Nuclear export of mRNAs requires loading the mRNP to the transporter Mex67/Mtr2 in the nucleoplasm, controlled access to the pore by the basket-localised TREX-2 complex and mRNA release at the cytoplasmic site by the DEAD-box RNA helicase Dbp5. Asymmetric localisation of nucleoporins (NUPs) and transport components as well as the ATP dependency of Dbp5 ensure unidirectionality of transport. Trypanosomes possess homologues of the mRNA transporter Mex67/Mtr2, but not of TREX-2 or Dbp5. Instead, nuclear export is likely fuelled by the GTP/GDP gradient created by the Ran GTPase. However, it remains unclear, how directionality is achieved since the current model of the trypanosomatid pore is mostly symmetric. We have revisited the architecture of the trypanosome nuclear pore complex using a novel combination of expansion microscopy, proximity labelling and streptavidin imaging. We could confidently assign the NUP76 complex, a known Mex67 interaction platform, to the cytoplasmic site of the pore and the NUP64/NUP98/NUP75 complex to the nuclear site. Having defined markers for both sites of the pore, we set out to map all 75 trypanosome proteins with known nuclear pore localisation to a subregion of the pore using mass spectrometry data from proximity labelling. This approach defined several further proteins with a specific localisation to the nuclear site of the pore, including proteins with predicted structural homology to TREX-2 components. We mapped the components of the Ran-based mRNA export system to the nuclear site (RanBPL), the cytoplasmic site (RanGAP, RanBP1) or both (Ran, MEX67). Lastly, we demonstrate, by deploying an auxin degron system, that NUP76 holds an essential role in mRNA export consistent with a possible functional orthology to NUP82/88. Altogether, the combination of proximity labelling with expansion microscopy revealed an asymmetric architecture of the trypanosome nuclear pore supporting inherent roles for directed transport. Our approach delivered novel nuclear pore associated components inclusive positional information, which can now be interrogated for functional roles to explore trypanosome-specific adaptions of the nuclear basket, export control, and mRNP remodelling.

Biocenter University of Würzburg Würzburg Germany

Department of Parasitology Faculty of Science Charles University Prague Biocev Vestec Prague Czech Republic

Zobrazit více v PubMed

Wente SR, Rout MP. The nuclear pore complex and nuclear transport. Cold Spring Harb Perspect Biol. 2010;2:a000562. doi: 10.1101/cshperspect.a000562 PubMed DOI PMC

Hampoelz B, Andres-Pons A, Kastritis P, Beck M. Structure and Assembly of the Nuclear Pore Complex. Annu Rev Biophys. 2019;48:1–22. doi: 10.1146/annurev-biophys-052118-115308 PubMed DOI

Schwartz TU. The Structure Inventory of the Nuclear Pore Complex. J Mol Biol. 2016;428:1986–2000. doi: 10.1016/j.jmb.2016.03.015 PubMed DOI PMC

Lin DH, Hoelz A. The Structure of the Nuclear Pore Complex (An Update). Annu Rev Biochem. 2019;88:1–59. doi: 10.1146/annurev-biochem-062917-011901 PubMed DOI PMC

Terry LJ, Wente SR. Flexible Gates: Dynamic Topologies and Functions for FG Nucleoporins in Nucleocytoplasmic Transport. Eukaryot Cell. 2009;8:1814–1827. doi: 10.1128/EC.00225-09 PubMed DOI PMC

Wing CE, Fung HYJ, Chook YM. Karyopherin-mediated nucleocytoplasmic transport. Nat Rev Mol Cell Biol. 2022;23:307–328. doi: 10.1038/s41580-021-00446-7 PubMed DOI PMC

Chen S, Jiang Q, Fan J, Cheng H. Nuclear mRNA export. Acta Biochim Biophys Sin. 2024. doi: 10.3724/abbs.2024145 PubMed DOI PMC

