KINDAI UNIVERSITY


*A space between the first name and last name, please enter

FUJIWARA Toshinobu

Profile

FacultyDepartment of Pharmacy / Graduate School of Medicine
PositionProfessor
Degree
Commentator Guidehttps://www.kindai.ac.jp/meikan/1343-fujiwara-toshinobu.html
URL
Mail
Last Updated :2020/09/30

Education and Career

Academic & Professional Experience

  •   2015 04 ,  - 現在, Professor, Laboratory of Biochemistry, Graduate School of Pharmaceutical Sciences, Kindai University
  •   2013 04 ,  - 2015 03 , Professor, Laboratory of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University
  •   2011 04 ,  - 2013 03 , Principal Investigator, Institute of Microbial Chemistry, Microbial Chemistry Research Foundation
  •   2010 04 ,  - 2011 03 , Associate Professor, Core Team, Organization of Advanced Science and Technology, Kobe Univ.
  •   2007 04 ,  - 2010 03 , Associate Professor, Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe Univ.
  •   2006 10 ,  - 2010 03 , Researcher, PREST, JST
  •   2003 02 ,  - 2007 03 , Assistant Professor, Department of Biosystems Scinece, Graduate School of Science and Technology, Kobe Univ.

Research Activities

Research Areas

  • Life sciences, Functional biochemistry, translation
  • Life sciences, Molecular biology, RNA

Research Interests

  • RNA engineering, Analyses of RNA binding proteins, The initiation of mammalian protein synthesis, RNA ribosome translation

