data; and P.B., E.C.S., J.A.W., J.P.S. and E.J.S. wrote the paper.

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Correspondence to Erik J Sontheimer.

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Bellare, P., Small, E., Huang, X. et al. A role for ubiquitin in the spliceosome assembly pathway. Nat Struct Mol Biol 15, 444–451 (2008). https://doi.org/10.1038/nsmb.1401

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