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Export of RNA from the nucleus is essential for all eukaryotic cells, with at least three major classes exported, mRNA, tRNA and rRNA. RNA export has emerged as a major step in the control of gene expression, with mRNA molecules required to complete a complex series of processing events and pass a quality control system to protect the cytoplasm from the expression of aberrant proteins. Many of these events are highly conserved across eukaryotes, reflecting their ancient origin, but significant deviation from a canonical pathway as described from animals and fungi has emerged in the trypanosomatids. With significant implications for the mechanisms that control gene expression and hence differentiation, responses to altered environments and fitness as a parasite, these deviations may also reveal additional, previously unsuspected, mRNA export pathways.

Erythroid nuclear condensation is a complex process in which compaction to one-tenth its original size occurs in an active nucleus simultaneously undergoing transcription and cell division. We previously found that the nuclear exportin Exportin7 (Xpo7), which is erythroid- specific and highly induced during terminal erythropoiesis, facilitates nuclear condensation. We also identified a previously unannotated, erythroid-specific isoform of Xpo7 (Xpo7B) containing a novel first exon Xpo7-1b expressed only in late Ter119

Over 90% of ovarian cancer cells exhibit p53 mutations or inactivation. In addition, p53 is exported outside of the nucleus by exportin-1 (XPO1), a protein that mediates the nuclear export of several cancer suppressor proteins. Overexpression of XPO1 is associated with resistance to chemotherapy, leading to poor prognosis in various cancers. The MDM2 inhibitor, RG-7388, is a known reactivator of p53 and has been tested with high interest as a therapeutic agent for cancer treatment. In addition, Selinexor, which is a second-generation selective inhibitor of nuclear export (SINE), is known to cause an accumulation of p53 in the nucleus and is also being explored as a therapy potentiating agent in combination treatments. This study was conducted to assess the efficacy of RG-7388 in combination with Selinexor for treating ovarian cancer. A combination of Selinexor and RG-7388 treatments was able to reduce the cell viability compared to individual treatments. In addition, the combination treatment revealed significant up-regulation of several cancer suppressor proteins in the whole lysate, cytoplasm, and nucleus. Finally, our results confirm that the combination of Selinexor with RG-7388 can induce a caspase-mediated apoptotic mechanism via up-regulation of p53 and p21.

For most genes, mRNA transcript abundance scales with cell size to ensure a constant concentration. Scaling of mRNA synthesis rates with cell size plays an important role, with regulation of the activity and abundance of RNA polymerase II (Pol II) now emerging as a key point of control. However, there is also considerable evidence for feedback mechanisms that kinetically couple the rates of mRNA synthesis, nuclear export, and degradation to allow cells to compensate for changes in one by adjusting the others. Researchers are beginning to integrate results from these different fields to reveal the mechanisms underlying transcript homeostasis. This will be crucial for moving beyond our current understanding of relative gene expression towards an appreciation of how absolute transcript levels are linked to other aspects of the cellular phenotype.

Actin cytoskeletal dynamics play a critical role in the regulation of myogenesis through mechanotransduction, Hippo signaling modulation, cell proliferation, and morphological changes. Although Twinfilin-1 (TWF1), a highly conserved actin-depolymerizing factor, is known to regulate actin filament assembly by sequestering actin monomer and capping barbed ends, the biological significance of TWF1 during the differentiation of myogenic progenitor cells has not been investigated. In this study, we unveiled the roles played by TWF1 in the proliferation and differentiation of C2C12 myoblasts. TWF1 was the predominant isoform in myoblasts, and its expression was induced during the early stage of differentiation. Knockdown of TWF1 by siRNA (siTWF1) induced the accumulation of actin filaments (F-actin) and promoted the nuclear translocation of Yes-associated protein (YAP) in the Hippo signaling pathway. TWF1 depletion activated transcription of YAP target genes and induced cell cycle and proliferation in myoblasts. Furthermore, TWF1 knockdown markedly reduced the expressions of myogenic regulatory factors, such as MyoD and MyoG, and drastically hindered myoblast differentiation, fusion, and myotube formation. Collectively, this study highlights the essential role of TWF1 in the myogenic differentiation of progenitor cells via modulation of F-actin and YAP, and suggests TWF1 as a potential therapeutic target for muscle wasting and myopathies.

Patients with chronic obstructive pulmonary disease (COPD) are characterized by an imbalance between oxidant enzymes and antioxidant enzymes. In the present study, we explored the protective effect of vitamin E on COPD and the underlying mechanisms. Targets of vitamin E were predicted by bioinformatics analysis. After establishing cigarette smoke (CS)-induced COPD rats, the expression levels of epidermal growth factor receptor (EGFR), cyclooxygenase 2 (COX2), and transcriptional activity of signal transducer and activator of transcription 3 (STAT3) were measured. Additionally, the effects of vitamin E on CS-induced COPD were explored by assessing inflammation, the reactive oxygen species (ROS), the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA), viability of human bronchial epithelioid (HBE) cells, and the expression of EGFR/MAPK pathway-related factors after loss- and gain- function assays. Vitamin E alleviated COPD. Vitamin E inhibited MAPK signaling pathway through decreasing EGFR expression. Additionally, vitamin E suppressed CS-induced HBE cell damage. Functionally, vitamin E attenuated CS-induced inflammation, apoptosis, and ROS by inhibiting the EGFR/MAPK axis, thereby inhibiting COX2-mediated p-STAT3 nuclear translocation. Moreover, overexpression of COX2 attenuated the protective effect of vitamin E on COPD rats. The present study shows that vitamin E inhibits the expression of COX2 by negatively regulating the EGFR/MAPK pathway, thereby inhibiting the translocation of phosphorylated STAT3 to the nucleus and relieving COPD.

S6K1 serves as an important signaling regulator of cell proliferation and growth in the mTOR signaling pathway. Excessive activation of the mTOR/S6K1 signaling pathway promotes abnormal cell growth and survival, thereby resulting in tumorigenesis. The roles of S6K1 in protein synthesis and metabolism are well known, but an additional role of S6K1 as a gene transcription regulator has not been much understood. Here, we demonstrated that S6K1 is dynamically distributed in the cytoplasm and nuclei of human cervical cancer cells. S6K1 nuclear localization was serum dependent and serum deprivation or rapamycin treatment inhibited S6K1 Thr389 phosphorylation and, thereby, S6K1 was retained in the cytoplasm. Furthermore, we found that endogenous S6K1 interacted with CREB in the cervical cancer cells. Additionally, S6K1 upregulated the CRE-driven promoter luciferase activity. The proto-oncogene c-JUN, which has several CREs, was attenuated in the S6K1 knockdown cervical cancer cells. The binding of CREB/S6K1 to the c-JUN promoter, altered by serum restimulation, was associated with active epigenetic markers. In HeLa cell, 891 promoter regions, to which S6K1 directly binds, were detected. Our findings suggested that active S6K1, which is dynamically translocated into the nucleus, directly binds to chromatin and could play a role in epigenetic mechanisms or transcription factor recruitment.