Magistris PD. The Great Escape: mRNA Export through the Nuclear Pore Complex. Int J Mol Sci. 2021;22:11767. doi: 10.3390/ijms222111767 PubMed DOI PMC

Ashkenazy-Titelman A, Shav-Tal Y, Kehlenbach RH. Into the basket and beyond: the journey of mRNA through the nuclear pore complex. Biochem J. 2020;477:23–44. doi: 10.1042/BCJ20190132 PubMed DOI

Xie Y, Ren Y. Mechanisms of nuclear mRNA export: A structural perspective. Traffic. 2019;20:829–840. doi: 10.1111/tra.12691 PubMed DOI PMC

Stankunas E, Köhler A. Docking a flexible basket onto the core of the nuclear pore complex. Nat Cell Biol. 2024;26:1504–1519. doi: 10.1038/s41556-024-01484-x PubMed DOI PMC

Jani D, Valkov E, Stewart M. Structural basis for binding the TREX2 complex to nuclear pores, GAL1 localisation and mRNA export. Nucleic Acids Res. 2014;42:6686–6697. doi: 10.1093/nar/gku252 PubMed DOI PMC

Umlauf D, Bonnet J, Waharte F, Fournier M, Stierle M, Fischer B, et al.. The human TREX-2 complex is stably associated with the nuclear pore basket. J Cell Sci. 2013;126:2656–2667. doi: 10.1242/jcs.118000 PubMed DOI

Stewart M. Macromolecular Protein Complexes II: Structure and Function. Subcell Biochem. 2020;93:461–470. doi: 10.1007/978-3-030-28151-9_15 DOI

Fernandez-Martinez J, Kim SJ, Shi Y, Upla P, Pellarin R, Gagnon M, et al.. Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform. Cell. 2016;167:1215–1228.e25. doi: 10.1016/j.cell.2016.10.028 PubMed DOI PMC

Bley CJ, Nie S, Mobbs GW, Petrovic S, Gres AT, Liu X, et al.. Architecture of the cytoplasmic face of the nuclear pore. Science. 2022;376:eabm9129. doi: 10.1126/science.abm9129 PubMed DOI PMC

Kim SJ, Fernandez-Martinez J, Nudelman I, Shi Y, Zhang W, Raveh B, et al.. Integrative structure and functional anatomy of a nuclear pore complex. Nature. 2018;555:475–482. doi: 10.1038/nature26003 PubMed DOI PMC

Allegretti M, Zimmerli CE, Rantos V, Wilfling F, Ronchi P, Fung HKH, et al.. In-cell architecture of the nuclear pore and snapshots of its turnover. Nature. 2020;586:796–800. doi: 10.1038/s41586-020-2670-5 PubMed DOI

Kosinski J, Mosalaganti S, von Appen A, Teimer R, DiGuilio AL, Wan W, et al.. Molecular architecture of the inner ring scaffold of the human nuclear pore complex. Science (New York, NY). 2016;352:363–365. doi: 10.1126/science.aaf0643 PubMed DOI PMC

Lin DH, Stuwe T, Schilbach S, Rundlet EJ, Perriches T, Mobbs G, et al.. Architecture of the symmetric core of the nuclear pore. Science (New York, NY). 2016;352:aaf1015. doi: 10.1126/science.aaf1015 PubMed DOI PMC

Mosalaganti S, Kosinski J, Albert S, Schaffer M, Strenkert D, Salomé PA, et al.. In situ architecture of the algal nuclear pore complex. Nat Commun. 2018;9:2361. doi: 10.1038/s41467-018-04739-y PubMed DOI PMC

Fernandez-Martinez J, Rout MP. One Ring to Rule them All? Structural and Functional Diversity in the Nuclear Pore Complex. Trends Biochem Sci. 2021;46:595–607. doi: 10.1016/j.tibs.2021.01.003 PubMed DOI PMC