Published Papers

  • Emerging Evidence of Translational Control by AU-Rich Element-Binding Proteins., Otsuka H, Fukao A, Funakami Y, Duncan KE, Fujiwara T, Frontiers in genetics, Frontiers in genetics, 10, 332, 2019 , Refereed
  • [Translational regulation is mediated by the cross-talk between the miRNA pathway and RNA binding proteins]., Aoyama T, Fukao A, Fujiwara T, Nihon yakurigaku zasshi. Folia pharmacologica Japonica, Nihon yakurigaku zasshi. Folia pharmacologica Japonica, 147(6), 346 - 350, Jun. 2016 , Refereed
  • Microbial fluorescence sensing for human neurotensin receptor type 1 using G alpha-engineered yeast cells, Jun Ishii, Asami Oda, Shota Togawa, Akira Fukao, Toshinobu Fujiwara, Chiaki Ogino, Akihiko Kondo, ANALYTICAL BIOCHEMISTRY, ANALYTICAL BIOCHEMISTRY, 446, 37 - 43, Feb. 2014 , Refereed
    Summary: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.
  • Staphylococcal Superantigen-like Protein 10 (SSL10) Inhibits Blood Coagulation by Binding to Prothrombin and Factor Xa via Their gamma-Carboxyglutamic Acid (Gla) Domain, Saotomo Itoh, Ryosuke Yokoyama, Go Kamoshida, Toshinobu Fujiwara, Hiromi Okada, Takemasa Takii, Tsutomu Tsuji, Satoshi Fujii, Hideki Hashizume, Kikuo Onozaki, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 288(30), 21569 - 21580, Jul. 2013 , Refereed
    Summary:The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 x 10(-7) m in surface plasmon resonance analysis. On the other hand, the resin failed to recover -carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca2+ and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.
  • Double Plant Homeodomain (PHD) Finger Proteins DPF3a and-3b Are Required as Transcriptional Co-activators in SWI/SNF Complex-dependent Activation of NF-kappa B RelA/p50 Heterodimer, Aya Ishizaka, Taketoshi Mizutani, Kazuyoshi Kobayashi, Toshio Tando, Kouhei Sakurai, Toshinobu Fujiwara, Hideo Iba, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 287(15), 11924 - 11933, Apr. 2012 , Refereed
    Summary:We have previously shown that DPF2 (requiem/REQ) functions as a linker protein between the SWI/SNF complex and RelB/p52 NF-kappa B heterodimer and plays important roles in NF-kappa B transactivation via its noncanonical pathway. Using sensitive 293FT reporter cell clones that had integrated a SWI/SNF-dependent NF-kappa B reporter gene, we find in this study that the overexpression of DPF1, DPF2, DPF3a, DPF3b, and PHF10 significantly potentiates the transactivating activity of typical NF-kappa B dimers. Knockdown analysis using 293FT reporter cells that endogenously express these five proteins at low levels clearly showed that DPF3a and DPF3b, which are produced from the DPF3 gene by alternative splicing, are the most critical for the RelA/p50 NF-kappa B heterodimer transactivation induced by TNF-alpha stimulation. Our data further show that this transactivation requires the SWI/SNF complex. DPF3a and DPF3b are additionally shown to interact directly with RelA, p50, and several subunits of the SWI/SNF complex in vitro and to be co-immunoprecipitated with RelA/p50 and the SWI/SNF complex from the nuclear fractions of cells treated with TNF-alpha. In ChIP experiments, we further found that endogenous DPF3a/b and the SWI/SNF complex are continuously present on HIV-1 LTR, whereas the kinetics of RelA/p50 recruitment after TNF-alpha treatment correlate well with the viral transcriptional activation levels. Additionally, re-ChIP experiments showed DPF3a/b and the SWI/SNF complex associate with RelA on the endogenous IL-6 promoter after TNF-alpha treatment. In conclusion, our present data indicate that by linking RelA/p50 to the SWI/SNF complex, DPF3a/b induces the transactivation of NF-kappa B target gene promoters in relatively inactive chromatin contexts.
  • [Biochemical selenocysteine synthesis and the phylogenic study]., Mizutani T, Osaka T, Fujiwara T, Shahidzzman M, Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan, Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan, 128(7), 989 - 996, Jul. 2008 , Refereed
  • [RNA binding proteins play the leading part of posttranscriptional regulation of gene expression in nerve]., Fujiwara T, Sakamoto H, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 51(16 Suppl), 2609 - 2616, Dec. 2006 , Refereed
  • Codon bias confers stability to human mRNAs., 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, EMBO reports, 20(11), e48220, Nov. 05 2019 , Refereed
    Summary: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.
  • ARF6 and AMAP1 are major targets of KRAS and TP53 mutations to promote invasion, PD-L1 dynamics, and immune evasion of pancreatic cancer, 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, Proceedings of the National Academy of Sciences of the United States of America, 116(35), 17450 - 17459, Aug. 27 2019 , Refereed
    Summary:© 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.
  • Translation of hepatitis A virus IRES is upregulated by a hepatic cell-specific factor, Akitoshi Sadahiro, Akira Fukao, Mio Kosaka, Yoshinori Funakami, Naoki Takizawa, Osamu Takeuchi, Kent E. Duncan, Toshinobu Fujiwara, Frontiers in Genetics, Frontiers in Genetics, 9(AUG), 307, Aug. 10 2018 , Refereed
    Summary:© 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.
