Evolution of a transcriptional regulator from a transmembrane nucleoporin.

Nuclear pore complexes (NPCs) emerged as nuclear transport channels in eukaryotic cells ∼1.5 billion years ago. While the primary role of NPCs is to regulate nucleo-cytoplasmic transport, recent research suggests that certain NPC proteins have additionally acquired the role of affecting gene expression at the nuclear periphery and in the nucleoplasm in metazoans. Here we identify a widely expressed variant of the transmembrane nucleoporin (Nup) Pom121 (named sPom121, for "soluble Pom121") that arose by genomic rearrangement before the divergence of hominoids. sPom121 lacks the nuclear membrane-anchoring domain and thus does not localize to the NPC. Instead, sPom121 colocalizes and interacts with nucleoplasmic Nup98, a previously identified transcriptional regulator, at gene promoters to control transcription of its target genes in human cells. Interestingly, sPom121 transcripts appear independently in several mammalian species, suggesting convergent innovation of Nup-mediated transcription regulation during mammalian evolution. Our findings implicate alternate transcription initiation as a mechanism to increase the functional diversity of NPC components.

Adenovirus entry: Stability, uncoating, and nuclear import.

Adenoviruses (AdVs) are widespread in vertebrates. They infect the respiratory and gastrointestinal tracts, the eyes, heart, liver, and kidney, and are lethal to immunosuppressed people. Mastadenoviruses infecting mammals comprise several hundred different types, and many specifically infect humans. Human adenoviruses are the most widely used vectors in clinical applications, including cancer treatment and COVID-19 vaccination. AdV vectors are physically and genetically stable and generally safe in humans. The particles have an icosahedral coat and a nucleoprotein core with a DNA genome. We describe the concept of AdV cell entry and highlight recent advances in cytoplasmic transport, uncoating, and nuclear import of the viral DNA. We highlight a recently discovered "linchpin" function of the virion protein V ensuring cytoplasmic particle stability, which is relaxed at the nuclear pore complex by cues from the E3 ubiquitin ligase Mind bomb 1 (MIB1) and the proteasome triggering disruption. Capsid disruption by kinesin motor proteins and microtubules exposes the linchpin and renders protein V a target for MIB1 ubiquitination, which dissociates V from viral DNA and enhances DNA nuclear import. These advances uncover mechanisms controlling capsid stability and premature uncoating and provide insight into nuclear transport of nucleic acids.

The Nuclear Pore Complex in Cell Type-Specific Chromatin Structure and Gene Regulation.

Nuclear pore complex (NPC)-mediated nucleocytoplasmic trafficking is essential for key cellular processes, such as cell growth, cell differentiation, and gene regulation. The NPC has also been viewed as a nuclear architectural platform that impacts genome function and gene expression by mediating spatial and temporal coordination between transcription factors, chromatin regulatory proteins, and transcription machinery. Recent findings have uncovered differential and cell type-specific expression and distinct chromatin-binding patterns of individual NPC components known as nucleoporins (Nups). Here, we examine recent studies that investigate the functional roles of NPCs and Nups in transcription, chromatin organization, and epigenetic gene regulation in the context of development and disease.

Channel nuclear pore protein 54 directs sexual differentiation and neuronal wiring of female reproductive behaviors in Drosophila.

BACKGROUND: Female reproductive behaviors and physiology change profoundly after mating. The control of pregnancy-associated changes in physiology and behaviors are largely hard-wired into the brain to guarantee reproductive success, yet the gene expression programs that direct neuronal differentiation and circuit wiring at the end of the sex determination pathway in response to mating are largely unknown. In Drosophila, the post-mating response induced by male-derived sex-peptide in females is a well-established model to elucidate how complex innate behaviors are hard-wired into the brain. Here, we use a genetic approach to further characterize the molecular and cellular architecture of the sex-peptide response in Drosophila females. RESULTS: Screening for mutations that affect the sensitivity to sex-peptide, we identified the channel nuclear pore protein Nup54 gene as an essential component for mediating the sex-peptide response, with viable mutant alleles leading to the inability of laying eggs and reducing receptivity upon sex-peptide exposure. Nup54 directs correct wiring of eight adult brain neurons that express pickpocket and are required for egg-laying, while additional channel Nups also mediate sexual differentiation. Consistent with links of Nups to speciation, the Nup54 promoter is a hot spot for rapid evolution and promoter variants alter nucleo-cytoplasmic shuttling. CONCLUSIONS: These results implicate nuclear pore functionality to neuronal wiring underlying the sex-peptide response and sexual differentiation as a response to sexual conflict arising from male-derived sex-peptide to direct the female post-mating response.

