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Find video protocols related to scientific articles indexed in Pubmed.
Structural basis for the selective nuclear import of the C2H2 zinc-finger protein Snail by importin ?.
Acta Crystallogr. D Biol. Crystallogr.
PUBLISHED: 01-14-2014
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Snail contributes to the epithelial-mesenchymal transition by suppressing E-cadherin in transcription processes. The Snail C2H2-type zinc-finger (ZF) domain functions both as a nuclear localization signal which binds to importin ? directly and as a DNA-binding domain. Here, a 2.5?Å resolution structure of four ZF domains of Snail1 complexed with importin ? is presented. The X-ray structure reveals that the four ZFs of Snail1 are required for tight binding to importin ? in the nuclear import of Snail1. The shape of the ZFs in the X-ray structure is reminiscent of a round snail, where ZF1 represents the head, ZF2-ZF4 the shell, showing a novel interaction mode, and the five C-terminal residues the tail. Although there are many kinds of C2H2-type ZFs which have the same fold as Snail, nuclear import by direct recognition of importin ? is observed in a limited number of C2H2-type ZF proteins such as Snail, Wt1, KLF1 and KLF8, which have the common feature of terminating in ZF domains with a short tail of amino acids.
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The role of Importin-?s in the maintenance and lineage commitment of mouse embryonic stem cells.
FEBS Open Bio
PUBLISHED: 01-01-2014
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Members of the Importin-? family recognize nuclear localization signals (NLS) and nuclear export signals (NES). These proteins play important roles in various nucleocytoplasmic transport processes in cells. Here, we examined the expression patterns of 21 identified Importin-? genes in mouse embryonic stem cells (mESCs), mouse embryonic fibroblast (MEF) and mESCs differentiated into neural ectoderm (NE) or mesoendoderm (ME). We observed striking differences in the Importin-? mRNA expression levels within these cell types. We also found that knockdown of selected Importin-? genes led to suppression of Nanog, and altered the balance of Oct4/Sox2 expression ratio, which is important for NE/ME lineage choice. Furthermore, we demonstrated that knockdown of XPO4, RanBP17, RanBP16, or IPO7 differentially affected the lineage selection of differentiating mESCs. More specifically, knockdown of XPO4 selectively stimulated the mESC differentiation towards definitive endoderm, while concomitantly inhibiting NE differentiation. RanBP17 knockdown also promoted endodermal differentiation with no effect on NE differentiation. RanBP16 knockdown caused differentiation into ME, while IPO7 knockdown inhibited NE differentiation, without obvious effects on the other lineages. Collectively, our results suggest that Importin-?s play important roles in cell fate determination processes of mESCs, such as in the maintenance of pluripotency or selection of lineage during differentiation.
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Downregulation of the small GTPase ras-related nuclear protein accelerates cellular ageing.
Biochim. Biophys. Acta
PUBLISHED: 06-21-2013
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The small GTPase Ran, Ras-related nuclear protein, plays important roles in multiple fundamental cellular functions such as nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation, by binding to either GTP or GDP as a molecular switch. Although it has been clinically demonstrated that Ran is highly expressed in multiple types of cancer cells and specimens, the physiological significance of Ran expression levels is unknown.
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Cell density-dependent nuclear accumulation of ELK3 is involved in suppression of PAI-1 expression.
Cell Struct. Funct.
PUBLISHED: 05-24-2013
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Cell-cell contact regulates the proliferation and differentiation of non-transformed cells, e.g., NIH/3T3 cells show growth arrest at high cell density. However, only a few reports described the dynamic behavior of transcription factors involved in this process. In this study, we showed that the mRNA levels of plasminogen activator inhibitor type 1 (PAI-1) decreased drastically at high cell density, and that ELK3, a member of the Ets transcription factor family, repressed PAI-1 expression. We also demonstrated that while ELK3 was distributed evenly throughout the cell at low cell density, it accumulated in the nucleus at high cell density, and that binding of DNA by ELK3 at the A domain facilitated its nuclear accumulation. Furthermore, we found that ETS1, a PAI-1 activator, occupied the ELK3-binding site within the PAI-1 promoter at low cell density, while it was released at high cell density. These results suggest that at high cell density, the switching of binding of transcription factors from ETS1 to ELK3 occurs at a specific binding site of the PAI-1 promoter, leading to the cell-density dependent suppression of PAI-1 expression.
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Quantitative regulation of nuclear pore complex proteins by O-GlcNAcylation.
Biochim. Biophys. Acta
PUBLISHED: 05-21-2013
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The nuclear pore complex (NPC) is a macromolecular assembly consisting of approximately 30 different proteins called nucleoporins. Several nucleoporins are O-GlcNAcylated, which is a post-translational modification in which the monosaccharide ?-N-acetylglucosamine (GlcNAc) is attached to serine or threonine residues within proteins. However, the biological significance of this modification on nucleoporins remains obscure. Here we found that Nup62 and Nup88 protein levels were significantly decreased upon knockdown of O-GlcNAc transferase (OGT), which catalyzes the O-GlcNAcylation of intracellular proteins. Although Nup88, unlike Nup62, was not recognized by an anti-O-GlcNAc antibody or WGA-HRP, knockdown of Nup62 caused a reduction in Nup88 protein levels, suggesting that the observed decrease in Nup88 in OGT knocked-down cells is due to a decrease in Nup62. Furthermore, we found that Nup88 was preferentially associated with O-GlcNAcylated Nup62 compared with non-O-GlcNAcylated Nup62. These results indicate that Nup62 protein levels are primarily maintained by O-GlcNAcylation and that Nup88 is quantitatively regulated through its interaction with O-GlcNAcylated Nup62.
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Human TREX component Thoc5 affects alternative polyadenylation site choice by recruiting mammalian cleavage factor I.
Nucleic Acids Res.
