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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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and Staley laboratories for advice and discussions, to S. Stevens (University of Texas, Austin) for communicating unpublished results and to M. French, D. Golden, J. Pellino, S. Prakash and J. Preall for critical reading of this manuscript.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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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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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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DOI: https://doi.org/10.1038/nsmb.1401
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