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.

High fluence low-level laser (HF-LLL), a mitochondria-targeted tumour phototherapy, results in oxidative damage and apoptosis of tumour cells, as well as damage to normal tissue. To circumvent this, the therapeutic effect of low fluence LLL (LFL), a non-invasive and drug-free therapeutic strategy, was identified for tumours and the underlying molecular mechanisms were investigated. We observed that LFL enhanced antigen-specific immune response of macrophages and dendritic cells by upregulating MHC class II, which was induced by mitochondrial reactive oxygen species (ROS)-activated signalling, suppressing tumour growth in both CD11c-DTR and C57BL/6 mice. Mechanistically, LFL upregulated MHC class II in an MHC class II transactivator (CIITA)-dependent manner. LFL-activated protein kinase C (PKC) promoted the nuclear translocation of CIITA, as inhibition of PKC attenuated the DNA-binding efficiency of CIITA to MHC class II promoter. CIITA mRNA and protein expression also improved after LFL treatment, characterised by direct binding of Src and STAT1, and subsequent activation of STAT1. Notably, scavenging of ROS downregulated LFL-induced Src and PKC activation and antagonised the effects of LFL treatment. Thus, LFL treatment altered the adaptive immune response via the mitochondrial ROS-activated signalling pathway to control the progress of neoplastic disease.

Nuclear composition determines nuclear function. The early embryos of many species begin life with large pools of maternally provided components that become rapidly imported into an increasing number of nuclei as the cells undergo repeated cleavage divisions. Because early cell cycles are too fast for nuclei to achieve steady-state nucleocytoplasmic partitioning, the composition of cleavage stage nuclei is likely dominated by nuclear import. The end of the rapid cleavage stage and onset of major zygotic transcription, known as the mid-blastula transition (MBT), is controlled by the ratio of nuclei/cytoplasm, indicating that changes in nuclear composition likely mediate MBT timing. Here, we explore how different nuclear import regimes can affect protein accumulation in the nucleus in the early Drosophila embryo. We find that nuclear import differs dramatically for a general nuclear cargo (NLS (nuclear localization signal)-mRFP) and a proposed MBT regulator (histone H3). We show that nuclear import rates of NLS-mRFP in a given nucleus remain relatively unchanged throughout the cleavage cycles, whereas those of H3 halve with each cycle. We model these two distinct modes of nuclear import as "nucleus-limited" and "import-limited" and examine how the two different modes can contribute to different protein accumulation dynamics. Finally, we incorporate these distinct modes of nuclear import into a model for cell-cycle regulation at the MBT and find that the import-limited H3 dynamics contribute to increased robustness and allow for stepwise cell-cycle slowing at the MBT.

Cell signaling and the following gene regulation are tightly regulated to keep homeostasis. NF-κB is a famous key transcription factor for inflammatory cell regulations that obtain a closed feedback loop with IκB. Similarly, we show here, NFAT is also tightly regulated via its downstream target, down syndrome critical region (DSCR)-1. In primary cultured endothelium, either shear stress or VEGF treatment revealed quick NFAT1 nuclear localization following the DSCR-1 transactivation, which in turn induced NFAT1 cytoplasm sequestration. Interestingly, both NFAT and DSCR-1 can be competitive substrates for calcineurin phosphatase and DSCR-1 is known to unstable protein, which caused NFAT1-nucleocytoplasmic damped oscillation via sustained shear stress or VEGF stimulation in endothelial cell (EC)s. To understand the molecular mechanism underlying the NFAT1 oscillation, we built a mathematical model of spatiotemporal regulation of NFAT1 combined with calcineurin and DSCR-1. Theoretically, manipulation of DSCR-1 expression in simulation predicted that DSCR-1 reduction would cause nuclear retention of dephosphorylated NFAT1 and disappearance of NFAT1 oscillation. To confirm this in ECs, DSCR-1 knockdown analysis was performed. DSCR-1 reduction indeed increased dephosphorylated NFAT1 in both the nucleus and cytoplasm, which eventually led to nuclear retention of NFAT1. Taken together, these studies suggest that DSCR-1 is a responsible critical factor for NFAT1 nucleocytoplasmic oscillation in shear stress or VEGF treated ECs. Our mathematical model successfully reproduced the experimental observations of NFAT1 dynamics. Combined mathematical and experimental approaches would provide a quantitative understanding way for the spatiotemporal NFAT1 feedback system.

In the nuclear pore complex, intrinsically disordered proteins (FG Nups), along with their interactions with more globular proteins called nuclear transport receptors (NTRs), are vital to the selectivity of transport into and out of the cell nucleus. Although such interactions can be modeled at different levels of coarse graining, in vitro experimental data have been quantitatively described by minimal models that describe FG Nups as cohesive homogeneous polymers and NTRs as uniformly cohesive spheres, in which the heterogeneous effects have been smeared out. By definition, these minimal models do not account for the explicit heterogeneities in FG Nup sequences, essentially a string of cohesive and noncohesive polymer units, and at the NTR surface. Here, we develop computational and analytical models that do take into account such heterogeneity in a minimal fashion and compare them with experimental data on single-molecule interactions between FG Nups and NTRs. Overall, we find that the heterogeneous nature of FG Nups and NTRs does play a role in determining equilibrium binding properties but is of much greater significance when it comes to unbinding and binding kinetics. Using our models, we predict how binding equilibria and kinetics depend on the distribution of cohesive blocks in the FG Nup sequences and of the binding pockets at the NTR surface, with multivalency playing a key role. Finally, we observe that single-molecule binding kinetics has a rather minor influence on the diffusion of NTRs in polymer melts consisting of FG-Nup-like sequences.

Background Nucleocytoplasmic transport is a crucial process for cell function. Previous studies have observed alterations in different molecules involved in it, relating them to ventricular function. However, there are no published data evaluating possible differences in the expression of these molecules in heart transplantation (HT) recipients. Our objective is to evaluate whether its levels are related to the appearance of cellular rejection (CR) during the first year after HT. Methods A prospective clinical cohort that included patients undergoing HT between January 2017 and January 2019 (n = 46). Blood samples for the analysis of importin 5 (IMP5), nucleoporin 153 (Nup153); RAN-GTPaseAP1 (RanGAP1), and sarcoplasmic reticulum calcium ATPase (ATP-aseCaTransp) were collected approximately 2 months post-HT. The levels obtained were correlated with the incidence of at least moderate CR during the first year of follow-up. Results Results showed that 17.39% of the patients had at least moderate CR during the first year of follow-up. Higher levels of IMP5, Nup153, and RanGAP1 were observed in this group. This difference was statistically significant in the case of Nup153 and RanGAP1 (15.94 ± 14.00 vs 28.62 ± 23.61, P = .048; 21.95 ± 15.97 vs 40.90 ± 27.16, P = .026, respectively); there was an opposite trend in the ATP-aseCaTransp case. Conclusion Patients with at least a moderate degree of CR during follow-up showed higher serum levels of IMP5, Nup153, and RanGAP1. The prognostic usefulness of the determination of these biomarkers and whether their elevation during follow-up would facilitate early, noninvasive identification of patients with CR remains to be clarified.

