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Plant cells enhance the tolerances to abiotic and biotic stresses via recognition of the stress, activation and nuclear import of signaling factors, up-regulation of defense genes, nuclear export of mRNA and translation of defense proteins. Nuclear pore-mediated transports should play critical roles in these processes, however, the regulatory mechanisms of nuclear-cytoplasmic transport during stress responses are largely unknown. In this study, a regulator of nuclear export of RNA and proteins, NbRanBP1-1 (Ran-binding protein1-1), was identified as an essential gene for the resistance of Show less

To explore the effect of microRNA-577 on the drug sensitivity of chronic myeloid leukemia (CML) and the underlying mechanism. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect the expression of microRNA-577 in peripheral blood of patients with chronic myeloid leukemia. Meanwhile, the expression of microRNA-577 was detected in CML cell line after imatinib treatment. Cell counting kit-8 (CCK-8) and flow cytometry assay were applied to verify the effect of microRNA-577 on cell proliferation and cycle. NUP160 was identified as a target gene of microRNA-577 by dual-luciferase reporter gene assay. Cell reverse test was performed to figure out whether microRNA-577 can enhance the sensitivity of CML to imatinib. QRT-PCR results revealed that microRNA-577 level was notably decreased in peripheral blood of patients with CML, and microRNA-577 could inhibit the proliferation and cycle of CML cells. In addition, the result of dual-luciferase reporting assay indicated that microRNA-577 had a binding relationship with NUP160, and up-regulation of microRNA-577 in CML cell lines reduced the expression of NUP160, and vice versa. Lastly, cell reverse experiments confirmed that microRNA-577 can alleviate the resistance of CML to imatinib. We found that microRNA-577 promotes the sensitivity of chronic myeloid leukemia cells to imatinib by down-regulating the expression of NUP160. Show less

The nuclear pore complex is comprised of approximately 30 proteins named nucleoporins (Nups) and tightly regulates nucleocytoplasmic transport of macromolecules across the nuclear membrane. Genetic alterations in many NUP genes are associated with many human maladies, such as neurological disease, autoimmune disorders and cancer. We reviewed the status quo of recent advancement of the knowledge of oncogenic role of nucleoporins in human carcinogenesis, focusing on major non-hematological malignancies in the recent literature. Both clinical study-derived and experiment-based reports were critically reviewed. We have also discussed the potential of nucleoporins as novel cancer biomarkers and promising therapeutic target against human malignancies. Several Nups such as Nup53, Nup88, Nup98, Nup160 and Nup214 modulated a plethora of cellular and physiological pathways involved in tumorigenesis such as GSK3β-Snail, Wnt/β-Catenin and RanGap1/RanBP2 signaling axes, DNA damage response, resistance to apoptosis and chemotherapy. Although classically, majority of studies have shown oncogenic roles of nucleoporins as genetic fusion partners in several types of leukemia, emerging evidence suggests that nucleoporins also modulate many cellular signaling pathways that are associated with several major non-hematological malignancies, such as carcinomas of skin, breast, lung, prostate and colon. Hence, nucleoporins are emerging as novel therapeutic targets in human tumors. Show less