PUBLISHED: 05-17-2013
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The transcription-export complex (TREX) couples mRNA transcription, processing and nuclear export. We found that CFIm68, a large subunit of a heterotetrameric protein complex mammalian cleavage factor I (CFIm), which is implicated in alternative polyadenylation site choice, co-purified with Thoc5, a component of human TREX. Immunoprecipitation using antibodies against different components of TREX indicated that most likely both complexes interact via an interaction between Thoc5 and CFIm68. Microarray analysis using human HeLa cells revealed that a subset of genes was differentially expressed on Thoc5 knockdown. Notably, the depletion of Thoc5 selectively attenuated the expression of mRNAs polyadenylated at distal, but not proximal, polyadenylation sites, which phenocopied the depletion of CFIm68. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) indicated that CFIm68 preferentially associated with the 5 regions of genes; strikingly, the 5 peak of CFIm68 was significantly and globally reduced on Thoc5 knockdown. We suggest a model in which human Thoc5 controls polyadenylation site choice through the co-transcriptional loading of CFIm68 onto target genes.
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Importin alpha subtypes determine differential transcription factor localization in embryonic stem cells maintenance.
Dev. Cell
PUBLISHED: 04-15-2013
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We recently demonstrated that the expression of the importin ? subtype is switched from ?2 to ?1 during neural differentiation in mouse embryonic stem cells (ESCs) and that this switching has a major impact on cell differentiation. In this study, we report a cell-fate determination mechanism in which importin ?2 negatively regulates the nuclear import of certain transcription factors to maintain ESC properties. The nuclear import of Oct6 and Brn2 was inhibited via the formation of a transport-incompetent complex of the cargo bound to a nuclear localization signal binding site in importin ?2. Unless this dominant-negative effect was downregulated upon ESC differentiation, inappropriate cell death was induced. We propose that although certain transcription factors are necessary for differentiation in ESCs, these factors are retained in the cytoplasm by importin ?2, thereby preventing transcription factor activity in the nucleus until the cells undergo differentiation.
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Differential role for transcription factor Oct4 nucleocytoplasmic dynamics in somatic cell reprogramming and self-renewal of embryonic stem cells.
J. Biol. Chem.
PUBLISHED: 04-11-2013
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Oct4 is a member of the POU family of transcription factors and plays a critical role in both maintenance of the undifferentiated state of embryonic stem (ES) cells and in the reprogramming of somatic cells to induced pluripotent stem cells. Oct4 is imported into the nucleus where it functions as a transcription factor; however, the spatiotemporal dynamic behavior of Oct4 remains largely unknown. In the present study we show that Oct4 is a nucleocytoplasmic shuttling protein. Furthermore, although Oct4 mutants with altered nuclear import/export activity were able to maintain the self-renewal of ES cells, they displayed limited potential for cellular reprogramming. These results indicate that the intracellular localization of Oct4, which is dependent on nucleocytoplasmic shuttling, must be more strictly regulated for cellular reprogramming, suggesting that Oct4 plays differential roles in the self-renewal of ES cells and in somatic cell reprogramming.
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The lipid mediator protectin D1 inhibits influenza virus replication and improves severe influenza.
Cell
PUBLISHED: 02-13-2013
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Influenza A viruses are a major cause of mortality. Given the potential for future lethal pandemics, effective drugs are needed for the treatment of severe influenza such as that caused by H5N1 viruses. Using mediator lipidomics and bioactive lipid screen, we report that the omega-3 polyunsaturated fatty acid (PUFA)-derived lipid mediator protectin D1 (PD1) markedly attenuated influenza virus replication via RNA export machinery. Production of PD1 was suppressed during severe influenza and PD1 levels inversely correlated with the pathogenicity of H5N1 viruses. Suppression of PD1 was genetically mapped to 12/15-lipoxygenase activity. Importantly, PD1 treatment improved the survival and pathology of severe influenza in mice, even under conditions where known antiviral drugs fail to protect from death. These results identify the endogenous lipid mediator PD1 as an innate suppressor of influenza virus replication that protects against lethal influenza virus infection.
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The nuclear import factor importin ?4 can protect against oxidative stress.
Biochim. Biophys. Acta
PUBLISHED: 01-09-2013
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The importin (IMP) superfamily of nuclear transport proteins is essential to key developmental pathways, including in the murine testis where expression of the 6 distinct IMP? proteins is highly dynamic. Present predominantly from the spermatocyte stage onwards, IMP?4 is unique in showing a striking nuclear localization, a property we previously found to be linked to maintenance of pluripotency in embryonic stem cells and to the cellular stress response in cultured cells. Here we examine the role of IMP?4 in vivo for the first time using a novel transgenic mouse model in which we overexpress an IMP?4-EGFP fusion protein from the protamine 1 promoter to recapitulate endogenous testicular germ cell IMP?4 expression in spermatids. IMP?4 overexpression did not affect overall fertility, testis morphology/weight or spermatogenic progression under normal conditions, but conferred significantly (>30%) increased resistance to oxidative stress specifically in the spermatid subpopulation expressing the transgene. Consistent with a cell-specific role for IMP?4 in protecting against oxidative stress, haploid germ cells from IMP?4 null mice were significantly (c. 30%) less resistant to oxidative stress than wild type controls. These results from two unique and complementary mouse models demonstrate a novel protective role for IMP?4 in stress responses specifically within haploid male germline cells, with implications for male fertility and genetic integrity.
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Nuclear retention of importin ? coordinates cell fate through changes in gene expression.
EMBO J.
PUBLISHED: 09-06-2011
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Various cellular stresses including oxidative stress induce a collapse of the Ran gradient, which causes accumulation of importin ? in the nucleus and a subsequent block of nuclear protein import. However, it is unknown whether accumulated importin ? performs roles in the nucleus after its migration in response to stress. In this study, we found that nuclear-retained importin ?2 binds with DNase I-sensitive nuclear component(s) and exhibits selective upregulation of mRNA encoding Serine/threonine kinase 35 (STK35) by microarray analysis. Chromatin immunoprecipitation and promoter analysis demonstrated that importin ?2 can access to the promoter region of STK35 and accelerate its transcription in response to hydrogen peroxide exposure. Furthermore, constitutive overexpression of STK35 proteins enhances caspase-independent cell death under oxidative stress conditions. These results collectively reveal that nuclear-localized importin ?2 influences gene expression and contributes directly to cell fate outcomes including non-apoptotic cell death.
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Cross-talk between distinct nuclear import pathways enables efficient nuclear import of E47 in conjunction with its partner transcription factors.
