A lineage-specific protein network at the trypanosome nuclear envelope.

The nuclear envelope (NE) separates translation and transcription and is the location of multiple functions, including chromatin organization and nucleocytoplasmic transport. The molecular basis for many of these functions have diverged between eukaryotic lineages. Trypanosoma brucei, a member of the early branching eukaryotic lineage Discoba, highlights many of these, including a distinct lamina and kinetochore composition. Here, we describe a cohort of proteins interacting with both the lamina and NPC, which we term lamina-associated proteins (LAPs). LAPs represent a diverse group of proteins, including two candidate NPC-anchoring pore membrane proteins (POMs) with architecture conserved with S. cerevisiae and H. sapiens, and additional peripheral components of the NPC. While many of the LAPs are Kinetoplastid specific, we also identified broadly conserved proteins, indicating an amalgam of divergence and conservation within the trypanosome NE proteome, highlighting the diversity of nuclear biology across the eukaryotes, increasing our understanding of eukaryotic and NPC evolution.

UBAP2L ensures homeostasis of nuclear pore complexes at the intact nuclear envelope.

Assembly of macromolecular complexes at correct cellular sites is crucial for cell function. Nuclear pore complexes (NPCs) are large cylindrical assemblies with eightfold rotational symmetry, built through hierarchical binding of nucleoporins (Nups) forming distinct subcomplexes. Here, we uncover a role of ubiquitin-associated protein 2-like (UBAP2L) in the assembly and stability of properly organized and functional NPCs at the intact nuclear envelope (NE) in human cells. UBAP2L localizes to the nuclear pores and facilitates the formation of the Y-complex, an essential scaffold component of the NPC, and its localization to the NE. UBAP2L promotes the interaction of the Y-complex with POM121 and Nup153, the critical upstream factors in a well-defined sequential order of Nups assembly onto NE during interphase. Timely localization of the cytoplasmic Nup transport factor fragile X-related protein 1 (FXR1) to the NE and its interaction with the Y-complex are likewise dependent on UBAP2L. Thus, this NPC biogenesis mechanism integrates the cytoplasmic and the nuclear NPC assembly signals and ensures efficient nuclear transport, adaptation to nutrient stress, and cellular proliferative capacity, highlighting the importance of NPC homeostasis at the intact NE.

SARS-CoV-2 Orf6 is positioned in the nuclear pore complex by Rae1 to inhibit nucleocytoplasmic transport.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accessory protein Orf6 works as an interferon antagonist, in part, by inhibiting the nuclear import activated p-STAT1, an activator of interferon-stimulated genes, and the export of the poly(A) RNA. Insight into the transport regulatory function of Orf6 has come from the observation that Orf6 binds to the nuclear pore complex (NPC) components: Rae1 and Nup98. To gain further insight into the mechanism of Orf6-mediated transport inhibition, we examined the role of Rae1 and Nup98. We show that Rae1 alone is not necessary to support p-STAT1 import or nuclear export of poly(A) RNA. Moreover, the loss of Rae1 suppresses the transport inhibitory activity of Orf6. We propose that the Rae1/Nup98 complex strategically positions Orf6 within the NPC where it alters FG-Nup interactions and their ability to support nuclear transport. In addition, we show that Rae1 is required for normal viral protein production during SARS-CoV-2 infection presumably through its role in supporting Orf6 function.

Two ancient membrane pores mediate mitochondrial-nucleus membrane contact sites.

Coordination between nucleus and mitochondria is essential for cell survival, and thus numerous communication routes have been established between these two organelles over eukaryotic cell evolution. One route for organelle communication is via membrane contact sites, functional appositions formed by molecular tethers. We describe a novel nuclear-mitochondrial membrane contact site in the protozoan Toxoplasma gondii. We have identified specific contacts occurring at the nuclear pore and demonstrated an interaction between components of the nuclear pore and the mitochondrial protein translocon, highlighting them as molecular tethers. Genetic disruption of the nuclear pore or the TOM translocon components, TgNup503 or TgTom40, respectively, result in contact site reduction, supporting their potential involvement in this tether. TgNup503 depletion further leads to specific mitochondrial morphology and functional defects, supporting a role for nuclear-mitochondrial contacts in mediating their communication. The discovery of a contact formed through interaction between two ancient mitochondrial and nuclear complexes sets the ground for better understanding of mitochondrial-nuclear crosstalk in eukaryotes.