Akey CW, Singh D, Ouch C, Echeverria I, Nudelman I, Varberg JM, et al.. Comprehensive structure and functional adaptations of the yeast nuclear pore complex. Cell. 2022;185:361–378.e25. doi: 10.1016/j.cell.2021.12.015 PubMed DOI PMC

Singh D, Soni N, Hutchings J, Echeverria I, Shaikh F, Duquette M, et al.. The molecular architecture of the nuclear basket. Cell. 2024. doi: 10.1016/j.cell.2024.07.020 PubMed DOI PMC

Niepel M, Strambio-de-Castillia C, Fasolo J, Chait BT, Rout MP. The nuclear pore complex–associated protein, Mlp2p, binds to the yeast spindle pole body and promotes its efficient assembly. J Cell Biol. 2005;170:225–235. doi: 10.1083/jcb.200504140 PubMed DOI PMC

Galy V, Gadal O, Fromont-Racine M, Romano A, Jacquier A, Nehrbass U. Nuclear retention of unspliced mRNAs in yeast is mediated by perinuclear Mlp1. Cell. 2004;116:63–73. doi: 10.1016/s0092-8674(03)01026-2 PubMed DOI

Padilla-Mejia NE, Field MC. Evolutionary, structural and functional insights in nuclear organisation and nucleocytoplasmic transport in trypanosomes. FEBS Lett. 2023;597:2501–2518. doi: 10.1002/1873-3468.14747 PubMed DOI PMC

Obado SO, Field MC, Rout MP. Comparative interactomics provides evidence for functional specialization of the nuclear pore complex. Nucleus (Austin, Tex). 2017;8:1–13. doi: 10.1080/19491034.2017.1313936 PubMed DOI PMC

DeGrasse JA, DuBois KN, Devos D, Siegel TN, Sali A, Field MC, et al.. Evidence for a Shared Nuclear Pore Complex Architecture That Is Conserved from the Last Common Eukaryotic Ancestor. Mol Cell Proteomics. 2009;8:2119–2130. doi: 10.1074/mcp.M900038-MCP200 PubMed DOI PMC

Obado SO, Brillantes M, Uryu K, Zhang W, Ketaren NE, Chait BT, et al.. Interactome Mapping Reveals the Evolutionary History of the Nuclear Pore Complex. Schwartz TU, editor. PLoS Biol. 2016;14:e1002365. doi: 10.1371/journal.pbio.1002365.s013 PubMed DOI PMC

Makarov AA, Padilla-Mejia NE, Field MC. Evolution and diversification of the nuclear pore complex. Biochem Soc Trans. 2021;49:1601–1619. doi: 10.1042/BST20200570 PubMed DOI PMC

Butterfield ER, Obado SO, Scutts SR, Zhang W, Chait BT, Rout MP, et al.. A lineage-specific protein network at the trypanosome nuclear envelope. Nucleus. 2024;15:2310452. doi: 10.1080/19491034.2024.2310452 PubMed DOI PMC

Hurwitz ME, Blobel G. NUP82 is an essential yeast nucleoporin required for poly(A)+ RNA export. J Cell Biol. 1995;130:1275–1281. doi: 10.1083/jcb.130.6.1275 PubMed DOI PMC

Grandi P, Emig S, Weise C, Hucho F, Pohl T, Hurt EC. A novel nuclear pore protein Nup82p which specifically binds to a fraction of Nsp1p. J Cell Biol. 1995;130:1263–1273. doi: 10.1083/jcb.130.6.1263 PubMed DOI PMC

Billington K, Halliday C, Madden R, Dyer P, Barker AR, Moreira-Leite FF, et al.. Genome-wide subcellular protein map for the flagellate parasite Trypanosoma brucei. Nat Microbiol. 2023;8:533–547. doi: 10.1038/s41564-022-01295-6 PubMed DOI PMC