To evaluate whether the levels of some molecules implicated in nucleocytoplasmic transport in human cardiomyocytes are related to the severity of heart failure (HF) in patients on the heart transplantation (HT) waiting list, and to determine whether there is a differential pattern of molecular alteration between ischemic cardiomyopathy (ICM) and non-ischemic dilated cardiomyopathy (DCM). Sixty-three blood samples collected before HT were analyzed to identify the levels of IMPORTIN5 (IMP5); IMPORTINalpha2; ATPaseCaTransp (ATPCa); NUCLEOPORIN153kDa (Nup153); NUCLEOPORIN160kDa (Nup160); RANGTPaseAP1 (RanGAP1) and EXPORTIN4 (EXP4). These data were then compared between patients with advanced HF with or without the need for ventricular support with extracorporeal membrane oxygenation (ECMO) as a bridge for HT, as well as between patients with non-ischemic DCM and patients with ICM. Thirty-three patients had ICM, 26 had non-ischemic DCM, and 4 had heart disease. Seventeen patients required ventricular assistance as a bridge to HT. The levels of ATPCa, RanGAP1, and IMP5 were significantly higher in patients with ECMO, while EXP4 was significantly higher in patients without ECMO. Patients with DCM showed higher levels of IMP5, RanGAP1, and Nup153 than those with ICM. Patients with advanced HF in critical condition (with ECMO as a bridge for HT) presented with significantly higher levels of ATPCa, RanGAP1, and IMP5, while patients with DCM had significantly higher levels of RanGAP1, IMP5, and Nup153. It remains to be clarified whether the determination of these molecules would facilitate the early identification of this group or if their alteration occurs as consequence of circulatory support with ECMO. Show less

Currently, cisplatin (DDP) is the first-line chemotherapeutic agent used for treatment of ovarian cancer, but gradually acquired drug resistance minimizes its therapeutic outcomes. We aimed to identify crucial genes associated with DDP resistance in ovarian cancer and uncover potential mechanisms. Two sets of gene expression data were downloaded from Gene Expression Omnibus, and bioinformatics analysis was conducted. In our study, the differentially expressed genes between DDP-sensitive and DDP-resistant ovarian cancer were screened in GSE15709 and GSE51373 database, and chromosome condensation 2 regulator (RCC2) and nucleoporin 160 were identified as 2 genes that significantly up-regulated in DDP-resistant ovarian cancer cell lines compared with DDP-sensitive cell lines. Moreover, RCC2, Ral small GTPase (RalA), and Ral binding protein-1 (RalBP1) expression was found to be significantly higher in DDP-resistant ovarian cancer tissues than in DDP-sensitive tissues. RCC2 plays a positive role in cell proliferation, apoptosis, and migration in DDP-resistant ovarian cancer cell lines in vitro and in vivo. Furthermore, RCC2 could interact with RalA, thus promoting its downstream effector RalBP1. RalA knockdown could reverse the effects of RCC2 overexpression on DDP-resistant ovarian cancer cell proliferation, apoptosis, and migration. Similarly, RalA overexpression could alleviate the effects of RCC2 knockdown in DDP-resistant ovarian cancer cells. Taken together, RCC2 may function as an oncogene, regulating the RalA signaling pathway, and intervention of RCC2 expression might be a promising therapeutic strategy for DDP-resistant ovarian cancer.-Gong, S., Chen, Y., Meng, F., Zhang, Y., Wu, H., Li, C., Zhang, G. RCC2, a regulator of the RalA signaling pathway, is identified as a novel therapeutic target in cisplatin-resistant ovarian cancer. Show less

Genetic mutations in dozens of monogenic genes can lead to serious podocyte dysfunction, which is a major cause of steroid-resistant nephrotic syndrome (SRNS). The NUP160 gene is expressed in both human kidney and mouse kidney. However, whether knockdown of NUP160 impairs podocytes has not yet been established. Therefore, we knocked down NUP160 by targeted short hairpin RNA (shRNA) in conditionally immortalized mouse podocytes and observed the effect of NUP160 knockdown on the proliferation, apoptosis, autophagy and cell migration of podocytes. We also investigated the effect of NUP160 knockdown on the expression and localization of podocyte associated molecules, such as nephrin, podocin, CD2AP and α-actinin-4. The knockdown of NUP160 significantly inhibited the proliferation of podocytes by decreasing the expression of both cyclin D1 and CDK4, increasing the expression of p27, and inducing S phase arrest. The knockdown of NUP160 promoted the apoptosis and autophagy of podocytes, and enhanced cell migration. The knockdown of NUP160 decreased the expression of nephrin, podocin and CD2AP in podocytes, and increased the expression of α-actinin-4. The knockdown of NUP160 also altered the subcellular localization of nephrin, podocin and CD2AP in podocytes. These results suggest that the knockdown of NUP160 impairs mouse podocytes, i.e. inhibiting cell proliferation, inducing apoptosis, autophagy and cell migration of mouse podocytes, and altering the expression and localization of podocyte associated molecules, including nephrin, podocin, CD2AP and α-actinin-4. Show less