The tumor microenvironment is a complex microenvironment that combines the biochemical and biophysical factors. When the cells are exposed to the microenvironment, the direct biophysical factor is the matrix hardness. As an auxiliary indicator of clinical disease diagnosis, it is still not clear how the matrix hardness induces cell malignant changes and the regulation mechanisms. In this study, we identified that hard matrix significantly promoted cancer cell migratory behaviors. Cell shape was closely associated with cancer cell malignancy, the high malignant cells were associated with high ratios of length/width and low circularity. F-actin networks were also linked with extracellular matrix, it was not regularly distributed when cells were in non-malignant tumor phases or under F-actin inhibition. F-actin might play the key role that transmitted the signal from extracellular matrix to the intracellular organelles. Further study confirmed that active YAP was translocated to nucleus on hard matrix. Cells on hard matrix with cytochalasin D reversed the cancer cell malignancy, meanwhile F-actin re-distributed to the membrane and YAP nucleus translocations were hindered. This work confirmed that F-actin and YAP were upstream-downstream cascade for the cellular and nucleus outside-in signal transductions. The above results demonstrated that hard matrix promoted breast cancer cell malignant behaviors through F-actin network and YAP activation. These results not only described the signal transductions from extracellular to intracellular that was initiated by the biophysical tumor microenvironment, but provided clinical intervention ideas for cancer treatments.

Purpose Autophagy inhibition is a novel therapeutic strategy suggested for patients with advanced cancer, especially those who have undergone radiation therapy. In the present study, we investigated whether autophagy inhibitors accelerate the progression of radiation-associated atherosclerosis (RAA). Methods and Materials Eight-week-old apolipoprotein (ApoE-/-) mice were fed a Western diet, and their left common carotid arteries were partially ligated to induce atherogenesis. Four weeks later, local ionizing radiation (IR) at a dose of 5 or 10 Gy was used to induce RAA in the left common carotid artery. After another 4 weeks, severe plaque burden associated with increased macrophage infiltration and lipid deposition, reduced smooth muscle cells, and decreased collagen expression was observed. In addition, these changes occurred in a dose-dependent manner. Improved autophagic flux caused by IR was observed in both macrophages of the atherosclerotic plaque and peritoneal macrophages in vitro. The inhibition of autophagic flux by chloroquine (50 mg/kg/d) further accelerated the progression of RAA in the left common carotid arteries of ApoE−/− mice. Furthermore, chloroquine treatment exacerbated IR-induced p65 nuclear translocation, IκBα degradation, and transcription of nuclear factor-κB (NF-κB) target genes in peritoneal macrophages. Conclusions IR promotes atherogenesis and increases autophagic flux. In addition, autophagy inhibition by chloroquine accelerates the progression of RAA lesions by stimulating NF-κB-mediated inflammatory responses in macrophages.

The binding of calmodulin (CaM) to NMDAR (N-methyl-D-aspartate receptor) GluN1 C-terminus is required for cacium (Ca2+)/calmodulin (CaM)-dependent inactivation of NMDAR. Previously, we found that GluN1 C-terminus translocated to nucleus, and regulated synaptic transmission. However, whether GluN1 C-terminus containing the nuclear localization signaling regulates the cellular distribution of CaM, and the CaM binding are not clear. In this study, we found that the 10 positive residues of GluN1 C-terminus determined the translocation of CaM to the nucleus. RNA sequencing data showed that CaM could regulate the genes expression of multiple cell surface membrane receptors. This was confirmed by electrophysiology data that the 10A mutation of GluN1 C-terminus increased the NMDAR/AMAPR-mediated synaptic transduction.

A mammalian embryo experiences the first cell segregation at the blastocyst stage, in which cells giving form to the embryo are sorted into two lineages; trophectoderm (TE) and inner cell mass (ICM). This first cell segregation process is governed by cell position-dependent Hippo signaling, which is a phosphorylation cascade determining whether Yes-associated protein 1 (YAP1), one of the key components of the Hippo signaling pathway, localizes within the nucleus or cytoplasm. YAP1 localization determines the transcriptional on/off switch of a key gene, Cdx2, required for TE differentiation. However, the control mechanisms involved in YAP1 nucleocytoplasmic shuttling post blastocyst formation remain unknown. This study focused on the mechanisms involved in YAP1 release from TE nuclei after blastocoel contraction in bovine blastocysts. The blastocysts contracted by blastocoel fluid aspiration showed that the YAP1 translocation from nucleus to cytoplasm in the TE cells was concomitant with the protruded actin cytoskeleton. This YAP1 release from TE nuclei in the contracted blastocysts was prevented by actin disruption and stabilization. In contrast, Y27632, which is a potent inhibitor of Rho-associated coiled-coil containing protein kinase 1/2 (ROCK) activity, was found to promote YAP1 nuclear localization in the TE cells of contracted blastocysts. Meanwhile, lambda protein phosphatase (LPP) treatment inducing protein dephosphorylation could not prevent YAP1 release from TE nuclei in the contracted blastocysts, indicating that YAP1 release from TE nuclei does not depend on the Hippo signaling pathway. These results suggested that blastocyst contraction causes YAP1 release from TE nuclei through actin cytoskeleton remodeling in a Hippo signaling-independent manner. Thus, the present study raised the possibility that YAP1 subcellular localization is controlled by actin cytoskeletal organization after the blastocyst formation. Our results demonstrate diverse regulatory mechanisms for YAP1 nucleocytoplasmic shuttling in TE cells.

Nuclear pore complexes (NPC) are highly conserved mega protein complexes that penetrate the double-layered nuclear membrane and form channels to allow bi-directional transport of macromolecules between the nucleus and the cytosol. Non-passive nucleocytoplasmic transport also requires nuclear transport receptors (NTR), which bind cargo molecules and shuttle them across the NPC. The NPC and NTRs constitute two fundamental layers of regulatory mechanisms that together determine the selective nuclear translocation of signal molecules and play essential roles in activating the precise response of a cell to environmental stimuli. Here we discuss recent findings in the NPC made by advanced structural biology approaches, and dissect distinct functions of different NPC components and NTRs in plants' responses to various biotic and abiotic stresses.

