Quantifying tagged mRNA export flux via nuclear pore complexes in single live cells

Control of organellar assembly and function is critical to eukaryotic homeostasis and survival. Gle1 is a highly conserved regulator of RNA-dependent DEAD-box ATPase proteins, with critical roles in both mRNA export and translation. In addition to its well-defined interaction with nuclear pore complexes, here we find that Gle1 is enriched at the centrosome and basal body. Gle1 assembles into the toroid-shaped pericentriolar material around the mother centriole. Reduced Gle1 levels are correlated with decreased pericentrin localization at the centrosome and microtubule organization defects. Of importance, these alterations in centrosome integrity do not result from loss of mRNA export. Examination of the Kupffer’s vesicle in Gle1-depleted zebrafish revealed compromised ciliary beating and developmental defects. We propose that Gle1 assembly into the pericentriolar material positions the DEAD-box protein regulator to function in localized mRNA metabolism required for proper centrosome function.

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RanBP2/Nup358 Provides a Major Binding Site for NXF1-p15 Dimers at the Nuclear Pore Complex and Functions in Nuclear mRNA Export

Molecular and Cellular Biology ◽

10.1128/mcb.24.3.1155-1167.2004 ◽

2004 ◽

Vol 24 (3) ◽

pp. 1155-1167 ◽

Cited By ~ 60

Author(s):

Daniel Forler ◽

Gwénaël Rabut ◽

Francesca D. Ciccarelli ◽

Andrea Herold ◽

Thomas Köcher ◽

...

Keyword(s):

Binding Site ◽

Nuclear Export ◽

Mrna Export ◽

Stable Complex ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Cytoplasmic Filaments ◽

Nuclear Levels ◽

Pore Complexes

ABSTRACT Metazoan NXF1-p15 heterodimers promote the nuclear export of bulk mRNA across nuclear pore complexes (NPCs). In vitro, NXF1-p15 forms a stable complex with the nucleoporin RanBP2/Nup358, a component of the cytoplasmic filaments of the NPC, suggesting a role for this nucleoporin in mRNA export. We show that depletion of RanBP2 from Drosophila cells inhibits proliferation and mRNA export. Concomitantly, the localization of NXF1 at the NPC is strongly reduced and a significant fraction of this normally nuclear protein is detected in the cytoplasm. Under the same conditions, the steady-state subcellular localization of other nuclear or cytoplasmic proteins and CRM1-mediated protein export are not detectably affected, indicating that the release of NXF1 into the cytoplasm and the inhibition of mRNA export are not due to a general defect in NPC function. The specific role of RanBP2 in the recruitment of NXF1 to the NPC is highlighted by the observation that depletion of CAN/Nup214 also inhibits cell proliferation and mRNA export but does not affect NXF1 localization. Our results indicate that RanBP2 provides a major binding site for NXF1 at the cytoplasmic filaments of the NPC, thereby restricting its diffusion in the cytoplasm after NPC translocation. In RanBP2-depleted cells, NXF1 diffuses freely through the cytoplasm. Consequently, the nuclear levels of the protein decrease and export of bulk mRNA is impaired.

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A conditional allele of the novel repeat-containing yeast nucleoporin RAT7/NUP159 causes both rapid cessation of mRNA export and reversible clustering of nuclear pore complexes.

The Journal of Cell Biology ◽

10.1083/jcb.129.4.939 ◽

1995 ◽

Vol 129 (4) ◽

pp. 939-955 ◽

Cited By ~ 119

Author(s):

L C Gorsch ◽

T C Dockendorff ◽

C N Cole

Keyword(s):

Messenger Rna ◽

Mrna Export ◽

Temperature Shift ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Temperature Sensitive ◽