  • Identification of matrix metalloproteinase 9-interacting sequences in staphylococcal superantigen-like protein 5, Katsuhiro Kohno, Saotomo Itoh, Akari Hanai, Takemasa Takii, Toshinobu Fujiwara, Kikuo Onozaki, Tsutomu Tsuji, Shigeaki Hida, Biochemical and Biophysical Research Communications, Biochemical and Biophysical Research Communications, 497(2), 713 - 718, Mar. 04 2018 , Refereed
    Summary:Staphylococcal superantigen like 5 (SSL5) is an exotoxin produced by S. aureus and has a strong inhibitory effect on MMP-9 enzymatic activity. However, the mechanism of inhibition remains unclear. We sought to identify the responsible regions of SSL5 for the interaction with MMP-9 by comparing a series of domain swap and deletion mutants of SSL5. Binding analyses revealed that SSL5 had two regions for binding to MMP-9 catalytic domain, β1-3 region (25SKELKNVTGY RYSKGGKHYL IFDKNRKFTR VQIFGK60) in N-terminal half and α4β9 region (138KELDFKLRQY LIQNFDLYKK FPKDSKIKVI MKD170) in C-terminal half. The collagen binding domain and zinc-chelating histidine residues of MMP-9 were not essential for the specific binding to SSL5. The domain swap mutants of SSL5 that conserved β1-3 but not α4β9 region inhibited the gelatinolysis by MMP-9, and the mutant of SSL7 that substituted β1-3 region to that of SSL5 acquired the binding and inhibitory activity. Furthermore, the polypeptide that harbored β1-3 region of SSL5 inhibited gelatinolysis by MMP-9. Taken together, SSL5 inhibits the MMP9 activity through binding to the catalytic domain, and the β1-3 region is responsible for the inhibition of proteolytic activity of MMP-9.
  • The CCR4-NOT deadenylase complex controls Atg7-dependent cell death and heart function., 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, Science signaling, 11(516), Feb. 06 2018 , Refereed
    Summary: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.
  • Kribellosides, novel RNA 5'-triphosphatase inhibitors from the rare actinomycete Kribbella sp MI481-42F6, Masayuki Igarashi, Ryuichi Sawa, Manabu Yamasaki, Chigusa Hayashi, Maya Umekita, Masaki Hatano, Toshinobu Fujiwara, Kiyohisa Mizumoto, Akio Nomoto, JOURNAL OF ANTIBIOTICS, JOURNAL OF ANTIBIOTICS, 70(5), 582 - 589, May 2017 , Refereed
    Summary:Yeast capping enzymes differ greatly from those of mammalian, both structurally and mechanistically. Yeast-type capping enzyme repressors are therefore candidate antifungal drugs. The 5'-guanine-N7 cap structure of mRNAs are an essential feature of all eukaryotic organisms examined to date and is the first co-transcriptional modification of cellular pre-messenger RNA. Inhibitors of the RNA 5'-triphosphatase in yeast are likely to show fungicidal effects against pathogenic yeast such as Candida. We discovered a new RNA 5'-triphosphatase inhibitor, designated as the kribellosides, by screening metabolites from actinomycetes. Kribellosides belong to the alkyl glyceryl ethers. These novel compounds inhibit the activity of Cet1p (RNA 5'-triphosphatase) from Saccharomyces cerevisiae in vitro with IC(50)s of 5-8 mu M and show antifungal activity with MICs ranging from 3.12 to 100 mu g ml(-1) against S. cerevisiae.
  • Spatial regulation of the KH domain RNA-binding protein Rnc1 mediated by a Crm1-independent nuclear export system in Schizosaccharomyces pombe, 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, MOLECULAR MICROBIOLOGY, 104(3), 428 - 448, May 2017 , Refereed
    Summary: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.
  • The coupled and uncoupled mechanisms by which trans-acting factors regulate mRNA stability and translation, Akira Fukao, Toshinobu Fujiwaray, JOURNAL OF BIOCHEMISTRY, JOURNAL OF BIOCHEMISTRY, 161(4), 309 - 314, Apr. 2017 , Refereed
    Summary: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.
  • Fine-tuning of translation initiation by RNA binding proteins, Toshinobu Fujiwara, Seikagaku, Seikagaku, 88(1), 135 - 138, 2016 , Refereed
  • The molecular mechanism of translational control via the communication between the microRNA pathway and RNA-binding proteins, Akira Fukao, Tomohiko Aoyama, Toshinobu Fujiwara, RNA BIOLOGY, RNA BIOLOGY, 12(9), 922 - 926, Sep. 2015 , Refereed
    Summary: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.
  • PI4K-beta and MKNK1 are regulators of hepatitis C virus IRES-dependent translation, Joachim Lupberger, Claudia Casanova, Benoit Fischer, Amelie Weiss, Isabel Fofana, Nelly Fontaine, Toshinobu Fujiwara, Mickael Renaud, Arnaud Kopp, Catherine Schuster, Laurent Brino, Thomas F. Baumert, Christian Thoma, SCIENTIFIC REPORTS, SCIENTIFIC REPORTS, 5, 13344, Sep. 2015 , Refereed
    Summary:Cellular translation is down-regulated by host antiviral responses. Picornaviridae and Flaviviridae including hepatitis C virus (HCV) evade this process using internal ribosomal entry sequences (IRESs). Although HCV IRES translation is a prerequisite for HCV replication, only few host factors critical for IRES activity are known and the global regulator network remains largely unknown. Since signal transduction is an import regulator of viral infections and the host antiviral response we combined a functional RNAi screen targeting the human signaling network with a HCV IRES-specific reporter mRNA assay. We demonstrate that the HCV host cell cofactors PI4K and MKNK1 are positive regulators of HCV IRES translation representing a novel pathway with a functional relevance for the HCV life cycle and IRES-mediated translation of viral RNA.