Until now, large-scale genome-wide association studies have identified 94 genes associated with Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Expression quantitative trait locus (eQTL) analysis showed that six genetic variants around six of these 94 genes could drive both disease susceptibility and altered expression of six nearby genes including CD33 (rs3865444), PILRB (rs1476679), NUP160 (rs10838725), LRRK2 (rs76904798), RGS1 (rs1323292), and METTL21B (rs701006). However, two of these six genetic variants rs1476679 and rs76904798 variants could regulate the expression of PILRB and LRRK2 only in the human monocyte-derived microglia-like (MDMi) cells, but not in human peripheral blood monocytes. Here, we aim to verify these findings using another two eQTL datasets in human peripheral blood immune cell CD14+ monocytes. The results that showed that rs1476679 and rs76904798 variants or their proxy variants could significantly regulate the expression of PILRB and LRRK2 in immune cell CD14+ monocytes and human peripheral blood. We believe that these findings provide important supplementary information about the regulatory mechanisms by which both variants influence PILRB and LRRK2 gene expression and neurodegenerative disease risk. Show less

Steroid-resistant nephrotic syndrome (SRNS) almost invariably progresses to end-stage renal disease. Although more than 50 monogenic causes of SRNS have been described, a large proportion of SRNS remains unexplained. Recently, it was discovered that mutations of NUP93 and NUP205, encoding 2 proteins of the inner ring subunit of the nuclear pore complex (NPC), cause SRNS. Here, we describe mutations in genes encoding 4 components of the outer rings of the NPC, namely NUP107, NUP85, NUP133, and NUP160, in 13 families with SRNS. Using coimmunoprecipitation experiments, we showed that certain pathogenic alleles weakened the interaction between neighboring NPC subunits. We demonstrated that morpholino knockdown of nup107, nup85, or nup133 in Xenopus disrupted glomerulogenesis. Re-expression of WT mRNA, but not of mRNA reflecting mutations from SRNS patients, mitigated this phenotype. We furthermore found that CRISPR/Cas9 knockout of NUP107, NUP85, or NUP133 in podocytes activated Cdc42, an important effector of SRNS pathogenesis. CRISPR/Cas9 knockout of nup107 or nup85 in zebrafish caused developmental anomalies and early lethality. In contrast, an in-frame mutation of nup107 did not affect survival, thus mimicking the allelic effects seen in humans. In conclusion, we discovered here that mutations in 4 genes encoding components of the outer ring subunits of the NPC cause SRNS and thereby provide further evidence that specific hypomorphic mutations in these essential genes cause a distinct, organ-specific phenotype. Show less

BACKGROUND We aimed to identify pivotal genes and pathways involved in pancreatic ductal adenocarcinoma (PDAC), and explore possible molecular markers for the early diagnosis of the disease. MATERIAL AND METHODS The array data of GSE74629, including 34 PDAC samples and 16 healthy samples, was downloaded from GEO (Gene Expression Omnibus) database. Then, the DEGs (differentially expressed genes) in PDAC samples were compared with healthy samples using limma (linear models for microarray). Gene functional interaction networks were analyzed with Cytoscape and ReactomeFIViz. PPI networks were constructed with Cytoscape software. In addition, PPI (protein-protein interaction) network clustering modules were analyzed with ClusterONE, and the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses for modules were performed. RESULTS A total of 630 upregulated and 1,002 downregulated DEGs were identified in PDAC samples compared with healthy samples. Some ribosomal protein genes with higher average correlation in module 0 were enriched in the ribosome pathway. NUP107 (nucleoporin 107 kDa) and NUP160 (nucleoporin 160 kDa) were enriched in module 3. HNRNPU (heterogeneous nuclear ribonucleoprotein U) with higher average correlation in module 8 was enriched in the spliceosome pathway. The ribosome pathway and the spliceosome pathway were significantly enriched in cluster 1 and cluster 2, respectively. CONCLUSIONS Ribosomal protein genes Nup170, Nup160, and HNRNPU, and the ribosome pathway as well as the spliceosome pathway may play important roles in PDAC progression. In addition, ribosomal protein genes Nup170, Nup160, and HNRNPU may be used as possible molecular markers for the early diagnosis of the disease. Show less

