Nuclear pore complexes (NPC) regulate nucleocytoplasmic transport and are anchored in the nuclear envelope by the transmembrane nucleoporin NDC1. NDC1 is essential for post-mitotic NPC assembly and the recruitment of ALADIN to the nuclear envelope. While no human disorder has been associated to one of the three transmembrane nucleoporins, biallelic variants in AAAS, encoding ALADIN, cause Triple-A syndrome (Allgrove syndrome). Triple A syndrome, characterized by alacrima, achalasia and adrenal insufficiency, often includes progressive demyelinating polyneuropathy and other neurological complaints. In this report, diagnostic exome and/or RNA-sequencing was performed in 7 individuals from 4 unrelated consanguineous families with AAAS negative triple A syndrome. Molecular and clinical studies followed to elucidate the pathogenic mechanism. The affected individuals presented with intellectual disability, motor impairment, severe demyelinating with secondary axonal polyneuropathy, alacrima and achalasia.. None of the affected individuals has adrenal insufficiency. All individuals presented with biallelic NDC1 in-frame deletions or missense variants, that affect amino acids and protein domains required for ALADIN binding. No other significant variants associated with the phenotypic features were reported. Skin fibroblasts derived from affected individuals show decreased recruitment of ALADIN to the NE and decreased post-mitotic NPC insertion, confirming pathogenicity of the variants. Taken together, our results implicate biallelic NDC1 variants in the pathogenesis of polyneuropathy and a Triple-A-like disorder without adrenal insufficiency, by interfering with physiological NDC1 functions, including the recruitment of ALADIN to the NPC.

The p53 family remains a captivating focus of an extensive number of current studies. Accumulating evidence indicates that p53 abnormalities rank among the most prevalent in cancer. Given the numerous existing studies, which mostly focus on the mutations, expression profiles, and functional perturbations exhibited by members of the p53 family across diverse malignancies, this review will concentrate more on less explored facets regarding p53 activation and stabilization by the nuclear pore complex (NPC) in cancer, drawing on several studies. p53 integrates a broad spectrum of signals and is subject to diverse regulatory mechanisms to enact the necessary cellular response. It is widely acknowledged that each stage of p53 regulation, from synthesis to degradation, significantly influences its functionality in executing specific tasks. Over recent decades, a large body of data has established that mechanisms of regulation, closely linked with protein activation and stabilization, involve intricate interactions with various cellular components. These often transcend canonical regulatory pathways. This new knowledge has expanded from the regulation of genes themselves to epigenomics and proteomics, whereby interaction partners increase in number and complexity compared with earlier paradigms. Specifically, studies have recently shown the involvement of the NPC protein in such complex interactions, underscoring the further complexity of p53 regulation. Furthermore, we also discuss therapeutic strategies based on recent developments in this field in combination with established targeted therapies.

SUMMARYCilia and the nucleus were two defining features of the last eukaryotic common ancestor. In early eukaryotic evolution, these structures evolved through the diversification of a common membrane-coating ancestor, the protocoatomer. While in cilia, the descendants of this protein complex evolved into parts of the intraflagellar transport complexes and BBSome, the nucleus gained its selectivity by recruiting protocoatomer-like proteins to the nuclear envelope to form the selective nuclear pore complexes. Recent studies show a growing number of proteins shared between the proteomes of the respective organelles, and it is currently unknown how ciliary transport proteins could acquire nuclear functions and vice versa. The nuclear functions of ciliary proteins are still observable today and remain relevant for the understanding of the disease mechanisms behind ciliopathies. In this work, we review the evolutionary history of cilia and nucleus and their respective defining proteins and integrate current knowledge into theories for early eukaryotic evolution. We postulate a scenario where both compartments co-evolved and that fits current models of eukaryotic evolution, explaining how ciliary proteins and nucleoporins acquired their dual functions.