Germinal center-associated nuclear protein (GANP) is a unique and multifunctional protein that plays a critical role in cell biology, neurodegenerative disorders, immunohematology, and oncogenesis. GANP is an orthologue of Saccharomyces Sac3, one of the components of the transcription export 2 (TREX-2) complex and a messenger RNA (mRNA) nuclear export factor. GANP is widely conserved in all mammals, including humans. Although GANP was originally discovered as a molecule upregulated in the germinal centers of secondary lymphoid follicles in peripheral lymphoid organs, it is expressed ubiquitously in many tissues. It serves numerous functions, including making up part of the mammalian TREX-2 complex; mRNA nuclear export via nuclear pores; prevention of R-loop formation, genomic instability, and hyper-recombination; and B-cell affinity maturation. In this review, we first overview the extensive analyses that have revealed the basic functions of GANP and its ancestor molecule Sac3, including mRNA nuclear export and regulation of R-loop formation. We then describe how aberrant expression of GANP is significantly associated with cancer development. Moreover, we discuss a crucial role for GANP in B-cell development, especially affinity maturation in germinal centers. Finally, we illustrate that overexpression of GANP in B cells leads to lymphomagenesis resembling Hodgkin lymphoma derived from germinal center B cells, and that GANP may be involved in transdifferentiation of B cells to macrophages, which strongly affects Hodgkin lymphomagenesis.

Aging is regulated by complex signaling networks, the details of which remain poorly understood. Here, we demonstrate that VPS-22/SNF8, a component of endosomal sorting complex required for transport-II (ESCRT-II), regulates the lifespan of C. elegans. In this study we show that worms with vps-22/snf8 gene knockdown had a shorter lifespan than wild-type worms. The expression pattern of VPS-22/SNF8 in C. elegans was highly similar to that of DAF-16. Knockout of daf-16 in C. elegans shortened the worms’ lifespan; however, reducing the expression of vps-22/snf8 in daf-16 null worms did not further shorten their lifespan, indicating that vps-22/snf8 and daf-16 may act in the same signaling pathway to regulate longevity. Over-expression of daf-16 rescued the short-lived phenotype of vps-22/snf8 knockdown worms. Moreover, down-regulation of vps-22/snf8 decreased the nuclear localization of DAF-16 and modulated the expression of daf-16 downstream genes that regulate longevity in C. elegans. In summary, our results indicate that vps-22/snf8 can regulate the longevity of C. elegans by partially modulating the activity of daf-16. These findings may help us to better understand the mechanisms of aging.

Neddylation is an ubiquitin-like modification of proteins that affects the activity, stability and protein-protein interaction of its substrates. Apart from its role as a promoter for Cullin ring E3 ligase to positively regulate the ubiquitylation process, other functional studies about neddylation are still lacking. In this study, we developed a system to explore the impact of neddylation on changes in the subcellular localization of proteins at the omics level. By applying a method combining subcellular protein extraction and immunoprecipitation-mass spectrometry (IP-MS), 81 proteins with a tendency to shuttle between the cytoplasm and nucleus due to different neddylation levels were obtained. Among the 81 candidates, transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) and growth arrest and DNA damage protein 45a (Gadd45a) were confirmed as novel substrates of Nedd8, and neddylation promotes TAK1 nuclear import as well as Gadd45a nuclear export.

Bovine adenovirus-3 (BAdV-3) is a non enveloped, icosahedral DNA virus containing a genome of 34446 bps. The intermediate region of BAdV-3 encodes pIX and IVa2 proteins. Here, we report the characterization of BAdV-3 IVa2. Anti-IVa2 serum detected a 50 kDa protein at 24–48 h post infection in BAdV-3 infected cells. The IVa2 localizes to nucleus and nucleolus of BAdV-3 infected cells. Analysis of mutant IVa2 demonstrated that amino acids 1–25 and 373–448 are required for nuclear and nucleolar localization of IVa2, respectively. The nuclear import of IVa2 utilize importin α -1 of importin nuclear import pathway. Although deletion/substitution of amino acids 4–18 is sufficient to abrogate the nuclear localization of IVa2, amino acids 1–25 are required for nuclear localization of a cytoplasmic protein. Furthermore, we demonstrate that amino acids 1–25 and 120–140 of IVa2 interact with importin α-1 and pV proteins, respectively in BAdV-3 infected cells.

Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505-527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515-519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.

The transcription factor forkhead box P3 (FOXP3) is a biomarker for regulatory T cells and can also be expressed in cancer cells, but its function in cancer appears to be divergent. The role of hepatocyte-expressed FOXP3 in hepatocellular carcinoma (HCC) is unknown. Here, we collected tumor samples and clinical information from 115 HCC patients and used five human cancer cell lines. We examined FOXP3 mRNA sequences for mutations, used a luciferase assay to assess promoter activities of FOXP3's target genes, and employed mouse tumor models to confirm in vitro results. We detected mutations in the FKH domain of FOXP3 mRNAs in 33% of the HCC tumor tissues, but in none of the adjacent nontumor tissues. None of the mutations occurred at high frequency, indicating that they occurred randomly. Notably, the mutations were not detected in the corresponding regions of FOXP3 genomic DNA, and many of them resulted in amino acid substitutions in the FKH region, altering FOXP3's subcellular localization. FOXP3 delocalization from the nucleus to the cytoplasm caused loss of transcriptional regulation of its target genes, inactivated its tumor-inhibitory capability, and changed cellular responses to histone deacetylase (HDAC) inhibitors. More complex FKH mutations appeared to be associated with worse prognosis in HCC patients. We conclude that mutations in the FKH domain of FOXP3 mRNA frequently occur in HCC and that these mutations are caused by errors in transcription and are not derived from genomic DNA mutations. Our results suggest that transcriptional mutagenesis of FOXP3 plays a role in HCC.

Post-translational modifications play essential roles in finely modulating eukaryotic circadian clock systems. In plants, the effects of O-glycosylation on the circadian clock and the underlying mechanisms remain largely unknown. The O-fucosyltransferase SPINDLY (SPY) and the O-GlcNAc transferase SECRET AGENT (SEC) are two prominent O-glycosylation enzymes in higher plants, with both overlapped and unique functions in plant growth and development. Unlike the critical role of O-GlcNAc in regulating the animal circadian clock, here we report that nuclear-localized SPY, but not SEC, specifically modulates the pace of the Arabidopsis circadian clock. By identifying the interactome of SPY, we identified PSEUDO-RESPONSE REGULATOR 5 (PRR5), one of the core circadian clock components, as a new SPY-interacting protein. PRR5 can be O-fucosylated by SPY in planta, while point mutation in the catalytic domain of SPY abolishes the O-fucosylation of PRR5. The protein abundance of PRR5 is strongly increased in spy mutants, while the degradation rate of PRR5 is much reduced, suggesting that PRR5 proteolysis is promoted by SPY-mediated O-fucosylation. Moreover, multiple lines of genetic evidence indicate that PRR5 is a major downstream target of SPY to specifically mediate its modulation of the circadian clock. Collectively, our findings provide novel insights into the specific role of the O-fucosyltransferase activity of SPY in modulating the circadian clock and implicate that O-glycosylation might play an evolutionarily conserved role in modulating the circadian clock system, via O-GlcNAcylation in mammals, but via O-fucosylation in higher plants.