Role of the San1 ubiquitin ligase in the heat stress-induced degradation of nonnative Nup1 in the nuclear pore complex.

The nuclear pore complex (NPC) mediates the selective exchange of macromolecules between the nucleus and the cytoplasm. Neurodegenerative diseases such as amyotrophic lateral sclerosis are characterized by mislocalization of nucleoporins (Nups), transport receptors, and Ras-related nuclear proteins into nucleoplasmic or cytosolic aggregates, underscoring the importance of precise assembly of the NPC. The assembly state of large protein complexes is strictly monitored by the protein quality control system. The ubiquitin-proteasome system may eliminate aberrant, misfolded, and/or orphan components; however, the involvement of the ubiquitin-proteasome system in the degradation of nonnative Nups in the NPC remains unclear. Here, we show that in Saccharomyces cerevisiae, although Nup1 (the FG-Nup component of the central core of the NPC) was stable, C-terminally green fluorescent protein-tagged Nup1, which had been incorporated into the NPC, was degraded by the proteasome especially under heat stress conditions. The degradation was dependent on the San1 ubiquitin ligase and Cdc48/p97, as well as its cofactor Doa1. We also demonstrate that San1 weakly but certainly contributes to the degradation of nontagged endogenous Nup1 in cells defective in NPC biogenesis by the deletion of NUP120. In addition, the overexpression of SAN1 exacerbated the growth defect phenotype of nup120Δ cells, which may be caused by excess degradation of defective Nups due to the deletion of NUP120. These biochemical and genetic data suggest that San1 is involved in the degradation of nonnative Nups generated by genetic mutation or when NPC biogenesis is impaired.

Nuclear pore dysfunction and disease: a complex opportunity.

The separation of genetic material from bulk cytoplasm has enabled the evolution of increasingly complex organisms, allowing for the development of sophisticated forms of life. However, this complexity has created new categories of dysfunction, including those related to the movement of material between cellular compartments. In eukaryotic cells, nucleocytoplasmic trafficking is a fundamental biological process, and cumulative disruptions to nuclear integrity and nucleocytoplasmic transport are detrimental to cell survival. This is particularly true in post-mitotic neurons, where nuclear pore injury and errors to nucleocytoplasmic trafficking are strongly associated with neurodegenerative disease. In this review, we summarize the current understanding of nuclear pore biology in physiological and pathological contexts and discuss potential therapeutic approaches for addressing nuclear pore injury and dysfunctional nucleocytoplasmic transport.

Mechanism of exportin retention in the cell nucleus.

Exportin receptors are concentrated in the nucleus to transport essential cargoes out of it. A mislocalization of exportins to the cytoplasm is linked to disease. Hence, it is important to understand how their containment within the nucleus is regulated. Here, we have studied the nuclear efflux of exportin2 (cellular apoptosis susceptibility protein or CAS) that delivers karyopherinα (Kapα or importinα), the cargo adaptor for karyopherinß1 (Kapß1 or importinß1), to the cytoplasm in a Ran guanosine triphosphate (RanGTP)-mediated manner. We show that the N-terminus of CAS attenuates the interaction of RanGTPase activating protein 1 (RanGAP1) with RanGTP to slow GTP hydrolysis, which suppresses CAS nuclear exit at nuclear pore complexes (NPCs). Strikingly, a single phosphomimetic mutation (T18D) at the CAS N-terminus is sufficient to abolish its nuclear retention and coincides with metastatic cellular behavior. Furthermore, downregulating Kapß1 disrupts CAS nuclear retention, which highlights the balance between their respective functions that is essential for maintaining the Kapα transport cycle. Therefore, NPCs play a functional role in selectively partitioning exportins in the cell nucleus.

