Advance

FUKAO Akira

    Department of Pharmacy　Lecturer
Last Updated :2024/04/25

Researcher Information

Degree

  • Doctor of Science（2011/03 Kobe University）

URL

J-Global ID

Research Areas

  • Life sciences / Molecular biology

Academic & Professional Experience

  • 2020/04 - Today  Kindai UniversityFaculty of Pharmacy講師
  • 2015/04 - 2020/03  Kindai UniversityFaculty of Pharmacy助教
  • 2014/04 - 2015/03  Nagoya City UniversityGraduate School of Pharmacy特任助教
  • 2011/04 - 2014/03  日本学術振興会特別研究員（PD）

Published Papers

  • Hikari Nishisaka; Takumi Tomohiro; Kako Fukuzumi; Akira Fukao; Yoshinori Funakami; Toshinobu Fujiwara
    Biochimie 221 20 - 26 2024/01 
    The RNA-binding protein HuD/ELAVL4 is essential for neuronal development and synaptic plasticity by governing various post-transcriptional processes of target mRNAs, including stability, translation, and localization. We previously showed that the linker region and poly(A)-binding domain of HuD play a pivotal role in promoting translation and inducing neurite outgrowth. In addition, we found that HuD interacts exclusively with the active form of Akt1, through the linker region. Although this interaction is essential for neurite outgrowth, HuD is not a substrate for Akt1, raising questions about the dynamics between HuD-mediated translational stimulation and its association with active Akt1. Here, we demonstrate that active Akt1 interacts with the cap-binding complex via HuD. We identify key amino acids in linker region of HuD responsible for Akt1 interaction, leading to the generation of two point-mutated HuD variants: one that is incapable of binding to Akt1 and another that can interact with Akt1 regardless of its phosphorylation status. In vitro translation assays using these mutants reveal that HuD-mediated translation stimulation is independent of its binding to Akt1. In addition, it is evident that the interaction between HuD and active Akt1 is essential for HuD-induced neurite outgrowth, whereas a HuD mutant capable of binding to any form of Akt1 leads to aberrant neurite development. Collectively, our results revisit the understanding of the HuD-Akt1 interaction in translation and suggest that this interaction contributes to HuD-mediated neurite outgrowth via a unique molecular mechanism distinct from translation regulation.
  • Hikari Nishisaka; Takumi Tomohiro; Akira Fukao; Yoshinori Funakami; Toshinobu Fujiwara
    Biological & pharmaceutical bulletin 46 (2) 158 - 162 2023 
    Translation initiation is the rate-limiting step of protein synthesis and is the main target of translation regulation. RNA-binding proteins (RBPs) are key mediators of the spatiotemporal control of translation and are critical for cell proliferation, development, and differentiation. We have previously shown that HuD, one of the neuronal RBPs, enhances cap-dependent translation through the direct interaction with eukaryotic initiation factor 4A (eIF4A) and poly(A) tail using a HeLa-derived in vitro translation system. We have also found that translation stimulation of HuD is essential for HuD-induced neurite outgrowth in PC12 cells. However, it remains unclear how HuD is involved in the regulation of translation initiation. Here, we report that HuD binds to eukaryotic initiation factor 3 (eIF3) via the eIF3b subunit, which belongs to the functional core of mammalian eIF3. eIF3 plays an essential role in recruiting the 40S ribosomal subunit onto mRNA in translation initiation. We hypothesize that the interaction between HuD and eIF3 stabilizes the translation initiation complex and increases translation efficiency. We also showed that the linker region of HuD is required for the interaction with eIF3b. Moreover, we found that eIF3b-binding region of HuD is conserved in all Hu proteins (HuB, HuC, HuD, and HuR). These data might also help to explain how Hu proteins stimulate translation in a cap- and poly(A)-dependent way.
  • Toru Suzuki; Miyuki Hoshina; Saori Nishijima; Naosuke Hoshina; Chisato Kikuguchi; Takumi Tomohiro; Akira Fukao; Toshinobu Fujiwara; Tadashi Yamamoto
    RNA biology 19 (1) 234 - 246 2022 
    CCR4-NOT complex-mediated mRNA deadenylation serves critical functions in multiple biological processes, yet how this activity is regulated is not fully understood. Here, we show that osmotic stress induces MAPKAPK-2 (MK2)-mediated phosphorylation of CNOT2. Programmed cell death is greatly enhanced by osmotic stress in CNOT2-depleted cells, indicating that CNOT2 is responsible for stress resistance of cells. Although wild-type (WT) and non-phosphorylatable CNOT2 mutants reverse this sensitivity, a phosphomimetic form of CNOT2, in which serine at the phosphorylation site is replaced with glutamate, does not have this function. We also show that mRNAs have elongated poly(A) tails in CNOT2-depleted cells and that introduction of CNOT2 WT or a non-phosphorylatable mutant, but not phosphomimetic CNOT2, renders their poly(A) tail lengths comparable to those in control HeLa cells. Consistent with this, the CCR4-NOT complex containing phosphomimetic CNOT2 exhibits less deadenylase activity than that containing CNOT2 WT. These data suggest that CCR4-NOT complex deadenylase activity is regulated by post-translational modification, yielding dynamic control of mRNA deadenylation.
  • Akira Fukao; Takumi Tomohiro; Toshinobu Fujiwara
    Cells 10 (7) 2021/07 
    Protein synthesis is tightly regulated at each step of translation. In particular, the formation of the basic cap-binding complex, eukaryotic initiation factor 4F (eIF4F) complex, on the 5' cap structure of mRNA is positioned as the rate-limiting step, and various cis-elements on mRNA contribute to fine-tune spatiotemporal protein expression. The cis-element on mRNAs is recognized and bound to the trans-acting factors, which enable the regulation of the translation rate or mRNA stability. In this review, we focus on the molecular mechanism of how the assembly of the eIF4F complex is regulated on the cap structure of mRNAs. We also summarize the fine-tuned regulation of translation initiation by various trans-acting factors through cis-elements on mRNAs.
  • Hiroshi Otsuka; Akira Fukao; Yoshinori Funakami; Kent E Duncan; Toshinobu Fujiwara
    Frontiers in genetics 12 715196 - 715196 2021 
    [This corrects the article DOI: 10.3389/fgene.2019.00332.].
  • Hiroshi Otsuka; Akira Fukao; Takumi Tomohiro; Shungo Adachi; Toru Suzuki; Akinori Takahashi; Yoshinori Funakami; Toru Natsume; Tadashi Yamamoto; Kent E Duncan; Toshinobu Fujiwara
    Biochimie 174 49 - 56 2020/07 [Refereed]
     