Shanmugasundram A, Starns D, Böhme U, Amos B, Wilkinson PA, Harb OS, et al.. TriTrypDB: An integrated functional genomics resource for kinetoplastida. PLoS Negl Trop Dis. 2023;17:e0011058. doi: 10.1371/journal.pntd.0011058 PubMed DOI PMC

Paysan-Lafosse T, Blum M, Chuguransky S, Grego T, Pinto BL, Salazar GA, et al.. InterPro in 2022. Nucleic Acids Res. 2022;51:D418–D427. doi: 10.1093/nar/gkac993 PubMed DOI PMC

Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015;10:845–858. doi: 10.1038/nprot.2015.053 PubMed DOI PMC

Wheeler RJ. A resource for improved predictions of Trypanosoma and Leishmania protein three-dimensional structure. PLoS ONE. 2021;16:e0259871. doi: 10.1371/journal.pone.0259871 PubMed DOI PMC

van Kempen M, Kim SS, Tumescheit C, Mirdita M, Lee J, Gilchrist CLM, et al.. Fast and accurate protein structure search with Foldseek. Nat Biotechnol. 2024;42:243–246. doi: 10.1038/s41587-023-01773-0 PubMed DOI PMC

Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al.. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–589. doi: 10.1038/s41586-021-03819-2 PubMed DOI PMC

Evans R, O’Neill M, Pritzel A, Antropova N, Senior A, Green T, et al.. Protein complex prediction with AlphaFold-Multimer. bioRxiv. 2022; 2021.10.04.463034. doi: 10.1101/2021.10.04.463034 DOI

Meng EC, Goddard TD, Pettersen EF, Couch GS, Pearson ZJ, Morris JH, et al.. UCSF ChimeraX: Tools for structure building and analysis. Protein Sci. 2023;32:e4792. doi: 10.1002/pro.4792 PubMed DOI PMC

Gu Z. Complex heatmap visualization iMeta. 2022;1:e43. doi: 10.1002/imt2.43 PubMed DOI PMC

Brun R, Schönenberger. Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 1979;36:289–292. PubMed

Dean S, Sunter J, Wheeler RJ, Hodkinson I, Gluenz E, Gull K. A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open Biol. 2015;5:140197. doi: 10.1098/rsob.140197 PubMed DOI PMC

Burkard GS, Jutzi P, Roditi I. Genome-wide RNAi screens in bloodstream form trypanosomes identify drug transporters. 2011;175:91–94. doi: 10.1016/j.molbiopara.2010.09.002 PubMed DOI

Rotureau B, Gego A, Carme B. Trypanosomatid protozoa: A simplified DNA isolation procedure. Exp Parasitol. 2005;111:207–209. doi: 10.1016/j.exppara.2005.07.003 PubMed DOI

Sunter J, Wickstead B, Gull K, Carrington M. A new generation of T7 RNA polymerase-independent inducible expression plasmids for Trypanosoma brucei. PLoS ONE. 2012;7:e35167. doi: 10.1371/journal.pone.0035167 PubMed DOI PMC

Gabiatti BP, Freire ER, Odenwald J, Holetz F, Carrington M, Kramer S, et al.. Intron-loss in Kinetoplastea correlates with a non-functional EJC and loss of NMD factors. BioRxive. 2024. Available from: https://www.biorxiv.org/content/10.1101/2024.03.25.586568v1. DOI

Bastin P, Bagherzadeh Z, Matthews KR, Gull K. A novel epitope tag system to study protein targeting and organelle biogenesis in Trypanosoma brucei. 1996;77:235–239. doi: 10.1016/0166-6851(96)02598-4 PubMed DOI

KOHL L, Sherwin T, Gull K. Assembly of the Paraflagellar Rod and the Flagellum Attachment Zone Complex During the Trypanosoma brucei Cell Cycle. J Eukaryot Microbiol. 1999;46:105–109. doi: 10.1111/j.1550-7408.1999.tb04592.x PubMed DOI