Sculpting nuclear envelope identity from the endoplasmic reticulum during the cell cycle.

The nuclear envelope (NE) regulates nuclear functions, including transcription, nucleocytoplasmic transport, and protein quality control. While the outer membrane of the NE is directly continuous with the endoplasmic reticulum (ER), the NE has an overall distinct protein composition from the ER, which is crucial for its functions. During open mitosis in higher eukaryotes, the NE disassembles during mitotic entry and then reforms as a functional territory at the end of mitosis to reestablish nucleocytoplasmic compartmentalization. In this review, we examine the known mechanisms by which the functional NE reconstitutes from the mitotic ER in the continuous ER-NE endomembrane system during open mitosis. Furthermore, based on recent findings indicating that the NE possesses unique lipid metabolism and quality control mechanisms distinct from those of the ER, we explore the maintenance of NE identity and homeostasis during interphase. We also highlight the potential significance of membrane junctions between the ER and NE.

The HIV capsid mimics karyopherin engagement of FG-nucleoporins.

HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus1,2. Remarkably, the intact HIV capsid is more than 1,000 times larger than the size limit prescribed by the diffusion barrier of the nuclear pore3. This barrier in the central channel of the nuclear pore is composed of intrinsically disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides. Through multivalent FG interactions, cellular karyopherins and their bound cargoes solubilize in this phase to drive nucleocytoplasmic transport4. By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG motifs from multiple nucleoporins and that this interaction licences capsids to penetrate FG-nucleoporin condensates. This karyopherin mimicry model addresses a key conceptual challenge for the role of the HIV capsid in nuclear entry and offers an explanation as to how an exogenous entity much larger than any known cellular cargo may be able to non-destructively breach the nuclear envelope.

HIV-1 capsids enter the FG phase of nuclear pores like a transport receptor.

HIV-1 infection requires nuclear entry of the viral genome. Previous evidence suggests that this entry proceeds through nuclear pore complexes (NPCs), with the 120 × 60 nm capsid squeezing through an approximately 60-nm-wide central channel1 and crossing the permeability barrier of the NPC. This barrier can be described as an FG phase2 that is assembled from cohesively interacting phenylalanine-glycine (FG) repeats3 and is selectively permeable to cargo captured by nuclear transport receptors (NTRs). Here we show that HIV-1 capsid assemblies can target NPCs efficiently in an NTR-independent manner and bind directly to several types of FG repeats, including barrier-forming cohesive repeats. Like NTRs, the capsid readily partitions into an in vitro assembled cohesive FG phase that can serve as an NPC mimic and excludes much smaller inert probes such as mCherry. Indeed, entry of the capsid protein into such an FG phase is greatly enhanced by capsid assembly, which also allows the encapsulated clients to enter. Thus, our data indicate that the HIV-1 capsid behaves like an NTR, with its interior serving as a cargo container. Because capsid-coating with trans-acting NTRs would increase the diameter by 10 nm or more, we suggest that such a 'self-translocating' capsid undermines the size restrictions imposed by the NPC scaffold, thereby bypassing an otherwise effective barrier to viral infection.

Nuclear pore protein POM121 regulates subcellular localization and transcriptional activity of PPARγ.