    Eukaryotic gene expression can be spatiotemporally tuned at the post-transcriptional level by cis-regulatory elements in mRNA sequences. An important example is the AU-rich element (ARE), which induces mRNA destabilization in a variety of biological contexts in mammals and can also mediate translational control. Regulation is mediated by trans-acting factors that recognize the ARE, such as Tristetraprolin (TTP) and BRF1/ZFP36L1. Although both proteins can destabilize their target mRNAs through the recruitment of the CCR4-NOT deadenylation complex, TTP also directly regulates translation. Whether ZFP36L1 can directly repress translation remains unknown. Here, we used an in vitro translation system derived from mammalian cell lines to address this key mechanistic issue in ARE regulation by ZFP36L1. Functional assays with mutant proteins reveal that ZFP36L1 can repress translation via AU-Rich elements independent of deadenylation. ZFP36L1-mediated translation repression requires interaction between ZFP36L1 and CNOT1, suggesting that it might use a repression mechanism similar to either TPP or miRISC. However, several lines of evidence suggest that the similarity ends there. Unlike, TTP, it does not efficiently interact with either 4E-HP or GIGYF2, suggesting it does not repress translation by recruiting these proteins to the mRNA cap. Moreover, ZFP36L1 could not repress ECMV-IRES driven translation and was resistant to pharmacological eIF4A inhibitor silvestrol, suggesting fundamental differences with miRISC repression via eIF4A. Collectively, our results reveal that ZFP36L1 represses translation directly and suggest that it does so via a novel mechanism distinct from other translational regulators that interact with the CCR4-NOT deadenylase complex.
  • Fabian Hia; Sheng Fan Yang; Yuichi Shichino; Masanori Yoshinaga; Yasuhiro Murakawa; Alexis Vandenbon; Akira Fukao; Toshinobu Fujiwara; Markus Landthaler; Tohru Natsume; Shungo Adachi; Shintaro Iwasaki; Osamu Takeuchi
    EMBO reports 20 (11) e48220  1469-221X 2019/11 [Refereed]
     
