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Nuclear export of pre-60S particles through the nuclear pore complex

Nature volume 618, pages 411–418 (2023)Cite this article

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Abstract

The nuclear pore complex (NPC) is the bidirectional gate that mediates the exchange of macromolecules or their assemblies between nucleus and cytoplasm1,2,3. The assembly intermediates of the ribosomal subunits, pre-60S and pre-40S particles, are among the largest cargoes of the NPC and the export of these gigantic ribonucleoproteins requires numerous export factors4,5. Here we report the cryo-electron microscopy structure of native pre-60S particles trapped in the channel of yeast NPCs. In addition to known assembly factors, multiple factors with export functions are also included in the structure. These factors in general bind to either the flexible regions or subunit interface of the pre-60S particle, and virtually form many anchor sites for NPC binding. Through interactions with phenylalanine-glycine (FG) repeats from various nucleoporins of NPC, these factors collectively facilitate the passage of the pre-60S particle through the central FG repeat network of the NPC. Moreover, in silico analysis of the axial and radial distribution of pre-60S particles within the NPC shows that a single NPC can take up to four pre-60S particles simultaneously, and pre-60S particles are enriched in the inner ring regions close to the wall of the NPC with the solvent-exposed surface facing the centre of the nuclear pore. Our data suggest a translocation model for the export of pre-60S particles through the NPC.

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Fig. 1: Native structure of pre-60S particle trapped in the NPC channel.
Fig. 2: Conformational changes in the pre-60S particle following nuclear export.
Fig. 3: Export receptors bind to the flexible regions of the pre-60S particle or to domain junctions of rRNA.
Fig. 4: Interaction networks between newly identified export factors and the pre-60S particle.
Fig. 5: Model of the pre-60S particle translocation through the NPC.

Data availability

The electron microscopy density map of the NPC-trapped pre-60S particle (EMD-34725), local refinement maps of export factors—including Mex67–Mtr2-1 (EMD-34638), Mex67–Mtr2-2 (EMD-34640), Mex67–Mtr2-3 (EMD-35767) and Crm1 (EMD-34641)—and Bud20 (EMD-35812) have been deposited in the Electron Microscopy Data Bank (http://www.ebi.ac.uk/pdbe/emdb/). Atomic coordinates of the NPC-trapped pre-60S particle (PDB 8HFR) and Mex67–Mtr2-1 (PDB 8HBN) have been deposited in the Protein Data Bank (http://www.rcsb.org).

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Acknowledgements

We thank the staff at both the cryo-EM centre of South University of Science and Technology and the Tsinghua University Branch of the National Protein Science Facility (Beijing) for their technical support on the cryo-EM and high-performance computation platforms. We thank Y. Zhang and Y. Li for helpful discussions. This work was supported by the National Natural Science Foundation of China (nos. 32071192/32271245 to S.-F.S. and 32230051 to N.G.) and by Tsinghua-Peking Center for Life Sciences (to L.Z. and Z.L.).

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  1. These authors contributed equally: Zongqiang Li, Shuaijiabin Chen

Authors and Affiliations

  1. School of Life Sciences, Southern University of Science and Technology, Shenzhen, China

    Zongqiang Li, Shuaijiabin Chen, Liang Zhao, Huiqin Xu, Xiaoyun Yang & Sen-Fang Sui

  2. State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, China

    Zongqiang Li, Shuaijiabin Chen, Liang Zhao, Guoqiang Huang & Sen-Fang Sui

  3. Cryo-EM Center, Southern University of Science and Technology, Shenzhen, China

    Peiyi Wang & Sen-Fang Sui

  4. State Key Laboratory of Membrane Biology, Peking-Tsinghua Joint Center for Life Sciences, School of Life Sciences, Peking University, Beijing, China

    Ning Gao

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Contributions

S.-F.S. supervised the project. Z.L. established the yeast strain and prepared samples for electron microscopy. L.Z., S.C., Z.L. and H.X. collected electron microscopy data. S.C. solved the structure of the pre-60S particle. S.C., L.Z. and G.H. performed electron microscopy analysis. S.C. and X.Y. analysed pre-60S particle distribution in the NPC channel. Z.L. and S.C. performed model building and structure refinement. S.C. performed AlphaFold-Multimer model generation. P.W. participated in electron microscopy data collection. Z.L., S.C., N.G. and S.-F.S. analysed the structure. Z.L. and S.C. jointly wrote the initial draft. N.G. and S.-F.S. edited the manuscript.

