bims-nucpor 2023-06-18 papers

bims-nucpor

Biomed News

on Nuclear pore complex and nucleoporins in stress, aging and disease

Issue of 2023–06–18
seven papers selected by
Sara Mingu, Johannes Gutenberg University

Y-complex nucleoporins independently contribute to nuclear pore assembly and gene regulation in neuronal progenitors.
Phase separation of intrinsically disordered FG-Nups is driven by highly dynamic FG motifs.
Differential affinity purification and mass spectrometry analysis of two nuclear pore complex isoforms in yeast S. cerevisiae.
Functional differentiation of Sec13 paralogues in the euglenozoan protists.
Protocol for imaging nuclear pore complexes on isolated nuclei of zebrafish early embryos by field emission scanning electron microscopy.
Co-translational binding of importins to nascent proteins.
The pediatric leukemia oncoprotein NUP98-KDM5A induces genomic instability that may facilitate malignant transformation.

J Cell Sci. 2023 06 01. pii: jcs261151. [Epub ahead of print]136(11):

Y-complex nucleoporins independently contribute to nuclear pore assembly and gene regulation in neuronal progenitors.

Clarisse Orniacki, Annalisa Verrico, Stéphane Pelletier, Benoit Souquet, Fanny Coulpier, Laurent Jourdren, Serena Benetti, Valérie Doye.

  Besides assembling nuclear pore complexes, the conduits of nuclear transport, many nucleoporins also contribute to chromatin organization and gene expression, with critical roles in development and pathologies. We previously reported that Nup133 and Seh1, two components of the Y-complex subassembly of the nuclear pore scaffold, are dispensable for mouse embryonic stem cell viability but required for their survival during neuroectodermal differentiation. Here, a transcriptomic analysis revealed that Nup133 regulates a subset of genes at early stages of neuroectodermal differentiation, including Lhx1 and Nup210l, which encodes a newly validated nucleoporin. These genes are also misregulated in Nup133ΔMid neuronal progenitors, in which nuclear pore basket assembly is impaired. However, a four-fold reduction of Nup133 levels, despite also affecting basket assembly, is not sufficient to alter Nup210l and Lhx1 expression. Finally, these two genes are also misregulated in Seh1-deficient neural progenitors, which only show a mild reduction in nuclear pore density. Together these data reveal a shared function of Y-complex nucleoporins in gene regulation during neuroectodermal differentiation, apparently independent of nuclear pore basket integrity.

Keywords:  Lhx1; Neuronal progenitors; Nucleoporin; Nup133; Nup210L; Seh1

DOI:  https://doi.org/10.1242/jcs.261151
Proc Natl Acad Sci U S A. 2023 Jun 20. 120(25): e2221804120

Phase separation of intrinsically disordered FG-Nups is driven by highly dynamic FG motifs.

Maurice Dekker, Erik Van der Giessen, Patrick R Onck.

  The intrinsically disordered FG-Nups in the central channel of the nuclear pore complex (NPC) form a selective permeability barrier, allowing small molecules to traverse by passive diffusion, while large molecules can only translocate with the help of nuclear transport receptors. The exact phase state of the permeability barrier remains elusive. In vitro experiments have shown that some FG-Nups can undergo phase separation into condensates that display NPC-like permeability barrier properties. Here, we use molecular dynamics simulations at amino acid resolution to study the phase separation characteristics of each of the disordered FG-Nups of the yeast NPC. We find that GLFG-Nups undergo phase separation and reveal that the FG motifs act as highly dynamic hydrophobic stickers that are essential for the formation of FG-Nup condensates featuring droplet-spanning percolated networks. Additionally, we study phase separation in an FG-Nup mixture that resembles the NPC stoichiometry and observe that an NPC condensate is formed containing multiple GLFG-Nups. We find that the phase separation of this NPC condensate is also driven by FG-FG interactions, similar to the homotypic FG-Nup condensates. Based on the observed phase separation behavior, the different FG-Nups of the yeast NPC can be divided into two classes: The FG-Nups (mostly GLFG-type) located in the central channel of the NPC form a highly dynamic percolated network formed by many short-lived FG-FG interactions, while the peripheral FG-Nups (mostly FxFG-type) at the entry and exit of the NPC channel likely form an entropic brush.

Keywords:  FG-Nups; intrinsically disordered proteins; nuclear pore complex; phase separation

DOI:  https://doi.org/10.1073/pnas.2221804120
STAR Protoc. 2023 Jun 15. pii: S2666-1667(23)00326-X. [Epub ahead of print]4(3): 102359

Differential affinity purification and mass spectrometry analysis of two nuclear pore complex isoforms in yeast S. cerevisiae.

Pierre Bensidoun, Daniel Zenklusen, Marlene Oeffinger.

  Two isoforms of the nuclear pore complex (NPC) have been identified in the yeast S. cerevisiae, which coexist at the periphery of the nucleus and differ by the presence or absence of a nuclear basket. Here, we present a protocol to isolate the two types of NPCs from the same cell extract and dissect their interactomes. We describe steps for powder preparation and magnetic bead conjunction and detail differential affinity purification and outcome evaluation through SDS-PAGE, silver staining, and mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Bensidoun et al.1.

Keywords:  Cell Biology; Mass Spectrometry; Molecular Biology

DOI:  https://doi.org/10.1016/j.xpro.2023.102359
Open Biol. 2023 Jun;13(6): 220364

Functional differentiation of Sec13 paralogues in the euglenozoan protists.