Mol. Biol. Cell
PUBLISHED: 08-10-2011
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Nuclear import of karyophilic proteins is carried out by a variety of mechanisms. We previously showed that two basic helix-loop-helix proteins, NeuroD1 and E47, synergistically affect each others nuclear import. In this study, we dissected the molecular pathways underlying nuclear import of the NeuroD1/E47 heterodimer. In vitro nuclear import assays indicated that importin ? family members are the major nuclear import receptors for E47. However, inhibition of importin ? resulted in cytoplasmic retention of E47 that could be rescued by its binding partner, NeuroD1, through heterodimerization. In addition, nuclear import of NeuroD1 was importin ? independent but importin ?1 dependent. In primary neurons, localization of endogenous E47 was not affected by importin ? inhibition, suggesting that neuronal E47 could be imported into the nucleus as a heterodimer with NeuroD1 by using importin ?1 alone. We also found that E47 had similar nuclear import characteristics in C2C12 cells, where E47 heterodimerized with MyoD, another helix-loop-helix protein, suggesting functional conservation within the same family of transcription factors. Collectively, our data reveal that E47 is imported into the nucleus via multiple pathways, depending on the molecular binding mode, establishing a previously uncharacterized cross-talk between two distinct nuclear import pathways.
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Generation and characterization of a monoclonal antibody against importin ?7/NPI-2.
Hybridoma (Larchmt)
PUBLISHED: 06-29-2011
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Many nuclear proteins are transported into the nucleus via the importin ?/?-mediated pathway. Importin ? comprises a multigene family. In this study, we generated and characterized a rat monoclonal antibody (MAb) 3F8 to importin ?7. The antibody was generated by the hybridization of mouse myeloma cells with lymph node cells from an immunized rat. The MAb 3F8 specifically recognized importin ?7 among importin ? isoforms as evidenced by immunoblotting analysis. Furthermore, MAb 3F8 detected exogenous importin ?7 in COS-7 cells by immunofluorescence. This MAb will be useful in the analysis of the isoform-specific function of importin ?7.
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Generation of rat monoclonal antibody specific for mouse importin ?8.
Hybridoma (Larchmt)
PUBLISHED: 06-29-2011
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The transport of proteins in and out of the nucleus plays important roles in major cellular processes, such as signal transduction and regulation of cell cycle. Proteins that contain a nuclear localization signal (NLS) are recognized by an importin ?/? heterodimer and targeted to the nucleus. Here, we report the generation of a rat monoclonal antibody (MAb) that recognizes a novel importin ? family member, importin ?8, which is expressed during oocyte maturation and early embryonic development. Immunoblot and immunolocalization analyses showed that this MAb was specific for mouse importin ?8 and not other importin ? family members. These data suggest that this MAb is useful for analyzing molecular functions of importin ?8.
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Cell type-dependent gene regulation by Staufen2 in conjunction with Upf1.
BMC Mol. Biol.
PUBLISHED: 06-15-2011
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dendritic mRNA transport machines. Although Stau2 is thought to be involved in the dendritic targeting of several mRNAs in neurons, the mechanism whereby Stau2 regulates these mRNAs is unknown. To elucidate the functions of Stau2, we screened for novel binding partners by affinity purification of GST-tagged Stau2 from 293F cells.
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Stimulation of CK2-dependent Grp94 phosphorylation by the nuclear localization signal peptide.
Mol. Cell. Biochem.
PUBLISHED: 06-13-2011
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The nuclear localization signal sequence (NLS) of SV40 Large T antigen is essential and sufficient for the nuclear translocation of the protein. Phosphorylation often modulates the intracellular distribution of signaling proteins. In this study, we investigated effects of the NLS-peptide of Large T antigen on protein phosphorylation. When crude cell lysates were incubated with [?-(32)P]ATP, phosphorylation of several endogenous substrates with molecular masses of 100, 80, 50, and 45 kDa by an endogenous kinase was stimulated by the addition of the wild type NLS-peptide (CPKKKRKVEDP). The mutated NLS-peptide (CPKTKRKVEDP) and the reversed NLS-peptide (PDEVKRKKKPC) are weak in the nuclear localization activity, and they only weakly stimulated phosphorylation of these substrates. The mobility of the 100 kDa phosphoprotein was indistinguishable with that of an endoplasmic reticulum (ER)-resident molecular chaperone glucose-regulated protein 94 (Grp94) belonging to the Hsp90 family, and purified Grp94 was phosphorylated by a kinase in cell lysates in an NLS-dependent fashion. The 100 kDa protein was identified as Grp94 by immunoprecipitation and reconstitution experiments. Purification of the NLS-dependent Grp94 kinase by sequential biochemical column chromatography steps resulted in isolation of two polypeptides with molecular masses of 42 and 27 kDa, which were identified as ? and ? subunit of protein kinase CK2, respectively, by western blotting analysis and biochemical characterization. Moreover, effect of an excess amount of GTP and V8 peptide mapping showed that the NLS-dependent Grp94 kinase in the cell lysate is identical with CK2. Surprisingly purified CK2 did phosphorylate Grp94 even without the NLS-peptide, suggesting that an additional suppressive factor is required for NLS-dependent phosphorylation of Grp94 by CK2. We suggest a possible general role for CK2-catalyzed phosphorylation in the regulation of NLS-dependent protein nuclear translocation.
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Cell type-specific transcriptional regulation of the gene encoding importin-?1.
Exp. Cell Res.
PUBLISHED: 05-25-2011
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Importin-?1 belongs to a receptor family that recognizes classical nuclear localization signals. Encoded by Kpna2, this receptor subtype is highly expressed in mouse embryonic stem (ES) cells. In this study, we identified a critical promoter region in Kpna2 and showed that the expression of this gene is differentially regulated in ES cells and NIH3T3 cells. Conserved CCAAT boxes are required for Kpna2 promoter activity in both ES and NIH3T3 cells. Interestingly, deletion of the region from nucleotide position -251 to -179 bp resulted in a drastic reduction in Kpna2 transcriptional activity only in ES cells. This region contains Krüppel-like factor (Klf) binding sequences and is responsible for transactivation of the gene by Klf2 and Klf4. Accordingly, endogenous Kpna2 mRNA levels decreased in response to depletion of Klf2 and Klf4 in ES cells. Our results suggest that Klf2 and Klf4 function redundantly to drive high level of Kpna2 expression in ES cells.
