Yoshihiro Yoneda

Osaka University, Suika, Ōsaka, Japan

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Publications (169)932.11 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: 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 β directly and as a DNA-binding domain. Here, a 2.5 Å resolution structure of four ZF domains of Snail1 complexed with importin β is presented. The X-ray structure reveals that the four ZFs of Snail1 are required for tight binding to importin β 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 β 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.
    Acta Crystallographica Section D Biological Crystallography 04/2014; 70(Pt 4):1050-60. · 12.67 Impact Factor
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    Yoichi Miyamoto, Kate L Loveland, Yoshihiro Yoneda
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    Percival Sangel, Masahiro Oka, Yoshihiro Yoneda
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    ABSTRACT: Members of the Importin-β 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-β 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-β mRNA expression levels within these cell types. We also found that knockdown of selected Importin-β 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-βs play important roles in cell fate determination processes of mESCs, such as in the maintenance of pluripotency or selection of lineage during differentiation.
    FEBS Journal 01/2014; 4(2014):112-120. · 4.25 Impact Factor
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    ABSTRACT: We recently demonstrated that the expression of the importin α subtype is switched from α2 to α1 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 α2 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 α2. 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 α2, thereby preventing transcription factor activity in the nucleus until the cells undergo differentiation.
    Developmental cell 07/2013; 26(2):123-35. · 13.36 Impact Factor
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    ABSTRACT: 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 β-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 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.
    Biochimica et Biophysica Acta 06/2013; · 4.66 Impact Factor
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    ABSTRACT: 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 IMPα proteins is highly dynamic. Present predominantly from the spermatocyte stage onwards, IMPα4 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 IMPα4 in vivo for the first time using a novel transgenic mouse model in which we overexpress an IMPα4-EGFP fusion protein from the protamine 1 promoter to recapitulate endogenous testicular germ cell IMPα4 expression in spermatids. IMPα4 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 IMPα4 in protecting against oxidative stress, haploid germ cells from IMPα4 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 IMPα4 in stress responses specifically within haploid male germline cells, with implications for male fertility and genetic integrity.
    Biochimica et Biophysica Acta 06/2013; · 4.66 Impact Factor
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    ABSTRACT: 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.
    Cell Structure and Function 05/2013; · 1.65 Impact Factor
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    ABSTRACT: 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.
    Nucleic Acids Research 05/2013; · 8.28 Impact Factor
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    ABSTRACT: 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 (iPS) 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, while 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.
    Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor
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    ABSTRACT: 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.
    Cell 03/2013; · 31.96 Impact Factor
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    ABSTRACT: 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 effect by adding them to CM. As a result, neither of them had pro-survival effect on pure glutamatergic neuronal population.
    Biochemical and Biophysical Research Communications 12/2012; · 2.41 Impact Factor
  • Masahiro Nagai, Yoshihiro Yoneda
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    ABSTRACT: BACKGROUND: 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. METHODS: During the long-term culture of normal mammalian cells, we found that the endogenous Ran level gradually reduced in a passage-dependent manner. To examine the physiological significance of Ran reduction, we first performed small interfering RNA (siRNA)-mediated abrogation of Ran in human diploid fibroblasts. RESULTS: Ran-depleted cells showed several senescent phenotypes. Furthermore, we found that nuclear accumulation of importin α, which was also observed in cells treated with siRNA against CAS, a specific export factor for importin α, occurred in the Ran-depleted cells before the cells showed senescent phenotypes. Further, the CAS-depleted cells also exhibited cellular senescence. Indeed, importin α showed predominant nuclear localisation in a passage-dependent manner. CONCLUSIONS: Reduction in Ran levels causes cytoplasmic decrease and nuclear accumulation of importin α leading to cellular senescence in normal cells. General Significance The amount of intracellular Ran may be critically related to cell fate determination, such as malignant transformation and senescence. The cellular aging process may proceed through gradual regression of Ran-dependent nucleocytoplasmic transport competency.
    Biochimica et Biophysica Acta 11/2012; · 4.66 Impact Factor
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    Yoichi Miyamoto, Kate L Loveland, Yoshihiro Yoneda
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    Yoichi Miyamoto, Kate L Loveland, Yoshihiro Yoneda
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    ABSTRACT: 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 α-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.
    Science 11/2012; 338(6107):666-71. · 31.20 Impact Factor
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    Toshihiro Sekimoto, Yoshihiro Yoneda
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    ABSTRACT: 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.
    Genes to Cells 06/2012; 17(7):525-35. · 2.73 Impact Factor
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    ABSTRACT: Nuclear transport is mediated by transport factors, including the importin β family members. The directionality of nuclear transport is governed by the asymmetrical distribution of the small GTPase Ran. Of note, importin α/β-mediated import of classical nuclear localization signal (cNLS)--containing cargo is more efficient than other Ran-dependent import pathways that do not require importin α. In this study, we characterized the role of importin α in nuclear transport by examining import efficiencies of cNLS-cargo/importin α/β complexes. We first depleted digitonin-permeabilized semi-intact cells of endogenous importin α and used the cells to show that the interaction between importin α and Nup153--a component of the nuclear pore complex (NPC)--is essential for efficient import of importin β-binding domain containing substrates, but not other cargoes that directly bind to importin β. Moreover, we found that the binding of importin α to Nup153 facilitates cNLS-mediated import, and demonstrated that importin α in import complexes and cargo-free importin α prebound to Nup153 promote efficient import of cNLS-containing proteins. This is the first in vitro study showing that in conjunction with Nup153, importin α contributes to directionally biased exit of cNLS-containing cargo to the nuclear side of NPCs.
    Traffic 04/2012; 13(7):934-46. · 4.65 Impact Factor
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    Yoichi Miyamoto, Kate L Loveland, Yoshihiro Yoneda
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    ABSTRACT: Importin α 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 α 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 α2 can access the promoter of Serine/threonine kinase 35 (STK35) and increase the levels of this transcript without requirement for importin β1. The nuclear accumulation of importin α 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 α can direct cell fate. There are now several reports of nuclear-localized importin α proteins in diverse cellular states, including cancer. Here we discuss the physiological significance of this novel functional capacity of nuclear importin α relationship to a variety of cellular states and fates.
    Communicative & integrative biology 03/2012; 5(2):220-2.
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    ABSTRACT: ABSTRACT: 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. RESULTS: Three RNA helicases, RNA helicase A, Upf1 and Mov10, were identified in Stau2-containing complexes. We focused our studies on Upf1, a key player in nonsense-mediated mRNA decay. Stau2 was found to bind directly to Upf1 in an RNA-independent manner in vitro. Tethering Stau2 to the 3'-untranslated region (UTR) of a reporter gene had little effect on its expression in HeLa cells. In contrast, when the same tethering assay was performed in 293F cells, we observed an increase in reporter protein levels. This upregulation of protein expression by Stau2 turned out to be dependent on Upf1. Moreover, we found that in 293F cells, Stau2 upregulates the reporter mRNA level in an Upf1-independent manner. CONCLUSIONS: These results indicate that the recruitment of Stau2 alone or in combination with Upf1 differentially affects the fate of mRNAs. Moreover, the results suggest that Stau2-mediated fate determination could be executed in a cell type-specific manner.
    BMC Molecular Biology 11/2011; 12:48. · 2.80 Impact Factor
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    ABSTRACT: Various cellular stresses including oxidative stress induce a collapse of the Ran gradient, which causes accumulation of importin α in the nucleus and a subsequent block of nuclear protein import. However, it is unknown whether accumulated importin α performs roles in the nucleus after its migration in response to stress. In this study, we found that nuclear-retained importin α2 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 α2 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 α2 influences gene expression and contributes directly to cell fate outcomes including non-apoptotic cell death.
    The EMBO Journal 09/2011; 31(1):83-94. · 9.82 Impact Factor