Odenwald J, Gabiatti B, Braune S, Shen S, Zoltner M, Kramer S. Detection of TurboID fusion proteins by fluorescent streptavidin outcompetes antibody signals and visualises targets not accessible to antibodies. Elife. 2024;13:RP95028. doi: 10.7554/eLife.95028 PubMed DOI PMC

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al.. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019 PubMed DOI PMC

Moreira CM, Kelemen CD, Obado SO, Zahedifard F, Zhang N, Holetz FB, et al.. Impact of inherent biases built into proteomic techniques: Proximity labeling and affinity capture compared. J Biol Chem. 2023;299:102726. doi: 10.1016/j.jbc.2022.102726 PubMed DOI PMC

Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M. Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ*. Mol Cell Proteomics. 2014;13:2513–2526. doi: 10.1074/mcp.M113.031591 PubMed DOI PMC

Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008;26:1367–1372. doi: 10.1038/nbt.1511 PubMed DOI

Aslett M, Aurrecoechea C, Berriman M, Brestelli J, Brunk BP, Carrington M, et al.. TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res. 2010;38:D457–D462. doi: 10.1093/nar/gkp851 PubMed DOI PMC

Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, et al.. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods. 2016;13:731–740. doi: 10.1038/nmeth.3901 PubMed DOI

Zoltner M, Pino RCD, Field MC. Sorting the Muck from the Brass: Analysis of Protein Complexes and Cell Lysates. Methods Mol Biol (Clifton N J). 2020;2116:645–653. doi: 10.1007/978-1-0716-0294-2_38 PubMed DOI

Perez-Riverol Y, Csordas A, Bai J, Bernal-Llinares M, Hewapathirana S, Kundu DJ, et al.. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 2019;47:D442–D450. doi: 10.1093/nar/gky1106 PubMed DOI PMC

Branon TC, Bosch JA, Sanchez AD, Udeshi ND, Svinkina T, Carr SA, et al.. Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol. 2018;36:880–887. doi: 10.1038/nbt.4201 PubMed DOI PMC

Tillberg PW, Chen F, Piatkevich KD, Zhao Y, Yu C-C, English BP, et al.. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat Biotechnol. 2016;34:987–992. doi: 10.1038/nbt.3625 PubMed DOI PMC

Gambarotto D, Zwettler FU, Guennec ML, Schmidt-Cernohorska M, Fortun D, Borgers S, et al.. Imaging cellular ultrastructures using expansion microscopy (U-ExM). Nat Methods. 2019;16:71–74. doi: 10.1038/s41592-018-0238-1 PubMed DOI PMC

Kalichava A, Ochsenreiter T. Ultrastructure expansion microscopy in Trypanosoma brucei. Open Biol. 2021;11:210132. doi: 10.1098/rsob.210132 PubMed DOI PMC

Gorilak P, Pružincová M, Vachova H, Olšinová M, Cernohorska MS, Varga V. Expansion microscopy facilitates quantitative super-resolution studies of cytoskeletal structures in kinetoplastid parasites. Open Biol. 2021;11:210131. doi: 10.1098/rsob.210131 PubMed DOI PMC

de Hernández MA, Peralta GM, Vena R, Alonso VL. Ultrastructural Expansion Microscopy in Three In Vitro Life Cycle Stages of Trypanosoma cruzi. J Vis Exp. 2023. doi: 10.3791/65381 PubMed DOI

Maishman L, Obado SO, Alsford S, Bart J-M, Chen W-M, Ratushny AV, et al.. Co-dependence between trypanosome nuclear lamina components in nuclear stability and control of gene expression. Nucleic Acids Res. 2016;44:10554–10570. doi: 10.1093/nar/gkw751 PubMed DOI PMC

Schwede A, Manful T, Jha BA, Helbig C, Bercovich N, Stewart M, et al.. The role of deadenylation in the degradation of unstable mRNAs in trypanosomes. Nucleic Acids Res. 2009;37:5511–5528. doi: 10.1093/nar/gkp571 PubMed DOI PMC

