Lin, Daniel H. and Correia, Ana R. and Cai, Sarah W. and Huber, Ferdinand M. and Jette, Claudia A. and Hoelz, André (2018) Structural and functional analysis of mRNA export regulation by the nuclear pore complex. Nature Communications, 9 . Art. No. 2319. ISSN 2041-1723. PMCID PMC5998080. doi:10.1038/s41467-018-04459-3. https://resolver.caltech.edu/CaltechAUTHORS:20180613-104057859
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Abstract
The nuclear pore complex (NPC) controls the passage of macromolecules between the nucleus and cytoplasm, but how the NPC directly participates in macromolecular transport remains poorly understood. In the final step of mRNA export, the DEAD-box helicase DDX19 is activated by the nucleoporins Gle1, Nup214, and Nup42 to remove Nxf1•Nxt1 from mRNAs. Here, we report crystal structures of Gle1•Nup42 from three organisms that reveal an evolutionarily conserved binding mode. Biochemical reconstitution of the DDX19 ATPase cycle establishes that human DDX19 activation does not require IP_6, unlike its fungal homologs, and that Gle1 stability affects DDX19 activation. Mutations linked to motor neuron diseases cause decreased Gle1 thermostability, implicating nucleoporin misfolding as a disease determinant. Crystal structures of human Gle1•Nup42•DDX19 reveal the structural rearrangements in DDX19 from an auto-inhibited to an RNA-binding competent state. Together, our results provide the foundation for further mechanistic analyses of mRNA export in humans.
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| Additional Information: | © 2018 The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Received 20 July 2017; Accepted 27 April 2018; Published 13 June 2018. Data availability: Atomic coordinates and related structure factors have been deposited in the Protein Data Bank with accession codes 6B4E, 6B4F, 6B4G, 6B4H, 6B4I, 6B4J, and 6B4K for the structures of S. cerevisiae Gle1CTD•Nup42GBM, H. sapiens Gle1CTD•Nup42GBM, C. thermophilum Gle1CTD•Nup42GBM, C. thermophilum Gle1CTD•Nup42GBM•IP6, H. sapiens Gle1CTD•Nup42GBM•DDX19∆N53(ADP), H. sapiens Gle1CTD•Nup42GBM•DDX19∆N53(AMP•PNP), and H. sapiens DDX19∆N53(AMP•PNP), respectively. Other data are available from the corresponding author upon reasonable request. We thank Alina Patke for critical reading of the manuscript, Karsten Thierbach for advice on yeast experiments, and Susan Wente for helpful discussions and sharing data prior to publication. We also acknowledge Jens Kaiser and the scientific staffs of SSRL Beamline 12-2, the National Institute of General Medical Sciences and National Cancer Institute Structural Biology Facility (GM/CA) at the Advanced Photon Source (APS), and the Frontier Microfocusing Macromolecular Crystallography (FMX) beamline at the National Synchrotron Light Source II (NSLS-II) for support with X-ray diffraction measurements. The operations at APS, SSRL, and NSLS-II are primarily supported by the Department of Energy. We acknowledge the Gordon and Betty Moore Foundation, the Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program for their support of the Molecular Observatory at the California Institute of Technology. GM/CA has been funded in whole or in part with federal funds from the National Cancer Institute (grant ACB-12002) and the National Institute of General Medical Sciences (grant AGM-12006). The crystal structure of ctGle1CTD•Nup42CTD•IP6 was determined at the CCP4/APS School for Macromolecular Crystallography (2016), which was supported by the National Cancer Institute (AGM-12006) and the National Institute of General Medical Science (ACB-12002). We acknowledge Navraj Pannu for his help. Valerie Altounian is acknowledged for creating the schematic illustration of the nuclear pore complex. D.H.L. and C.A.J. were supported by an NIH Research Service Award (5 T32 GM07616). D.H.L. and F.M.H. were supported by an Amgen Graduate Fellowship through the Caltech-Amgen Research Collaboration. S.W.C. was supported by a Margaret Leighton Summer Undergraduate Research Fellowship and a John Stauffer Summer Undergraduate Research Fellowship through the Caltech Student-Faculty Programs Office. A.H. is a Faculty Scholar of the Howard Hughes Medical Institute and an Investigator of the Heritage Medical Research Institute. A.H. was also supported by NIH grant R01-GM117360, Caltech startup funds, and a Teacher-Scholar Award of the Camille & Henry Dreyfus Foundation. Author Contributions: A.H. and D.H.L. conceived the project, designed the experiments, and analyzed all data. D.H.L., A.R.C., and S.W.C. purified proteins, performed biochemical analyses, and determined crystal structures. D.H.L., S.W.C., A.R.C., and F.M.H. generated yeast strains and performed yeast analyses. C.A.J. purified proteins and performed biochemical analysis of Nup155 amino acid substitution variants. A.H. supervised all experimental work. A.H. and D.H.L. wrote the manuscript with comments from all authors. The authors declare no competing interests. | ||||||||||||||||||||||||||
| Group: | Heritage Medical Research Institute | ||||||||||||||||||||||||||
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| PubMed Central ID: | PMC5998080 | ||||||||||||||||||||||||||
| DOI: | 10.1038/s41467-018-04459-3 | ||||||||||||||||||||||||||
| Record Number: | CaltechAUTHORS:20180613-104057859 | ||||||||||||||||||||||||||
| Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20180613-104057859 | ||||||||||||||||||||||||||
| Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | ||||||||||||||||||||||||||
| ID Code: | 87061 | ||||||||||||||||||||||||||
| Collection: | CaltechAUTHORS | ||||||||||||||||||||||||||
| Deposited By: | Tony Diaz | ||||||||||||||||||||||||||
| Deposited On: | 13 Jun 2018 17:57 | ||||||||||||||||||||||||||
| Last Modified: | 10 Mar 2022 16:49 |
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