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Nucleocytoplasmic transport of microRNAs and related small RNAs.
Traffic
PUBLISHED: 05-18-2011
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Small RNAs with lengths of 20-30 nucleotides, such as microRNAs (miRNAs), play important regulatory roles in various cellular processes. In conventional linear processing pathways, precursors of miRNAs are transported out of the nucleus by the specific nuclear transport receptor, exportin-5. The exported precursors are further processed and eventually incorporated into the RNA-induced silencing complex (RISC), which silences the expression of the target genes by posttranscriptional mechanisms in the cytoplasm. Subsequent identification and characterization of P-element induced wimpy testis (PIWI)-interacting small RNAs (piRNAs) and endogenous small interfering RNAs (endo-siRNAs) revealed that the processing mechanisms of these newly emerging small RNAs differ from those of miRNAs. Moreover, cumulative experimental evidence indicates that the nuclear functions of the small RNAs, such as transcriptional gene silencing, could be widespread in divergent species. These observations appended other interesting features in the biogenesis and nucleocytoplasmic transport mechanisms of these small RNAs. In this review, we discuss the mechanisms and biological significance of the intracellular trafficking of small RNAs in animal cells.
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Targeted disruption of one of the importin ? family members leads to female functional incompetence in delivery.
FEBS J.
PUBLISHED: 03-22-2011
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Importin?? mediates the nuclear import of proteins through nuclear pore complexes in eukaryotic cells, and is common to all eukaryotes. Previous reports identified at least six importin ? family genes in mice. Although these isoforms show differential binding to various import cargoes in vitro, the in vivo physiological roles of these mammalian importin ? isoforms remain unknown. Here, we generated and examined importin ?5 knockout (imp?5(-/-)) mice. These mice developed normally, and showed no gross histological abnormalities in most major organs. However, the ovary and uterus of imp?5(-/-) female mice exhibited hypoplasia. Furthermore, we found that imp?5(-/-) female mice had a 50% decrease in serum progesterone levels and a 57% decrease in progesterone receptor mRNA levels in the ovary. Additionally, imp?5(-/-) uteruses that were treated with exogenous gonadotropins displayed hypertrophy, similarly to progesterone receptor-deficient mice. Although these mutant female mice could become pregnant, the total number of pups was significantly decreased, and some of the pups were dead at birth. These results suggest that importin ?5 has essential roles in the mammalian female reproductive organs.
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Importin alpha protein acts as a negative regulator for Snail protein nuclear import.
J. Biol. Chem.
PUBLISHED: 03-17-2011
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Snail, a zinc finger-containing transcriptional regulator, migrates into the nucleus where it controls gene expression. We demonstrated previously that importin ?1 directly recognizes the zinc finger domain of Snail and transports it into the nucleus. Here, using in vitro and in vivo assays, we show that importin ?, an adaptor protein for importin ?1, negatively regulates the nuclear import of Snail mediated by importin ?1. In vitro binding assays indicated that importin ? interacted with the zinc finger domain of Snail to compete with the binding of importin ?1 and that Snail did not form a ternary complex with importin ?/importin ?1. Overexpression of importin ? in A549 cells reduced the endogenous Snail protein level, which was restored by inhibitors of the proteasome and glycogen synthase kinase 3?. Furthermore, knockdown of importin ? by siRNA treatment increased the endogenous Snail protein level in several cancer cell lines. This study provides a novel regulatory mechanism of the nuclear protein import process by importin ? and gives an implication to control Snail activity by inhibiting its nuclear localization.
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Proteomic analysis of importin ?-interacting proteins in adult mouse brain.
Cell Struct. Funct.
PUBLISHED: 02-05-2011
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Many transport factors, such as importins and exportins, have been identified, and the molecular mechanisms underlying nucleocytoplasmic transport have been characterized. The specific molecules that are carried by each transport factor and the temporal profiles that characterize the movements of various proteins into or out of the nucleus, however, have yet to be elucidated. Here, we used a proteomic approach to identify molecules that are transported into the nuclei of adult mouse brain cells via importin ?5. We identified 48 proteins in total, among which we chose seven to characterize more extensively: acidic (leucine-rich) nuclear phosphoprotein 32 family member A (Anp32a), far upstream element binding protein 1 (FUBP1), thyroid hormone receptor ?1 (TR?1), transaldolase 1, CDC42 effector protein 4 (CDC42-ep4), Coronin 1B, and brain-specific creatine kinase (CK-B). Analyses using green fluorescent protein (GFP)-fused proteins showed that Anp32a, FUBP1, and TR?1 were localized in the nucleus, whereas transaldolase 1, CDC42-ep4, CK-B, and Coronin 1B were distributed in both the cytoplasm and nucleus. Using a digitonin-permeabilized in vitro transport assay, we demonstrated that, with the exception of CK-B, these proteins were transported into the nucleus by importin ?5 together with importin ? and Ran. Further, we found that leptomycin B (LMB) treatment increased nuclear CK-B-GFP signals, suggesting that CK-B enters the nucleus and is then exported in a CRM1-dependent manner. Thus, we identified a comprehensive set of candidate proteins that are transported into the nucleus in a manner dependent on importin ?5, which enhances our understanding of nucleocytoplasmic signaling in neural cells.
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Mice lacking Ran binding protein 1 are viable and show male infertility.
FEBS Lett.
PUBLISHED: 01-31-2011
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The small GTPase Ran plays important roles in multiple aspects of cellular function. Maximal RanGAP activity is achieved with the aid of RanBP1 and/or presumably of RanBP2. Here, we show that RanBP1-knockout mice are unexpectedly viable, and exhibit male infertility due to a spermatogenesis arrest, presumably caused by down-regulation of RanBP2 during spermatogenesis. Indeed, siRNA-mediated depletion of RanBP2 caused severe cell death only in RanBP1-deficient MEFs, indicating that simultaneous depletion of RanBP1 and RanBP2 severely affects normal cell viability. Collectively, we conclude that the dramatic decrease in "RanBP" activity impairs germ cell viability and affects spermatogenesis decisively in RanBP1-knockout mice.