Nuclear pore complexes (NPCs) are gateways through the nuclear envelope. How they form into a structure containing three rings and integrate into the nuclear envelope remains a challenging paradigm for coordinated assembly of macro-complexes. In vertebrates, the cytoplasmic and nucleoplasmic rings of NPCs are mostly formed by multiple copies of the Nup107-Nup160 complex, whereas the central, or inner ring is composed of Nup53, Nup93, Nup155 and the two paralogues Nup188 and Nup205. Inner ring assembly is only partially understood. Using Show less

This study was aimed at elucidating the fate of three important nuclear envelope components - lamins B and A/C and nucleoporin Nup160, during meiotic maturation of mouse oocytes. These proteins were localized by epifluorescence and confocal microscopy using specific antibodies in oocytes at different stages from prophase I (germinal vesicle) to metaphase II. In immature germinal vesicle oocytes, all three proteins were detected at the nuclear periphery. In metaphase I and metaphase II, lamin B co-localized with the meiotic spindle, lamin A/C was found in a diffuse halo surrounding the spindle and to a lesser degree throughout the cytoplasm, and Nup160 was concentrated to the spindle poles. To our knowledge, this is the first report on nucleoporin localization in mammalian oocytes and the first successful detection of lamins in mature oocytes. While the distribution patterns of both lamins closely paralleled the respective stages of mitosis, Nup160 localization in metaphase oocytes corresponded to that in mitotic prometaphase rather than metaphase. The peculiar distribution of this nucleoporin in oocytes may reflect its role in meiosis-specific mechanisms of spindle assembly and its regulation. Show less

Microglia are emerging as a key cell type in neurodegenerative diseases, yet human microglia are challenging to study in vitro. We developed an in vitro cell model system composed of human monocyte-derived microglia-like (MDMi) cells that recapitulated key aspects of microglia phenotype and function. We then used this model system to perform an expression quantitative trait locus (eQTL) study examining 94 genes from loci associated with Alzheimer's disease, Parkinson's disease, and multiple sclerosis. We found six loci ( Show less

Several nucleoporins in the nuclear pore complex (NPC) have been reported to be involved in abiotic stress responses in plants. However, the molecular mechanism of how NPC regulates abiotic stress responses, especially the expression of stress responsive genes remains poorly understood. From a forward genetics screen using an abiotic stress-responsive luciferase reporter (RD29A-LUC) in the sickle-1 (sic-1) mutant background, we identified a suppressor caused by a mutation in NUCLEOPORIN 85 (NUP85), which exhibited reduced expression of RD29A-LUC in response to ABA and salt stress. Consistently, the ABA and salinity induced expression of several stress responsive genes such as RD29A, COR15A and COR47 was significantly compromised in nup85 mutants and other nucleoporin mutants such as nup160 and hos1. Subsequently, Immunoprecipitation and mass spectrometry analysis revealed that NUP85 is potentially associated with HOS1 and other nucleoporins within the nup107-160 complex, along with several mediator subunits. We further showed that there is a direct physical interaction between MED18 and NUP85. Similar to NUP85 mutations, MED18 mutation was also found to attenuate expression of stress responsive genes. Taken together, we not only revealed the involvement of NUP85 and other nucleoporins in regulating ABA and salt stress responses, but also uncovered a potential relation between NPC and mediator complex in modulating the gene expression in plants. Show less