  • Regnase-1 and Roquin Regulate a Common Element in Inflammatory mRNAs by Spatiotemporally Distinct Mechanisms, 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, 161(5), 1058 - 1073, May 2015 , Refereed
    Summary: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.
  • MicroRNAs Trigger Dissociation of eIF4AI and eIF4AII from Target mRNAs in Humans, Akira Fukao, Yuichiro Mishima, Naoki Takizawa, Shigenori Oka, Hiroaki Imataka, Jerry Pelletier, Nahum Sonenberg, Christian Thoma, Toshinobu Fujiwara, MOLECULAR CELL, MOLECULAR CELL, 56(1), 79 - 89, Oct. 2014 , Refereed
    Summary: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.
  • Efficient and cost effective production of active-form human PKB using silkworm larvae, Ryoko Maesaki, Ryosuke Satoh, Masato Taoka, Teppei Kanaba, Tsunaki Asano, Chiharu Fujita, Toshinobu Fujiwara, Yutaka Ito, Toshiaki Isobe, Toshio Hakoshima, Katsumi Maenaka, Masaki Mishima, SCIENTIFIC REPORTS, SCIENTIFIC REPORTS, 4, 6016, Aug. 2014 , Refereed
    Summary:Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKB alpha using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKB alpha protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1 mg PKB alpha/20 larvae was recorded. Kinase assays showed that the recombinant PKB alpha possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKB alpha with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.
  • Structural Insights into the Recruitment of SMRT by the Corepressor SHARP under Phosphorylative Regulation, Suzuka Mikami, Teppei Kanaba, Naoki Takizawa, Ayaho Kobayashi, Ryoko Maesaki, Toshinobu Fujiwara, Yutaka Ito, Masaki Mishima, STRUCTURE, STRUCTURE, 22(1), 35 - 46, Jan. 2014 , Refereed
    Summary:The transcriptional corepressors SMRT/NCoR, components of histone deacetylase complexes, interact with nuclear receptors and many other transcription factors. SMRT is a target for the ubiquitously expressed protein kinase CK2, which is known to phosphorylate a wide variety of substrates. Increasing evidence suggests that CK2 plays a regulatory role in many cellular events, particularly, in transcription. However, little is known about the precise mode of action involved. Here, we report the three-dimensional structure of a SMRT/HDAC1-associated repressor protein (SHARP) in complex with phosphorylated SMRT, as determined by solution NMR. Phosphorylation of the CK2 site on SMRT significantly increased affinity for SHARP. We also confirmed the significance of CK2 phosphorylation by reporter assay and propose a mechanism involving the process of phosphorylation acting as a molecular switch. Finally, we propose that the SPOC domain functions as a phosphorylation binding module.
  • The Essential Role for the RNA Triphosphatase Cet1p in Nuclear Import of the mRNA Capping Enzyme Cet1p-Ceg1p Complex of Saccharomyces cerevisiae, Naoki Takizawa, Toshinobu Fujiwara, Manabu Yamasaki, Ayako Saito, Akira Fukao, Akio Nomoto, Kiyohisa Mizumoto, PLOS ONE, PLOS ONE, 8(10), e78000, Oct. 2013 , Refereed
    Summary: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.
  • Structure of the second RRM domain of Nrd1, a fission yeast MAPK target RNA binding protein, and implication for its RNA recognition and regulation, Ayaho Kobayashi, Teppei Kanaba, Ryosuke Satoh, Toshinobu Fujiwara, Yutaka Ito, Reiko Sugiura, Masaki Mishima, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 437(1), 12 - 17, Jul. 2013 , Refereed
    Summary:Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and thereby suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed. (c) 2013 Elsevier Inc. All rights reserved.
  • Targeting cancer cell-specific RNA interference by siRNA delivery using a complex carrier of affibody-displaying bio-nanocapsules and liposomes, Yuya Nishimura, Hiroaki Mieda, Jun Ishii, Chiaki Ogino, Toshinobu Fujiwara, Akihiko Kondo, JOURNAL OF NANOBIOTECHNOLOGY, JOURNAL OF NANOBIOTECHNOLOGY, 11, 19, Jun. 2013 , Refereed
    Summary:Background: Small interfering RNA (siRNA) has attracted attention in the field of nucleic acid medicine as a RNA interference (RNAi) application that leads to gene silencing due to specific messenger RNA (mRNA) destruction. However, since siRNA is unstable in blood and unable to cross the cell membrane, encapsulation of siRNA into a carrier is required. Results: In this study, we used a carrier that combined Z(HER2)-displaying bio-nanocapsule (derived from hepatitis B virus surface antigen) and liposomes in a complex in order to investigate the feasibility of effective and target-cell-specific RNAi applications. As a result, by observing RNAi only in HER2-expressing breast cancer cells, using our proposed methodology, we successfully demonstrated target-cell-specific delivery and effective function expression of siRNA. Conclusions: These findings show that, in the field of nucleic acid medicine, Z(HER2)-BNC/ LP can be a useful carrier for siRNA delivery, and could also become a useful tool for gene silencing and to accomplish protein knock-down.