Nuclear Pore Complexes ◽

Reporter Protein ◽

Direct Role ◽

Pore Complexes

In a screen for Saccharomyces cerevisiae genes required for nucleocytoplasmic transport of messenger RNA, we identified the RAT7 gene (ribonucleic acid trafficking), which encodes an essential protein of 1,460 amino acids. Rat7p is located at the nuclear rim in a punctate pattern characteristic of nucleoporins. Furthermore, the central third of Rat7p contains 22 XXFG and three XFXFG degenerate repeats that are similar to signature GLFG and XFXFG repeats present in a majority of yeast and some mammalian nucleoporins sequenced to date. Shift of a strain bearing the temperature-sensitive rat7-1 allele from 23 degrees C to 37 degrees C resulted in rapid (within 15 minutes) cessation of mRNA export, but did not cause concomitant cytoplasmic accumulation of a reporter protein bearing a nuclear localization signal. This suggests that Rat7p may play a direct role in nucleocytoplasmic export of RNA. Immunofluorescence and thin section electron microscopy revealed that in rat7-1 cells grown at 23 degrees C, the majority of nuclear pore complexes (NPCs) were clustered on one side of the nucleus. No ultrastructural abnormalities of the nuclear envelope were seen. Interestingly, shifting rat7-1 cells to 37 degrees C for 1 h caused the NPCs to disperse, restoring near wild-type NPC distribution. After this temperature shift, the mutant Rat7p was no longer detectable by immunofluorescence.

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Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

10.1101/685263 ◽

2019 ◽

Cited By ~ 1

Author(s):

Vasilisa Aksenova ◽

Hang Noh Lee ◽

Alexandra Smith ◽

Shane Chen ◽

Prasanna Bhat ◽

...

Keyword(s):

Gene Expression ◽

Nuclear Export ◽

Protein Modification ◽

Mrna Export ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Nucleocytoplasmic Trafficking ◽

Pore Complexes ◽

Slow Turnover

AbstractNuclear pore complexes (NPCs) are important for many processes beyond nucleocytoplasmic trafficking, including protein modification, chromatin remodeling, transcription, mRNA processing and mRNA export. The multi-faceted nature of NPCs and the slow turnover of their components has made it difficult to understand the role of basket nucleoporins (Nup153, Nup50 and Tpr) in these diverse processes. To address this question, we used anAuxin-InducedDegron (AID) system to distinguish roles of basket nucleoporins: Loss of individual nucleoporins caused distinct alteration in patterns of nucleocytoplasmic trafficking and gene expression. Importantly, Tpr elimination caused rapid and pronounced changes in transcriptomic profiles within two hours of auxin addition. These changes were dissimilar to shifts observed after loss of Nup153 or Nup50, but closely related to changes after depletion of mRNA export receptor NXF1 or the GANP subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex. Moreover, GANP association to NPCs was specifically disrupted upon TPR depletion. Together, our findings demonstrate a unique and pivotal role of Tpr in regulating gene expression through GANP- and/or NXF1-dependent mRNA nuclear export.

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Nuclear Pore Complex Acetylation Regulates mRNA Export and Cell Cycle Commitment in Budding Yeast

10.1101/2021.09.01.458533 ◽

2021 ◽

Author(s):

Mercè Gomar-Alba ◽

Vasilisa Pozharskaia ◽

Celia Schaal ◽

Arun Kumar ◽

Basile Jacquel ◽

...

Keyword(s):

Gene Expression ◽

Nuclear Export ◽

Mrna Export ◽

Cell Types ◽

Nuclear Pore ◽

Specific Cell ◽

Nuclear Pore Complexes ◽

Daughter Cells ◽

Associated Proteins ◽

Pore Complexes

AbstractNuclear pore complexes (NPCs) mediate communication between the nucleus and the cytoplasm and regulate gene expression by interacting with transcription and mRNA export factors. Lysine acetyl-transferases (KATs) promote transcription through acetylation of chromatin-associated proteins. We find that Esa1, the KAT subunit of the yeast NuA4 complex, also acetylates the nuclear pore basket component Nup60 to promote mRNA export. Acetylation of Nup60 recruits mRNA export factors to the nuclear basket, including the scaffolding subunit of the Transcription and Export 2 (TREX-2) complex, Sac3. Esa1-dependent nuclear export of mRNAs promotes entry into S phase, and is inhibited by the Hos3 deacetylase in G1 daughter cells to restrain their premature commitment to a new cell division cycle. This mechanism also inhibits expression of the nutrient-regulated GAL1 gene specifically in daughter cells. These results reveal how acetylation contributes to the functional plasticity of NPCs in specific cell types, and demonstrate how the evolutionarily conserved NuA4 complex regulates gene expression dually at the level of transcription and mRNA export, by modifying the nucleoplasmic entrance to nuclear pores.