    Codon bias has been implicated as one of the major factors contributing to mRNA stability in several model organisms. However, the molecular mechanisms of codon bias on mRNA stability remain unclear in humans. Here, we show that human cells possess a mechanism to modulate RNA stability through a unique codon bias. Bioinformatics analysis showed that codons could be clustered into two distinct groups-codons with G or C at the third base position (GC3) and codons with either A or T at the third base position (AT3): the former stabilizing while the latter destabilizing mRNA. Quantification of codon bias showed that increased GC3-content entails proportionately higher GC-content. Through bioinformatics, ribosome profiling, and in vitro analysis, we show that decoupling the effects of codon bias reveals two modes of mRNA regulation, one GC3- and one GC-content dependent. Employing an immunoprecipitation-based strategy, we identify ILF2 and ILF3 as RNA-binding proteins that differentially regulate global mRNA abundances based on codon bias. Our results demonstrate that codon bias is a two-pronged system that governs mRNA abundance.
  • Shigeru Hashimoto; Shotaro Furukawa; Ari Hashimoto; Akio Tsutaho; Akira Fukao; Yurika Sakamura; Gyanu Parajuli; Yasuhito Onodera; Yutaro Otsuka; Haruka Handa; Tsukasa Oikawa; Soichiro Hata; Yoshihiro Nishikawa; Yusuke Mizukami; Yuzo Kodama; Masaaki Murakami; Toshinobu Fujiwara; Satoshi Hirano; Hisataka Sabe
    Proceedings of the National Academy of Sciences of the United States of America 116 (35) 17450 - 17459 0027-8424 2019/08 [Refereed]
     
    © 2019 National Academy of Sciences. All rights reserved. Although KRAS and TP53 mutations are major drivers of pancreatic ductal adenocarcinoma (PDAC), the incurable nature of this cancer still remains largely elusive. ARF6 and its effector AMAP1 are often overexpressed in different cancers and regulate the intracellular dynamics of integrins and E-cadherin, thus promoting tumor invasion and metastasis when ARF6 is activated. Here we show that the ARF6-AMAP1 pathway is a major target by which KRAS and TP53 cooperatively promote malignancy. KRAS was identified to promote eIF4A-dependent ARF6 mRNA translation, which contains a quadruplex structure at its 5′-untranslated region, by inducing TEAD3 and ETV4 to suppress PDCD4; and also eIF4E-dependent AMAP1 mRNA translation, which contains a 5′- terminal oligopyrimidine-like sequence, via up-regulating mTORC1. TP53 facilitated ARF6 activation by platelet-derived growth factor (PDGF), via its known function to promote the expression of PDGF receptor β (PDGFRβ) and enzymes of the mevalonate pathway (MVP). The ARF6-AMAP1 pathway was moreover essential for PDGF-driven recycling of PD-L1, in which KRAS, TP53, eIF4A/4Edependent translation, mTOR, and MVP were all integral. We moreover demonstrated that the mouse PDAC model KPC cells, bearing KRAS/TP53 mutations, express ARF6 and AMAP1 at high levels and that the ARF6-based pathway is closely associated with immune evasion of KPC cells. Expression of ARF6 pathway components statistically correlated with poor patient outcomes. Thus, the cooperation among eIF4A/4E-dependent mRNA translation and MVP has emerged as a link by which pancreatic driver mutations may promote tumor cell motility, PD-L1 dynamics, and immune evasion, via empowering the ARF6-based pathway and its activation by external ligands.
  • Otsuka H; Fukao A; Funakami Y; Duncan KE; Fujiwara T
    Frontiers in genetics 10 332 - 332 2019 [Refereed]
     
    RNA-binding proteins (RBPs) are key regulators of posttranscriptional gene expression and control many important biological processes including cell proliferation, development, and differentiation. RBPs bind specific motifs in their target mRNAs and regulate mRNA fate at many steps. The AU-rich element (ARE) is one of the major cis-regulatory elements in the 3' untranslated region (UTR) of labile mRNAs. Many of these encode factors requiring very tight regulation, such as inflammatory cytokines and growth factors. Disruption in the control of these factors' expression can cause autoimmune diseases, developmental disorders, or cancers. Therefore, these mRNAs are strictly regulated by various RBPs, particularly ARE-binding proteins (ARE-BPs). To regulate mRNA metabolism, ARE-BPs bind target mRNAs and affect some factors on mRNAs directly, or recruit effectors, such as mRNA decay machinery and protein kinases to target mRNAs. Importantly, some ARE-BPs have stabilizing roles, whereas others are destabilizing, and ARE-BPs appear to compete with each other when binding to target mRNAs. The function of specific ARE-BPs is modulated by posttranslational modifications (PTMs) including methylation and phosphorylation, thereby providing a means for cellular signaling pathways to regulate stability of specific target mRNAs. In this review, we summarize recent studies which have revealed detailed molecular mechanisms of ARE-BP-mediated regulation of gene expression and also report on the importance of ARE-BP function in specific physiological contexts and how this relates to disease. We also propose an mRNP regulatory network based on competition between stabilizing ARE-BPs and destabilizing ARE-BPs.
  • Akitoshi Sadahiro; Akira Fukao; Mio Kosaka; Yoshinori Funakami; Naoki Takizawa; Osamu Takeuchi; Kent E. Duncan; Toshinobu Fujiwara
    Frontiers in Genetics 9 (AUG) 307  2018/08 [Refereed]
     