Manipulation of the subcellular localization of transcription factors by preventing their shuttling via the nuclear pore complex (NPC) emerges as a novel therapeutic strategy against cancer. One transmembrane component of the NPC is POM121, encoded by a tandem gene locus POM121A/C on chromosome 7. Overexpression of POM121 is associated with metabolic diseases (e.g., diabetes) and unfavorable clinical outcome in patients with colorectal cancer (CRC). Peroxisome proliferator-activated receptor-gamma (PPARγ) is a transcription factor with anti-diabetic and anti-tumoral efficacy. It is inhibited by export from the nucleus to the cytosol via the RAS-RAF-MEK1/2-ERK1/2 signaling pathway, a major oncogenic driver of CRC. We therefore hypothesized that POM121 participates in the transport of PPARγ across the NPC to regulate its transcriptional activity on genes involved in metabolic and tumor control. We found that POM121A/C mRNA was enriched and POM121 protein co-expressed with PPARγ in tissues from CRC patients conferring poor prognosis. Its interactome was predicted to include proteins responsible for tumor metabolism and immunity, and in-silico modeling provided insights into potential 3D structures of POM121. A peptide region downstream of the nuclear localization sequence (NLS) of POM121 was identified as a cytoplasmic interactor of PPARγ. POM121 positivity correlated with the cytoplasmic localization of PPARγ in patients with KRAS mutant CRC. In contrast, POM121A/C silencing by CRISPR/Cas9 sgRNA or siRNA enforced nuclear accumulation of PPARγ and activated PPARγ target genes promoting lipid metabolism and cell cycle arrest resulting in reduced proliferation of human CRC cells. Our data suggest the POM121-PPARγ axis as a potential drugable target in CRC.

HIV-1 capsid shape, orientation, and entropic elasticity regulate translocation into the nuclear pore complex.

Nuclear import and uncoating of the viral capsid are critical steps in the HIV-1 life cycle that serve to transport and release genomic material into the nucleus. Viral core import involves translocating the HIV-1 capsid at the nuclear pore complex (NPC). Notably, the central channel of the NPC appears to often accommodate and allow passage of intact HIV-1 capsid, though mechanistic details of the process remain to be fully understood. Here, we investigate the molecular interactions that operate in concert between the HIV-1 capsid and the NPC that regulate capsid translocation through the central channel. To this end, we develop a "bottom-up" coarse-grained (CG) model of the human NPC from recently released cryo-electron tomography structure and then construct composite membrane-embedded CG NPC models. We find that successful translocation from the cytoplasmic side to the NPC central channel is contingent on the compatibility of the capsid morphology and channel dimension and the proper orientation of the capsid approach to the channel from the cytoplasmic side. The translocation dynamics is driven by maximizing the contacts between phenylalanine-glycine nucleoporins at the central channel and the capsid. For the docked intact capsids, structural analysis reveals correlated striated patterns of lattice disorder likely related to the intrinsic capsid elasticity. Uncondensed genomic material inside the docked capsid augments the overall lattice disorder of the capsid. Our results suggest that the intrinsic "elasticity" can also aid the capsid to adapt to the stress and remain structurally intact during translocation.

The nuclear pore Y-complex functions as a platform for transcriptional regulation of FLOWERING LOCUS C in Arabidopsis.

The nuclear pore complex (NPC) has multiple functions beyond the nucleo-cytoplasmic transport of large molecules. Subnuclear compartmentalization of chromatin is critical for gene expression in animals and yeast. However, the mechanism by which the NPC regulates gene expression is poorly understood in plants. Here we report that the Y-complex (Nup107-160 complex, a subcomplex of the NPC) self-maintains its nucleoporin homeostasis and modulates FLOWERING LOCUS C (FLC) transcription via changing histone modifications at this locus. We show that Y-complex nucleoporins are intimately associated with FLC chromatin through their interactions with histone H2A at the nuclear membrane. Fluorescence in situ hybridization assays revealed that Nup96, a Y-complex nucleoporin, enhances FLC positioning at the nuclear periphery. Nup96 interacted with HISTONE DEACETYLASE 6 (HDA6), a key repressor of FLC expression via histone modification, at the nuclear membrane to attenuate HDA6-catalyzed deposition at the FLC locus and change histone modifications. Moreover, we demonstrate that Y-complex nucleoporins interact with RNA polymerase II to increase its occupancy at the FLC locus, facilitating transcription. Collectively, our findings identify an attractive mechanism for the Y-complex in regulating FLC expression via tethering the locus at the nuclear periphery and altering its histone modification.