The nuclear pore complex (NPC), a gateway for nucleocytoplasmic trafficking, is composed of ∼30 different proteins called nucleoporins. It remains unknown whether the NPCs within a species are homogeneous or vary depending on the cell type or physiological condition. Here, we present evidence for compositionally distinct NPCs that form within a single cell in a binucleated ciliate. In Show less

In both animals and plants, messenger (m)RNA export has been shown to contribute to immune response regulation. The Arabidopsis nuclear protein MOS11, along with the nucleoporins MOS3/Nup96/SAR3 and Nup160/SAR1 are components of the mRNA export machinery and contribute to immunity mediated by nucleotide binding leucine-rich repeat immune receptors (NLR). The human MOS11 ortholog CIP29 is part of a small protein complex with three additional members: the RNA helicase DDX39, ALY, and TAF15b. We systematically assessed the biological roles of the Arabidopsis homologs of these proteins in toll interleukin 1 receptor-type NLR (TNL)-mediated immunity using reverse genetics. Although mutations in ALY and DDX39 did not result in obvious defects, taf15b mutation partially suppressed the autoimmune phenotypes of a gain-of-function TNL mutant, snc1. An additive effect on snc1 suppression was observed in mos11-1 taf15b snc1 triple mutant plants, suggesting that MOS11 and TAF15b have independent functions. TAF15b-GFP fusion protein, which fully complemented taf15b mutant phenotypes, localized to nuclei similarly to MOS11. However, it was also targeted to cytosolic granules identified as processing bodies. In addition, we observed no change in SNC1 mRNA levels, whereas less SNC1 protein accumulated in taf15b mutant, suggesting that TAF15b contributes to SNC1 homeostasis through posttranscriptional mechanisms. In summary, this study highlights the importance of posttranscriptional RNA processing mediated by TAF15b in the regulation of TNL-mediated immunity. Show less

A growing body of evidence suggests that microRNAs (miRNAs) are involved in Alzheimer's disease (AD) and that some disease-associated genetic variants are located within miRNA binding sites. In the present study, we sought to characterize functional polymorphisms in miRNA target sites within the loci defined in earlier genome-wide association studies (GWAS). The main objectives of this study were to (1) facilitate the identification of the gene or genes responsible for the GWAS signal within a locus of interest and (2) determine how functional polymorphisms might be involved in the AD process (e.g., by affecting miRNA-mediated variations in gene expression). Stringent in silico analyses were developed to select potential polymorphisms susceptible to impairment of miRNA-mediated repression, and subsequent functional assays were performed in HeLa and HEK293 cells. Two polymorphisms were identified and further analyzed in vitro. The AD-associated rs7143400-T allele (located in 3' untranslated region [3'-UTR] of FERMT2) cotransfected with miR-4504 resulted in lower protein levels relative to the rs7143400-G allele cotransfected with the same miRNA. The AD-associated rs9909-C allele in the 3'-UTR of NUP160 abolished the miR-1185-1-3p-regulated expression observed for the rs9909-G allele. When considered in conjunction with the findings of previous association studies, our results suggest that decreased expression of FERMT2 might be a risk factor in the etiopathology of AD, whereas increased expression of NUP160 might protect against the disease. Our data therefore provide new insights into AD by highlighting two new proteins putatively involved in the disease process. Show less