Renpenning syndrome belongs to a group of X-linked intellectual disability disorders. The Renpenning syndrome–associated protein PQBP1 (polyglutamine-binding protein 1) is intrinsically disordered, associates with several splicing factors, and is involved in pre-mRNA splicing. PQBP1 uses its C-terminal YxxPxxVL motif for binding to the splicing factor TXNL4A (thioredoxin like 4A), but the biological function of this interaction has yet to be elucidated. In this study, using recombinant protein expression, in vitro binding assays, and immunofluorescence microscopy in HeLa cells, we found that a recently reported X-linked intellectual disability–associated missense mutation, resulting in the PQBP1-P244L variant, disrupts the interaction with TXNL4A. We further show that this interaction is critical for the subcellular location of TXNL4A. In combination with other PQBP1 variants lacking a functional nuclear localization signal required for recognition by the nuclear import receptor karyopherin β2, we demonstrate that PQBP1 facilitates the nuclear import of TXNL4A via a piggyback mechanism. These findings expand our understanding of the molecular basis of the PQBP1–TXNL4A interaction and of the etiology and pathogenesis of Renpenning syndrome and related disorders.

Interferon regulatory factors (IRFs) are a family of transcription factors essential to the control of antiviral immune response, cell growth, differentiation and apoptosis. IRF10 was originally found in chicken, which was induced by the v-Rel oncoprotein in lymphoid cell lines and involved in the upregulation of major histocompatibility complex (MHC) class I and guanylate-binding protein. In fish, IRF10 plays negative roles in regulation of the interferon (IFN) response. Here, we identified a splice variant of IRF10, named as EcIRF10-SF in orange spotted grouper, which shares the first three exons with the long form (EcIRF10-LF) and retains part of intron 3, creating a premature termination codon. Furthermore, we observed that the EcIRF10-SF exhibits similar expression pattern compared to its native counterparts. Functional studies demonstrate that the two EcIRF10 isoforms repress DrIFNϕ1 and DrIFNϕ3 promoter activity and negatively regulate fish antiviral gene expression. Subcellular localization analysis shows that the amino acids from 57 to 86 within DBD are required for IRF10 nuclear import. Overall, our description of transcript diversification of IRF10 in the grouper provides a coherent framework to further dissect its roles in immune response.

Crosstalk exists when two or more post-translational modifications, nearby in sequence or 3D space, affect each other or a protein's interactions. Saccharomyces cerevisiae protein Npl3p has six repeats of sequence SRGG, in a disordered domain, which can carry arginine methylation and serine phosphorylation. Crosstalk of the modifications controls Npl3p interactions with nuclear import, export, and other proteins. Here, we asked whether repeated SRGG motifs existed in other S. cerevisiae proteins and whether they serve a related function. Two other proteins had multiple SRGG motifs: Nop1p (fibrillarin) and Gar1p, both nucleolar proteins, which had nine and four motifs, respectively. For Nop1p, we first showed it to be extensively methylated in vivo. We then showed that the Nop1p SRGG motif is subjected to methylation by Hmt1p, phosphorylation by Sky1p, and Glc7p dephosphorylation and that there is crosstalk whereby phosphorylation blocks methylation. This is consistent with our recent motif analysis of Hmt1p, which revealed a negative specificity for acidic residues at −1 and −2 positions. On knockout of HMT1, Nop1p-GFP localization was not typically nucleolar. Conditional two-hybrid analysis, of Nop1p with C/D box small ribonuclear proteins Nop56p and Nop58p, suggested this may be associated with decreased protein-protein interactions on loss of arginine methylation. The effect of SRGG phosphorylation on the interactions of Nop1p remains unknown yet was predicted to cause a structural disorder-to-order transition in the Nop1p N-terminal domain. The SRGG motif is one of very few examples of modification crosstalk that has related functions in multiple proteins from the same species.

Optical manipulations are widely used to analyze neuronal functions in vivo. Blue light is frequently used to activate channelrhodopsins or LOV domains, although the degrees of its absorption and scattering are higher than those of longer wavelength light. High spatial resolution of optical manipulation is easily achieved in vitro, while the light is unevenly scattered and absorbed in tissues due to many factors. It is difficult to spatially measure a blue light transmission area in vivo. Here, we propose a genetic method to visualize blue light transmission in the brain and other organs using light-induced nuclear translocation of fluorescent proteins with a LOV domain. A light-inducible nuclear localization signal (LINuS) consists of a LOV2 domain fused with a nuclear localization signal (NLS). We confirmed that blue light illumination induced reversible translocation of NES-tdTomato-LINuS from the cytosol to the nucleus within 30 min in HEK293 cells. By employing a PHP.eb capsid that can penetrate the blood-brain barrier, retro-orbital sinus injection of adeno-associated virus (AAV) vectors induced scattered expression of nuclear export signal (NES)-tdTomato-LINuS in the brain. We confirmed that 30-min transcranial blue light illumination induced nuclear translocation of NES-tdTomato-LINuS in the cortex, the hippocampus, and even the paraventricular nucleus of the thalamus. We also found that mice exposed to blue light in a shaved abdominal area exhibited a substantial increase in nuclear translocation in the ventral surface lobe of the liver. These results provide a simple way to obtain useful information on light transmission in tissues without any transgenic animals or skillful procedures.

Exposure of cells to bortezomib (BTZ) could activate heat shock protein (HSP) expression, which is regulated mainly by heat shock factor 1 (HSF1). We determined the role of Apg-1 (HSPA4L, a member of the HSP110 family) in HSF1 activation and bortezomib sensitivity by silencing HSF1 in multiple myeloma (MM) cells. We observed that the Apg-1 protein level was upregulated as BTZ concentration increased. To investigate the mechanism underlying Apg-1 induction, we evaluated the HSF1 translocation and found BTZ-inducible transposition to the nucleus of HSF1. In addition, cleaved caspase 3 and PARP might account for increased BTZ sensitivity on Apg-1 silencing. Furthermore, silencing HSF1 with shRNA or triptolide resulted in significant BTZ sensitivity. It had a more profound effect on cell death caused by BTZ when myeloma cells were adherent to bone marrow stromal cell lines (Hs-5). In summary, we found that Apg-1 knockdown sensitized myeloma cells to bortezomib treatment, which may provide a new approach in multiple myeloma treatment.

This Invited Review is a kind of scientific autobiography based on the presentation at the Symposium "Viruses: Discovering Big in Small" held in honor of the author's 90th birthday (Moscow, March 2019).

In cancer, aberrant growth factor receptor signaling reprograms cellular metabolism and global gene transcription to drive aggressive growth, but the underlying mechanisms are not well-understood. Here we show that in the highly lethal brain tumor glioblastoma (GBM), mTOR complex 2 (mTORC2), a critical core component of the growth factor signaling system, couples acetyl-CoA production with nuclear translocation of histone-modifying enzymes including pyruvate dehydrogenase and class IIa histone deacetylases to globally alter histone acetylation. Integrated analyses in orthotopic mouse models and in clinical GBM samples reveal that mTORC2 controls iron metabolisms via histone H3 acetylation of the iron-related gene promoter, promoting tumor cell survival. These results nominate mTORC2 as a critical epigenetic regulator of iron metabolism in cancer.