  • Functional and direct interaction between the RNA binding protein HuD and active Akt1, 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, NUCLEIC ACIDS RESEARCH, 40(5), 1944 - 1953, Mar. 2012 , Refereed
    Summary: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.
  • Translational inhibition by deadenylation-independent mechanisms is central to microRNA-mediated silencing in zebrafish, 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, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 109(4), 1104 - 1109, Jan. 2012 , Refereed
    Summary: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.
  • Mechanisms of translational regulation by a human eIF5-mimic protein, 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, NUCLEIC ACIDS RESEARCH, 39(19), 8314 - 8328, Oct. 2011 , Refereed
    Summary: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.
  • Microtubule association of a neuronal RNA-binding protein HuD through its binding to the light chain of MAP1B, Yuki Fujiwara, Katsumi Kasashima, Kuniaki Saito, Miho Fukuda, Akira Fukao, Yumi Sasano, Kunio Inoue, Toshinobu Fujiwara, Hiroshi Sakamoto, BIOCHIMIE, BIOCHIMIE, 93(5), 817 - 822, May 2011 , Refereed
    Summary: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.
  • Single-Embryo Metabolomics and Systematic Prediction of Developmental Stage in Zebrafish, Shunsuke Hayashi, Mako Yoshida, Toshinobu Fujiwara, Shingo Maegawa, Eiichiro Fukusaki, ZEITSCHRIFT FUR NATURFORSCHUNG SECTION C-A JOURNAL OF BIOSCIENCES, ZEITSCHRIFT FUR NATURFORSCHUNG SECTION C-A JOURNAL OF BIOSCIENCES, 66(3-4), 191 - 198, Mar. 2011 , Refereed
    Summary:Metabolites, the end products of gene expression in living organisms, are tightly correlated with an organism's development and growth. Thus, metabolic profiling is a potentially important tool for understanding the events that have occurred in cells, tissues, and individual organisms. Here, we present a method for predicting the developmental stage of zebrafish embryos using novel metabolomic non-target fingerprints of "single-embryos". With this method, we observed the rate of development at different temperatures. Our results suggest that this method allows us to analyse the condition, or distinguish the genotype, of single-embryos before expression of their ultimate phenotype.
  • A small nucleolar RNA functions in rRNA processing in Caenorhabditis elegans, Yusuke Hokii, Yumi Sasano, Mayu Sato, Hiroshi Sakamoto, Kazumi Sakata, Ryuzo Shingai, Akito Taneda, Shigenori Oka, Hyouta Himeno, Akira Muto, Toshinobu Fujiwara, Chisato Ushida, NUCLEIC ACIDS RESEARCH, NUCLEIC ACIDS RESEARCH, 38(17), 5909 - 5918, Sep. 2010 , Refereed
    Summary:CeR-2 RNA is one of the newly identified Caenorhabditis elegans noncoding RNAs (ncRNAs). The characterization of CeR-2 by RNomic studies has failed to classify it into any known ncRNA family. In this study, we examined the spatiotemporal expression patterns of CeR-2 to gain insight into its function. CeR-2 is expressed in most cells from the early embryo to adult stages. The subcellular localization of this RNA is analogous to that of fibrillarin, a major protein of the nucleolus. It was observed that knockdown of C/D small nucleolar ribonucleoproteins (snoRNPs), but not of H/ACA snoRNPs, resulted in the aberrant nucleolar localization of CeR-2 RNA. A mutant worm with a reduced amount of cellular CeR-2 RNA showed changes in its pre-rRNA processing pattern compared with that of the wild-type strain N2. These results suggest that CeR-2 RNA is a C/D snoRNA involved in the processing of rRNAs.
  • The ELAV Protein HuD Stimulates Cap-Dependent Translation in a Poly(A)- and eIF4A-Dependent Manner, Akira Fukao, Yumi Sasano, Hiroaki Imataka, Kunio Inoue, Hiroshi Sakamoto, Nahum Sonenberg, Christian Thoma, Toshinobu Fujiwara, MOLECULAR CELL, MOLECULAR CELL, 36(6), 1007 - 1017, Dec. 2009 , Refereed
    Summary: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.
  • DAZL Relieves miRNA-Mediated Repression of Germline mRNAs by Controlling Poly(A) Tail Length in Zebrafish, Yasuaki Takeda, Yuichiro Mishima, Toshinobu Fujiwara, Hiroshi Sakamoto, Kunio Inoue, PLOS ONE, PLOS ONE, 4(10), e7513, Oct. 2009 , Refereed
    Summary:Background: During zebrafish embryogenesis, microRNA (miRNA) miR-430 contributes to restrict Nanos1 and TDRD7 to primordial germ cells (PGCs) by inducing mRNA deadenylation, mRNA degradation, and translational repression of nanos1 and tdrd7 mRNAs in somatic cells. The nanos1 and tdrd7 3'UTRs include cis-acting elements that allow activity in PGCs even in the presence of miRNA-mediated repression. Methodology/Principal Findings: Using a GFP reporter mRNA that was fused with tdrd7 3'UTR, we show that a germline-specific RNA-binding protein DAZ-like (DAZL) can relieve the miR-430-mediated repression of tdrd7 mRNA by inducing poly(A) tail elongation (polyadenylation) in zebrafish. We also show that DAZL enhances protein synthesis via the 3'UTR of dazl mRNA, another germline mRNA targeted by miR-430. Conclusions/Significance: Our present study indicated that DAZL acts as an "anti-miRNA factor'' during vertebrate germ cell development. Our data also suggested that miRNA-mediated regulation can be modulated on specific target mRNAs through the poly(A) tail control.