Obado SO, Stein M, Hegedűsová E, Zhang W, Hutchinson S, Brillantes M, et al.. Mex67 paralogs mediate division of labor in trypanosome RNA processing and export. bioRxiv. 2022; 2022.06.27.497849. doi: 10.1101/2022.06.27.497849 DOI

Obado SO, Rout MP, Field MC. Sending the message: specialized RNA export mechanisms in trypanosomes. Trends Parasitol. 2022;38:854–867. doi: 10.1016/j.pt.2022.07.008 PubMed DOI PMC

Brasseur A, Bayat S, Chua XL, Zhang Y, Zhou Q, Low BC, et al.. The bi-lobe-associated LRRP1 regulates Ran activity in Trypanosoma brucei. J Cell Sci. 2014;127:4846–4856. doi: 10.1242/jcs.148015 PubMed DOI

Li Z, Chen S, Zhao L, Huang G, Xu H, Yang X, et al.. Nuclear export of pre-60S particles through the nuclear pore complex. Nature. 2023;618:411–418. doi: 10.1038/s41586-023-06128-y PubMed DOI

Wu J, Matunis MJ, Kraemer D, Blobel G, Coutavas E. Nup358, a Cytoplasmically Exposed Nucleoporin with Peptide Repeats, Ran-GTP Binding Sites, Zinc Fingers, a Cyclophilin A Homologous Domain, and a Leucine-rich Region *. J Biol Chem. 1995;270:14209–14213. doi: 10.1074/jbc.270.23.14209 PubMed DOI

Mahajan R, Delphin C, Guan T, Gerace L, Melchior F. A Small Ubiquitin-Related Polypeptide Involved in Targeting RanGAP1 to Nuclear Pore Complex Protein RanBP2. Cell. 1997;88:97–107. doi: 10.1016/s0092-8674(00)81862-0 PubMed DOI

Lindsay ME, Plafker K, Smith AE, Clurman BE, Macara IG. Npap60/Nup50 Is a Tri-Stable Switch that Stimulates Importin-α:β-Mediated Nuclear Protein Import. Cell. 2002;110:349–360. doi: 10.1016/s0092-8674(02)00836-x PubMed DOI

Nakielny S, Shaikh S, Burke B, Dreyfuss G. Nup153 is an M9-containing mobile nucleoporin with a novel Ran-binding domain. EMBO J. 1999;18:1982–1995. doi: 10.1093/emboj/18.7.1982 PubMed DOI PMC

Mackmull M, Klaus B, Heinze I, Chokkalingam M, Beyer A, Russell RB, et al.. Landscape of nuclear transport receptor cargo specificity. Mol Syst Biol. 2017;13:962. doi: 10.15252/msb.20177608 PubMed DOI PMC

Kose S, Furuta M, Imamoto N. Hikeshi, a Nuclear Import Carrier for Hsp70s, Protects Cells from Heat Shock-Induced Nuclear Damage. Cell. 2012;149:578–589. doi: 10.1016/j.cell.2012.02.058 PubMed DOI

Buhlmann M, Walrad P, Rico E, Ivens A, Capewell P, Naguleswaran A, et al.. NMD3 regulates both mRNA and rRNA nuclear export in African trypanosomes via an XPOI-linked pathway. Nucleic Acids Res. 2015. doi: 10.1093/nar/gkv330 PubMed DOI PMC

Alsford S, Turner D, Obado S, Sanchez-Flores A, Glover L, Berriman M, et al.. High throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res. 2011. doi: 10.1101/gr.115089.110 PubMed DOI PMC

Güther MLS, Urbaniak MD, Tavendale A, Prescott A, Ferguson MAJ. High-confidence glycosome proteome for procyclic form Trypanosoma brucei by epitope-tag organelle enrichment and SILAC proteomics. J Proteome Res. 2014;13:2796–2806. doi: 10.1021/pr401209w PubMed DOI PMC