Protocol for live-cell super-resolution imaging of transport of pre-ribosomal subunits through the nuclear pore complex.

Here, we present a protocol for single-molecule super-resolution imaging of the nuclear export of pre-ribosomal subunits pre-40S and pre-60S through nuclear pore complexes. We describe steps for plating cells and co-transfecting cells. We then detail steps for using single-point edge-excitation sub-diffraction microscopy, allowing visualization of real-time dynamics of the pre-ribosomal subunits. For complete details on the use and execution of this protocol, please refer to Junod et al. (2023).1.

Nuclear pore pathology underlying multisystem proteinopathy type 3-related inclusion body myopathy.

OBJECTIVE: Multisystem proteinopathy type 3 (MSP3) is an inherited, pleiotropic degenerative disorder caused by a mutation in heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which can affect the muscle, bone, and/or nervous system. This study aimed to determine detailed histopathological features and transcriptomic profile of HNRNPA1-mutated skeletal muscles to reveal the core pathomechanism of hereditary inclusion body myopathy (hIBM), a predominant phenotype of MSP3. METHODS: Histopathological analyses and RNA sequencing of HNRNPA1-mutated skeletal muscles harboring a c.940G > A (p.D314N) mutation (NM_031157) were performed, and the results were compared with those of HNRNPA1-unlinked hIBM and control muscle tissues. RESULTS: RNA sequencing revealed aberrant alternative splicing events that predominantly occurred in myofibril components and mitochondrial respiratory complex. Enrichment analyses identified the nuclear pore complex (NPC) and nucleocytoplasmic transport as suppressed pathways. These two pathways were linked by the hub genes NUP50, NUP98, NUP153, NUP205, and RanBP2. In immunohistochemistry, these nucleoporin proteins (NUPs) were mislocalized to the cytoplasm and aggregated mostly with TAR DNA-binding protein 43 kDa and, to a lesser extent, with hnRNPA1. Based on ultrastructural observation, irregularly shaped myonuclei with deep invaginations were frequently observed in atrophic fibers, consistent with the disorganization of NPCs. Additionally, regarding the expression profiles of overall NUPs, reduced expression of NUP98, NUP153, and RanBP2 was shared with HNRNPA1-unlinked hIBMs. INTERPRETATION: The shared subset of altered NUPs in amyotrophic lateral sclerosis (ALS), as demonstrated in prior research, HNRNPA1-mutated, and HNRNPA1-unlinked hIBM muscle tissues may provide evidence regarding the underlying common nuclear pore pathology of hIBM, ALS, and MSP.

NUA positively regulates plant immunity by coordination with ESD4 to deSUMOylate TPR1 in Arabidopsis.

Nuclear pore complex (NPC) is composed of multiple nucleoporins (Nups). A plethora of studies have highlighted the significance of NPC in plant immunity. However, the specific roles of individual Nups are poorly understood. NUCLEAR PORE ANCHOR (NUA) is a component of NPC. Loss of NUA leads to an increase in SUMO conjugates and pleiotropic developmental defects in Arabidopsis thaliana. Herein, we revealed that NUA is required for plant defense against multiple pathogens. NUCLEAR PORE ANCHOR associates with the transcriptional corepressor TOPLESS-RELATED1 (TPR1) and contributes to TPR1 deSUMOylation. Significantly, NUA-interacting protein EARLY IN SHORT DAYS 4 (ESD4), a SUMO protease, specifically deSUMOylates TPR1. It has been previously established that the SUMO E3 ligase SAP AND MIZ1 DOMAIN-CONTAINING LIGASE 1 (SIZ1)-mediated SUMOylation of TPR1 represses the immune-related function of TPR1. Consistent with this notion, the hyper-SUMOylated TPR1 in nua-3 leads to upregulated expression of TPR1 target genes and compromised TPR1-mediated disease resistance. Taken together, our work uncovers a mechanism by which NUA positively regulates plant defense responses by coordination with ESD4 to deSUMOylate TPR1. Our findings, together with previous studies, reveal a regulatory module in which SIZ1 and NUA/ESD4 control the homeostasis of TPR1 SUMOylation to maintain proper immune output.