  • A novel application of metabolomics in vertebrate development, Shunsuke Hayashi, Shinichi Akiyama, Yutaka Tamaru, Yasuaki Takeda, Toshinobu Fujiwara, Kunio Inoue, Akio Kobayashi, Shingo Maegawa, Eiichiro Fukusaki, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 386(1), 268 - 272, Aug. 2009 , Refereed
    Summary:Many Studies have demonstrated the functions of individual genes associated with embryogenesis and have determined the genome sequences of several organisms. Despite the availability of enormous amount of genetic information, dynamic changes that occur during embryogenesis have not yet been completely understood. In order to understand the dynamic processes involved in embryogenesis, we employed the metabolomic approach. The results of our study indicated that there is a close correlation between metabolomes and developmental stages. Our method enables the identification of embryonic stages using metabolomes as "fingerprints." In this manner, we could successfully predict embryonic development on the basis of metabolomic fingerprints. This is the first report describing a model for predicting vertebrate development by using metabolomics. (C) 2009 Elsevier Inc. All rights reserved.
  • WDR55 Is a Nucleolar Modulator of Ribosomal RNA Synthesis, Cell Cycle Progression, and Teleost Organ Development, Norimasa Iwanami, Tomokazu Higuchi, Yumi Sasano, Toshinobu Fujiwara, Vu Q. Hoa, Minoru Okada, Sadiqur R. Talukder, Sanae Kunimatsu, Jie Li, Fumi Saito, Chitralekha Bhattacharya, Angabin Matin, Takashi Sasaki, Nobuyoshi Shimizu, Hiroshi Mitani, Heinz Himmelbauer, Akihiro Momoi, Hisato Kondoh, Makoto Furutani-Seiki, Yousuke Takahama, PLOS GENETICS, PLOS GENETICS, 4(8), e1000171, Aug. 2008 , Refereed
    Summary:The thymus is a vertebrate-specific organ where T lymphocytes are generated. Genetic programs that lead to thymus development are incompletely understood. We previously screened ethylnitrosourea-induced medaka mutants for recessive defects in thymus development. Here we report that one of those mutants is caused by a missense mutation in a gene encoding the previously uncharacterized protein WDR55 carrying the tryptophan- aspartate-repeat motif. We find that WDR55 is a novel nucleolar protein involved in the production of ribosomal RNA ( rRNA). Defects in WDR55 cause aberrant accumulation of rRNA intermediates and cell cycle arrest. A mutation in WDR55 in zebrafish also leads to analogous defects in thymus development, whereas WDR55-null mice are lethal before implantation. These results indicate that WDR55 is a nuclear modulator of rRNA synthesis, cell cycle progression, and embryonic organogenesis including teleost thymus development.
  • Tissue-specific splicing regulator Fox-1 induces exon skipping by interfering E complex formation on the downstream intron of human F1 gamma gene, Kazuhiro Fukumura, Ayako Kato, Yui Jin, Takashi Ideue, Tetsuro Hirose, Naoyuki Kataoka, Toshinobu Fujiwara, Hiroshi Sakamoto, Kunio Inoue, NUCLEIC ACIDS RESEARCH, NUCLEIC ACIDS RESEARCH, 35(16), 5303 - 5311, Aug. 2007 , Refereed
    Summary:Fox-1 is a regulator of tissue-specific splicing, via binding to the element (U) GCAUG in mRNA precursors, in muscles and neuronal cells. Fox-1 can regulate splicing positively or negatively, most likely depending on where it binds relative to the regulated exon. In cases where the (U) GCAUG element lies in an intron upstream of the alternative exon, Fox-1 protein functions as a splicing repressor to induce exon skipping. Here we report the mechanism of exon skipping regulated by Fox-1, using the hF1 gamma gene as a model system. We found that Fox-1 induces exon 9 skipping by repressing splicing of the downstream intron 9 via binding to the GCAUG repressor elements located in the upstream intron 8. In vitro splicing analyses showed that Fox-1 prevents formation of the pre-spliceosomal early (E) complex on intron 9. In addition, we located a region of the Fox-1 protein that is required for inducing exon skipping. Taken together, our data show a novel mechanism of how RNA-binding proteins regulate alternative splicing.
  • Differential regulation of germline mRNAs in soma and germ cells by zebrafish miR-430, Yuichlro Mishima, Antonio J. Giraldez, Yasuaki Takeda, Toshinobu Fujiwara, Hiroshi Sakamoto, Alexander F. Schier, Kunio Inoue, CURRENT BIOLOGY, CURRENT BIOLOGY, 16(21), 2135 - 2142, Nov. 2006 , Refereed
    Summary:Early in development, primordial germ cells (PGCs) are set aside from somatic cells and acquire a unique gene-expression program [1].The mechanisms underlying germline-specific gene expression are largely unknown. Nanos expression is required during germline development [2-5] and is posttranscriptionally restricted to PGCs [4,6-8]. Here we report that the microRNA miR-430 targets the 3' untranslated region (UTR) of nanos1 during zebrafish embryogenesis. A miR430 target site within the nanos1 3' UTR reduces poly(A) tail length, mRNA stability, and translation. Repression is disrupted in maternal-zygotic dicer mutants (MZdicer), which lack mature miRNAs [9], and is restored by injection of processed miR-430. Although miR-430 represses other genes equally in germline and soma, specific regions in the nanos1 31 UTR compensate for microRNA-mediated repression in PGCs and allow germline-specific expression. We show that the 3' UTR of an additional PGC-specific gene, TDRD7, is also targeted by miR-430. These results indicate that miR-430 targets the 3' UTRs of germline genes and suggest that differential susceptibility to microRNAs contributes to tissue-specific gene expression.