Lo Y-H, Sobhany M, Hsu AL, Ford BL, Krahn JM, Borgnia MJ, et al.. Cryo-EM structure of the essential ribosome assembly AAA-ATPase Rix7. Nat Commun. 2019;10:513. doi: 10.1038/s41467-019-08373-0 PubMed DOI PMC

Anderson J, Phan L, Hinnebusch AG. The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 2000;97:5173–5178. doi: 10.1073/pnas.090102597 PubMed DOI PMC

Kramarz K, Schirmeisen K, Boucherit V, Saada AA, Lovo C, Palancade B, et al.. The nuclear pore primes recombination-dependent DNA synthesis at arrested forks by promoting SUMO removal. Nat Commun. 2020;11:5643. doi: 10.1038/s41467-020-19516-z PubMed DOI PMC

Schneider M, Hellerschmied D, Schubert T, Amlacher S, Vinayachandran V, Reja R, et al.. The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression. Cell. 2015;162:1016–1028. doi: 10.1016/j.cell.2015.07.059 PubMed DOI PMC

Papai G, Frechard A, Kolesnikova O, Crucifix C, Schultz P, Ben-Shem A. Structure of SAGA and mechanism of TBP deposition on gene promoters. Nature. 2020;577:711–716. doi: 10.1038/s41586-020-1944-2 PubMed DOI

Ellisdon AM, Dimitrova L, Hurt E, Stewart M. Structural basis for the assembly and nucleic acid binding of the TREX-2 transcription-export complex. Nat Struct Mol Biol. 2012;19:328–336. doi: 10.1038/nsmb.2235 PubMed DOI PMC

Yaseen NR, Blobel G. Two distinct classes of Ran-binding sites on the nucleoporin Nup-358. Proc Natl Acad Sci U S A. 1999;96:5516–5521. doi: 10.1073/pnas.96.10.5516 PubMed DOI PMC

Higa MM, Alam SL, Sundquist WI, Ullman KS. Molecular Characterization of the Ran-binding Zinc Finger Domain of Nup153*. J Biol Chem. 2007;282:17090–17100. doi: 10.1074/jbc.M702715200 PubMed DOI

Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, et al.. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024;630:493–500. doi: 10.1038/s41586-024-07487-w PubMed DOI PMC

Bernad R, van der Velde H, Fornerod M, Pickersgill H. Nup358/RanBP2 Attaches to the Nuclear Pore Complex via Association with Nup88 and Nup214/CAN and Plays a Supporting Role in CRM1-Mediated Nuclear Protein Export. Mol Cell Biol. 2004;24:2373–2384. doi: 10.1128/MCB.24.6.2373-2384.2004 PubMed DOI PMC

Gabernet-Castello C, O’Reilly AJ, Dacks JB, Field MC. Evolution of Tre-2/Bub2/Cdc16 (TBC) Rab GTPase-activating proteins. Mol Biol Cell. 2013;24:1574–1583. doi: 10.1091/mbc.E12-07-0557 PubMed DOI PMC

Matunis MJ, Wu J, Blobel G. SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex. J Cell Biol. 1998;140:499–509. doi: 10.1083/jcb.140.3.499 PubMed DOI PMC

Mahajan R, Gerace L, Melchior F. Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association. J Cell Biol. 1998;140:259–270. doi: 10.1083/jcb.140.2.259 PubMed DOI PMC

Matunis MJ, Coutavas E, Blobel G. A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J Cell Biol. 1996;135:1457–1470. doi: 10.1083/jcb.135.6.1457 PubMed DOI PMC

Xu XM, Meulia T, Meier I. Anchorage of Plant RanGAP to the Nuclear Envelope Involves Novel Nuclear-Pore-Associated Proteins. Curr Biol. 2007;17:1157–1163. doi: 10.1016/j.cub.2007.05.076 PubMed DOI