    © 2018 Sadahiro, Fukao, Kosaka, Funakami, Takizawa, Takeuchi, Duncan and Fujiwara. Many viruses strongly prefer to infect certain cell types, a phenomenon known as "tropism." Understanding tropism's molecular basis is important for the design of vaccines and antiviral therapy. A common mechanism involves viral protein interactions with cell-specific surface receptors, but intracellular mechanisms involving translation have also been described. In this report, we focus on Hepatitis A Virus (HAV) tissue tropism from the standpoint of the translational machinery. HAV genomic RNA, like other positive stranded RNA viruses, is devoid of a cap structure and its translation is driven by highly structured RNA sequences termed internal ribosome entry site (IRES) in the 5' untranslated region (UTR). Unlike most viral IRESs, HAV IRES-mediated translation requires eIF4E and the 3' end of HAV RNA is polyadenylated. However, the molecular mechanism of HAV IRES-mediated translation initiation remains poorly understood. We analyzed HAV-IRES-mediated translation in a cell-free system derived from either non-hepatic cells (HeLa) or hepatoma cells (Huh-7) that enables investigation of the contribution of the cap and the poly(A) tail. This revealed that HAV IRES-mediated translation activity in hepatoma cell extracts is higher as compared to extracts derived from a non-hepatic line. Our data suggest that HAV IRES-mediated translation is upregulated by a hepatic cell-specific activator in a poly(A) tail-independent manner.
  • Tomokazu Yamaguchi; Takashi Suzuki; Teruki Sato; Akinori Takahashi; Hiroyuki Watanabe; Ayumi Kadowaki; Miyuki Natsui; Hideaki Inagaki; Satoko Arakawa; Shinji Nakaoka; Yukio Koizumi; Shinsuke Seki; Shungo Adachi; Akira Fukao; Toshinobu Fujiwara; Tohru Natsume; Akinori Kimura; Masaaki Komatsu; Shigeomi Shimizu; Hiroshi Ito; Yutaka Suzuki; Josef M Penninger; Tadashi Yamamoto; Yumiko Imai; Keiji Kuba
    Science signaling 11 (516) 1945-0877 2018/02 [Refereed]
     
    Shortening and removal of the polyadenylate [poly(A)] tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression 4-negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In Cnot3-depleted hearts, Atg7 expression was posttranscriptionally increased. Genetic ablation of Atg7, but not Atg5, increased survival and partially restored cardiac function of Cnot1 or Cnot3 knockout mice. We further showed that in Cnot3-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death-promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis.
  • Satoh Ryosuke; Matsumura Yasuhiro; Tanaka Akitomo; Takada Makoto; Ito Yuna; Hagihara Kanako; Inari Masahiro; Kita Ayako; Fukao Akira; Fujiwara Toshinobu; Hirai Shinya; Tani Tokio; Sugiura Reiko
    Molecular microbiology 104 (3) 428-448  1365-2958 2017/05 [Refereed]
     
    Here, we analyzed the spatial regulation of Rnc1 and discovered a putative nuclear export signal (NES)Rnc1 , which dictates the cytoplasmic localization of Rnc1 in a Crm1-independent manner. Intriguingly, the Rnc1 NES mutant destabilized Pmp1 mRNA, suggesting the functional importance of the Rnc1 cytoplasmic localization. Mutation in Rae1, but not Mex67 deletion or overproduction, induced Rnc1 accumulation in the nucleus, suggesting that Rnc1 is exported from the nucleus to the cytoplasm via the mRNA export pathway involving Rae1. Importantly, mutations in the Rnc1 KH-domains abolished the mRNA-binding ability and induced nuclear localization, suggesting that Rnc1 may be exported from the nucleus together with its target mRNAs. Collectively, the functional Rae1-dependent mRNA export system may influence the cytoplasmic localization and function of Rnc1.
  • Ryosuke Satoh; Yasuhiro Matsumura; Akitomo Tanaka; Makoto Takada; Yuna Ito; Kanako Hagihara; Masahiro Inari; Ayako Kita; Akira Fukao; Toshinobu Fujiwara; Shinya Hirai; Tokio Tani; Reiko Sugiura
    MOLECULAR MICROBIOLOGY WILEY 104 (3) 428 - 448 0950-382X 2017/05 [Refereed]
     