BACKGROUND: The piRNA pathway in animal gonads functions as an \'RNA-based immune system\', serving to silence transposable elements and prevent inheritance of novel invaders. In Drosophila, this pathway relies on three gonad-specific Argonaute proteins (Argonaute-3, Aubergine and Piwi) that associate with 23-28 nucleotide piRNAs, directing the silencing of transposon-derived transcripts. Transposons constitute a primary driver of genome evolution, yet the evolution of piRNA pathway factors has not received in-depth exploration. Specifically, channel nuclear pore proteins, which impact piRNA processing, exhibit regions of rapid evolution in their promoters. Consequently, the question arises whether such a mode of evolution is a general feature of transposon silencing pathways.
RESULTS: By employing genomic analysis of coding and promoter regions within genes that function in transposon silencing in Drosophila, we demonstrate that the promoters of germ cell-specific piRNA factors are undergoing rapid evolution. Our findings indicate that rapid promoter evolution is a common trait among piRNA factors engaged in germline silencing across insect species, potentially contributing to gene expression divergence in closely related taxa. Furthermore, we observe that the promoters of genes exclusively expressed in germ cells generally exhibit rapid evolution, with some divergence in gene expression.
CONCLUSIONS: Our results suggest that increased germline promoter evolution, in partnership with other factors, could contribute to transposon silencing and evolution of species through differential expression of genes driven by invading transposons.

Nuclear pore complexes (NPCs) regulate the entry and exit of molecules from the cell nucleus. Small molecules pass through NPCs by diffusion while large molecules enter and exit the nucleus by karyopherins, which serve as transport factors. Exportin-1 (XPO1) is a protein that is an important member of the karyopherin family and carries macromolecules from the nucleus to the cytoplasm. XPO1 is responsible for nuclear-cytoplasmic transport of protein, ribosomal RNA and certain required mRNAs for ribosomal biogenesis. Furthermore, XPO1-mediated nuclear export is associated with various types of disease, such as cancer, inflammation and viral infection. The key role of XPO1 in carcinogenesis and its potential as a therapeutic target has been demonstrated by previous studies. Clinical use of novel developed generation-specific XPO1 inhibitors and their combination with other agents to block XPO1-mediated nuclear export are a promising new treatment strategy. The aim of the present study was to explain the working mechanism of XPO1 and inhibitors that block XPO1-mediated nuclear export.

Cell division presents a challenge for eukaryotic cells: how can chromosomes effectively segregate within the confines of a membranous nuclear compartment? Different organisms have evolved diverse solutions by modulating the degree of nuclear compartmentalization, ranging from complete nuclear envelope breakdown to complete maintenance of nuclear compartmentalization via nuclear envelope expansion. Many intermediate forms exist between these extremes, suggesting that nuclear dynamics during cell division are surprisingly plastic. In this review, we highlight the evolutionary diversity of nuclear divisions, focusing on two defining characteristics: (1) chromosome compartmentalization and (2) nucleocytoplasmic transport. Further, we highlight recent evidence that nuclear behavior during division can vary within different cellular contexts in the same organism. The variation observed within and between organisms underscores the dynamic evolution of nuclear divisions tailored to specific contexts and cellular requirements. In-depth investigation of diverse nuclear divisions will enhance our understanding of the nucleus, both in physiological and pathological states.

In metazoans, the nuclear envelope (NE) disassembles during the prophase and reassembles around segregated chromatids during the telophase. The process of NE formation has been extensively studied using live-cell imaging. At the early step of NE reassembly in human cells, specific pattern-like localization of inner nuclear membrane (INM) proteins, connected to the nuclear pore complex (NPC), was observed in the so-called \"core\" region and \"noncore\" region on telophase chromosomes, which corresponded to the \"pore-free\" region and the \"pore-rich\" region, respectively, in the early G1 interphase nucleus. We refer to these phenomena as NE subdomain formation. To biochemically investigate this process, we aimed to develop an in vitro NE reconstitution system using digitonin-permeabilized semi-intact mitotic human cells coexpressing two INM proteins, emerin and lamin B receptor, which were labeled with fluorescent proteins. The targeting and accumulation of INM proteins to chromosomes before and after anaphase onset in semi-intact cells were observed using time-lapse imaging. Our in vitro NE reconstitution system recapitulated the formation of the NE subdomain, as in living cells, although chromosome segregation and cytokinesis were not observed. This in vitro NE reconstitution required the addition of a mitotic cytosolic fraction supplemented with a cyclin-dependent kinase inhibitor and energy sources. The cytoplasmic soluble factor(s) dependency of INM protein targeting differed among the segregation states of chromosomes. Furthermore, the NE reconstituted on segregated chromosomes exhibited active nucleocytoplasmic transport competency. These results indicate that the chromosome status changes after anaphase onset for recruiting NPC components.