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Axotomy induces axonogenesis in hippocampal neurons by a mechanism dependent on importin ?.
Biochem. Biophys. Res. Commun.
PUBLISHED: 01-22-2011
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We characterize the previously unrecognized phenomenon of axotomy-induced axonogenesis in rat embryonic hippocampal neurons in vitro and elucidate the underlying mechanism. New neurites arose from cell bodies after axotomy and grew. These neurites were Tau-1-positive, and the injured axons showed negative immunoreactivity for Tau-1. Axonogenesis was delayed in these neurons by inhibiting the dynein-dynactin complex through the overexpression of p50. Importin ?, which was locally translated after axotomy, was associated with the dynein-importin ? complex and was required for axonogenesis. Taken together, these results suggest that retrograde transport of injury-induced signals in injured axons play key roles in the axotomy-induced axonogenesis of hippocampal neurons.
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Nup358, a nucleoporin, functions as a key determinant of the nuclear pore complex structure remodeling during skeletal myogenesis.
FEBS J.
PUBLISHED: 01-04-2011
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The nuclear pore complex (NPC) is the only gateway for molecular trafficking across the nuclear envelope. The NPC is not merely a static nuclear-cytoplasmic transport gate; the functional analysis of nucleoporins has revealed dynamic features of the NPC in various cellular functions, such as mitotic spindle formation and protein modification. However, it is not known whether the NPC undergoes dynamic changes during biological processes such as cell differentiation. In the present study, we evaluate changes in the expression levels of several nucleoporins and show that the amount of Nup358/RanBP2 within individual NPCs increases during muscle differentiation in C2C12 cells. Using atomic force microscopy, we demonstrate structural differences between the cytoplasmic surfaces of myoblast and myotube NPCs and a correlation between the copy number of Nup358 and the NPC structure. Furthermore, small interfering RNA-mediated depletion of Nup358 in myoblasts suppresses myotube formation without affecting cell viability, suggesting that NUP358 plays a role in myogenesis. These findings indicate that the NPC undergoes dynamic remodeling during muscle cell differentiation and that Nup358 is prominently involved in the remodeling process.
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Specific monoclonal antibody against the nuclear pore complex protein, Nup96.
Hybridoma (Larchmt)
PUBLISHED: 12-06-2010
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Nup96 is a component of the Nup107-160 complex, the largest subunit of the nuclear pore complex. Nup96 is generated as a precursor protein with Nup98. However, the mechanism by which Nup96 contributes to cell function is not clear. We report here on the preparation of a monoclonal antibody (MAb) directed against mouse Nup96. The antibody was produced by the hybridization of mouse myeloma cells with lymph node cells from an immunized rat. The antibody, MAb 4H5, specifically recognized Nup96, as evidenced by immunoblotting using the whole cell lysates. In immunostaining using MAb 4H5, a nuclear rim staining pattern was observed. This antibody will be useful in immunoblotting and immunolocalization experiments, as well as further analyses of the biological function and cellular dynamics of this protein.
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Nuclear import of serum response factor in airway smooth muscle.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 12-03-2010
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We have previously shown that the transcription-promoting activity of serum response factor (SRF) is partially regulated by its extranuclear redistribution. In this study, we examined the cellular mechanisms that facilitate SRF nuclear entry in canine tracheal smooth muscle cells. We used in vitro pull-down assays to determine which karyopherin proteins bound SRF and found that SRF binds KPNA1 and KPNB1 through its nuclear localization sequence. Immunoprecipitation studies also demonstrated direct SRF-KPNA1 interaction in HEK293 cells. Import assays demonstrated that KPNA1 and KPNB1 together were sufficient to mediate rapid nuclear import of SRF-GFP. Our studies also suggest that SRF is able to gain nuclear entry through an auxiliary, nuclear localization sequence-independent mechanism.
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The timing of retroviral silencing correlates with the quality of induced pluripotent stem cell lines.
Biochim. Biophys. Acta
PUBLISHED: 06-28-2010
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Induced pluripotent stem (iPS) cells can be generated from somatic cells by introducing the four transcription factors Oct4, Sox2, Klf4, and c-Myc. Given that iPS cell technology may be useful for medical applications, the quality of iPS cells needs to be maintained during prolonged cultivation. However, it is unclear whether there are any differences in stability among different iPS clones.
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Effective culture conditions for the induction of pluripotent stem cells.
Biochim. Biophys. Acta
PUBLISHED: 04-12-2010
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Induced pluripotent stem (iPS) cells, which are functionally comparable to embryonic stem (ES) cells, can be generated from mouse fibroblasts by expression of a defined set of transcription factors Oct4, Sox2, Klf4, and c-Myc. Since iPS cells are generated from somatic cells, they provide an invaluable source of pluripotent stem cells for cell transplantation therapy that does not present ethical problems. However, the reprogramming efficiency is extremely low, and optimal culture conditions for iPS cell derivation have not been clearly defined.
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The mobile FG nucleoporin Nup98 is a cofactor for Crm1-dependent protein export.
Mol. Biol. Cell
PUBLISHED: 04-07-2010
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Nup98 is a mobile nucleoporin that forms distinct dots in the nucleus, and, although a role for Nup98 in nuclear transport has been suggested, its precise function remains unclear. Here, we show that Nup98 plays an important role in Crm1-mediated nuclear protein export. Nuclear, but not cytoplasmic, dots of EGFP-tagged Nup98 disappeared rapidly after cell treatment with leptomycin B, a specific inhibitor of the nuclear export receptor, Crm1. Mutational analysis demonstrated that Nup98 physically and functionally interacts with Crm1 in a RanGTP-dependent manner through its N-terminal phenylalanine-glycine (FG) repeat region. Moreover, the activity of the Nup98-Crm1 complex was modulated by RanBP3, a known cofactor for Crm1-mediated nuclear export. Finally, cytoplasmic microinjection of anti-Nup98 inhibited the Crm1-dependent nuclear export of proteins, concomitant with the accumulation of anti-Nup98 in the nucleus. These results clearly demonstrate that Nup98 functions as a novel shuttling cofactor for Crm1-mediated nuclear export in conjunction with RanBP3.