    RNA-binding proteins (RBPs) play important roles in the posttranscriptional regulation of gene expression, including mRNA stability, transport and translation. Fission yeast rnc1(+) encodes a K Homology (KH)-type RBP, which binds and stabilizes the Pmp1 MAPK phosphatase mRNA thereby suppressing the Cl- hypersensitivity of calcineurin deletion and MAPK signaling mutants. Here, we analyzed the spatial regulation of Rnc1 and discovered a putative nuclear export signal (NES)(Rnc1), which dictates the cytoplasmic localization of Rnc1 in a Crm1-independent manner. Notably, mutations in the NESRnc1 altered nucleocytoplasmic distribution of Rnc1 and abolished its function to suppress calcineurin deletion, although the Rnc1 NES mutant maintains the ability to bind Pmp1 mRNA. Intriguingly, the Rnc1 NES mutant destabilized Pmp1 mRNA, suggesting the functional importance of the Rnc1 cytoplasmic localization. Mutation in Rae1, but not Mex67 deletion or overproduction, induced Rnc1 accumulation in the nucleus, suggesting that Rnc1 is exported from the nucleus to the cytoplasm via the mRNA export pathway involving Rae1. Importantly, mutations in the Rnc1 KH-domains abolished the mRNA-binding ability and induced nuclear localization, suggesting that Rnc1 may be exported from the nucleus together with its target mRNAs. Collectively, the functional Rae1-dependent mRNA export system may influence the cytoplasmic localization and function of Rnc1.
  • Akira Fukao; Toshinobu Fujiwaray
    JOURNAL OF BIOCHEMISTRY OXFORD UNIV PRESS 161 (4) 309 - 314 0021-924X 2017/04 [Refereed]
     
    In mammals, spatiotemporal control of protein synthesis plays a key role in the post-transcriptional regulation of gene expression during cell proliferation, development and differentiation and RNA-binding proteins (RBPs) and microRNAs (miRNAs) are required for this phenomenon. RBPs and miRNAs control the levels of mRNA protein products by regulating mRNA stability and translation. Recent studies have shown that RBPs and miRNAs simultaneously regulate mRNA stability and translation, and that the differential functions of RBPs and miRNAs are dependent on their interaction partners. Here, we summarize the coupled-and uncoupled mechanisms by which trans-acting factors regulate mRNA stability and translation.
  • Aoyama T; Fukao A; Fujiwara T
    Nihon yakurigaku zasshi. Folia pharmacologica Japonica 147 (6) 346 - 350 0015-5691 2016/06 [Refereed]
  • Akira Fukao; Tomohiko Aoyama; Toshinobu Fujiwara
    RNA BIOLOGY TAYLOR & FRANCIS INC 12 (9) 922 - 926 1547-6286 2015/09 [Refereed]
     
    MicroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs found in most plants and animals. The miRNA pathway regulates posttranscriptional gene expression through the deadenylation and translation repression of target mRNAs. Recent studies revealed that the early step of translation initiation is the target of pure translation repression by the miRNA pathway. Moreover, particularly in animals, the miRNA pathway is required for neuronal development, differentiation, and plasticity. In addition, some functions of miRNAs are regulated by RNA-binding proteins (RBPs) in neuronal cells. This review summarizes new insights about the molecular mechanisms of pure translation repression by miRNA pathway and the communication between the miRNA pathway and RBPs in neuronal local translation.
  • Takashi Mino; Yasuhiro Murakawa; Akira Fukao; Alexis Vandenbon; Hans-Hermann Wessels; Daisuke Ori; Takuya Uehata; Sarang Tartey; Shizuo Akira; Yutaka Suzuki; Carola G. Vinuesa; Uwe Ohler; Daron M. Standley; Markus Landthaler; Toshinobu Fujiwara; Osamu Takeuchi
    CELL CELL PRESS 161 (5) 1058 - 1073 0092-8674 2015/05 [Refereed]
     