Biallelic Variants in the Nuclear Pore Complex Protein NUP93 Are Associated with Non-progressive Congenital Ataxia.

Nuclear pore complexes (NPCs) are the gateways of the nuclear envelope mediating transport between cytoplasm and nucleus. They form huge complexes of 125 MDa in vertebrates and consist of about 30 different nucleoporins present in multiple copies in each complex. Here, we describe pathogenic variants in the nucleoporin 93 (NUP93) associated with an autosomal recessive form of congenital ataxia. Two rare compound heterozygous variants of NUP93 were identified by whole exome sequencing in two brothers with isolated cerebellar atrophy: one missense variant (p.R537W) results in a protein which does not localize to NPCs and cannot functionally replace the wild type protein, whereas the variant (p.F699L) apparently supports NPC assembly. In addition to its recently described pathological role in steroid-resistant nephrotic syndrome, our work identifies NUP93 as a candidate gene for non-progressive congenital ataxia.

The nuclear pore complex: a strategic platform for regulating cell signaling.

Contents Summary 25 I Introduction 25 II. Structural organization of the NPC 26 III. The role of NPCs in immune signaling 26 IV. The role of NPCs in hormone signaling 28 V. Conclusions 29 Acknowledgements 29 References 29 SUMMARY: Nuclear pore complexes (NPCs) are fundamental components of the eukaryotic cell. They perforate the nuclear envelope and serve as highly selective transport gates that enable bi-directional macromolecule exchange between the nucleus and cytoplasm. Recent studies illustrate that the NPC is not a static structural channel but a flexible environment and strategic player during nuclear signaling. The constitutional and conformational dynamics of the NPC allow it to tailor nucleocytoplasmic transport activities and define specific signaling output in response to various cellular and environmental cues. In this Insight, we review the roles of NPC constituents in immune activation and hormone signaling in plants, and discuss the possible role of the NPC as a legitimate platform for regulating cell signaling.

RNA Nuclear Export: From Neurological Disorders to Cancer.

The presence of a nuclear envelope, also known as nuclear membrane, defines the structural framework of all eukaryotic cells by separating the nucleus, which contains the genetic material, from the cytoplasm where the synthesis of proteins takes place. Translation of proteins in Eukaryotes is thus dependent on the active transport of DNA-encoded RNA molecules through pores embedded within the nuclear membrane. Several mechanisms are involved in this process generally referred to as RNA nuclear export or nucleocytoplasmic transport of RNA. The regulated expression of genes requires the nuclear export of protein-coding messenger RNA molecules (mRNAs) as well as non-coding RNAs (ncRNAs) together with proteins and pre-assembled ribosomal subunits. The nuclear export of mRNAs is intrinsically linked to the co-transcriptional processing of nascent transcripts synthesized by the RNA polymerase II. This functional coupling is essential for the survival of cells allowing for timely nuclear export of fully processed transcripts, which could otherwise cause the translation of abnormal proteins such as the polymeric repeat proteins produced in some neurodegenerative diseases. Alterations of the mRNA nuclear export pathways can also lead to genome instability and to various forms of cancer. This chapter will describe the molecular mechanisms driving the nuclear export of RNAs with a particular emphasis on mRNAs. It will also review their known alterations in neurological disorders and cancer, and the recent opportunities they offer for the potential development of novel therapeutic strategies.

Components of the Arabidopsis nuclear pore complex play multiple diverse roles in control of plant growth.

The nuclear pore complex (NPC) is a multisubunit protein conglomerate that facilitates movement of RNA and protein between the nucleus and cytoplasm. Relatively little is known regarding the influence of the Arabidopsis NPC on growth and development. Seedling development, flowering time, nuclear morphology, mRNA accumulation, and gene expression changes in Arabidopsis nucleoporin mutants were investigated. Nuclear export of mRNA is differentially affected in plants with defects in nucleoporins that lie in different NPC subcomplexes. This study reveals differences in the manner by which nucleoporins alter molecular and plant growth phenotypes, suggesting that nuclear pore subcomplexes play distinct roles in nuclear transport and reveal a possible feedback relationship between the expression of genes involved in nuclear transport.

Targeting colorectal cancer at the level of nuclear pore complex.

