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Structural evidence for consecutive Hel308-like modules in the spliceosomal ATPase Brr2

Abstract

Brr2 is a DExD/H-box helicase responsible for U4/U6 unwinding during spliceosomal activation. Brr2 contains two helicase-like domains, each of which is followed by a Sec63 domain with unknown function. We determined the crystal structure of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA helicase Hel308. This, together with sequence similarities between Brr2's helicase-like domains and domains 1–3 of Hel308, led us to hypothesize that Brr2 contains two consecutive Hel308-like modules (Hel308-I and Hel308-II). Our structural model and mutagenesis data suggest that Brr2 shares a similar helicase mechanism with Hel308. We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo. We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2–Prp8-CTR complex to U4/U6. Our results have important implications for the mechanism and regulation of Brr2's activity in splicing.

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Figure 1: The structure of yeast Brr2.
Figure 2: ATPase and helicase activities of Brr2 mutants.
Figure 3: Deletions of domains in Hel308-II lead to growth and splicing defects.
Figure 4: The interaction of Brr2 with RNA and the effect of Prp8-CTR on these interactions.
Figure 5: Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo.

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Acknowledgements

We are grateful to J. Staley (University of Chicago) and S. Stevens (University of Texas Austin) for providing yeast strains and plasmids; J. Beggs (University of Edinburgh) for anti-Brr2 antibody; and P. Fabrizio (Max Planck Institute for Biophysical Chemistry) for anti-Snu114 antibody. We thank B. Chong for help generating preliminary crystals. We thank the X-ray facility (supported in part by the University of Colorado Cancer Center) at the University of Colorado Denver. R.Z. is supported by a Kimmel Scholar award. 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 (NIH) to R.Z. (GM080334), an American Cancer Society Research Scholar grant to R.Z. (RSG-06-165-01-GMC), a NIH grant to C.G. (GM21119), an American Heart Association postdoctoral fellowship to L.Z. (0820036Z) and a National Institutes of General Medical Sciences postdoctoral fellowship to C.M. (F32GM077844).

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L.Z., T.X., C.M., L.-O.B., C.G., J.A.P. and R.Z. designed and analyzed the experiments; L.Z., T.X., C.M., L.-O.B., J.S., J.N., J.A.P. and R.Z. performed the experiments; R.Z. wrote the paper.

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Correspondence to Rui Zhao.

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Zhang, L., Xu, T., Maeder, C. et al. Structural evidence for consecutive Hel308-like modules in the spliceosomal ATPase Brr2. Nat Struct Mol Biol 16, 731–739 (2009). https://doi.org/10.1038/nsmb.1625

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