    Regnase-1 and Roquin are RNA binding proteins essential for degradation of inflammation-related mRNAs and maintenance of immune homeostasis. However, their mechanistic relationship has yet to be clarified. Here, we show that, although Regnase-1 and Roquin regulate an overlapping set of mRNAs via a common stem-loop structure, they function in distinct subcellular locations: ribosome/endoplasmic reticulum and processing-body/stress granules, respectively. Moreover, Regnase-1 specifically cleaves and degrades translationally active mRNAs and requires the helicase activity of UPF1, similar to the decay mechanisms of nonsense mRNAs. In contrast, Roquin controls translationally inactive mRNAs, independent of UPF1. Defects in both Regnase-1 and Roquin lead to large increases in their target mRNAs, although Regnase-1 tends to control the early phase of inflammation when mRNAs are more actively translated. Our findings reveal that differential regulation of mRNAs by Regnase-1 and Roquin depends on their translation status and enables elaborate control of inflammation.
  • Akira Fukao; Yuichiro Mishima; Naoki Takizawa; Shigenori Oka; Hiroaki Imataka; Jerry Pelletier; Nahum Sonenberg; Christian Thoma; Toshinobu Fujiwara
    MOLECULAR CELL CELL PRESS 56 (1) 79 - 89 1097-2765 2014/10 [Refereed]
     
    In animals, key functions of microRNA-induced silencing complex (miRISC) are translational repression and deadenylation followed by mRNA decay. While miRISC represses translation initiation, it is poorly understood how miRISC exerts this function. Here we assessed the effect of miRISC on synergistic recruitment of translation initiation factors to target mRNAs by using direct biochemical assays. We show that miRISC promotes eIF4AI and eIF4AII release from target mRNAs prior to dissociation of eIF4E and eIF4G in a deadenylation-independent manner. Strikingly, miRISC-induced release of eIF4AI and eIF4AII from target mRNAs and miRISC-induced inhibition of cap-dependent translation can both be counteracted by the RNA-binding protein HuD via a direct interaction of HuD with eIF4A. Furthermore, the pharmacological eIF4A inhibitor silvestrol, which locks eIF4A on mRNAs, conferred resistance to miRNA-mediated translational repression. In summary, we propose that both eIF4AI and eIF4AII are functionally important targets in miRISC-mediated translation control.
  • Jun Ishii; Asami Oda; Shota Togawa; Akira Fukao; Toshinobu Fujiwara; Chiaki Ogino; Akihiko Kondo
    ANALYTICAL BIOCHEMISTRY ACADEMIC PRESS INC ELSEVIER SCIENCE 446 37 - 43 0003-2697 2014/02 [Refereed]
     
    Neurotensin receptor type-I (NTSR1) is a member of the G-protein-coupled receptor (GPCR) family. The natural ligand of NTSR1 is neurotensin (NT), a neuromodulator of the central nervous system. Because NT is also involved in many oncogenic actions, the signaling mediator NTSR1 is a significant molecular target in medicinal and therapeutic fields. In the current study, we constructed a fluorescence-based microbial yeast biosensor that can monitor the activation of human NTSR1 signaling responding to its agonist. To increase the sensitivity of the biosensor, a yeast strain with the green fluorescent protein (GFP) reporter gene was genetically engineered to enhance binding with human NTSRI expressed on the membrane. Following previous reports, the 5 carboxy-terminal amino acid residues of the guanine nucleotide binding protein a-subunit (G alpha) in yeast Gpalp were substituted with the equivalent human G alpha, sequences (Gpal/G alpha(q) transplant). After optimizing the assay conditions, the G alpha-engineered yeast demonstrated significantly improved sensing for NTSR1 signaling. Because detection using a GFP fluorescence reporter considerably simplifies the measurement procedure, this microbial fluorescence sensor holds promise for use in the diagnosis of NTSR1-associated diseases and the development of agonists. (C) 2013 Elsevier Inc. All rights reserved.
  • Naoki Takizawa; Toshinobu Fujiwara; Manabu Yamasaki; Ayako Saito; Akira Fukao; Akio Nomoto; Kiyohisa Mizumoto
    PLOS ONE PUBLIC LIBRARY SCIENCE 8 (10) e78000  1932-6203 2013/10 [Refereed]
     