BACKGROUND: Nuclear pore complexes (NPCs) are the architectures entrenched in nuclear envelop of a cell that regulate the nucleo-cytoplasmic transportation of materials, such as proteins and RNAs for proper functioning of a cell. The appropriate localization of proteins and RNAs within the cell is essential for its normal functionality. For such a complex transportation of materials across the NPC, around 60 proteins are involved comprising nucleoporins, karyopherins and RAN system proteins that play a vital role in NPC's structure formation, cargo translocation across NPC, and cargoes' rapid directed transportation respectively. In various cancers, the structure and function of NPC is often exaggerated, following altered expressions of its nucleoporins and karyopherins, affecting other proteins of associated signaling pathways. Some inhibitors of karyopherins at present, have potential to regulate the altered level/expression of these karyopherin molecules. AIM OF REVIEW: This review summarizes the data from 1990 to 2023, mainly focusing on recent studies that illustrate the structure and function of NPC, the relationship and mechanisms of nucleoporins and karyopherins with colorectal cancer, as well as therapeutic values, in order to understand the pathology and underlying basis of colorectal cancer associated with NPC. This is the first review to our knowledge elucidating the detailed updated studies targeting colorectal cancer at NPC. The review also aims to target certain karyopherins, Nups and their possible inhibitors and activators molecules as a therapeutic strategy. KEY SCIENTIFIC CONCEPTS OF REVIEW: NPC structure provides understanding, how nucleoporins and karyopherins as key molecules are responsible for appropriate nucleocytoplasmic transportation. Many studies provide evidences, describing the role of disrupted nucleoporins and karyopherins not only in CRC but also in other non-hematological and hematological malignancies. At present, some inhibitors of karyopherins have therapeutic potential for CRC, however development of more potent inhibitors may provide more effective therapeutic strategies for CRC in near future.

Analysis of Tau/Nucleoporin Interactions by Surface Plasmon Resonance Spectroscopy.

Tau, a soluble and predominantly neuronal protein, is best known for its microtubule (MT)-binding function in the cytosol, where it decisively contributes to stability as well as modulation of MT dynamics. In Alzheimer's disease and other tauopathies, Tau is altered into forming intracellular neurofibrillary tangles; additionally, also a mislocalization from the cytosol to the nucleus has been observed where interactions of Tau with the nucleus become possible. Using surface plasmon resonance (SPR), it was recently shown that Tau can directly interact with certain nucleoporins (e.g., Nup98), components of the nuclear pore complex (NPC). The NPC constitutes large regulated pores in the nuclear envelope that facilitate the bidirectional exchange of proteins, nucleic acids, and other biomolecules between the inner section of the nucleus and the cytosol, the nucleocytoplasmic transport. The mechanism of Tau/Nup interactions is as yet unknown, and a systematic interaction analysis of Tau with different Nups can be of high value to decipher the molecular binding mechanism of Tau to Nups. SPR is a useful tool to analyze binding affinities and kinetic parameters in a label-free environment. While one interaction partner is immobilized on a sensor chip, the second is supplied within a constant flow of buffer. Binding of mobile molecules to immobilized ones changes the refractive index of the medium close to the sensor surface with the signal being proportional to the bound mass. In this chapter, we describe the application of the SPR technique for the investigation of Tau binding to nucleoporins.

Unraveling the impact of disrupted nucleocytoplasmic transport systems in C9orf72-associated ALS.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two adult-onset neurodegenerative diseases that are part of a common disease spectrum due to clinical, genetic, and pathological overlap. A prominent genetic factor contributing to both diseases is a hexanucleotide repeat expansion in a non-coding region of the C9orf72 gene. This mutation in C9orf72 leads to nuclear depletion and cytoplasmic aggregation of Tar DNA-RNA binding protein 43 (TDP-43). TDP-43 pathology is characteristic of the majority of ALS cases, irrespective of disease causation, and is present in ~50% of FTD cases. Defects in nucleocytoplasmic transport involving the nuclear pore complex, the Ran-GTPase cycle, and nuclear transport factors have been linked with the mislocalization of TDP-43. Here, we will explore and discuss the implications of these system abnormalities of nucleocytoplasmic transport in C9orf72-ALS/FTD, as well as in other forms of familial and sporadic ALS.

Transformation of Chaetomium thermophilum and Affinity Purification of Native Thermostable Protein Complexes.

