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ATP-dependent unwinding of U4/U6 snRNAs by the Brr2 helicase requires the C terminus of Prp8

Nature Structural & Molecular Biology volume 16, pages 42–48 (2009)Cite this article

Abstract

The spliceosome is a highly dynamic machine requiring multiple RNA-dependent ATPases of the DExD/H-box family. A fundamental unanswered question is how their activities are regulated. Brr2 function is necessary for unwinding the U4/U6 duplex, a step essential for catalytic activation of the spliceosome. Here we show that Brr2-dependent dissociation of U4/U6 snRNAs in vitro is activated by a fragment from the C terminus of the U5 snRNP protein Prp8. In contrast to its helicase-stimulating activity, this fragment inhibits Brr2 U4/U6-dependent ATPase activity. Notably, U4/U6 unwinding activity is not stimulated by fragments carrying alleles of prp8 that in humans confers an autosomal dominant form of retinitis pigmentosa. Because Brr2 activity must be restricted to prevent premature catalytic activation, our results have important implications for fidelity maintenance in the spliceosome.

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Figure 1: A C-terminal fragment of Prp8 was able to directly interact with Brr2.
Figure 2: Retinitis pigmentosa mutants of Prp8 have variable effects on growth and ability of Prp8 to bind Brr2.
Figure 3: Unwinding of U4/U6 by Brr2 is stimulated by addition of Prp8-CTF.
Figure 4: ATPase activity of RNA-dependent Brr2 is inhibited by Prp8-CTF.
Figure 5: ATPase activity of RNA-dependent Brr2 is affected differently by RP mutations.

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Acknowledgements

We would like to thank S. Stevens (University of Texas, Austin) for the Brr2-TAP overexpression plasmid; L. Rice for assistance with MS; J. Credle for assistance with protein purification; R. Luhrmann (Max-Planck-Institute for Biophysical Chemistry) for the gift of anti-GST antibody; and J. Weissman (University of California, San Francisco) for the gift of anti-CBP antibody. We also thank J. Abelson and J. Pleiss for helpful discussions and members of the Guthrie laboratory for critical reading of the manuscript. C.G. is an American Cancer Society Research Professor of Molecular Genetics. This work was supported by a grant from the US National Institutes of Health to C.G. (GM21119), a National Institutes of General Medical Sciences postdoctoral fellowship to C.M. (F32GM077844), an American Cancer Society postdoctoral fellowship (PF-01-236-01-GMC) and Boyer Funds to A.K.K.

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  1. Alan K Kutach

    Present address: Present address: Roche Palo Alto, 3431 Hillview Ave, Palo Alto, California 94394, USA.,

  2. Corina Maeder and Alan K Kutach: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of California, San Francisco, 600 16th Street, Genentech Hall, San Francisco, 94143, California, USA

    Corina Maeder, Alan K Kutach & Christine Guthrie

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  1. Corina Maeder
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C.M. and A.K.K. performed and analyzed the experiments; C.M., A.K.K. and C.G. designed the experiments and wrote the paper.

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Correspondence to Christine Guthrie.

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Maeder, C., Kutach, A. & Guthrie, C. ATP-dependent unwinding of U4/U6 snRNAs by the Brr2 helicase requires the C terminus of Prp8. Nat Struct Mol Biol 16, 42–48 (2009). https://doi.org/10.1038/nsmb.1535

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