The Herpes Simplex Virus pUL16 and pUL21 Proteins Prevent Capsids from Docking at Nuclear Pore Complexes.

After entry into cells, herpes simplex virus (HSV) nucleocapsids dock at nuclear pore complexes (NPCs) through which viral genomes are released into the nucleoplasm where viral gene expression, genome replication, and early steps in virion assembly take place. After their assembly, nucleocapsids are translocated to the cytoplasm for final virion maturation. Nascent cytoplasmic nucleocapsids are prevented from binding to NPCs and delivering their genomes to the nucleus from which they emerged, but how this is accomplished is not understood. Here we report that HSV pUL16 and pUL21 deletion mutants accumulate empty capsids at the cytoplasmic face of NPCs late in infection. Additionally, prior expression of pUL16 and pUL21 prevented incoming nucleocapsids from docking at NPCs, delivering their genomes to the nucleus and initiating viral gene expression. Both pUL16 and pUL21 localized to the nuclear envelope, placing them in an appropriate location to interfere with nucleocapsid/NPC interactions.

Nucleoporin downregulation modulates progenitor differentiation independent of nuclear pore numbers.

Nucleoporins (NUPs) comprise nuclear pore complexes, gateways for nucleocytoplasmic transport. As primary human keratinocytes switch from the progenitor state towards differentiation, most NUPs are strongly downregulated, with NUP93 being the most downregulated NUP in this process. To determine if this NUP downregulation is accompanied by a reduction in nuclear pore numbers, we leveraged Stochastic Optical Reconstruction Microscopy. No significant changes in nuclear pore numbers were detected using three independent NUP antibodies; however, NUP reduction in other subcellular compartments such as the cytoplasm was identified. To investigate how NUP reduction influences keratinocyte differentiation, we knocked down NUP93 in keratinocytes in the progenitor-state culture condition. NUP93 knockdown diminished keratinocytes' clonogenicity and epidermal regenerative capacity, without drastically affecting nuclear pore numbers or permeability. Using transcriptome profiling, we identified that NUP93 knockdown induces differentiation genes related to both mechanical and immune barrier functions, including the activation of known NF-κB target genes. Consistently, keratinocytes with NUP93 knockdown exhibited increased nuclear localization of the NF-κB p65/p50 transcription factors, and increased NF-κB reporter activity. Taken together, these findings highlight the gene regulatory roles contributed by differential NUP expression levels in keratinocyte differentiation, independent of nuclear pore numbers.

From the sideline: Tissue-specific nucleoporin function in health and disease, an update.

The subcellular compartmentalisation of eukaryotic cells requires selective exchange between the cytoplasm and the nucleus. Intact nucleocytoplasmic transport is vital for normal cell function and mutations in the executing machinery have been causally linked to human disease. Central players in nucleocytoplasmic exchange are nuclear pore complexes (NPCs), which are built from ~30 distinct proteins collectively termed nucleoporins. Aberrant nucleoporin expression was detected in human cancers and autoimmune diseases since quite some time, while it was through the increasing use of next generation sequencing that mutations in nucleoporin genes associated with mainly rare hereditary diseases were revealed. The number of newly identified mutations is steadily increasing, as is the number of diseases. Mutational hotspots have emerged: mutations in the scaffold nucleoporins seemingly affect primarily inner organs, such as heart, kidney, and ovaries, whereas genetic alterations in peripheral, cytoplasmic nucleoporins affect primarily the central nervous system and development. In this review, we summarise latest insights on altered nucleoporin function in the context of human hereditary disorders, with a focus on those where mechanistic insights are beginning to emerge.