Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform

The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating hybrid proteomic and structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14‐MDa Nup82‐Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC. Support or Funding Information Simons Foundation grant 349247 (Simons Electron Microscopy Center, NYSBC), the NSERC, Canadian Institutes of Health Research grant MOP‐232642, and the Canadian Foundation for Innovation (D.Z.), as well as NSF graduate research fellowship 1650113 (I.E.C.) and NIH grants U54 GM103511(B.T.C., A.S., and M.P.R.), R01 GM112108 (M.P.R.), P41 GM109824 (M.P.R., A.S., and B.T.C.), P41 GM103314 (B.T.C.), and R01 GM083960 (A.S.).Highlights • Integrative structure at 9 Å precision of the endogenous Nup82 holo‐complex • Molecular architecture of the conserved 1.8‐MDa cytoplasmic mRNA export platform • Structural framework to understand the mRNP remodeling and export processes • mRNP remodeling machinery is positioned over the NPC's central channel, not in filaments1Integrative Structure of the Core Nup82 Holo‐complex (A) Three views of the localization probability density map corresponding to the Nup82 holo‐complex ensemble are shown (light gray), with a single representative ribbon structure embedded; the proteins, subunits, and different structural features of the complex are indicated. (B) Exploded view of the Nup82 holo‐complex subunits and protein components2Nup82 Holo‐complex Structure Validation (A) Circos‐XL plots showing the distribution of all DSS (top plot) or EDC (bottom plot) cross‐links mapping within the core of the Nup82 holo‐complex. (B) Structure of the Nup82 holo‐complex showing the cross‐links falling within the expected C α ‐C α maximum distance threshold (blue) or outside of that threshold (orange). (C) GFP mass‐tagging analysis of the Nup82 holo‐complex. (D) SAXS analysis of the Nup82 (572–690) fragment3Molecular Architecture of the Cytoplasmic mRNA Export and Remodeling Platform(A) Structure of the Nup82‐Nup84 complex assembly.(B) Molecular architecture of the cytoplasmic mRNA export and remodeling platform.4mRNA Export Phenotype in Nup84 Complex Mutants Is Associated with Defective Incorporation of the Nup82 Holo‐complex into the NPC (A) The mRNA export defect phenotype was quantified and plotted (B) Mapping of the color code described in (A) into the Nup84 complex components. (C) The severity of nuclear mRNA accumulation phenotype (detailed in A and B) (D) Subcellular localization of Nup82‐GFP in Nup84 complex truncation mutants.5Position of the Nup82‐Nup84 Complex Assembly within the NPC (A) Fitting to the yeast NPC map. (B) Comparison with the human NPC tomographic cryo‐EM map (EMDB: 2444) (C) Comparison with the mutant human NPC tomographic cryo‐EM map (EMDB: 3104)6The Nup82‐Nup84 Complex Assembly Acts as a Scaffold to Organize the FG Region and mRNP Remodeling Sites in the NPCTop: model for the arrangement of the FG regions associated to the Nup82 holo‐complex. FG regions were modeled using molecular dynamics.