Drahomíra Faktorová, Kristína Záhonová, Corinna Benz, Joel B Dacks, Mark C Field, Julius Lukeš.

  The β-propeller protein Sec13 plays roles in at least three distinct processes by virtue of being a component of the COPII endoplasmic reticulum export vesicle coat, the nuclear pore complex (NPC) and the Seh1-associated (SEA)/GATOR nutrient-sensing complex. This suggests that regulatory mechanisms coordinating these cellular activities may operate via Sec13. The NPC, COPII and SEA/GATOR are all ancient features of eukaryotic cells, and in the vast majority of eukaryotes, a single Sec13 gene is present. Here we report that the Euglenozoa, a lineage encompassing the diplonemid, kinetoplastid and euglenid protists, possess two Sec13 paralogues. Furthermore, based on protein interactions and localization studies we show that in diplonemids Sec13 functions are divided between the Sec13a and Sec13b paralogues. Specifically, Sec13a interacts with COPII and the NPC, while Sec13b interacts with Sec16 and components of the SEA/GATOR complex. We infer that euglenozoan Sec13a is responsible for NPC functions and canonical anterograde transport activities while Sec13b acts within nutrient and autophagy-related pathways, indicating a fundamentally distinct organization of coatomer complexes in euglenozoan flagellates.

Keywords:  Diplonema; SEA/GATOR complex; coatomer; membrane trafficking; nuclear pore complex; paralogue expansion

DOI:  https://doi.org/10.1098/rsob.220364
STAR Protoc. 2023 Jun 13. pii: S2666-1667(23)00308-8. [Epub ahead of print]4(3): 102341

Protocol for imaging nuclear pore complexes on isolated nuclei of zebrafish early embryos by field emission scanning electron microscopy.

Weimin Shen, Anming Meng.

  Here, we present a protocol to observe the three-dimensional surface of nuclear pore complexes (NPCs) of vertebrate early embryos by field emission scanning electron microscopy (FESEM). We describe steps from zebrafish early embryo collection and nuclei exposure to FESEM sample preparation and final NPC state analysis. This approach provides an easy way to observe surface morphology of NPCs from the cytoplasmic side. Alternatively, additional purification steps after nuclei exposure supply intact nuclei for further mass spectrometry analysis or other utilization. For complete details on the use and execution of this protocol, please refer to Shen et al.1.

Keywords:  Cell Biology; Cell separation/fractionation; Developmental biology

DOI:  https://doi.org/10.1016/j.xpro.2023.102341
Nat Commun. 2023 Jun 09. 14(1): 3418

Co-translational binding of importins to nascent proteins.

Maximilian Seidel, Natalie Romanov, Agnieszka Obarska-Kosinska, Anja Becker, Nayara Trevisan Doimo de Azevedo, Jan Provaznik, Sankarshana R Nagaraja, Jonathan J M Landry, Vladimir Benes, Martin Beck.

Various cellular quality control mechanisms support proteostasis. While, ribosome-associated chaperones prevent the misfolding of nascent chains during translation, importins were shown to prevent the aggregation of specific cargoes in a post-translational mechanism prior the import into the nucleoplasm. Here, we hypothesize that importins may already bind ribosome-associated cargo in a co-translational manner. We systematically measure the nascent chain association of all importins in Saccharomyces cerevisiae by selective ribosome profiling. We identify a subset of importins that bind to a wide range of nascent, often uncharacterized cargoes. This includes ribosomal proteins, chromatin remodelers and RNA binding proteins that are aggregation prone in the cytosol. We show that importins act consecutively with other ribosome-associated chaperones. Thus, the nuclear import system is directly intertwined with nascent chain folding and chaperoning.

DOI: https://doi.org/10.1038/s41467-023-39150-9
Cell Death Dis. 2023 Jun 10. 14(6): 357

The pediatric leukemia oncoprotein NUP98-KDM5A induces genomic instability that may facilitate malignant transformation.

Joan Domingo-Reinés, Rosa Montes, Adrián Garcia-Moreno, Amador Gallardo, Jose Manuel Sanchez-Manas, Iván Ellson, Mar Lamolda, Chiara Calabro, Jose Antonio López-Escamez, Purificación Catalina, Pedro Carmona-Sáez, Pedro J Real, David Landeira, Verónica Ramos-Mejia.

Pediatric Acute Myeloid Leukemia (AML) is a rare and heterogeneous disease characterized by a high prevalence of gene fusions as driver mutations. Despite the improvement of survival in the last years, about 50% of patients still experience a relapse. It is not possible to improve prognosis only with further intensification of chemotherapy, as come with a severe cost to the health of patients, often resulting in treatment-related death or long-term sequels. To design more effective and less toxic therapies we need a better understanding of pediatric AML biology. The NUP98-KDM5A chimeric protein is exclusively found in a particular subgroup of young pediatric AML patients with complex karyotypes and poor prognosis. In this study, we investigated the impact of NUP98-KDM5A expression on cellular processes in human Pluripotent Stem Cell models and a patient-derived cell line. We found that NUP98-KDM5A generates genomic instability through two complementary mechanisms that involve accumulation of DNA damage and direct interference of RAE1 activity during mitosis. Overall, our data support that NUP98-KDM5A promotes genomic instability and likely contributes to malignant transformation.

DOI: https://doi.org/10.1038/s41419-023-05870-5