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Specialization of chromatin-bound nuclear pore complexes promotes yeast aging

10.1101/2021.06.28.450139 ◽

2021 ◽

Author(s):

Anne C Meinema ◽

Theo Aspert ◽

Sung Sik Lee ◽

Gilles Charvin ◽

Yves Barral

Keyword(s):

Quality Control ◽

Nuclear Pore Complex ◽

Mrna Export ◽

Yeast Cells ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Replicative Lifespan ◽

Yeast Aging ◽

Key Drivers ◽

Pore Complexes

The nuclear pore complex (NPC) mediates nearly all exchanges between nucleus and cytoplasm, and changes composition in many species as the organism ages. However, how these changes arise and whether they contribute themselves to aging is poorly understood. We show that in replicatively aging yeast cells attachment of DNA circles to NPCs drives the displacement of the NPCs’ nuclear basket and cytoplasmic complexes. Remodeling of the NPC resulted from the regulation of basket components by SAGA, rather than from damages. These changes affected NPC interaction with mRNA export factors, without affecting the residence of import factors or engaging the NPC quality control machinery. Mutations preventing NPC remodeling extended the replicative lifespan of the cells. Thus, our data indicate that DNA circles accumulating in the mother cell drive aging at least in part by triggering NPC specialization. We suggest that antagonistic pleiotropic effects of NPC specialization are key drivers of aging.

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Nucleoporin TPR is an integral component of the TREX-2 mRNA export pathway

Nature Communications ◽

10.1038/s41467-020-18266-2 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 3

Author(s):

Vasilisa Aksenova ◽

Alexandra Smith ◽

Hangnoh Lee ◽

Prasanna Bhat ◽

Caroline Esnault ◽

...

Keyword(s):

Gene Expression ◽

Mrna Export ◽

Mrna Turnover ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Nucleocytoplasmic Trafficking ◽

Cellular Functions ◽

Pore Complexes ◽

Slow Turnover

Abstract Nuclear pore complexes (NPCs) are important for cellular functions beyond nucleocytoplasmic trafficking, including genome organization and gene expression. This multi-faceted nature and the slow turnover of NPC components complicates investigations of how individual nucleoporins act in these diverse processes. To address this question, we apply an Auxin-Induced Degron (AID) system to distinguish roles of basket nucleoporins NUP153, NUP50 and TPR. Acute depletion of TPR causes rapid and pronounced changes in transcriptomic profiles. These changes are dissimilar to shifts observed after loss of NUP153 or NUP50, but closely related to changes caused by depletion of mRNA export receptor NXF1 or the GANP subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex. Moreover, TPR depletion disrupts association of TREX-2 subunits (GANP, PCID2, ENY2) to NPCs and results in abnormal RNA transcription and export. Our findings demonstrate a unique and pivotal role of TPR in gene expression through TREX-2- and/or NXF1-dependent mRNA turnover.

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Interphase Nuclei of Many Mammalian Cell Types Contain Deep, Dynamic, Tubular Membrane-bound Invaginations of the Nuclear Envelope

The Journal of Cell Biology ◽

10.1083/jcb.136.3.531 ◽

1997 ◽

Vol 136 (3) ◽

pp. 531-544 ◽

Cited By ~ 260

Author(s):

Mark Fricker ◽

Michael Hollinshead ◽

Nick White ◽

David Vaux

Keyword(s):

Nuclear Envelope ◽

Time Lapse ◽

Cell Types ◽

Live Cells ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Nuclear Surface ◽