Circular RNAs (circRNAs) are upregulated during neurogenesis. Where and how circRNAs are localized and what roles they play during this process have remained elusive. Comparing the nuclear and cytoplasmic circRNAs between H9 cells and H9-derived forebrain (FB) neurons, we identify that a subset of adenosine (A)-rich circRNAs are restricted in H9 nuclei but exported to cytosols upon differentiation. Such a subcellular relocation of circRNAs is modulated by the poly(A)-binding protein PABPC1. In the H9 nucleus, newly produced (A)-rich circRNAs are bound by PABPC1 and trapped by the nuclear basket protein TPR to prevent their export. Modulating (A)-rich motifs in circRNAs alters their subcellular localization, and introducing (A)-rich circRNAs in H9 cytosols results in mRNA translation suppression. Moreover, decreased nuclear PABPC1 upon neuronal differentiation enables the export of (A)-rich circRNAs, including circRTN4(2,3), which is required for neurite outgrowth. These findings uncover subcellular localization features of circRNAs, linking their processing and function during neurogenesis.

Nucleocytoplasmic transport of macromolecules is essential in eukaryotic cells. In this process, the karyopherins play a central role when they transport cargoes across the nuclear pore complex. Importin 4 belongs to the karyopherin β family. Many studies have focused on finding substrates for importin 4, but no direct mechanism studies of its precise transport function have been reported. Therefore, this paper mainly aimed to study the mechanism of nucleoporins in mediating nuclear import and export of importin 4. To address this question, we constructed shRNAs targeting Nup358, Nup153, Nup98, and Nup50. We found that depletion of Nup98 resulted in a shift in the subcellular localization of importin 4 from the cytoplasm to the nucleus. Mutational analysis demonstrated that Nup98 physically and functionally interacts with importin 4 through its N-terminal phenylalanine-glycine (FG) repeat region. Mutation of nine of these FG motifs to SG motifs significantly attenuated the binding of Nup98 to importin 4, and we further confirmed the essential role of the six FG motifs in amino acids 121-360 of Nup98 in binding with importin 4. In vitro transport assay also confirmed that VDR, the substrate of importin 4, could not be transported into the nucleus after Nup98 knockdown. Overall, our results showed that Nup98 is required for efficient importin 4-mediated transport. This is the first study to reveal the mechanism of importin 4 in transporting substrates into the nucleus.

To integrate with host chromatin and establish a productive infection, HIV-1 must translocate the viral Ribonucleoprotein (RNP) complex through the nuclear pore complex (NPC). Current assay to measure HIV-1 nuclear import relies on a transient byproduct of HIV-1 integration failure called 2-LTR circles. However, 2-LTR circles require complete or near-complete reverse transcription and association with the non-homologous end joining (NHEJ) machinery in the nucleus, which can complicate interpretation of 2-LTR circle formation as a measure of nuclear import kinetics. Here, we describe an approach to measure nuclear import of infectious HIV-1 particles. This involves chemically induced dimerization of Nup62, a central FG containing nucleoporin. Using this technique, nuclear import of infectious particles can be monitored in both primary and cell culture models. In response to host factor depletion or restriction factors, changes in HIV-1 nuclear import can be effectively measured using the nuclear import kinetics (NIK) assay.