Alternatively activated or M2 macrophages, as opposed to the well characterized pro-inflammatory or M1 macrophages, vitally regulate anti-inflammation, wound healing, and tissue repair to maintain tissue homeostasis. Although ubiquitous presence of macrophages in diverse tissues, exposed to different physical environments, infers distinct immune responses of M2 macrophages with high phenotypic heterogeneity, the underlying mechanism of how the varying extracellular mechanical conditions alter their immunological activation remains unclear. Here, we demonstrate that M2 activation requires a threshold mechanical cue from the extracellular microenvironment, and matrix rigidity-dependent macrophage spreading is mediated by the F-actin formation that is essential to regulate mechanosensitive M2 activation of macrophages. We identified a new mechanosensing function of STAT6 (signal transducer and activator of transcription 6), a key transcription factor for M2 activation, whose intranuclear transportation is promoted by the rigid matrix that facilitates the F-actin formation. Our findings further highlight the critical role of mechanosensitive M2 activation of macrophages in long-term adaptation to the extracellular microenvironment by bridging nuclear mechanosensation and immune responses.

Importin β1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in the detection of importin complex cargos and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable biotinylation by antibody recognition analysis of importin β1 interactomes. The monoclonal antibody recognizes an epitope comprising residues 301-320 of human KPBN1 and strikingly is highly specific for cytoplasmic KPNB1 in diverse applications, with little reaction with KPNB1 in the nucleus. Biotinylation by antibody recognition with this novel antibody revealed numerous new interactors of importin β1, expanding the KPNB1 interactome to cytoplasmic and signaling complexes that highlight potential new functions for the importins complex beyond nucleocytoplasmic transport. Data are available via ProteomeXchange with identifier PXD032728.

de Almeida et al. (2021) developed a temporally controlled CRISPR-Cas9 screen to identify mechanisms controlling MYC levels and discovered that intact proteasomes are imported into the nucleus by AKIRIN2 binding to proteasomes at one end and a nuclear import receptor at the other.

The onion-derived metabolite, S-1-propenylmercaptocysteine (CySSPe), protects against oxidative stress and exhibits anti-inflammatory effects by modulating cellular redox homeostasis. We sought to establish whether CySSPe activates nuclear factor erythroid 2-related factor 2 (Nrf2) and whether activation of Nrf2 by CySSPe involves modification of the Kelch-like ECH-associated protein-1 (Keap1) to manifest these effects. We found that CySSPe stabilized Nrf2 protein and facilitated nuclear translocation to induce expression of antioxidant enzymes, including NQO1, HO-1, and GCL. Moreover, CySSPe attenuated tert-butyl hydroperoxide-induced cytotoxicity and dose-dependently inhibited reactive oxygen species production. Silencing experiments using Nrf2-siRNA confirmed that CySSPe conferred protection against oxidative stress by activating Nrf2. CySSPe enhanced cellular pool of reduced glutathione (GSH) and improved GSH:GSSG ratio. Pretreatment of cells with l-buthionine-S,R-sulfoximine (BSO) confirmed that CySSPe increases de novo synthesis of GSH by upregulating expression of the GSH-synthesizing enzyme GCL. Treatment of cells with CySSPe elevated hydrogen sulfide (H2S) production. Inhibition of H2S-synthesizing enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS), by pretreating cells with propargylglycine (PAG) and oxyaminoacetic acid (AOAA) revealed that H2S production was partially dependent on a CSE/CBS-catalyzed β-elimination reaction with CySSPe that likely produced 1-propenyl persulfide (RSSH). Depleting cells of their GSH pool by exposure to BSO and diethylmaleate attenuated H2S production, suggesting a GSH-dependent formation of H2S, likely via the reduction of RSSH by GSH. Finally, treatment of cells with CySSPe persulfidated Keap1, which may be the mechanism involved for the stabilization of Nrf2 by CySSPe. Taken together, our results showed that attenuation of oxidative stress by CySSPe is associated with its ability to produce H2S or RSSH, which persulfidates Keap1 and activates Nrf2 signaling. This study provides insights on the potential of CySSPe as an onion-derived dietary agent that modulates redox homeostasis and combats oxidative stress.

The nuclear export protein (NEP) of the influenza A virus exports viral ribonucleoproteins to the host cell cytoplasm following nuclear transcription. In this work conservation analysis of 3000 protein sequences and molecular modelling of full-length NEP identified ligand binding sites overlapping with high sequence conservation. Two binding hot spots were identified close to the first nuclear export signal and several hot spots overlapped with highly conserved amino acids such as Arg42, Asp43, Lys39, Ile80, Gln101 and Val109. Virtual screening with ~43,000 compounds against a binding site showed affinities of up to −8.95 kcal/mol, while ~1700 approved drugs showed affinities of up to −8.31 kcal/mol. A drug-like compounds predicted was ZINC01564229 that could be used as probe to investigate NEP function or as a new drug lead. The approved drugs Nandrolone phenylpropionate and Estropipate were predicted to bind with high affinity and may be investigated for repurposing as anti-influenza drugs. Importance The influenza A virus causes respiratory illness in humans and farm animals annually across the world. Antigenic shifts and drifts in the surface proteins lead to genome diversity and unpredictable pandemics and epidemics. The high evolution rate of the RNA genome can also limit the effectiveness of antivirals and is the cause of emerging resistance. From a human health perspective, it is important that compounds identified as potential influenza replication inhibitors remain effective long-term. This work presents results which are based on computational predictions that reveal interactions between available compounds and regions of the influenza A nuclear export protein which display high conservation. Due to a low probability of highly conserved regions undergoing genomic changes, these compounds may serve as ideal leads for new antivirals.

The three canonical members of the family of homeodomain-interacting protein (HIP) kinases fulfill overlapping and distinct roles in cellular stress response pathways. Here we systematically compared all three endogenous HIPKs for their intracellular distribution and mutual interactions. The endogenous HIPKs are contained in high molecular weight complexes of ~700 kDa but do not directly interact physically. Under basal conditions, HIPK1 was mostly cytoplasmic, while HIPK3 was found in the nucleus and HIPK2 occurred in both compartments. Inhibition of nuclear export by leptomycin B resulted in the nuclear accumulation of mainly HIPK1 and HIPK2, indicating constitutive dynamic nucleocytoplasmic shuttling. The carcinogenic chemical stressor sodium arsenite caused the induction of HIPK2-dependent cell death and also resulted in a rapid and complete nuclear translocation of HIPK2, showing that the intracellular distribution of this kinase can undergo dynamic regulation.