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Identification of importin alpha1 as a novel constituent of RNA stress granules.
Biochim. Biophys. Acta
PUBLISHED: 03-24-2010
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Importin alpha is a nuclear transport receptor well established for its ability to mediate importin beta-mediated nuclear import of proteins that possess classical nuclear localization signal (cNLS). Previously, we reported that importin alpha rapidly accumulates to the nucleus in response to H2O2-induced oxidative stress, which implies a role for this protein in stress response. In this study, we show that importin alpha1 (also known as KPNA2 or Rch1), a major subtype of the importin alpha family, localizes to RNA stress granules (SGs), large cytoplasmic bodies that are thought to function as RNA triage sites during stress response. The recruitment of importin alpha1 to SGs was compatible with its nuclear accumulation during heat shock. Depletion of endogenous importin alpha1 using siRNA showed that importin alpha1 regulates the dynamics of SG assembly, and that it promotes cell survival in arsenite-treated cells. These data revealed, for the first time, the involvement of importin alpha in the assembly of RNA granules and its pro-survival role during stress response.
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Cytoplasmic polyadenylation element-like sequences are involved in dendritic targeting of BDNF mRNA in hippocampal neurons.
FEBS Lett.
PUBLISHED: 02-12-2010
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Several mRNAs are known to be targeted to dendrites in hippocampal neurons. In this study, we show that brain-derived neurotrophic factor (BDNF) mRNA has two distinct cis-acting dendritic targeting elements in the short 3 untranslated region (UTR): a constitutive element and an activity-dependent one. Moreover, deletion of serial cytoplasmic polyadenylation element (CPE)-like sequences in the short 3UTR suppressed both constitutive and activity-dependent dendritic targeting. In addition to the interaction with cytoplasmic polyadenylation element binding protein-1 (CPEB-1), depolarization enhanced CPEB-1 recruitment to the activity-dependent targeting element. These results suggest that CPE-like sequences are involved in the activity-dependent as well as constitutive dendritic targeting of BDNF mRNA.
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Two isoforms of Npap60 (Nup50) differentially regulate nuclear protein import.
Mol. Biol. Cell
PUBLISHED: 12-16-2009
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Npap60 (Nup50) is a nucleoporin that binds directly to importin alpha. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. In this study, we provide both in vitro and in vivo evidence that Npap60L and Npap60S function differently in nuclear protein import. In vitro binding assays revealed that Npap60S stabilizes the binding of importin alpha to classical NLS-cargo, whereas Npap60L promotes the release of NLS-cargo from importin alpha. In vivo time-lapse experiments showed that when the Npap60 protein level is controlled, allowing CAS to efficiently promote the dissociation of the Npap60/importin alpha complex, Npap60S and Npap60L suppress and accelerate the nuclear import of NLS-cargo, respectively. These results demonstrate that Npap60L and Npap60S have opposing functions and suggest that Npap60L and Npap60S levels must be carefully controlled for efficient nuclear import of classical NLS-cargo in humans. This study provides novel evidence that nucleoporin expression levels regulate nuclear import efficiency.
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A high-resolution structure of the pre-microRNA nuclear export machinery.
Science
PUBLISHED: 12-08-2009
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Nuclear export of microRNAs (miRNAs) by exportin-5 (Exp-5) is an essential step in miRNA biogenesis. Here, we present the 2.9 angstrom structure of the pre-miRNA nuclear export machinery formed by pre-miRNA complexed with Exp-5 and a guanine triphosphate (GTP)-bound form of the small nuclear guanine triphosphatase (GTPase) Ran (RanGTP). The x-ray structure shows that Exp-5:RanGTP recognizes the 2-nucleotide 3 overhang structure and the double-stranded stem of the pre-miRNA. Exp-5:RanGTP shields the pre-miRNA stem from degradation in a baseball mitt-like structure where it is held by broadly distributed weak interactions, whereas a tunnel-like structure of Exp-5 interacts strongly with the 2-nucleotide 3 overhang through hydrogen bonds and ionic interactions. RNA recognition by Exp-5:RanGTP does not depend on RNA sequence, implying that Exp-5:RanGTP can recognize a variety of pre-miRNAs.
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Microscopic dissection of the process of stress granule assembly.
Biochim. Biophys. Acta
PUBLISHED: 05-26-2009
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Stress granules (SGs) are mRNA triage sites that are formed in response to a variety of cellular stress. To study how SGs bring about the massive spatial compartmentalization, we monitored the localization of various RNA-binding proteins (RBPs) targeted to SGs upon exposure to stress. We discovered that concomitant with the onset of eIF2alpha phosphorylation, RBPs accumulate locally in the cytoplasm, which leads to increased inter-molecular interactions and the formation of robustly detergent-resistant foci. Subsequently, microtubules (MTs) mediate 1) the ordered spatial organization of SGs and 2) the recruitment of a set of nuclear-localized SG components to the cytoplasm. Meanwhile, MTs did not appear to be required for the maintenance of SG distribution after its assembly. Our data suggest that the process of SG formation is composed of MT-independent and -dependent pathways, which take place sequentially during stress response.
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The role of the nuclear transport system in cell differentiation.
Semin. Cell Dev. Biol.
PUBLISHED: 04-24-2009
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The eukaryotic cell nuclear transport system selectively mediates molecular trafficking to facilitate the regulation of cellular processes. The components of this system include diverse transport factors such as importins and nuclear pore components that are precisely organized to coordinate cellular events. A number of studies have demonstrated that the nuclear transport system is indispensible in many types of cellular responses. In particular, the nuclear transport machinery has been shown to be an important regulator of development, organogenesis, and tissue formation, wherein altered nuclear transport of key transcription factors can lead to disease. Importantly, precise switching between distinct forms of importin alpha is central to neural lineage specification, consistent with the hypothesis that importin expression can be a key mediator of cell differentiation.
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Synergistic nuclear import of NeuroD1 and its partner transcription factor, E47, via heterodimerization.
Exp. Cell Res.