Rose A, Meier I. A domain unique to plant RanGAP is responsible for its targeting to the plant nuclear rim. Proc Natl Acad Sci U S A. 2001;98:15377–15382. doi: 10.1073/pnas.261459698 PubMed DOI PMC

Hopper AK, Traglia HM, Dunst RW. The yeast RNA1 gene product necessary for RNA processing is located in the cytosol and apparently excluded from the nucleus. J Cell Biol. 1990;111:309–321. doi: 10.1083/jcb.111.2.309 PubMed DOI PMC

Melchior F, Weber K, Gerke V. A functional homologue of the RNA1 gene product in Schizosaccharomyces pombe: purification, biochemical characterization, and identification of a leucine-rich repeat motif. Mol Biol Cell. 1993;4:569–581. doi: 10.1091/mbc.4.6.569 PubMed DOI PMC

Hutchins JR, Moore WJ, Clarke PR. Dynamic localisation of Ran GTPase during the cell cycle. BMC Cell Biol. 2009;10:66. doi: 10.1186/1471-2121-10-66 PubMed DOI PMC

Kramer S, Kimblin NC, Carrington M. Genome-wide in silico screen for CCCH-type zinc finger proteins of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. BMC Genomics. 2010;11:283. doi: 10.1186/1471-2164-11-283 PubMed DOI PMC

Dostalova A, Käser S, Cristodero M, Schimanski B. The nuclear mRNA export receptor Mex67-Mtr2 of Trypanosoma brucei contains a unique and essential zinc finger motif. Mol Microbiol. 2013;88:728–739. doi: 10.1111/mmi.12217 PubMed DOI

Holzer G, Antonin W. Nup50 plays more than one instrument. Cell Cycle. 2022;21:1785–1794. doi: 10.1080/15384101.2022.2074742 PubMed DOI PMC

Seewald MJ, Kraemer A, Farkasovsky M, Kürner C, Wittinghofer A, Vetter IR. Biochemical Characterization of the Ran-RanBP1-RanGAP System: Are RanBP Proteins and the Acidic Tail of RanGAP Required for the Ran-RanGAP GTPase Reaction? Mol Cell Biol. 2003;23:8124–8136. doi: 10.1128/MCB.23.22.8124-8136.2003 PubMed DOI PMC

Anderson J, Phan L, Cuesta R, Carlson BA, Pak M, Asano K, et al.. The essential Gcd10p–Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA. Genes Dev. 1998;12:3650–3662. doi: 10.1101/gad.12.23.3650 PubMed DOI PMC

Tang J, Jia P, Xin P, Chu J, Shi D-Q, Yang W-C. The Arabidopsis TRM61/TRM6 complex is a bona fide tRNA N1-methyladenosine methyltransferase. J Exp Bot. 2020;71:3024–3036. doi: 10.1093/jxb/eraa100 PubMed DOI PMC

Simos G, Tekotte H, Grosjean H, Segref A, Sharma K, Tollervey D, et al.. Nuclear pore proteins are involved in the biogenesis of functional tRNA. EMBO J. 1996;15:2270–2284. PubMed PMC

Clark MW, Abelson J. The subnuclear localization of tRNA ligase in yeast. J Cell Biol. 1987;105:1515–1526. doi: 10.1083/jcb.105.4.1515 PubMed DOI PMC

Hang J, Dasso M. Association of the Human SUMO-1 Protease SENP2 with the Nuclear Pore*. J Biol Chem. 2002;277:19961–19966. doi: 10.1074/jbc.M201799200 PubMed DOI

Nie M, Boddy MN. Pli1PIAS1 SUMO Ligase Protected by the Nuclear Pore-associated SUMO Protease Ulp1SENP1/2 *. J Biol Chem. 2015;290:22678–22685. doi: 10.1074/jbc.M115.673038 PubMed DOI PMC