Human phospholipid scramblase 1 (PLSCR1) is strongly expressed in response to interferon (IFN) treatment and viral infection, and PLSCR1 has been suggested to play an important role in IFN-dependent antiviral responses. In this study, we showed that the basal expression of PLSCR1 was significantly elevated in Epstein-Barr virus (EBV)–infected nasopharyngeal carcinoma (NPC). PLSCR1 was observed to directly interact with the EBV immediate-early transactivator BZLF1 in vitro and in vivo, and this interaction repressed the BZLF1-mediated transactivation of an EBV lytic BMRF1 promoter construct. In addition, PLSCR1 expression decreased the BZLF1-mediated up-regulation of lytic BMRF1 mRNA and protein expression in WT and PLSCR1-knockout EBV-infected NPC cells. Furthermore, we showed that PLSCR1 represses the interaction between BZLF1 and CREB-binding protein (CBP), which enhances the BZLF1-mediated transactivation of EBV lytic promoters. These results reveal for the first time that PLSCR1 specifically interacts with BZLF1 and negatively regulates its transcriptional regulatory activity by preventing the formation of the BZLF1-CBP complex. This interaction may contribute to the establishment of latent EBV infection in EBV-infected NPC cells.

Polychlorinated biphenyls (PCBs) are classic persistent organic pollutants (POPs) and are associated with the progression of many cancers, including liver cancer. The present study investigated the effect of 2,3'4,4',5-pentachlorobiphenyl (PCB118) on hepatocellular carcinoma cell proliferation and its underlying mechanisms. The results indicated that PCB118 exposure promotes the proliferation and glycolysis of hepatocellular carcinoma SMMC-7721 cells. Moreover, PCB118 exposure increased the expression level of pyruvate kinase M2 (PKM2) and its nuclear translocation, whereas treatment with PKM2 shRNA suppressed the induction of cell proliferation and glycolysis by PCB118. PCB118 stimulated reactive oxygen species (ROS) production by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Treatment with the antioxidants N-acetyl-L-cysteine (NAC) and superoxide dismutase (SOD) prevented PCB118-induced effects on PKM2, cell proliferation and glycolysis. Furthermore, we found that PCB118 activated NADPH oxidase through the aryl hydrocarbon receptor (AhR) in SMMC-7721 cells. Consistently, treatment with AhR shRNA suppressed PCB118-induced effects on PKM2, cell proliferation and glycolysis. Overall, these results indicated that PCB118 promotes HCC cell proliferation via PKM2-dependent upregulation of glycolysis, which is mediated by AhR/NADPH oxidase-induced ROS production.

Sirtuin 7 (SIRT7) is an NAD+-dependent lysine deacetylase that regulates diverse biological processes. We recently observed that SIRT7 deficiency suppresses the nuclear accumulation of p65, which is a component of nuclear factor kappa B. However, the underlying molecular mechanism remains elusive. In this study, we demonstrated that SIRT7 interacts with a small GTPase, Ras-related nuclear antigen (Ran), and deacetylates Ran at K37. The nuclear export of p65 was facilitated in SIRT7-deficient fibroblast cells, while the nuclear export was inhibited in SIRT7-deficient cells expressing K37R-Ran (deacetylation-mimicking mutant). Additionally, the nuclear export of p65 in wild-type fibroblast cells was promoted by K37Q-Ran (acetylation-mimicking mutant). K37Q-Ran exhibited an increased ability to bind to chromosome region maintenance 1 (CRM1), which is a major nuclear receptor that mediates the export of cargo proteins, and enhanced the binding between p65 and CRM1. These data suggest that SIRT7 is a lysine deacetylase that targets the K37 residue of Ran to suppress the nuclear export of p65.

To characterize the immuno-stimulating ingredient from the Korean citrus, Cheongkyool, a crude polysaccharide (CCE-0) was isolated from the pectinase digests of Cheongkyool peels, from which the complex polysaccharide CCE-I was purified to homogeneity by gel filtration. CCE-I highly enhanced the production of IL-6, TNF-α, and NO in RAW 264.7 cell lines. It augmented the mRNA expression of IL-6, TNF-α, and iNOS in a dose-dependent manner. Moreover, CCE-I dose-dependently induced phosphorylation of MAPKs and NF-κB related proteins and led to the nuclear translocation of p65. The effect of CCE-I on NO and IL-6 production was suppressed by treatment with specific antibodies for TLR2, TLR4, and scavenger receptors. Conversely, the primary structure of CCE-I that exhibited potent immunostimulatory activity was characterized by sugar composition, linkage analysis, and oligosaccharide analysis after β-elimination. The results suggested that CCE-I may be a rhamnogalacturonan-I type, highly branched polysaccharide with short arabinan and galactan side chains.

Ginsenoside Rg1 is the active ingredient of Chinese herbal medicine ginseng and sanqi, which has remarkable effects on anti-inflammation and anti-diabetes. In this study, we explored the molecular mechanism of ginsenoside Rg1 against diabetes in rat subjected to insulin resistance induced by high-fat and high-sugar (HFHS). Biochemical analysis revealed that ginsenoside Rg1 significantly decreased the serum levels of alanine transaminase, aspartate transaminase, alkaline phosphatase, total cholesterol, triglyceride, low-density lipoprotein and increased the serum levels of high-density lipoprotein, which indicated ginsenoside Rg1 improved the extent of hepatic steatosis. Furthermore, ginsenoside Rg1 suppressed the expression of IL-1β, IL-6,TNF-α,NF-κB and G6Pase, however, p-Akt was up-regulated. These results suggested that ginsenosideRg1 improved insulin resistance through suppressing inflammatory response and glucose output, which may be a potential therapeutic strategy in protecting hepatic steatosis.

Central to eukaryotic cell function, transport into and out of the nucleus is largely mediated by members of the Importin (IMP) superfamily of transporters of α- and β-types. The first inhibitor of nuclear transport, leptomycin B (LMB), was shown to be a specific inhibitor of the IMPβ homologue Exportin 1 (EXP1) almost 20 years ago, but it has only been in the last five or so years that new inhibitors of nuclear export as well as import have been identified and characterised. Of utility in biological research, these inhibitors include those that target-specific EXPs/IMPs, with accompanying toxicity profiles, as well as agents that specifically target particular nuclear import cargoes. Both types of inhibitors have begun to be tested in preclinical/clinical studies, with particular focus on limiting various types of cancer or treating viral infection, and the most advanced agent targeting EXP1 (Selinexor) has progressed successfully through >40 clinical trials for a range of high-grade cancers and is approaching FDA approval for a number of indications. Selectively inhibiting the nucleocytoplasmic trafficking of specific proteins of interest remains a challenge, but progress in the area of the host-pathogen interface holds promise for the future.

The mRNA molecule roams through the nucleus on its way out to the cytoplasm. mRNA encounters and is bound by many protein factors, from the moment it begins to emerge from RNA polymerase II and during its travel in the nucleoplasm, where it will come upon chromatin and nuclear bodies. Some of the protein factors that engage with the mRNA can process it, until finally reaching a mature state fit for export through the nuclear pore complex (NPC). Examining the lifecycle of mRNAs in living cells using mRNA tagging techniques opens a window into our understanding of the rules that drive the dynamics of gene expression from transcription to mRNA export.