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Correspondence to Sen-Fang Sui.

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Extended data figures and tables

Extended Data Fig. 1 Cryo-EM data analysis of the NPC-trapped pre-60S ribosome.

a, A representative raw micrograph of the pre-60S containing NPC particles from similar 187,076 micrographs and representative 2D class averages of pre-60S are shown on the right from selected 279,900 particles. b, Local resolution map for a central slice (left) and the overall 3D reconstruction (right). c, Angular distribution of particles used for the 3D reconstruction. d, FSC curves for the cryo-EM map of NPC-trapped pre-60S particle. The threshold of 0.143 was used to determine the overall resolution of the map. e, Flowcharts for cryo-EM data processing. See “Methods” for details.

Extended Data Fig. 2 Density presentation of the NPC-trapped pre-60S particle.

Representative density maps of proteins and rRNAs from the NPC-trapped pre-60S particle. Different fashions of interactions between magnesium ions and pre-60S particle, and newly identified regions from ribosomal proteins are shown. Magnesium ions are displayed as spheres.

Extended Data Fig. 3 Identifications of Nmd3-NTD and Ecm1.

a, The position of Nmd3 in the NPC-trapped pre-60S particle is colored as orange, boxed by black frame and enlarged in (b). b, Fitting of Nmd3 into the map. The density is displayed as transparent surface representation. The NTD, CTD and the linker between NTD and CTD are color coded. c, Schematic representation of the domain structures of Nmd3. Domains are color coded as in (b). d, The extra density of two helices is located between L1 stalk and H34, boxed by red frame and enlarged in (e). e, Fitting of Ecm1 (aa 107 to 159) into the map. The density is displayed as transparent surface representation and bulky side chains of several residues are shown as models. f-g, Interactions between Ecm1 and uL2 and eL43 are shown as transparent surface representation (f) and cartoon (g). These results are consistent with the previous mass spectrometry identification49.

Extended Data Fig. 4 Changed regions of the 25S rRNA upon nuclear export of pre-60S particle.

Compared with the structures of pre-60S particles before nuclear export (left) and after nuclear export (right), four regions of rRNA (red) suffer dramatic conformational change including H38, L1 stalk, H69-71 and ES27. The unchanged bulk of rRNA is colored cyan.

Extended Data Fig. 5 Export factors and disease-related mutations.

a–g, Conserved cancer related mutations of the human export factors are mapped in the interfaces between pre-60S particle and Mex67-Mtr2-1 (a), Mex67-Mtr2-2 (b), Mex67-Mtr2-3 (c), Nmd3 (d), Arx1 (e), Ecm1 (f) and Tma16 (g). Mutation positions are shown as spheres.

Extended Data Fig. 6 Interaction between Gle2 and Rlp24 predicted by AlphaFold-Multimer.

a, The structure of Gle2 contains seven WD40 domains. b, Surface electrostatic potentials of Gle2 displays a positively charged patch. c-d, The predicted Gle2-Rlp24 structure (d) and the associated PAE (predicted alignment error) value (c). Confidence level of PAE: Dark green is good (low error), light green is bad (high error). e, The predicted Gle2-Rlp24 structure shows that the negatively charged loop of Rlp24 is embedded into the positively charged patch of Gle2. f, Representative top5 models of the predicted Gle2-Rlp24 structures from S.cerevisiae (left column) and the top5 models of the predicted Gle2 and C-terminal of Rlp24 from H.sapiens (middle column) and M.musculus (right column), suggesting that a conserved negatively charged motif in the C-terminal of Rlp24 is responsible for binding to Gle2. g, Structural comparison of NPC-trapped pre-60S particle (left panel: skyblue, Rlp24: limon, Gle2: teal) and pre-60S-Drg1 complex (middle panel: lightblue, Drg1 hexamer: salmon). The positional relationship of Rlp24, Gle2 and Drg1 hexamer is enlarged in top view (left) and side view (right, central slice) at the bottom.