  • Heterologous expression of Aquifex aeolicus ribosome recycling factor in Escherichia coli is dominant lethal by forming a complex that lacks functional co-ordination for ribosome disassembly, T Yamami, K Ito, T Fujiwara, Y Nakamura, MOLECULAR MICROBIOLOGY, MOLECULAR MICROBIOLOGY, 55(1), 150 - 161, Jan. 2005 , Refereed
    Summary:Recycling the post-termination ribosomal complex requires the co-ordinated effort of the ribosome, ribosome recycling factor (RRF) and elongation factor EF-G. Although Aquifex aeolicus RRF (aaRRF) binds Escherichia coli ribosomes as efficiently as E. coli RRF, the resulting complex is non-functional and dominant lethal in E. coli, even in the presence of homologous A. aeolicus EF-G. These findings suggest that the E. coli post-termination ribosomal complex with aaRRF lacks functional co-ordination with EF-G required for ribosome recycling. A chimeric EF-G (E. coli domains I-III, A. aeolicus domains IV-V) or an A. aeolicus EF-G with distinct mutations in the domain I-II interface could activate aaRRF. Furthermore, novel mutations that localize to one surface of the L-shape structure of aaRRF restored activity in E. coli. These aaRRF mutations are spatially distinct from mutations previously described and suggest a novel active centre for coupling EF-G's G domain motor action to ribosome disassembly.
  • Ribosome recycling factor disassembles the post-termination ribosomal complex independent of the ribosomal translocase activity of elongation factor G, T Fujiwara, K Ito, T Yamami, Y Nakamura, MOLECULAR MICROBIOLOGY, MOLECULAR MICROBIOLOGY, 53(2), 517 - 528, Jul. 2004 , Refereed
    Summary:Ribosome recycling factor (RRF) disassembles post-termination ribosomal complexes in concert with elongation factor EF-G freeing the ribosome for a new round of polypeptide synthesis. How RRF interacts with EF-G and disassembles post-termination ribosomes is unknown. RRF is structurally similar to tRNA and is therefore thought to bind to the ribosomal A site and be translocated by EF-G during ribosome disassembly as a mimic of tRNA. However, EF-G variants that remain active in GTP hydrolysis but are defective in tRNA translocation fully activate RRF function in vivo and in vitro. Furthermore, RRF and the GTP form of EF-G do not co-occupy the terminating ribosome in vitro; RRF is ejected by EF-G from the preformed complex. These findings suggest that RRF is not a functional mimic of tRNA and disassembles the post-termination ribosomal complex independently of the translocation activity of EF-G.
  • RBD-1, a nucleolar RNA-binding protein, is essential for Caenorhabditis elegans early development through 18S ribosomal RNA processing, E Saijou, T Fujiwara, T Suzaki, K Inoue, H Sakamoto, NUCLEIC ACIDS RESEARCH, NUCLEIC ACIDS RESEARCH, 32(3), 1028 - 1036, Feb. 2004 , Refereed
    Summary:RBD-1 is the Caenorhabditis elegans homolog of Mrd1p, which was recently shown to be required for 18S ribosomal RNA (rRNA) processing in yeast. To gain insights into the relationship between ribosome biogenesis and the development of multicellular organisms, we examined the expression and function of RBD-1. Maternal RBD-1 in the fertilized egg disappears immediately after cleavage starts, whereas zygotic RBD-1 first appears in late embryos and is localized in the nucleolus in most cells, although zygotic transcription of pre-rRNA is known to be initiated as early as the one-cell stage. RNA interference of the rbd-1gene severely inhibits the processing of 18S rRNA in association with various developmental abnormalities, indicating its essential role in pre-rRNA processing and development in C. elegans. These results provide evidence for the linkage between ribosome biogenesis and the control of development and imply unexpected uncoupling of transcription and processing of pre-rRNA in early C. elegans embryos.
  • Elongation factor G participates in ribosome disassembly by interacting with ribosome recycling factor at their tRNA-mimicry domains, K Ito, T Fujiwara, T Toyoda, Y Nakamura, MOLECULAR CELL, MOLECULAR CELL, 9(6), 1263 - 1272, Jun. 2002 , Refereed
    Summary:Elongation factor G (EF-G) is a G protein with motor function that drives two target molecules, a tRNA in the translating ribosome and the ribosome recycling factor (RRF) in the posttermination complex. How G protein motor action is transmitted to RRF is unknown. Thermus thermophilus RRF is nonfunctional in Escherichia coli. It became functional upon introducing a plasmid expressing E. coli EF-G with surface changes in its tRNA-mimic domain or by replacing the E. coli EF-G tRNA-mimic domain by the Thermus domain. Thermus RRF could also be activated by introducing surface substitutions in its anticodon arm-mimic region. These gain-of-function phenotypes depend on the combination of heterologous EF-G and RRF alleles. These mutational studies suggest that EF-G motor action is transmitted to RRF by specific surface contacts between the domains that mimic the anticodon arm.