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. Show less

NFAT5 is an osmoregulated transcription factor that particularly increases expression of genes involved in protection against hypertonicity. Transcription factors often contain unstructured regions that bind co-regulatory proteins that are crucial for their function. The NH2-terminal region of NFAT5 contains regions predicted to be intrinsically disordered. We used peptide aptamer-based affinity chromatography coupled with mass spectrometry to identify protein preys pulled down by one or more overlapping 20 amino acid peptide baits within a predicted NH2-terminal unstructured region of NFAT5. We identify a total of 467 unique protein preys that associate with at least one NH2-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from HEK293 cells treated with elevated, normal, or reduced NaCl concentrations. Different sets of proteins are pulled down from nuclear vs. cytoplasmic extracts. We used GeneCards to ascertain known functions of the protein preys. The protein preys include many that were previously known, but also many novel ones. Consideration of the novel ones suggests many aspects of NFAT5 regulation, interaction and function that were not previously appreciated, for example, hypertonicity inhibits NFAT5 by sumoylating it and the NFAT5 protein preys include components of the CHTOP complex that desumoylate proteins, an action that should contribute to activation of NFAT5. Show less

The BioID method uses a promiscuous biotin ligase to detect protein-protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein-protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius. Show less

Two genes encoding protein components of the nuclear pore complex Nup160 and Nup96 cause lethality in F2-like hybrid genotypes between Drosophila simulans and Drosophila melanogaster. In particular, D. simulans Nup160 and Nup96 each cause inviability when hemizygous or homozygous in species hybrids that are also hemizygous (or homozygous) for the D. melanogaster X chromosome. The hybrid lethality of Nup160, however, is genetically complex, depending on one or more unknown additional factors in the autosomal background. Here we study the genetics and evolution of Nup160-mediated hybrid lethality in three ways. First, we test for variability in Nup160-mediated hybrid lethality within and among the three species of the D. simulans clade- D. simulans, D. sechellia, and D. mauritiana. We show that the hybrid lethality of Nup160 is fixed in D. simulans and D. sechellia but absent in D. mauritiana. Second, we explore how the hybrid lethality of Nup160 depends on other loci in the autosomal background. We find that D. simulans Nup160-mediated hybrid lethality does not depend on the presence of D. melanogaster Nup96, and we find that D. simulans and D. mauritiana are functionally differentiated at Nup160 as well as at other autosomal factor(s). Finally, we use population genetics data to show that Nup160 has experienced histories of recurrent positive selection both before and after the split of the three D. simulans clade species ∼240,000 years ago. Our genetic results suggest that a hybrid lethal Nup160 allele evolved before the split of the three D. simulans clade species, whereas the other autosomal factor(s) evolved more recently. Show less

Nuclear pore complexes (NPCs) form the gateway to the nucleus, mediating virtually all nucleocytoplasmic trafficking. Assembly of a nuclear pore complex requires the organization of many soluble sub-complexes into a final massive structure embedded in the nuclear envelope. By use of a LacI/LacO reporter system, we were able to assess nucleoporin (Nup) interactions, show that they occur with a high level of specificity, and identify nucleoporins sufficient for initiation of the complex process of NPC assembly in vivo. Eleven nucleoporins from different sub-complexes were fused to LacI-CFP and transfected separately into a human cell line containing a stably integrated LacO DNA array. The LacI-Nup fusion proteins, which bound to the array, were examined for their ability to recruit endogenous nucleoporins to the intranuclear LacO site. Many could recruit nucleoporins of the same sub-complex and a number could also recruit other sub-complexes. Strikingly, Nup133 and Nup107 of the Nup107/160 subcomplex and Nup153 and Nup50 of the nuclear pore basket recruited a near full complement of nucleoporins to the LacO array. Furthermore, Nup133 and Nup153 efficiently targeted the LacO array to the nuclear periphery. Our data support a hierarchical, seeded assembly pathway and identify Nup133 and Nup153 as effective "seeds" for NPC assembly. In addition, we show that this system can be applied to functional studies of individual nucleoporin domains as well as to specific nucleoporin disease mutations. We find that the R391H cardiac arrhythmia/sudden death mutation of Nup155 prevents both its subcomplex assembly and nuclear rim targeting of the LacO array. Show less