PUBLISHED: 02-13-2009
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The transition from undifferentiated pluripotent cells to terminally differentiated neurons is coordinated by a repertoire of transcription factors. NeuroD1 is a type II basic helix loop helix (bHLH) transcription factor that plays critical roles in neuronal differentiation and maintenance in the central nervous system. Its dimerization with E47, a type I bHLH transcription factor, leads to the transcriptional regulation of target genes. Mounting evidence suggests that regulating the localization of transcription factors contributes to the regulation of their activity during development as defects in their localization underlie a variety of developmental disorders. In this study, we attempted to understand the nuclear import mannerisms of NeuroD1 and E47. We found that the nuclear import of NeuroD1 and E47 is energy-dependent and involves the Ran-mediated pathway. Herein, we demonstrate that NeuroD1 and E47 can dimerize inside the cytoplasm before their nuclear import. Moreover, this dimerization promotes nuclear import as the nuclear accumulation of NeuroD1 was enhanced in the presence of E47 in an in vitro nuclear import assay, and NLS-deficient NeuroD1 was successfully imported into the nucleus upon E47 overexpression. NeuroD1 also had a similar effect on the nuclear accumulation of NLS-deficient E47. These findings suggest a novel role for dimerization that may promote, at least partially, the nuclear import of transcription factors allowing them to function efficiently in the nucleus.
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Selective localization of PCBP2 to cytoplasmic processing bodies.
Biochim. Biophys. Acta
PUBLISHED: 01-29-2009
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Processing bodies (P-bodies) are cytoplasmic domains that have been implicated in critical steps of the regulation of gene expression, including mRNA decay and post-transcriptional gene silencing. Previously, we reported that PCBP2 (Poly-(rC) Binding Protein 2), a facilitator of IRES-mediated translation, is a novel P-body component. Interestingly, PCBP2 is recruited to only a subset of Dcp1a-positive P-bodies, which may reflect functional diversity among these structures. In this study, we examined the selective P-body localization of PCBP2 in detail. Co-localization studies between Dcp1a and PCBP2 revealed that PCBP2 is present in approximately 40% of P-bodies. While PCBP2 was more likely to reside in larger P-bodies, P-body size did not seem to be the sole determinant, and puromycin-induced enlargement of P-bodies only modestly increased the percentage of PCBP2-positive P-bodies. Photobleaching experiments demonstrated that the accumulation of PCBP2 to specific P-bodies is a dynamic process, which does not involve the proteins transcription-dependent nucleo-cytoplasmic shuttling activity. Finally, we found that PCBP1, a close relative of PCBP2, localizes to P-bodies in a similar manner to PCBP2. Taken together, these results establish the compositional diversity among P-bodies, and that PCBP2, probably in complex with other mRNP factors, may dynamically recognize such differences and accumulate to specific P-bodies.
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Infrared laser-mediated gene induction in targeted single cells in vivo.
Nat. Methods
PUBLISHED: 01-24-2009
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We developed infrared laser-evoked gene operator (IR-LEGO), a microscope system optimized for heating cells without photochemical damage. Infrared irradiation causes reproducible temperature shifts of the in vitro microenvironment in a power-dependent manner. When applied to living Caenorhabditis elegans, IR-LEGO induced heat shock-mediated expression of transgenes in targeted single cells in a more efficient and less deleterious manner than a 440-nm dye laser and elicited physiologically relevant phenotypic responses.
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Adaptor Aly and co-adaptor Thoc5 function in the Tap-p15-mediated nuclear export of HSP70 mRNA.
EMBO J.
PUBLISHED: 01-05-2009
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In metazoans, nuclear export of bulk mRNA is mediated by Tap-p15, a conserved heterodimeric export receptor that cooperates with adaptor RNA-binding proteins. In this article, we show that Thoc5, a subunit of the mammalian TREX complex, binds to a distinct surface on the middle (Ntf2-like) domain of Tap. Notably, adaptor protein Aly and Thoc5 can simultaneously bind to non-overlapping binding sites on Tap-p15. In vivo, Thoc5 was not required for bulk mRNA export. However, nuclear export of HSP70 mRNA depends on both Thoc5 and Aly. Consistent with a function as a specific export adaptor, Thoc5 exhibits in vitro RNA-binding activity and is associated with HSP70 mRNPs in vivo as a component of the stable THO complex. Thus, through the combinatorial use of an adaptor (e.g., Aly) and co-adapter (e.g., Thoc5), Tap-p15 could function as an export receptor for different classes of mRNAs.
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mDia2 shuttles between the nucleus and the cytoplasm through the importin-{alpha}/{beta}- and CRM1-mediated nuclear transport mechanism.
J. Biol. Chem.
PUBLISHED: 01-02-2009
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Mammalian homolog of Drosophila diaphanous (mDia) consisting of three isoforms, mDia1, mDia2, and mDia3, is an effector of Rho GTPases that catalyzes actin nucleation and polymerization. Although the mDia actions on actin dynamics in the cytoplasm have been well studied, whether mDia accumulates and functions in the nucleus remains largely unknown. Given the presence of actin and actin-associated proteins in the nucleus, we have examined nuclear localization of mDia isoforms. We expressed each of mDia isoforms as a green fluorescent protein fusion protein and examined their localization. Although all the mDia isoforms were localized predominantly in the cytoplasm under the steady-state conditions, mDia2 and not mDia1 or mDia3 accumulated extensively in the nucleus upon treatment with leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export. The LMB-induced nuclear accumulation was confirmed for endogenous mDia2 by using an antibody specific to mDia2. Studies using green fluorescent protein fusions of various truncation mDia2 mutants and point mutants of some of these proteins identified a functional nuclear localization signal in the N terminus of mDia2 and at least one functional nuclear export signal in the C terminus. The nuclear localization signal of mDia2 bound to importin-alpha and was imported into the nucleus by importin-alpha/beta complex in an in vitro transport assay. Consistently, depletion of importin-beta with RNA interference suppressed the LMB-induced nuclear localization of endogenous mDia2. These results suggest that mDia2 continuously shuttles between the nucleus and the cytoplasm using specific nuclear transport machinery composing of importin-alpha/beta and CRM1.