Extended Data Fig. 7 Export factors bind to the flexible or protruding regions of rRNA and connect different domains of rRNAs.

a, Mex67-Mtr2-1 connects the domain IV and domain VI through interactions with ES27 and H101ES41. b, Mex67-Mtr2-2 simultaneously interacts with 5S RNA, H38 from domain II and the L1 stalk from domain V. c, The domain I, domain III and the 5.8S RNA are bridged by Mex67-Mtr2-3. d-e, Nmd3 is enclosed by domain II, domain IV and domain V through extensive interactions with H38, H68, H75, H80-81, H89 and H93. f, Ecm1 connects scaffolds domain II, domain IV and domain V by binding to H75, H62, H66, H67 and L1 stalk. g, Tma16 bridges the domain II, domain V and 5S RNA to prevent the pre-maturation of the CP and P0 stalk. h, Alb1 links the 5.8S RNA, domain I and domain VI through interactions with H2, H19, H24, H25, H94 and H98. i, Bud20 is clamped between the H61-H63 from domain IV and H96 from domain VI. j, Arx1 is sandwiched by the flexible ES27 from domain IV, H12 and H24 from domain I and H59 from domain III. Export factors and rRNAs are shown in surface and cartoon representation, respectively. Different export factors and rRNA domains are color coded.

Extended Data Fig. 8 Export factors interact with NPC.

a, The map of NPC-trapped pre-60S particle displayed in a low threshold, highlighting numerous extra densities connected to export factors. These densities could be assigned to the central FG Nups and/or NTFs with/without cargoes (CFNC) including FG-repeats omitted from the wall of NPC. The eL19 and YBL028C obviously interact with the extra density, suggesting a possible role in direct interactions with the NPC. b–e, Local refinements of Gle2 (b), Mex67-Mtr2-2 (c), Mex67-Mtr2-3 (d) and Bud20 (e) further show the interactions between these export factors and the NPC.

Extended Data Fig. 9 Heterogeneity analysis, distribution and remapping of representative pre-60S particles and three bound Mex67-Mtr2 respectively.

a-b, Skip-align classifications of the three Mex67-Mtr2 modules (a) and the entire pre-60S particles (b). c–e, The radial distribution of Mex67-Mtr2-1 (c), Mex67-Mtr2-2 (d) and Mex67-Mtr2-3 (e) within the NPC. f, The pre-60S particles and three Mex67-Mtr2 are remapped back into the same NPC showing in top-view. The relative position of the pre-60S particles (red arrow), Mex67-Mtr2-1 (blue arrow), Mex67-Mtr2-2 (yellow arrow) and Mex67-Mtr2-3 (black arrow) are indicated.

Extended Data Fig. 10 Measurement diagram of the relative position of pre-60S particles in the NPC.

d1 represents the projection distance between the center of NPC and the center of pre-60S in the micrograph, d2 is the projection of d1 on the symmetry axis of NPC, that is, the distance from the center of pre-60S to the equatorial plane of NPC, and d3 is the distance from the center of pre-60S to the symmetry axis of NPC.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table

Supplementary information

Supplementary Tables

The file contains information on model building for NPC-trapped pre-60S particles (Supplementary Table 1) and conserved mutants of nuclear exporters in cancers (Supplementary Table 2).

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Li, Z., Chen, S., Zhao, L. et al. Nuclear export of pre-60S particles through the nuclear pore complex. Nature 618, 411–418 (2023). https://doi.org/10.1038/s41586-023-06128-y

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