Angiosarcoma is a malignant vascular tumor originating from endothelial cells of blood vessels or lymphatic vessels. The specific driver mutations in angiosarcoma remain unknown. In this study, we investigated this issue by transcriptome sequencing of patient-derived angiosarcoma cells (ISO-HAS), identifying a novel fusion gene NUP160-SLC43A3 found to be expressed in 9 of 25 human angiosarcoma specimens that were examined. In tumors harboring the fusion gene, the duration between the onset of symptoms and the first hospital visit was significantly shorter, suggesting more rapid tumor progression. Stable expression of the fusion gene in nontransformed human dermal microvascular endothelial cells elicited a gene-expression pattern mimicking ISO-HAS cells and increased cell proliferation, an effect traced in part to NUP160 truncation. Conversely, RNAi-mediated attenuation of NUP160 in ISO-HAS cells decreased cell number. Confirming the oncogenic effects of the fusion protein, subcutaneous implantation of NUP160-SLC43A3-expressing fibroblasts induced tumors resembling human angiosarcoma. Collectively, our findings advance knowledge concerning the genetic causes of angiosarcoma, with potential implications for new diagnostic and therapeutic approaches. Show less

In metazoa, nuclear pore complexes (NPCs) are assembled from constituent nucleoporins by two distinct mechanisms: in the re-forming nuclear envelope at the end of mitosis and into the intact nuclear envelope during interphase. Here, we show that the nucleoporin Nup153 is required for NPC assembly during interphase but not during mitotic exit. It functions in interphasic NPC formation by binding directly to the inner nuclear membrane via an N-terminal amphipathic helix. This binding facilitates the recruitment of the Nup107-160 complex, a crucial structural component of the NPC, to assembly sites. Our work further suggests that the nuclear transport receptor transportin and the small GTPase Ran regulate the interaction of Nup153 with the membrane and, in this way, direct pore complex assembly to the nuclear envelope during interphase. Show less

Nup98 is a glycine-leucine-phenylalanine-glycine (GLFG) repeat-containing nucleoporin that, in addition to nuclear transport, contributes to multiple aspects of gene regulation. Previous studies revealed its dynamic localization within intranuclear structures known as GLFG bodies. Here we show that the mammalian Nup107-160 complex (Y-complex), a major scaffold module of the nuclear pore, together with its partner Elys, colocalizes with Nup98 in GLFG bodies. The frequency and size of GLFG bodies vary among HeLa sublines, and we find that an increased level of Nup98 is associated with the presence of bodies. Recruitment of the Y-complex and Elys into GLFG bodies requires the C-terminal domain of Nup98. During cell division, Y-Nup-containing GLFG bodies are disassembled in mitotic prophase, significantly ahead of nuclear pore disassembly. FRAP studies revealed that, unlike at nuclear pores, the Y-complex shuttles into and out of GLFG bodies. Finally, we show that within the nucleoplasm, a fraction of Nup107, a key component of the Y-complex, displays reduced mobility, suggesting interaction with other nuclear components. Together our data uncover a previously neglected intranuclear pool of the Y-complex that may underscore a yet-uncharacterized function of these nucleoporins inside the nucleus, even in cells that contain no detectable GLFG bodies. Show less

In interspecific hybrids between Drosophila melanogaster and Drosophila simulans, the D. simulans nucleoporin-encoding Nup96(sim) and Nup160(sim) can cause recessive lethality if the hybrid does not also inherit the D. simulans X chromosome. In addition, Nup160(sim) leads to recessive female sterility in the D. melanogaster genetic background. Here, we conducted carefully controlled crosses to better understandthe relationship between Nup96(sim) and Nup160(sim). Nup96(sim) did not lead to female sterility in the D. melanogaster genetic background, and double introgression of Nup96(sim) and Nup160(sim) did not generally lead to lethality when one was heterozygous and the other homozygous (hemizygous). It appears that introgression of additional autosomal D. simulans genes is necessary to cause lethality and that the effect of the introgression is dominant to D. melanogaster alleles. Interestingly, the genetic background affected dominance of Nup96(sim), and double introgression carrying homozygous Nup96(sim) and hemizygous Nup160(sim) resulted in lethality. Thus, Nup96(sim) and Nup160(sim) seem to be two components of the same incompatibility. Show less