    mRNA capping is the first cotranscriptional modification of mRNA in the nucleus. In Saccharomyces cerevisiae, the first two steps of mRNA capping are catalyzed by the RNA triphosphatase Cet1p and the RNA guanylyltransferase Ceg1p. Cet1p and Ceg1p interact to form a mRNA capping enzyme complex and the guanylyltransferase activity of Ceg1p is stimulated by binding with Cet1p. The Cet1p-Ceg1p complex needs to be transported into the nucleus, where mRNA capping occurs. However, the molecular mechanism of nuclear transport of the Cet1p-Ceg1p complex is not known. Here, we show that Cet1p is responsible and that the Cet1p-Ceg1p interaction is essential for the nuclear localization of the Cet1p-Ceg1p complex. The results indicate that the Cet1p-Ceg1p interaction is important not only for the activation of Ceg1p, but also for nuclear import of the complex.
  • Toshinobu Fujiwara; Akira Fukao; Yumi Sasano; Hidenori Matsuzaki; Ushio Kikkawa; Hiroaki Imataka; Kunio Inoue; Shogo Endo; Nahum Sonenberg; Christian Thoma; Hiroshi Sakamoto
    NUCLEIC ACIDS RESEARCH OXFORD UNIV PRESS 40 (5) 1944 - 1953 0305-1048 2012/03 [Refereed]
     
    The RNA binding protein HuD plays essential roles in neuronal development and plasticity. We have previously shown that HuD stimulates translation. Key for this enhancer function is the linker region and the poly(A) binding domain of HuD that are also critical for its function in neurite outgrowth. Here, we further explored the underlying molecular interactions and found that HuD but not the ubiquitously expressed HuR interacts directly with active Akt1. We identify that the linker region of HuD is required for this interaction. We also show by using chimeric mutants of HuD and HuR, which contain the reciprocal linker between RNA-binding domain 2 (RBD2) and RBD3, respectively, and by overexpressing a dominant negative mutant of Akt1 that the HuD-Akt1 interaction is functionally important, as it is required for the induction of neurite outgrowth in PC12 cells. These results suggest the model whereby RNA-bound HuD functions as an adapter to recruit Akt1 to trigger neurite outgrowth. These data might also help to explain how HuD enhances translation of mRNAs that encode proteins involved in neuronal development.
  • Yuichiro Mishima; Akira Fukao; Tomoyoshi Kishimoto; Hiroshi Sakamoto; Toshinobu Fujiwara; Kunio Inoue
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA NATL ACAD SCIENCES 109 (4) 1104 - 1109 0027-8424 2012/01 [Refereed]
     
    MicroRNA (miRNA) is a class of small noncoding RNA approximately 22 nt in length. Animal miRNA silences complementary mRNAs via translational inhibition, deadenylation, and mRNA degradation. However, the underlying molecular mechanisms remain unclear. A key question is whether these three outputs are independently induced by miRNA through distinct mechanisms or sequentially induced within a single molecular pathway. Here, we successfully dissected these intricate outputs of miRNA-mediated repression using zebrafish embryos as a model system. Our results indicate that translational inhibition and deadenylation are independent outputs mediated by distinct domains of TNRC6A, which is an effector protein in the miRNA pathway. Translational inhibition by TNRC6A is divided into two mechanisms: PAM2 motif-mediated interference of poly(A)-binding protein (PABP), and inhibition of 5' cap- and poly(A) tail-independent step(s) by a previously undescribed P-GL motif. Consistent with these observations, we show that, in zebrafish embryos, miRNA inhibits translation of the target mRNA in a deadenylation- and PABP-independent manner at early time points. These results indicate that miRNA exerts multiple posttranscriptional outputs via physically and functionally independent mechanisms and that direct translational inhibition is central to miRNA-mediated repression.
  • Chingakham Ranjit Singh; Ryosuke Watanabe; Donghui Zhou; Martin D. Jennings; Akira Fukao; Bumjun Lee; Yuka Ikeda; John A. Chiorini; Susan G. Campbell; Mark P. Ashe; Toshinobu Fujiwara; Ronald C. Wek; Graham D. Pavitt; Katsura Asano
    NUCLEIC ACIDS RESEARCH OXFORD UNIV PRESS 39 (19) 8314 - 8328 0305-1048 2011/10 [Refereed]
     
    The translation factor eIF5 is an important partner of eIF2, directly modulating its function in several critical steps. First, eIF5 binds eIF2/GTP/Met-tRNA(i)(Met) ternary complex (TC), promoting its recruitment to 40S ribosomal subunits. Secondly, its GTPase activating function promotes eIF2 dissociation for ribosomal subunit joining. Finally, eIF5 GDP dissociation inhibition (GDI) activity can antagonize eIF2 reactivation by competing with the eIF2 guanine exchange factor (GEF), eIF2B. The C-terminal domain (CTD) of eIF5, a W2-type HEAT domain, mediates its interaction with eIF2. Here, we characterize a related human protein containing MA3- and W2-type HEAT domains, previously termed BZW2 and renamed here as eIF5-mimic protein 1 (5MP1). Human 5MP1 interacts with eIF2 and eIF3 and inhibits general and gene-specific translation in mammalian systems. We further test whether 5MP1 is a mimic or competitor of the GEF catalytic subunit eIF2B epsilon or eIF5, using yeast as a model. Our results suggest that 5MP1 interacts with yeast eIF2 and promotes TC formation, but inhibits TC binding to the ribosome. Moreover, 5MP1 is not a GEF but a weak GDI for yeast eIF2. We propose that 5MP1 is a partial mimic and competitor of eIF5, interfering with the key steps by which eIF5 regulates eIF2 function.
  • Yuki Fujiwara; Katsumi Kasashima; Kuniaki Saito; Miho Fukuda; Akira Fukao; Yumi Sasano; Kunio Inoue; Toshinobu Fujiwara; Hiroshi Sakamoto
    BIOCHIMIE ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER 93 (5) 817 - 822 0300-9084 2011/05 [Refereed]
     
    RNA-binding proteins (RBPs) play a vital role in the post-transcriptional regulation of gene expression during neuronal differentiation and synaptic plasticity. One such RBP family, the neuronal Hu protein family, serves as an early marker of neuronal differentiation and targets several mRNAs containing adenine/uridine-rich elements. Recently, we reported that one of the neuronal Hu proteins, HuD stimulates cap-dependent translation through interactions with eIF4A and poly (A) tail. Nevertheless, little is known with respect to how neuronal Hu proteins contribute to the local translation of target mRNAs in neuronal differentiation. Here, we found that neuronal Hu proteins, but not the ubiquitously expressed HuR protein, directly interact with the light chain of microtubule-associated proteins MAP1B (LC1). We also show that HuD simultaneously binds both RNA and LC1 in vitro and that it tightly associates with microtubules in cells in an LC1-dependent manner, raising the possibility that HuD recruits target mRNAs to microtubules. These results uncover the neuronal binding partners for neuron-specific Hu proteins and suggest the involvement of Hu proteins in microtubule-mediated regulation of mRNA expression within neuronal processes. (C) 2011 Elsevier Masson SAS. All rights reserved.
  • Akira Fukao; Yumi Sasano; Hiroaki Imataka; Kunio Inoue; Hiroshi Sakamoto; Nahum Sonenberg; Christian Thoma; Toshinobu Fujiwara
    MOLECULAR CELL CELL PRESS 36 (6) 1007 - 1017 1097-2765 2009/12 [Refereed]
     
    The RNA-binding protein HuD promotes neuronal differentiation by an unknown mechanism. Here we identify an enhancer function of HuD in translation. Translation stimulation by HuD requires both a 3' poly(A) tail and a 5' m(7)G cap structure. We also show that HuD directly interacts with eIF4A. This interaction and the poly(A)-binding activity of HuD are critical for its translational enhancer function because HuD-eIF4A- and HuD-poly(A)-binding mutants fail to stimulate translation. We show that translation of HCV IRES mRNA, which is eIF4A independent, is not stimulated by HuD. We also find that the eIF4A and poly(A)-binding activities of HuD are not only important for stimulating translation but also are essential for HuD-induced neurite outgrowth in PC12 cells. This example of cap-dependent translational regulation might explain at least in part how HuD triggers the induction of neuronal differentiation.

MISC

Research Grants & Projects

  • 無細胞in vitro翻訳システムを用いた脱アデニル化複合体によるmRNA分解を介さない翻訳抑制機構の解析
    公益財団法人内等記念科学振興財団：内藤記念科学奨励金・研究助成
    Date (from‐to) : 2017/12 -2019/09 
    Author : 深尾亜喜良
  • 神経特異的RNA結合蛋白質HuDが担う部位特異的翻訳制御機構の解析
    日本学術振興会：科学研究費補助金（若手研究（B））
    Date (from‐to) : 2015/04 -2018/03 
    Author : 深尾亜喜良
  • ヒトGPCR標的RNAアプタマーの創製とその応用による新規腫瘍診断薬の開発
    日本学術振興会：特別研究員（PD）
    Date (from‐to) : 2011/04 -2014/03 
    Author : 深尾亜喜良

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