Chaetomium thermophilum, a eukaryotic thermophile, is an aspiring organism holding great potential for various biochemical and biotechnological applications. Prerequisite for genetic manipulation is a reliable transformation system for target genes combined with selection markers operating at the high growth temperatures of the fungus. Here, we present a detailed protocol for Chaetomium thermophilum protoplast transformation to allow stable chromosomal integration of constructs into its genome, rendering this eukaryotic thermophile a valuable resource for affinity purification of native thermostable protein complexes, like nuclear pore subcomplexes.

Purification of Cdk-CyclinB-Kinase-Targeted Phosphopeptides from Nuclear Envelope.

We describe a method for rapid identification of protein kinase substrates within the nuclear envelope. Open mitosis in higher eukaryotes is characterized by nuclear envelope breakdown (NEBD) concerted with disassembly of the nuclear lamina and dissociation of nuclear pore complexes (NPCs) into individual subcomplexes. Evidence indicates that reversible phosphorylation events largely drive this mitotic NEBD. These posttranslational modifications likely disrupt structurally significant interactions among nucleoporins (Nups), lamina and membrane proteins of the nuclear envelope (NE). It is therefore critical to determine when and where these substrates are phosphorylated. One likely regulator is the mitotic kinase: Cdk1-Cyclin B. We employed an "analog-sensitive" Cdk1 to bio-orthogonally and uniquely label its substrates in the NE with a phosphate analog tag. Subsequently, peptides covalently modified with the phosphate analogs are rapidly purified by a tag-specific covalent capture and release methodology. In this manner, we were able to confirm the identity of known Cdk1 targets in the NE and discover additional candidates for regulation by mitotic phosphorylation.

Nucleocytoplasmic Communication in Healthy and Diseased Plant Tissues.

The double membrane of the nuclear envelope (NE) constitutes a selective compartment barrier that separates nuclear from cytoplasmic processes. Plant viability and responses to a changing environment depend on the spatial communication between both compartments. This communication is based on the bidirectional exchange of proteins and RNAs and is regulated by a sophisticated transport machinery. Macromolecular traffic across the NE depends on nuclear transport receptors (NTRs) that mediate nuclear import (i.e. importins) or export (i.e. exportins), as well as on nuclear pore complexes (NPCs) that are composed of nucleoporin proteins (NUPs) and span the NE. In this review, we provide an overview of plant NPC- and NTR-directed cargo transport and we consider transport independent functions of NPCs and NE-associated proteins in regulating plant developmental processes and responses to environmental stresses.

The Portal Vertex of KSHV Promotes Docking of Capsids at the Nuclear Pores.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-related herpesvirus. Like other herpesviruses, the KSHV icosahedral capsid includes a portal vertex, composed of 12 protein subunits encoded by open reading frame (ORF) 43, which enables packaging and release of the viral genome into the nucleus through the nuclear pore complex (NPC). Capsid vertex-specific component (CVSC) tegument proteins, which directly mediate docking at the NPCs, are organized on the capsid vertices and are enriched on the portal vertex. Whether and how the portal vertex is selected for docking at the NPC is unknown. Here, we investigated the docking of incoming ORF43-null KSHV capsids at the NPCs, and describe a significantly lower fraction of capsids attached to the nuclear envelope compared to wild-type (WT) capsids. Like WT capsids, nuclear envelope-associated ORF43-null capsids co-localized with different nucleoporins (Nups) and did not detach upon salt treatment. Inhibition of nuclear export did not alter WT capsid docking. As ORF43-null capsids exhibit lower extent of association with the NPCs, we conclude that although not essential, the portal has a role in mediating the interaction of the CVSC proteins with Nups, and suggest a model whereby WT capsids can dock at the nuclear envelope through a non-portal penton vertex, resulting in an infection 'dead end'.

Regulation of Plant Immunity by Nuclear Membrane-Associated Mechanisms.

Unlike animals, plants do not have specialized immune cells and lack an adaptive immune system. Instead, plant cells rely on their unique innate immune system to defend against pathogens and coordinate beneficial interactions with commensal and symbiotic microbes. One of the major convergent points for plant immune signaling is the nucleus, where transcriptome reprogramming is initiated to orchestrate defense responses. Mechanisms that regulate selective transport of nuclear signaling cargo and chromatin activity at the nuclear boundary play a pivotal role in immune activation. This review summarizes the current knowledge of how nuclear membrane-associated core protein and protein complexes, including the nuclear pore complex, nuclear transport receptors, and the nucleoskeleton participate in plant innate immune activation and pathogen resistance. We also discuss the role of their functional counterparts in regulating innate immunity in animals and highlight potential common mechanisms that contribute to nuclear membrane-centered immune regulation in higher eukaryotes.