The nuclear pore complex (NPC) is an enormous proteinaceous complex composed of multiple copies of about 30 different proteins called nucleoporins. In this study, we analyzed the composition of the NPC in the model organism Schizosaccharomyces pombe using strains in which individual nucleoporins were tagged with GFP. We identified 31 proteins as nucleoporins by their localization to the nuclear periphery. Gene disruption analysis in previous studies coupled with gene disruption analysis in the present study indicates that 15 of these nucleoporins are essential for vegetative cell growth and the other 16 nucleoporins are non-essential. Among the 16 non-essential nucleoporins, 11 are required for normal progression through meiosis and their disruption caused abnormal spore formation or poor spore viability. Based on fluorescence measurements of GFP-fused nucleoporins, we estimated the composition of the NPC in S. pombe and found that the organization of the S. pombe NPC is largely similar to that of other organisms; a single NPC was estimated as being 45.8-47.8 MDa in size. We also used fluorescence measurements of single NPCs and quantitative western blotting to analyze the composition of the Nup107-Nup160 subcomplex, which plays an indispensable role in NPC organization and function. Our analysis revealed low amounts of Nup107 and Nup131 and high amounts of Nup132 in the Nup107-Nup160 subcomplex, suggesting that the composition of this complex in S. pombe may differ from that in S. cerevisiae and humans. Comparative analysis of NPCs in various organisms will lead to a comprehensive understanding of the functional architecture of the NPC. Show less

Ran is a small ras-related GTPase that controls the nucleocytoplasmic exchange of macromolecules across the nuclear envelope. It binds to chromatin early during nuclear formation and has important roles during the eukaryotic cell cycle, where it regulates mitotic spindle assembly, nuclear envelope formation and cell cycle checkpoint control. Like other GTPases, Ran relies on the cycling between GTP-bound and GDP-bound conformations to interact with effector proteins and regulate these processes. In nucleocytoplasmic transport, Ran shuttles across the nuclear envelope through nuclear pores. It is concentrated in the nucleus by an active import mechanism where it generates a high concentration of RanGTP by nucleotide exchange. It controls the assembly and disassembly of a range of complexes that are formed between Ran-binding proteins and cellular cargo to maintain rapid nuclear transport. Ran also has been identified as an essential protein in nuclear envelope formation in eukaryotes. This mechanism is dependent on importin-β, which regulates the assembly of further complexes important in this process, such as Nup107-Nup160. A strong body of evidence is emerging implicating Ran as a key protein in the metastatic progression of cancer. Ran is overexpressed in a range of tumors, such as breast and renal, and these perturbed levels are associated with local invasion, metastasis and reduced patient survival. Furthermore, tumors with oncogenic KRAS or PIK3CA mutations are addicted to Ran expression, which yields exciting future therapeutic opportunities. Show less

A key building block of the nuclear pore complex (NPC) is the Nup84 subcomplex that has been structurally analyzed predominantly in the yeast system. To expand this analysis and gain insight into the evolutionary conservation of its structure, we reconstituted an octameric Nup84 complex using the subunits from a thermophile, Chaetomium thermophilum (ct). This assembly carries Nup37 and Elys, which are characteristic subunits of the orthologous human Nup107-Nup160 complex but absent from the yeast Saccharomyces cerevisiae. We found that Elys binds cooperatively to the complex requiring both Nup37 and Nup120. Unexpectedly, the reconstituted ctNup84 complex formed a striking dimer structure with an unpredicted side-to-side arrangement of two molecules. Finally, crosslinking mass spectrometry allowed the mapping of key protein interfaces within the Y-shaped complex. Thus, the thermophilic Nup84 complex can serve as a structural model for higher eukaryotic Nup107-Nup160 assemblies to gain insight into its possible configuration within the NPC scaffold. Show less