Publication Stats

4k Citations
932.11 Total Impact Points

Institutions

  • 1985–2014
    • Osaka University
      • • Graduate School of Frontier Biosciences
      • • Division of Biochemistry
      • • Division of Neurobiology and Cell Biology
      • • Institute for Protein Research
      • • Division of Cellular and Molecular Biology
      Suika, Ōsaka, Japan
  • 2007–2012
    • Universität Heidelberg
      • Center for Biochemistry (BZH)
      Heidelberg, Baden-Wuerttemberg, Germany
  • 2010
    • The University of Tokyo
      • College of Art and Science & Graduate School of Arts and Sciences
      Tokyo, Tokyo-to, Japan
  • 2006
    • Tokyo Metropolitan Institute of Medical Science
      Edo, Tōkyō, Japan
  • 2002–2006
    • Osaka City University
      • • Graduate School of Engineering
      • • Graduate School of Medicine
      Ōsaka-shi, Osaka-fu, Japan
  • 2005
    • Kagoshima University
      • Graduate School of Medical and Dental Sciences
      Kagoshima-shi, Kagoshima-ken, Japan
  • 2004–2005
    • RIKEN
      • Laboratory for Cell Signaling Dynamics
      Wako, Saitama-ken, Japan
  • 1997–2000
    • Saitama Cancer Center
      Saitama, Saitama, Japan
  • 1987
    • National Institute for Basic Biology
      Okazaki, Aichi, Japan