Nucleocytoplasmic transport of intrinsically disordered proteins studied by high-speed super-resolution microscopy.

Both natively folded and intrinsically disordered proteins (IDPs) destined for the nucleus need to transport through the nuclear pore complexes (NPCs) in eukaryotic cells. NPCs allow for passive diffusion of small folded proteins while barricading large ones, unless they are facilitated by nuclear transport receptors. However, whether nucleocytoplasmic transport of IDPs would follow these rules remains unknown. By using a high-speed super-resolution fluorescence microscopy, we have measured transport kinetics and 3D spatial locations of transport routes through native NPCs for various IDPs. Our data revealed that the rules executed for folded proteins are not well followed by the IDPs. Instead, both large and small IDPs can passively diffuse through the NPCs. Furthermore, their diffusion efficiencies and routes are differentiated by their content ratio of charged (Ch) and hydrophobic (Hy) amino acids. A Ch/Hy-ratio mechanism was finally suggested for nucleocytoplasmic transport of IDPs.

The Role of Torsin AAA+ Proteins in Preserving Nuclear Envelope Integrity and Safeguarding Against Disease.

Torsin ATPases are members of the AAA+ (ATPases associated with various cellular activities) superfamily of proteins, which participate in essential cellular processes. While AAA+ proteins are ubiquitously expressed and demonstrate distinct subcellular localizations, Torsins are the only AAA+ to reside within the nuclear envelope (NE) and endoplasmic reticulum (ER) network. Moreover, due to the absence of integral catalytic features, Torsins require the NE- and ER-specific regulatory cofactors, lamina-associated polypeptide 1 (LAP1) and luminal domain like LAP1 (LULL1), to efficiently trigger their atypical mode of ATP hydrolysis. Despite their implication in an ever-growing list of diverse processes, the specific contributions of Torsin/cofactor assemblies in maintaining normal cellular physiology remain largely enigmatic. Resolving gaps in the functional and mechanistic principles of Torsins and their cofactors are of considerable medical importance, as aberrant Torsin behavior is the principal cause of the movement disorder DYT1 early-onset dystonia. In this review, we examine recent findings regarding the phenotypic consequences of compromised Torsin and cofactor activities. In particular, we focus on the molecular features underlying NE defects and the contributions of Torsins to nuclear pore complex biogenesis, as well as the growing implications of Torsins in cellular lipid metabolism. Additionally, we discuss how understanding Torsins may facilitate the study of essential but poorly understood processes at the NE and ER, and aid in the development of therapeutic strategies for dystonia.

Extra-Low-Frequency Pulse Stimulated Conformational Change in Blood-Cell Proteins and Consequent Immune Activity Transformation.

Objective: investigation of the extra-low-frequency (ELF) stimulation effect on blood-cell proteins, that causes variation in its electrostatic-state. A hypothesis that this results in the conformational change in the blood-cell proteins which could enhance immune activity is explored. Since HIV-1 and host-cell engage through charge-charge interactions, an electrical-pulse may cause charge redistribution, hypothetically resulting in host-cell proteins to be isolated from viral access. Methods: Buffy coat samples were exposed to ELF square waveform pulses of 5Hz, 10Hz and 1MHz, for 2-hours, and were then examined using immunofluorescence technique. The expression of glycoprotein CD4, and co-receptor protein CCR5, were investigated. Also, the binding activity of the N-terminal domain of CCR5 and the distribution of the nuclear-pore-complex (NPC) transport factor, FGNup153 were investigated. Comparison with control samples were carried out. Results: Increased CD4 count, which could enhance the immune system. In addition, the inability of N-terminus-specific antibody 3A9 to bind to CCR5 N-terminal, could be due to the interactions with the ELF electric-field, which may also hypothetically inhibit HIV-1 attachment. Furthermore, the electrostatic interactions between the ELF pulse and the FGNup153 induces redistribution in its disorder sequence and possibly causes conformational change. This could possibly prevent large virus particle transport through the NPC. Conclusion: Novel concept of ELF stimulation of blood cellular proteins has been developed leading to transformation of immune activity. Clinical-Impact: The translational aspect is the use of ELF as an avenue of electro-medicine and the results are a possible foundation for the clinical application of ELF stimulation in immune response.

Identification of new transmembrane proteins concentrated at the nuclear envelope using organellar proteomics of mesenchymal cells.

The double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) - the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) - a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins.