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The Ran-GTP/importin β pathway has been implicated in a diverse array of mitotic functions in somatic mitosis; however, the possible meiotic roles of Ran-GTP/importin β in mammalian oocyte meiosis are still not fully understood. In the present study, importazole (IPZ), a small molecule inhibitor of the interaction between Ran and importin β was used to explore the potential meiotic roles of Ran-GTP/importin β in porcine oocytes undergoing meiosis. After IPZ treatment, the extrusion rate of the first polar body (PB1) was significantly decreased, and a higher proportion of the oocytes were arrested at the germinal vesicle breakdown (GVBD) stage. Moreover, IPZ treatment led to severe defects in metaphase I (MI) spindle assembly and chromosome alignment during the germinal vesicle (GV)-to-MI stage, as well as failure of metaphase II (MII) spindle reassembly and homologous chromosome segregation during the MI-to-MII stage. Notably, IPZ treatment decreased TPX2 expression and abnormal subcellular localization. Furthermore, the expression levels of aurora kinase A (AURKA) and transforming acidic coiled-coil 3 (TACC3) were significantly reduced after IPZ treatment. Collectively, these data indicate that the interaction of Ran-GTP and importin β is essential for proper spindle assembly and successful chromosome segregation during two consecutive meiotic divisions in porcine oocytes, and regulation of this complex might be related to its effect on the TPX2 signaling cascades.

Background. The role and expression level change in circ_TNPO1 (hsa_circ_0072951) in atherosclerosis (AS) and VSMC dysfunction remain unknown. In this study, we try to explore the effects of circ_TNPO1 on oxidized low-density lipoprotein (ox-LDL)-induced human vascular smooth muscle cell (VSMC) excessive proliferation and migration, and the potential molecular mechanism. Methods. Quantitative real-time polymerase chain reaction (RT-qPCR) and western blot experiment were used to detect the serum samples from AS patients and healthy controls. CCK-8, Transwell, and the dual-luciferase reporter gene assay were used to detect the cell biology. Results. In human AS serum and ox-LDL-induced VSMCs, circ_TNPO1 was increased, whereas miR-181b was decreased. Silencing circ_TNPO1 inhibited proliferation and migration activity and reduced protein expression of PCNA, Ki-67, MMP2, and E-cadherin and promoted N-cadherin protein expression in ox-LDL induced VSMCs. Remarkably, miR-181b knockdown or Notch1 overexpression could efficiently offset the proliferation and migration inhibiting effect of circ_TNPO1 knockdown in ox-LDL-induced VSMCs. Furthermore, a molecular mechanism study pointed out that circ_TNPO1 and Notch1 are direct-acting targets of miR-181b. Conclusions. In conclusion, our study indicated that circ_TNPO1 promotes the proliferation and migration progression of VSMCs in atherosclerosis through the miR-181b/Notch1 axis.

After a meal, excess nutrients are stored within adipose tissue as triglycerides in structures called lipid droplets. Previous genome-wide RNAi screens have identified that mRNA splicing factor genes are required for normal lipid droplet formation in Drosophila cells. We have previously shown that mRNA splicing factors called serine/arginine-rich (SR) proteins are important for triglyceride storage in the Drosophila fat body. SR proteins shuttle in and out of the nucleus with the help of proteins called Transportins (Tnpo-SR); however, whether this transport is important for SR protein-mediated regulation of lipid storage is unknown. The purpose of this study is to characterize the role of Tnpo-SR proteins in regulating lipid storage in the Drosophila fat body. Decreasing Tnpo-SR in the adult fat body resulted in an increase in triglyceride storage and consistent with this phenotype, Tnpo-SR-RNAi flies also have increased starvation resistance. In addition, the lipid accumulation in Tnpo-SR-RNAi flies is the result of increased triglyceride stored in each fat body cell and not due to increased food consumption. Interestingly, the splicing of CPT1, an enzyme important for the β-oxidation of fatty acids, is altered in Tnpo-SR-RNAi fat bodies. The isoform that produces the less catalytically active form of CPT1 accumulates in fat bodies where Tnpo-SR levels are decreased, suggesting a decrease in lipid breakdown, potentially causing the excess triglyceride storage observed in these flies. Together, these data suggest that the transport of splicing proteins in and out of the nucleus is important for proper triglyceride storage in the Drosophila fat body.

AML cells are arranged in a hierarchy with stem/progenitor cells giving rise to more differentiated bulk cells. Despite the importance of stem/progenitors in the pathogenesis of AML, the determinants of the AML stem/progenitor state are not fully understood. Through a comparison of genes that are significant for growth and viability of AML cells by way of a CRISPR screen, with genes that are differentially expressed in leukemia stem cells (LSC), we identified importin 11 (IPO11) as a novel target in AML. Importin 11 (IPO11) is a member of the importin β family of proteins that mediate transport of proteins across the nuclear membrane. In AML, knockdown of IPO11 decreased growth, reduced engraftment potential of LSC, and induced differentiation. Mechanistically, we identified the transcription factors BZW1 and BZW2 as novel cargo of IPO11. We further show that BZW1/2 mediate a transcriptional signature that promotes stemness and survival of LSC. Thus, we demonstrate for the first time how specific cytoplasmic-nuclear regulation supports stem-like transcriptional signature in relapsed AML.

Introduction: Karyopherin alpha 2 (KPNA2) and karyopherin beta 1 (KPNB1) constitute nuclear transport protein complexes involved in nuclear import and are significant in tumor progression. Although high KPNA2 expression was associated with poor prognosis in solid tumors, the relationship between KPNA2 and KPNB1 expression and their prognostic role in gastric cancer (GC) remains unclear. Methods: Immunohistochemistry was used to correlate the expression of KPNA2 and KPNB1 with various features, including clinicopathological characteristics in 130 patients with GC and survival in 94 patients with invasive lesions extending to the submucosa or deeper. Results: High expression of KPNA2 and KPNB1 was found in 25% and 36% of the patients, respectively. Both were significantly related to tumor depth, lymph node metastasis, lymphatic invasion, venous invasion, and Ki-67 expression. KPNA2 expression was significantly related to that of KPNB1 (p < 0.001). Patients with high KPNB1 expression had poorer prognosis than those with low expression (p = 0.027), as was also observed in case of KPNA2 (p < 0.001). Patients with high expression of both KPNA2 and KPNB1 accounted for 18% and had a poorer prognosis than those with high expression of either and those with low expression of both (p = 0.001). Multivariate analysis revealed that high expression of both KPNA2 and KPNB1 was an independent prognostic factor in patients with GC (hazard ratio, 3.46; 95% confidence interval, 1.64–2.73, p = 0.001). Conclusion: KPNA2 expression was correlated with KPNB1 expression, and high co-expression of KPNA2 and KPNB1 may represent a strong prognostic biomarker in GC.

Background: This study is a retrospective study. The purpose of this study is to construct and validate an early warning model of lung cancer through machine learning. Methods: The CDKN2A gene expression profile and clinical information were downloaded from The Cancer Genome Atlas (TCGA) database and divided into a tumor group and a normal group (n = 57). The top 5 somatic mutation-related genes were extracted from 567 somatic mutation data downloaded from TCGA database using random forest algorithm. Cox proportional hazard model and nomogram were constructed combining CDKN2A, 5 somatic mutation-related genes, gender, and smoking index. Patients were divided into high-risk and low-risk groups according to risk score. The predictability of the model in the prognosis of lung cancer was estimated by Kaplan–Meier survival analysis and receiver operating characteristics curve. Results: We constructed a prognostic model consisting of 5 somatic mutation-related genes (sphingosine 1-phosphate receptor 1 [S1PR1], dedicator of cytokinesis 7 [DOCK7], DEAD-box helicase 4 [DDX4], laminin subunit beta 3 [LAMB3], and importin 5 [IPO5]), cyclin-dependent kinase inhibitor 2A (CDKN2A), gender, and smoking indicators. The high-risk group had a lower overall survival rate compared to the low-risk group (hazard ratio = 2.14, P = 0 .0323). The area under the curve predicted for 3-year, 5-year, and 10-year survival rates are 0.609, 0.673, and 0.698, respectively. The accuracy, sensitivity, and specificity of the model for predicting the 10-year survival rate of lung cancer are 76.19%, 56.71%, and 86.23%. Conclusion: The lung cancer early warning model and nomogram may provide an essential reference for patients with lung cancer management in the clinic.

Appropriate nucleo-cytoplasmic partitioning of proteins is a vital regulatory mechanism in phytohormone signaling and plant development. However, how this is achieved remains incompletely understood. The Karyopherin (KAP) superfamily is critical for separating the biological processes in the nucleus from those in the cytoplasm. The KAP superfamily is divided into Importin α (IMPα) and Importin β (IMPβ) families and includes the core components in mediating nucleocytoplasmic transport. Recent reports suggest the KAPs play crucial regulatory roles in Arabidopsis development and stress response by regulating the nucleo-cytoplasmic transport of members in hormone signaling. However, the KAP members and their associated molecular mechanisms are still poorly understood in maize. Therefore, we first identified seven IMPα and twenty-seven IMPβ genes in the maize genome and described their evolution traits and the recognition rules for substrates with nuclear localization signals (NLSs) or nuclear export signals (NESs) in plants. Next, we searched for the protein interaction partners of the ZmKAPs and selected the ones with Arabidopsis orthologs functioning in auxin biosynthesis, transport, and signaling to predict their potential function. Finally, we found that several ZmKAPs share similar expression patterns with their interacting proteins, implying their function in root development. Overall, this article focuses on the Karyopherin superfamily in maize and starts with this entry point by systematically comprehending the KAP-mediated nucleo-cytoplasmic transport process in plants, and then predicts the function of the ZmKAPs during maize development, with a perspective on a closely associated regulatory mechanism between the nucleo-cytoplasmic transport and the phytohormone network.

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.

Importin-(Imp)β family nucleocytoplasmic transport receptors (NTRs) are supposed to bind to their cargoes through interaction between a confined interface on an NTR and a nuclear localization or export signal (NLS/NES) on a cargo. Although consensus NLS/NES sequence motifs have been defined for cargoes of some NTRs, many experimentally identified cargoes of those NTRs lack those motifs, and consensus NLSs/NESs have been reported for only a few NTRs. Crystal structures of NTR–cargo complexes have exemplified 3D structure-dependent binding of cargoes lacking a consensus NLS/NES to different sites on an NTR. Since only a limited number of NTR–cargo interactions have been studied, whether most cargoes lacking a consensus NLS/NES bind to the same confined interface or to various sites on an NTR is still unclear. Addressing this issue, we generated four mutants of transportin-(Trn)SR, of which many cargoes lack a consensus NLS, and eight mutants of Imp13, where no consensus NLS has been defined, and we analyzed their binding to as many as 40 cargo candidates that we previously identified by a nuclear import reaction-based method. The cargoes bind differently to the NTR mutants, suggesting that positions on an NTR contribute differently to the binding of respective cargoes.

To explore the genetic basis for a neonate featuring developmental delay.Clinical examination and laboratory tests were carried out for the patient. Peripheral venous blood samples of the proband and his parents were extracted and subjected to target capture next generation sequencing. Candidate variant was verified by Sanger sequencing.The patient, a four-month-old male, has presented with developmental delay and weakness of limbs. Genetic testing revealed that he had harbored a novel c.1432CT variant of the TNPO3 gene, which was inherited from his mother. The nonsense variant has resulted in premature termination of protein translation and was predicted to be pathogenic by bioinformatics analysis.The heterozygous c.1432CT variant of the TNPO3 gene probably underlay the limb-girdle muscular dystrophies form 1F in this patient. Above finding has enriched the variation spectrum of the TNPO3 gene.

Core histones package chromosomal DNA and regulate genomic transactions, with their import and deposition involving a dedicated repertoire of molecular chaperones. Histones H3 and H4 have been predominantly characterised as obligate heterodimers, however, recent findings have alluded to the existence of a significant pool of monomeric histone H3 in the nucleoplasm. Using a combination of in vitro and in vivo experiments, here we show that monomeric H3 and H4 use an Importin 5 (Imp5) dependent pathway for their nuclear import, distinct from Importin 4 (Imp4) previously described for H3-H4 dimers. Using mutants that disrupt the histone fold, we show monomeric H3 loses its interaction with Imp4, but retains interactions with Imp5 and the chaperone NASP. H4 monomeric mutants similarly bind Imp5 and not Imp4, however, they lose interaction with NASP, retaining their interaction with the HAT1-RBBP7 complex instead. In vitro experiments revealed that Imp5 and NASP are mutually exclusive in their binding, suggesting a facilitated hand-off mechanism. Furthermore, new H3 accumulates rapidly in a NASP-bound complex after nuclear translocation. NASP can assemble into three distinct co-chaperoning complexes, including a novel complex containing NASP, H3 and the putative ubiquitin ligase UBR7, a NASP-H3-H4-RBBP7 subcomplex and the previously characterised NASP-H3-H4-ASF1-HAT1-RBBP7 multi-chaperoning complex. Here we propose an alternative import pathway and folding mechanism for monomeric H3 and H4 that involves Imp5, rather than Imp4, and hands off to nuclear chaperones NASP, RBBP7 and HAT1. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/464968v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@462172org.highwire.dtl.DTLVardef@3df5c5org.highwire.dtl.DTLVardef@1d28f01org.highwire.dtl.DTLVardef@aa8fb0_HPS_FORMAT_FIGEXP M_FIG C_FIG

The shortest dystrophins, Dp71 and Dp40, are transcribed from the DMD gene through an internal promoter located in intron 62. These proteins are the main product of the DMD gene in the nervous system and have been involved in various functions related to cellular differentiation and proliferation as well as other cellular processes. Dp71 mRNA undergoes alternative splicing that results in different Dp71 protein isoforms. The subcellular localization of some of these isoforms in the PC12 cell line has been previously reported, and a differential subcellular distribution was observed, which suggests a particular role for each isoform. With the aim of obtaining information on their function, this study identified factors involved in the nuclear transport of Dp71 and Dp40 isoforms in the PC12 cell line. Cell cultures were treated with specific nuclear import/export inhibitors to determine the Dp71 isoform transport routes. The results showed that all isoforms of Dp71 and Dp40 included in the analysis have the ability to enter the cell nucleus through α/β importin, and the main route of nuclear export for Dp71 isoforms is through the exportin CRM1, which is not the case for Dp40.

Uncontrolled growth, distant metastasis and chemoresistance are critical characteristics of pancreatic ductal adenocarcinoma (PDAC), and they result in high mortality; however, the mechanisms triggering these effects have not been fully investigated. In this study, we analysed a dataset in the Cancer Genome Atlas (TCGA) and identified PCDH1, a rarely studied transmembrane protein, as a novel prognostic marker in PDAC patients. We demonstrated that PCDH1 expression was upregulated in PDAC tissues, and its expression levels were associated with the depth of tumour invasion and lymph node metastasis. Patients with high PCDH1 levels showed poor overall survival (OS). We also investigated the biological significance of PCDH1 in PDAC cell growth, metastasis, and side population (SP) phenotype acquisition and explored the internal molecular mechanisms of PCDH1 action. Our results demonstrated that PCDH1 enhanced p65 nuclear localization by interacting with KPNB1, a well-characterized nuclear transporter, thereby activating the NF-κB signalling pathway and increasing its functional effects during PDAC progression. Hence, our results indicate that PCDH1 can be used as a negative prognostic marker and may be a potential therapeutic target for PDAC patients.

Background More than twenty years after its discovery, the role of the importin beta superfamily member Ran GTP-binding protein (RanBP) 17 is still ill defined. Previously, we observed notable RanBP17 RNA expression levels in head and neck squamous cell carcinoma (HNSCC) cell lines with disruptive TP53 mutations. Methods We deployed HNSCC cell lines as well as cell lines from other tumor entities such as HCT116, MDA-MB-231 and H460, which were derived from colon, breast and lung cancers respectively. RNAi was used to evaluate the effect of RanBP17 on cell proliferation. FACS analysis was used for cell sorting according to their respective cell cycle phase and for BrdU assays. Immunocytochemistry was deployed for colocalization studies of RanBP17 with Nucleolin and SC35 (nuclear speckles) domains. TCGA analysis was performed for prognostic assessment and correlation analysis of RanBP17 in HNSCC patients. Results RNAi knockdown of RanBP17, significantly reduced cell proliferation in HNSCC cell lines. This effect was also seen in the HNSCC unrelated cell lines HCT116 and MDA-MB-231. Similarly, inhibiting cell proliferation with cisplatin reduced RanBP17 in keratinocytes but lead to induction in tumor cell lines. A similar observation was made in tumor cell lines after treatment with the EGFR kinase inhibitor AG1478. In addition to previous reports, showing colocalization of RanBP17 with SC35 domains, we observed colocalization of RanBP17 to nuclear bodies that are distinct from nucleoli and SC35 domains. Interestingly, for HPV positive but not HPV negative HNSCC, TCGA data base analysis revealed a strong positive correlation of RanBP17 RNA with patient survival and CDKN2A. Conclusions Our data point to a role of RanBP17 in proliferation of HNSCC and other epithelial cells. Furthermore, RanBP17 could potentially serve as a novel prognostic marker for HNSCC patients. However, we noted a major discrepancy between RanBP17 RNA and protein expression levels with the used antibodies. These observations could be explained by the presence of additional RanBP17 splice isoforms and more so of non-coding circular RanBP17 RNA species. These aspects need to be addressed in more detail by future studies.

Clear cell renal cell carcinoma (ccRCC) is a common malignant tumour of the urinary tract. The major causes of poor prognosis are the lack of early diagnosis and metastasis. Accumulating research reveals that circular RNAs (circRNAs) can play key roles in the development and the progression of cancer. However, the role of circRNAs in ccRCC is still uncertain.The circRNAs microarray (n = 4) was performed to investigate the circRNAs with differential expression in ccRCC tissues. The candidate circRNA was selected based on the cut-off criteria, such as circRNA expression abundance, circRNA size and the design of divergent primers. The circ-transportin-3 (TNPO3) levels in ccRCC tissues were tested by quantitative real-time (qRT)-PCR (n = 110). The characteristics and subcellular localization of circ-TNPO3 were identified via RNase R assay, qRT-PCR and fluorescence in situ hybridization (FISH). Then, we explored the biological roles of circ-TNPO3 in ccRCC via the function experiments in vitro and in vivo. RNA pull-down, RNA immunoprecipitation, bioinformatic analysis, RNA-FISH assays and rescue assays were applied to validate the interactions between circ-TNPO3, insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and serpin family H member 1 (SERPINH1) to uncover the underlying molecular mechanisms of circ-TNPO3.We detected the obvious downregulation of circ-TNPO3 in ccRCC compared to matched adjacent normal tissues (n = 110). The lower circ-TNPO3 expression was found in ccRCC patients with distant metastasis, higher World Health Organization/International Society of Urologic Pathologists (WHO/ISUP) grade and more advanced tumour T stage. In vitro and in vivo, circ-TNPO3 significantly suppressed the proliferation and migration of ccRCC cells. Mechanistically, we elucidated that circ-TNPO3 directly bound to IGF2BP2 protein and then destabilized SERPINH1 mRNA. Moreover, IGF2BP2/SERPINH1 axis was responsible for circ-TNPO3's function of inhibiting ccRCC metastasis. Epithelial splicing regulatory protein 1 (ESRP1) was probably involved in the biogenesis of circ-TNPO3.Circ-TNPO3 can suppress ccRCC progression and metastasis via directly binding to IGF2BP2 protein and destabilizing SERPINH1 mRNA. Circ-TNPO3 may act as a potential target for ccRCC treatment.

NFAT5 is the only known mammalian tonicity-responsive transcription factor with an essential role in cellular adaptation to hypertonic stress. It is also implicated in diverse physiological and pathological processes. NFAT5 activity is tightly regulated by extracellular tonicity, but the underlying mechanisms remain elusive. Here, we demonstrate that NFAT5 enters the nucleus via the nuclear pore complex. We found that NFAT5 utilizes a unique nuclear localization signal (NFAT5-NLS) for nuclear import. siRNA screening revealed that only karyopherin β1 (KPNB1), but not karyopherin α, is responsible for the nuclear import of NFAT5 via direct interaction with the NFAT5-NLS. Proteomics analysis and siRNA screening further revealed that nuclear export of NFAT5 under hypotonicity is driven by exportin-T (XPOT), where the process requires RuvB-like AAA-type ATPase 2 (RUVBL2) as an indispensable chaperone. Our findings have identified an unconventional tonicity-dependent nucleocytoplasmic trafficking pathway for NFAT5 that represents a critical step in orchestrating rapid cellular adaptation to change in extracellular tonicity. These findings offer an opportunity for the development of novel NFAT5 targeting strategies that are potentially useful for the treatment of diseases associated with NFAT5 dysregulation.

Recent studies have revealed that cells utilize liquid–liquid phase separation (LLPS) as a mechanism in assembly of membrane-less organelles, such as RNP granules. The nucleus is a well-known membrane-bound organelle surrounded by the nuclear envelope; the nuclear pore complex on the nuclear envelope likely applies LLPS in the central channel to facilitate selective biological macromolecule exchange. Karyopherin-β family proteins exclusively pass through the central channel with cargos by dissolving the phase separated hydrogel formed by the phenylalanine-glycine (FG) repeats-containing nucleoporins. Karyopherin-βs also exhibit dissolution activity for the phase separation of cargo proteins. Many cargos, including RNA-binding proteins containing intrinsically disordered regions (IDRs), undergo phase separation; however, aberrant phase separation is linked to fatal neurodegenerative diseases. Multiple weak interactions between karyopherin-βs and phase separation-prone proteins, such as FG repeats-containing nucleoporins or IDR-containing karyopherin-β cargos, are likely to be important for passing through the nuclear pore complex and maintaining the soluble state of cargo, respectively. In this review, we discuss how karyopherin-βs regulate phase separation to function.

Importin 8, encoded by IPO8, is a ubiquitously expressed member of the importin-beta protein family that translocates cargo molecules such as proteins, RNAs, and ribonucleoprotein complexes into the nucleus in a RanGTP-dependent manner. Current knowledge of the cargoes of importin 8 is limited, but TGF-beta signaling components such as SMAD1-4 have been suggested to be among them. Here, we report that bi-allelic loss-of-function variants in IPO8 cause a syndromic form of thoracic aortic aneurysm(TAA) with clinical overlap with Loeys-Dietz and Shprintzen-Goldberg syndromes. Seven individuals from six unrelated families showed a consistent phenotype with early-onset TAA, motor developmental delay, connective tissue findings, and craniofacial dysmorphic features. A C57BL/6N Ipo8 knockout mouse model recapitulates TAA development from 8-12 weeks onward in both sexes but most prominently shows ascending aorta dilatation with a propensity for dissection in males. Compliance assays suggest augmented passive stiffness of the ascending aorta in male Ipo8(-/-) mice throughout life. Immunohistological investigation of mutant aortic walls reveals elastic fiber disorganization and fragmentation along with a signature of increased TGF-beta signaling, as evidenced by nuclear pSmad2 accumulation. RT-qPCR assays of the aortic wall in male Ipo8(-/-) mice demonstrate decreased Smad6/7 and increased Mmp2 and Ccn2 (Ctgf) expression, reinforcing a role for dysregulation of the TGF-beta signaling pathway in TAA development. Because importin 8 is the most downstream TGF-beta-related effector implicated in TAA pathogenesis so far, it offers opportunities for future mechanistic studies and represents a candidate drug target for TAA.

Despite modest improvement in patient outcomes from recent advances in pharmacotherapy targeting fibrogenic signaling pathways, idiopathic pulmonary fibrosis (IPF) remains a major unsolved clinical problem. One reason for this is that available antifibrotic agents slow down but do not arrest fibrotic progression. To arrest fibrotic progression, its obligatory drivers need to be identified. We previously discovered that fibrogenic mesenchymal progenitor cells (MPCs) are key drivers of fibrotic progression in IPF, serving as cells of origin for disease-mediating myofibroblasts. IPF MPCs have high levels of nuclear S100A4, which interacts with the proteasome to promote p53 degradation and self-renewal. However, the mechanism underlying S100A4 accumulation in the nucleus of IPF MPCs remains unknown. Here we show that hyaluronan (HA) is present in the fibroblastic focus together with CD44-expressing MPCs and that ligation of CD44 by HA triggers S100A4 nuclear translocation to support IPF MPC self-renewal. The mechanism involves HA-mediated formation of a CD44/S100A4/transportin 1 complex, which promotes S100A4 nuclear import. In a humanized mouse model of pulmonary fibrosis, IPF MPC fibrogenicity was significantly attenuated by 1) knockdown of CD44 or 2) introduction of an S100A4 mutant construct that prevents S100A4 nuclear import. These data indicate that signaling through the HA/CD44/S100A4 axis is an integral component of IPF MPC fibrogenicity.

We previously found that a deletion in γ-coronavirus

Glioma stem cells (GSCs) are a special kind of cells in GBM showing tumor initiation, self-renewal, and multi-lineage differentiation abilities. Finding novel circRNAs related to GSCs is of great significance for the study of glioma. qPCR, western blotting, and immunohistochemistry were used to detect the expression levels of circKPNB1, SPI1, DGCR8, and TNF-α. The expression of these molecules in GSCs was regulated by lentiviral-based infection. RNA immunoprecipitation assay, RNA pull-down, dual-luciferase reporter, and chromatin immunoprecipitation assays were used to study the direct regulation mechanisms among these molecules. All the MTS, EDU, transwell, neurosphere formation assays, ELDA assays, and xenograft experiments were used to detect the malignant phenotype of GSCs. We found a novel circRNA circKPNB1 was overexpressed in GBM and associated with GBM patients’ poor prognosis. CircKPNB1 overexpression can promote the cell viabilities, proliferation, invasion, neurospheres formation abilities, and stemness of GSCs. Mechanistically, circKPNB1 regulates the protein stability and nuclear translocation of SPI1. SPI1 promotes the malignant phenotype of GSCs via TNF-α mediated NF-κB signaling. SPI1 can also transcriptionally upregulate DGCR8 expression, and the latter can maintain the stability of circKPNB1 and forms a positive feedback loop among DGCR8, circKPNB1 and SPI1. Our study found circKPNB1 was a novel oncogene in GBM and of great significance in the diagnosis and prognosis prediction of GBM and maybe a novel target for molecular targeted therapy.

Karyopherin beta 1 (Kpnβ1) is the principal nuclear importer of cargo proteins and plays a role in many cellular processes. Its expression is upregulated in cancer and essential for cancer cell viability, thus the identification of its binding partners might help in the discovery of anti-cancer therapeutic targets and cancer biomarkers. Herein, we applied immunoprecipitation coupled to mass spectrometry (IP-MS) to identify Kpnβ1 binding partners in normal and cancer cells. IP-MS identified 100 potential Kpnβ1 binding partners in non-cancer hTERT-RPE1, 179 in HeLa cervical cancer, 147 in WHCO5 oesophageal cancer and 176 in KYSE30 oesophageal cancer cells, including expected and novel interaction partners. 38 binding proteins were identified in all cell lines, with the majority involved in RNA metabolism. 18 binding proteins were unique to the cancer cells, with many involved in protein translation. Western blot analysis validated the interaction of known and novel binding partners with Kpnβ1 and revealed enriched interactions between Kpnβ1 and select proteins in cancer cells, including proteins involved in cancer development, such as Kpnα2, Ran, CRM1, CCAR1 and FUBP1. Together, this study shows that Kpnβ1 interacts with numerous proteins, and its enhanced interaction with certain proteins in cancer cells likely contributes to the cancer state.

Transcription factor EBF1 (early B cell factor 1) acts as a key regulator of B cell specification. The transcriptional network in which EBF1 operates has been extensively studied; however, the regulation of EBF1 function remains poorly defined. By mass spectrometric analysis of proteins associated with endogenous EBF1 in pro-B cells, we identified the nuclear import receptor Transportin-3 (Tnpo3) and found that it interacts with the immunoglobulin-like fold domain of EBF1. We delineated glutamic acid 271 of EBF1 as a critical residue for the association with Tnpo3. EBF1E271A showed normal nuclear localization; however, it had an impaired B cell programming ability in conditions of Notch signaling, as determined by retroviral transduction of Ebf1−/− progenitors. By RNA-seq analysis of EBF1E271A-expressing progenitors, we found an up-regulation of T lineage determinants and down-regulation of early B genes, although similar chromatin binding of EBF1E271A and EBF1wt was detected in pro-B cells expressing activated Notch1. B lineage-specific inactivation of Tnpo3 in mice resulted in a block of early B cell differentiation, accompanied by a down-regulation of B lineage genes and up-regulation of T and NK lineage genes. Taken together, our observations suggest that Tnpo3 ensures B cell programming by EBF1 in nonpermissive conditions.

Subcellular localization of the deubiquitinating enzyme BAP1 is deterministic for its tumor suppressor activity. While the monoubiquitination of BAP1 by an atypical E2/E3-conjugated enzyme UBE2O and BAP1 auto-deubiquitination are known to regulate its nuclear localization, the molecular mechanism by which BAP1 is imported into the nucleus has remained elusive. Here, we demonstrated that transportin-1 (TNPO1, also known as Karyopherin β2 or Kapβ2) targets an atypical C-terminal proline-tyrosine nuclear localization signal (PY-NLS) motif of BAP1 and serves as the primary nuclear transporter of BAP1 to achieve its nuclear import. TNPO1 binding dissociates dimeric BAP1 and sequesters the monoubiquitination sites flanking the PY-NLS of BAP1 to counteract the function of UBE2O that retains BAP1 in the cytosol. Our findings shed light on how TNPO1 regulates the nuclear import, self-association, and monoubiquitination of BAP1 pertinent to oncogenesis.

Influenza A viruses (IAVs) are a major global health threat and in the future, may cause the next pandemic. Although studies have partly uncovered the molecular mechanism of IAV–host interaction, it requires further research. In this study, we explored the roles of transportin-3 (TNPO3) in IAV infection. We found that TNPO3-deficient cells inhibited infection with four different IAV strains, whereas restoration of TNPO3 expression in knockout (KO) cells restored IAV infection. TNPO3 overexpression in wild-type (WT) cells promoted IAV infection, suggesting that TNPO3 is involved in the IAV replication. Furthermore, we found that TNPO3 depletion restrained the uncoating in the IAV life cycle, thereby inhibiting the process of viral ribonucleoprotein (vRNP) entry into the nucleus. However, KO of TNPO3 did not affect the virus attachment, endocytosis, or endosomal acidification processes. Subsequently, we found that TNPO3 can colocalize and interact with viral proteins M1 and M2. Taken together, the depletion of TNPO3 inhibits IAV uncoating, thereby inhibiting IAV replication. Our study provides new insights and potential therapeutic targets for unraveling the mechanism of IAV replication and treating influenza disease.

Faithful degradation of mRNAs is a critical step in gene expression, and eukaryotes share a major conserved mRNA decay pathway. In this major pathway, the two rate-determining steps in mRNA degradation are the initial gradual removal of the poly(A) tail, followed by removal of the cap structure. Removal of the cap structure is carried out by the decapping enzyme, containing the Dcp2 catalytic subunit. Although the mechanism and regulation of mRNA decay is well understood, the consequences of defects in mRNA degradation are less clear. Dcp2 has been reported as either essential or nonessential. Here, we clarify that Dcp2 is not absolutely required for spore germination and extremely slow growth, but in practical terms it is impossible to continuously culture dcp2∆ under laboratory conditions without suppressors arising. We show that null mutations in at least three different genes are each sufficient to restore growth to a dcp2∆, of which kap123∆ and tl(gag)g∆ appear the most specific. We show that kap123∆ and tl(gag)g∆ suppress dcp2 by mechanisms that are different from each other and from previously isolated dcp2 suppressors. The suppression mechanism for tL(GAG)G is determined by the unique GAG anticodon of this tRNA, and thus likely by translation of some CUC or CUU codons. Unlike previously reported suppressors of decapping defects, these suppressors do not detectably restore decapping or mRNA decay to normal rates, but instead allow survival while only modestly affecting RNA homeostasis. These results provide important new insight into the importance of decapping, resolve previously conflicting publications about the essentiality of DCP2, provide the first phenotype for a tl(gag)g mutant, and show that multiple distinct mechanisms can bypass Dcp2 requirement.

In addition to the role of programmed cell death ligand 1 (PD-L1) in facilitating tumour cells escape from immune surveillance, it is considered as a crucial effector in transducing intrinsic signals to promote tumour development. Our previous study has pointed out that PD-L1 promotes non-small cell lung cancer (NSCLC) cell proliferation, but the mechanism remains elusive. Here we first demonstrated that PD-L1 expression levels were positively correlated with p-MerTK levels in patient samples and NSCLC cell lines. In addition, PD-L1 knockdown led to the reduced phosphorylation level of MerTK in vitro. We next showed that PD-L1 regulated NSCLC cell proliferation via Gas6/MerTK signaling pathway in vitro and in vivo. To investigate the underlying mechanism, we unexpectedly found that PD-L1 translocated into the nucleus of cancer cells which was facilitated through the binding of Karyopherin β1 (KPNB1). Nuclear PD-L1 (nPD-L1), coupled with transcription factor Sp1, regulated the synthesis of Gas6 mRNA and promoted Gas6 secretion to activate MerTK signaling pathway. Taken together, our results shed light on the novel role of nPD-L1 in NSCLC cell proliferation and reveal a new molecular mechanism underlying nPD-L1-mediated Gas6/MerTK signaling activation. All above findings provide the possible combinational implications for PD-L1 targeted immunotherapy in the clinic.