Excitotoxic levels of glutamate represent a physiological stress that is strongly linked to amyotrophic lateral sclerosis (ALS) and other neurological disorders. Emerging evidence indicates a role for neurodegenerative disease–linked RNA-binding proteins (RBPs) in the cellular stress response. However, the relationships between excitotoxicity, RBP function, and disease have not been explored. Here, using primary cortical and motor neurons, we found that excitotoxicity induced the translocation of select ALS-linked RBPs from the nucleus to the cytoplasm within neurons. RBPs affected by excitotoxicity included TAR DNA–binding protein 43 (TDP-43) and, most robustly, fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS). We noted that FUS is translocated through a calcium-dependent mechanism and that its translocation coincides with striking alterations in nucleocytoplasmic transport. Furthermore, glutamate-induced up-regulation of glutamate ionotropic receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)–type subunit 2 (GRIA2) in neurons depended on FUS expression, consistent with a functional role for FUS in excitotoxic stress. These findings reveal molecular links among prominent factors in neurodegenerative diseases, namely excitotoxicity, disease-associated RBPs, and nucleocytoplasmic transport.

Nicotinamide phosphoribosyltransferase (NAMPT) is located in both the nucleus and cytoplasm and has multiple biological functions including catalyzing the rate-limiting step in NAD synthesis. Moreover, up-regulated NAMPT expression has been observed in many cancers. However, the determinants and regulation of NAMPT's nuclear transport are not known. Here, we constructed a GFP–NAMPT fusion protein to study NAMPT's subcellular trafficking. We observed that in unsynchronized 3T3-L1 preadipocytes, 25% of cells had higher GFP–NAMPT fluorescence in the cytoplasm, and 62% had higher GFP–NAMPT fluorescence in the nucleus. In HepG2 hepatocytes, 6% of cells had higher GFP–NAMPT fluorescence in the cytoplasm, and 84% had higher GFP–NAMPT fluorescence in the nucleus. In both 3T3-L1 and HepG2 cells, GFP–NAMPT was excluded from the nucleus immediately after mitosis and migrated back into it as the cell cycle progressed. In HepG2 cells, endogenous, untagged NAMPT displayed similar changes with the cell cycle, and in nonmitotic cells, GFP–NAMPT accumulated in the nucleus. Similarly, genotoxic, oxidative, or dicarbonyl stress also caused nuclear NAMPT localization. These interventions also increased poly(ADP-ribosyl) polymerase and sirtuin activity, suggesting an increased cellular demand for NAD. We identified a nuclear localization signal in NAMPT and amino acid substitution in this sequence ((424)RSKK to ASGA), which did not affect its enzymatic activity, blocked nuclear NAMPT transport, slowed cell growth, and increased histone H(3) acetylation. These results suggest that NAMPT is transported into the nucleus where it presumably increases NAD synthesis required for cell proliferation. We conclude that specific inhibition of NAMPT transport into the nucleus might be a potential avenue for managing cancer.

Stella is a maternal gene required for oogenesis and early embryogenesis. Stella overexpression in somatic cells causes global demethylation. As we have recently shown, Stella sequesters nuclear ubiquitin-like with PHD and RING finger domains 1 (UHRF1), a RING finger–type E3 ubiquitin ligase essential for DNA methylation mediated by DNA methyltransferase 1 and triggers global demethylation. Here, we report an overexpressed mutant Stella protein without nuclear export activity surprisingly retained its ability to cause global demethylation. By combining biochemical interaction assays, isothermal titration calorimetry, immunostaining, and live-cell imaging with fluorescence recovery after photobleaching, we found that Stella disrupts UHRF1's association with chromatin by directly binding to the plant homeodomain of UHRF1 and competing for the interaction between UHRF1 and the histone H3 tail. Consistently, overexpression of Stella mutants that do not directly interact with UHRF1 fails to cause genome-wide demethylation. In the presence of nuclear Stella, UHRF1 could not bind to chromatin and exhibited increased dynamics in the nucleus. Our results indicate that Stella employs a multilayered mechanism to achieve robust UHRF1 inhibition, which involves the dissociation from chromatin and cytoplasmic sequestration of UHRF1.

The transcriptional cofactor nascent polypeptide-associated complex and co-regulator α (NACA) regulates osteoblast maturation and activity. NACA functions, at least in part, by binding to Jun proto-oncogene, AP-1 transcription factor subunit (cJUN) and potentiating the transactivation of AP-1 targets such as osteocalcin (Bglap) and matrix metallopeptidase 9 (Mmp9). NACA activity is modulated by phosphorylation carried out by several kinases, but a phosphatase regulating NACA's activity remains to be identified. Here, we used affinity purification with MS in HEK293T cells to isolate NACA complexes and identified protein phosphatase 1 catalytic subunit α (PP1A) as a NACA-associated Ser/Thr phosphatase. NACA interacted with multiple components of the PP1A holoenzyme complex: the PPP1CA catalytic subunit and the regulatory subunits PPP1R9B, PPP1R12A and PPP1R18. MS analysis revealed that NACA co-expression with PPP1CA causes dephosphorylation of NACA at Thr-89, Ser-151, and Thr-174. NACA Ser/Thr-to-alanine variants displayed increased nuclear localization, and NACA dephosphorylation was associated with specific recruitment of novel NACA interactants, such as basic transcription factor 3 (BTF3) and its homolog BTF3L4. NACA and PP1A cooperatively potentiated cJUN transcriptional activity of the AP-1–responsive MMP9-luciferase reporter, which was abolished when Thr-89, Ser-151, or Thr-174 were substituted with phosphomimetic aspartate residues. We confirmed the NACA–PP1A interaction in MC3T3-E1 osteoblastic cells and observed that NACA phosphorylation status at PP1A-sensitive sites is important for the regulation of AP-1 pathway genes and for osteogenic differentiation and matrix mineralization. These results suggest that PP1A dephosphorylates NACA at specific residues, impacting cJUN transcriptional activity and osteoblast differentiation and function.

Ultraviolet-C (UVC) electromagnetic radiation is the most damaging type of the UV radiation and causes many cellular and physiological responses. UVC has been using for sterilization and disinfection, and the risk of exposure to the UVC is increasing. Here, we determined the effect of the UVC on the cellular circadian clock system. UVC irradiation synchronized the biological clock system and induced time-dependent expression of clock genes including Clock, Cry1, and Per1. The rhythmic expression of clock genes is also followed by time-dependent mRNA degradation or non-canonical translation initiation of clock genes. Furthermore, we show a translocation of PERIOD1 (PER1) protein after UVC irradiation, which mediates the rhythmic feedback loop of clock genes. Our results suggest that UVC can synchronize the circadian clock system, and induces rhythmic expression of clock genes via time-dependent transcription, post-transcription, and post-translational modification.