Topological Features ◽

Cytoplasmic Transport ◽

Pore Complexes

The nuclear envelope consists of a doublemembraned extension of the rough endoplasmic reticulum. In this report we describe long, dynamic tubular channels, derived from the nuclear envelope, that extend deep into the nucleoplasm. These channels show cell-type specific morphologies ranging from single short stubs to multiple, complex, branched structures. Some channels transect the nucleus entirely, opening at two separate points on the nuclear surface, while others terminate at or close to nucleoli. These channels are distinct from other topological features of the nuclear envelope, such as lobes or folds. The channel wall consists of two membranes continuous with the nuclear envelope, studded with features indistinguishable from nuclear pore complexes, and decorated on the nucleoplasmic surface with lamins. The enclosed core is continuous with the cytoplasm, and the lumenal space between the membranes contains soluble ER-resident proteins (protein disulphide isomerase and glucose-6-phosphatase). Nuclear channels are also found in live cells labeled with the lipophilic dye DiOC6. Time-lapse imaging of DiOC6-labeled cells shows that the channels undergo changes in morphology and spatial distribution within the interphase nucleus on a timescale of minutes. The presence of a cytoplasmic core and nuclear pore complexes in the channel walls suggests a possible role for these structures in nucleo–cytoplasmic transport. The clear association of a subset of these structures with nucleoli would also be consistent with such a transport role.

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In cell architecture of the nuclear pore complex and snapshots of its turnover

10.1101/2020.02.04.933820 ◽

2020 ◽

Cited By ~ 4

Author(s):

Matteo Allegretti ◽

Christian E. Zimmerli ◽

Vasileios Rantos ◽

Florian Wilfling ◽

Paolo Ronchi ◽

...

Keyword(s):

Nuclear Envelope ◽

Mrna Export ◽

Light And Electron Microscopy ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Formidable Challenge ◽

Cellular Context ◽

Pore Complexes

SummaryNuclear pore complexes (NPCs) mediate exchange across the nuclear envelope. They consist of hundreds of proteins called nucleoporins (Nups) that assemble in multiple copies to fuse the inner and outer nuclear membranes. Elucidating the molecular function and architecture of NPCs imposes a formidable challenge and requires the convergence of in vitro and in situ approaches. How exactly NPC architecture accommodates processes such as mRNA export or NPC assembly and turnover inside of cells remains poorly understood. Here we combine integrated in situ structural biology, correlative light and electron microscopy with yeast genetics to structurally analyze NPCs within the native context of Saccharomyces cerevisiae cells under conditions of starvation and exponential growth. We find an unanticipated in situ layout of nucleoporins with respect to overall dimensions and conformation of the NPC scaffold that could not have been predicted from previous in vitro analysis. Particularly striking is the configuration of the Nup159 complex, which appears critical to spatially accommodate not only mRNA export but also NPC turnover by selective autophagy. We capture structural snapshots of NPC turnover, revealing that it occurs through nuclear envelope herniae and NPC-containing nuclear vesicles. Our study provides the basis for understanding the various membrane remodeling events that happen at the interface of the nuclear envelope with the autophagy apparatus and emphasizes the need of investigating macromolecular complexes in their cellular context.

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Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex

The Journal of Cell Biology ◽

10.1083/jcb.200704174 ◽

2007 ◽

Vol 178 (7) ◽

pp. 1121-1132 ◽

Cited By ~ 84

Author(s):

Laura J. Terry ◽

Susan R. Wente

Keyword(s):

Messenger Rna ◽

Large Scale ◽

Protein Import ◽

Global Analysis ◽

Mrna Export ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Versus Protein ◽

Transport Receptors ◽

Pore Complexes

Trafficking of nucleic acids and large proteins through nuclear pore complexes (NPCs) requires interactions with NPC proteins that harbor FG (phenylalanine-glycine) repeat domains. Specialized transport receptors that recognize cargo and bind FG domains facilitate these interactions. Whether different transport receptors utilize preferential FG domains in intact NPCs is not fully resolved. In this study, we use a large-scale deletion strategy in Saccharomyces cerevisiae to generate a new set of more minimal pore (mmp) mutants that lack specific FG domains. A comparison of messenger RNA (mRNA) export versus protein import reveals unique subsets of mmp mutants with functional defects in specific transport receptors. Thus, multiple functionally independent NPC translocation routes exist for different transport receptors. Our global analysis of the FG domain requirements in mRNA export also finds a requirement for two NPC substructures—one on the nuclear NPC face and one in the NPC central core. These results pinpoint distinct steps in the mRNA export mechanism that regulate NPC translocation efficiency.

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