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Roles of the TREX complex in nuclear export of mRNA.
RNA Biol
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Recent studies have established that nuclear export of mRNAs is functionally coupled to different steps in gene expression processes, such as transcription, splicing, 3-end formation and even translation. It is suggested that the manner in which individual mRNAs are transcribed and processed in the nucleus affects nuclear export and eventually their cytoplasmic fates, thus such inter-dependence could be especially important for the efficient expression of newly transcribed mRNAs, which are encoded on genes activated by different stimuli including various environmental stresses and developmental cues. Here we describe recently uncovered molecular mechanisms of the nuclear export of mRNA, in which the TREX (TRanscription-EXport) complex plays important roles of functionally connecting transcription, mRNA packaging and nuclear export.
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Improvement in protocol to generate homogeneous glutamatergic neurons from mouse embryonic stem cells reduced apoptosis.
Biochem. Biophys. Res. Commun.
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Obtaining a homogenous population of central nervous system neurons has been a significant challenge in neuroscience research; however, a recent study established a retinoic acid-treated embryoid bodies-based differentiation protocol that permits the effective generation of highly homogeneous glutamatergic cortical pyramidal neurons from embryonic stem cells. We were able to reproduce this protocol regarding the purity of glutamatergic neurons, but these neurons were not sufficiently healthy for long-term observation under the same conditions that were originally described. Here, we achieved a substantial improvement in cell survival by applying a simple technique: We changed the medium for glutamatergic neurons from the original complete medium to commercially available SBM (the Nerve-Cell Culture Medium manufactured by Sumitomo Bakelite Co. Ltd.) and finally succeeded in maintaining healthy neurons for at least 3 weeks without decreasing their purity. Because SBM contains glial conditioned medium, we postulated that brain-derived neurotrophic factor or basic fibroblast growth factor is the key components responsible for pro-survival effect of SBM on neurons, and examined their effects by adding them to CM. As a result, neither of them had pro-survival effect on pure glutamatergic neuronal population.
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Synchronizing nuclear import of ribosomal proteins with ribosome assembly.
Science
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Ribosomal proteins are synthesized in the cytoplasm, before nuclear import and assembly with ribosomal RNA (rRNA). Little is known about coordination of nucleocytoplasmic transport with ribosome assembly. Here, we identify a transport adaptor, symportin 1 (Syo1), that facilitates synchronized coimport of the two 5S-rRNA binding proteins Rpl5 and Rpl11. In vitro studies revealed that Syo1 concomitantly binds Rpl5-Rpl11 and furthermore recruits the import receptor Kap104. The Syo1-Rpl5-Rpl11 import complex is released from Kap104 by RanGTP and can be directly transferred onto the 5S rRNA. Syo1 can shuttle back to the cytoplasm by interaction with phenylalanine-glycine nucleoporins. X-ray crystallography uncovered how the ?-solenoid symportin accommodates the Rpl5 amino terminus, normally bound to 5S rRNA, in an extended groove. Symportin-mediated coimport of Rpl5-Rpl11 could ensure coordinated and stoichiometric incorporation of these proteins into pre-60S ribosomes.
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Nuclear importin ? and its physiological importance.
Commun Integr Biol
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Importin ? is recognized as a classical nuclear localization signal (cNLS) receptor which mediates nucleocytoplasmic transport. However, it rapidly accumulates in the nucleus in response to cellular stresses, including oxidative stress, causing a blockade of the classical nuclear import pathway. We set out to determine whether importin ? performs roles in the nucleus after cellular exposure to stresses and discovered that it can act directly to modulate gene expression. With remarkable selectivity, importin ?2 can access the promoter of Serine/threonine kinase 35 (STK35) and increase the levels of this transcript without requirement for importin ?1. The nuclear accumulation of importin ? occurred following exposure to stresses which decreased intracellular ATP levels and was followed by non-apoptotic cell death. Hence the gene regulatory function of nuclear importin ? can direct cell fate. There are now several reports of nuclear-localized importin ? proteins in diverse cellular states, including cancer. Here we discuss the physiological significance of this novel functional capacity of nuclear importin ? relationship to a variety of cellular states and fates.
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Intrinsic and extrinsic negative regulators of nuclear protein transport processes.
Genes Cells
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The nuclear-cytoplasmic protein transport is a critical process in cellular events. The identification of transport signals (nuclear localization signal and nuclear export signal) and their receptors has facilitated our understanding of this expanding field. Nuclear transport must be appropriately regulated to deliver proteins through the nuclear pore when their functions are required in the nucleus, and to export them into the cytoplasm when they are not needed in the nucleus. Altered nuclear transport processes have been observed in stressed cells, which would change gene expressions. Some viruses interfere with nuclear transport in host cells to evade immune defense. Moreover, certain transport factors negatively regulate nuclear protein transport in cells. Understanding the regulatory mechanisms of nuclear-cytoplasmic trafficking not only provides important information about cellular processes, but also is of use for developing specific inhibitors for transport pathways.
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The interaction between importin-? and Nup153 promotes importin-?/?-mediated nuclear import.
Traffic
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Nuclear transport is mediated by transport factors, including the importin ? family members. The directionality of nuclear transport is governed by the asymmetrical distribution of the small GTPase Ran. Of note, importin ?/?-mediated import of classical nuclear localization signal (cNLS)--containing cargo is more efficient than other Ran-dependent import pathways that do not require importin ?. In this study, we characterized the role of importin ? in nuclear transport by examining import efficiencies of cNLS-cargo/importin ?/? complexes. We first depleted digitonin-permeabilized semi-intact cells of endogenous importin ? and used the cells to show that the interaction between importin ? and Nup153--a component of the nuclear pore complex (NPC)--is essential for efficient import of importin ?-binding domain containing substrates, but not other cargoes that directly bind to importin ?. Moreover, we found that the binding of importin ? to Nup153 facilitates cNLS-mediated import, and demonstrated that importin ? in import complexes and cargo-free importin ? prebound to Nup153 promote efficient import of cNLS-containing proteins. This is the first in vitro study showing that in conjunction with Nup153, importin ? contributes to directionally biased exit of cNLS-containing cargo to the nuclear side of NPCs.
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