Abstract
Superfamily 1 and superfamily 2 RNA helicases are ubiquitous messenger-RNA–protein complex (mRNP) remodelling enzymes that have critical roles in all aspects of RNA metabolism1,2. The superfamily 2 DEAD-box ATPase Dbp5 (human DDX19) functions in mRNA export and is thought to remodel mRNPs at the nuclear pore complex (NPC)3,4,5,6,7,8. Dbp5 is localized to the NPC via an interaction with Nup159 (NUP214 in vertebrates)3,4,5,8,9 and is locally activated there by Gle1 together with the small-molecule inositol hexakisphosphate (InsP6)10,11. Local activation of Dbp5 at the NPC by Gle1 is essential for mRNA export in vivo10,12; however, the mechanistic role of Dbp5 in mRNP export is poorly understood and it is not known how Gle1InsP6 and Nup159 regulate the activity of Dbp5. Here we report, from yeast, structures of Dbp5 in complex with Gle1InsP6, Nup159/Gle1InsP6 and RNA. These structures reveal that InsP6 functions as a small-molecule tether for the Gle1–Dbp5 interaction. Surprisingly, the Gle1InsP6–Dbp5 complex is structurally similar to another DEAD-box ATPase complex essential for translation initiation, eIF4G–eIF4A, and we demonstrate that Gle1InsP6 and eIF4G both activate their DEAD-box partner by stimulating RNA release. Furthermore, Gle1InsP6 relieves Dbp5 autoregulation and cooperates with Nup159 in stabilizing an open Dbp5 intermediate that precludes RNA binding. These findings explain how Gle1InsP6, Nup159 and Dbp5 collaborate in mRNA export and provide a general mechanism for DEAD-box ATPase regulation by Gle1/eIF4G-like activators.
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Protein Data Bank
Data deposits
Coordinates and structure factors for Dbp5–CTD(L327V)/InsP6/Gle1(H337R), Dbp5–CTD(L327V)/InsP6/Gle1(WT), Δ90Dbp5(L327V)/RNA/ADP•BeF;3, Δ90Dbp5/RNA/ADP•BeF;3, Δ90Dbp5(L327V)/InsP6/Gle1(H337R)/ADP and Δ90Dbp5(L327V)/InsP6/Gle1(H337R)/ADP/Nup159 have been deposited in the protein data bank under accession numbers 3PEU, 3PEV, 3PEW, 3PEY, 3PEX and 3PEZ, respectively.
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Acknowledgements
We thank J. Kirsch, J. Thorner, M. Welch, D. Wemmer, B. Krantz, J. Zorn and all J.M.B. and K.W. laboratory members for discussions and advice. We also thank J. Kuriyan, M. Welch and S. Marqusee for access to equipment and workspace; J. Tanamachi, G. Meigs and J. Holton at ALS beamline 8.3.1; and N. Echols for assistance with programs from the Yale Morph Server. Research was supported by the G Harold and Leila Y Mathers foundation (J.M.B.) and the NIH (K.W., R01GM58065 and RC1GM91533).
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B.M., N.D.T., J.M.B. and K.W. designed the experiments. Protein purifications for crystallization and biochemical assays were performed by B.M. Crystal screening, optimization and data collection were performed by B.M. and N.D.T. Data processing, structure solution and model building was performed by N.D.T. In vivo tests and in vitro RNA binding, ATPase assays and other biochemical assays were performed by B.M. with assistance from M.A.S. and K.J.H. B.M., N.D.T., J.M.B. and K.W. both analysed and interpreted the data and wrote the manuscript.
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Supplementary information
Supplementary Information
This file contains Supplementary Figures 1-6 with legends, Supplementary Tables 1-5 and additional references. (PDF 7788 kb)
Supplementary Movie 1
This movie shows an energy-minimized, linear interpolation of Dbp5 and Gle1 between the RNA-Δ90Dbp5 and Gle1-Δ90Dbp5 structural states. The movie begins in the RNA-Δ90Dbp5 state, with Gle1 and IP6 separated from the Dbp5 complex. The interpolation then transitions to the Gle1-Δ90Dbp5 state, in which the RNA and BeF3 are moved far from the ligand binding sites in order to mimic their release. The interpolation then plays in reverse to mimic RNA and ATP dependent closing of Dbp5 and release of Gle1. Note the large rigid body movement of the two RecA-like domains. Colouring is consistent with Fig. 1. (MOV 3881 kb)
Supplementary Movie 2
This movie shows the same interpolation as in Supplementary Movie 1, but focusing on the RNA binding site. Gle1-induced separation of the two RecA-like domains causes a significant structural change at the RNA binding site, disrupting the bipartite nucleic acid binding cleft and pulling RNA binding sidechains far apart. Colouring is consistent with Fig. 1. (MOV 2551 kb)
Supplementary Movie 3
This movie shows an energy-minimized, linear interpolation of Dbp5 between the RNA-Δ90Dbp5, Gle1-Δ90Dbp5, and Nup159-Δ90Dbp5-Gle1 structural states. The movie begins with Dbp5 in the RNA-Δ90Dbp5 state, then the interpolation transitions to the Gle1-Δ90Dbp5 state followed the Nup159-Δ90Dbp5-Gle1 state. The interpolation then plays in reverse. Note the large rigid body movement of the two RecA-like domains, which proceed along a common trajectory between the three discrete structural states. Colouring is consistent with Fig. 4. (MOV 1565 kb)
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Montpetit, B., Thomsen, N., Helmke, K. et al. A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP6 in mRNA export. Nature 472, 238–242 (2011). https://doi.org/10.1038/nature09862
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DOI: https://doi.org/10.1038/nature09862
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