Misc

  • ヒトにおけるmicroRNAが蛋白質合成を制御する機序の解明, FUKAO AKIRA, FUJIWARA TOSHINOBU, 月刊臨床免疫・アレルギー科, 64, 3, 307, 312,   2015 09 25 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201502220863477824
  • パラダイムシフトする翻訳制御研究 microRNA機構とRNA結合タンパク質による遺伝子発現制御, FUKAO AKIRA, FUJIWARA TOSHINOBU, FUJIWARA TOSHINOBU, 細胞工学, 34, 8, 767, 771,   2015 07 22 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201502215030905667
  • RNA結合タンパク質HuDが仲介するmRNA分解と翻訳との共役機構, TAKECHI MIWA, OSHIMA TAKUYA, FUKAO AKIRA, FUJIWARA TOSHINOBU, 日本RNA学会年会要旨集, 17th, 114,   2015 07 15 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201502201407074250
  • RNA結合タンパク質HuDによる翻訳促進機構の素過程の解析, OTSUKA HIROSHI, FUKAO AKIRA, FUJIWARA TOSHINOBU, 日本RNA学会年会要旨集, 17th, 251,   2015 07 15 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201502201526800180
  • RNA結合性マルチドメインタンパク質Nrd1の溶液状態における構造解析, KOBAYASHI SAIHO, SATO RYOSUKE, FUJIWARA TOSHINOBU, ITO TAKASHI, SUGIURA REIKO, MISHIMA MASAKI, 日本蛋白質科学会年会プログラム・要旨集, 15th, 92,   2015 05 26 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201502261342230390
  • カイコ幼虫を用いた活性型PKBの発現と精製, MAESAKI RYOKO, SATO RYOSUKE, TAOKA MANGO, KANEBA TEPPEI, ASANO TSUNAKI, FUJITA CHIHARU, FUJIWARA TOSHINOBU, ITO TAKASHI, ISOBE TOSHIAKI, HAKOSHIMA TOSHIO, MAENAKA KATSUMI, MISHIMA MASAKI, 日本蛋白質科学会年会プログラム・要旨集, 15th, 123,   2015 05 26 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201502289925029962
  • 溶液状態におけるRNA結合タンパク質Nrd1の構造解析, KOBAYASHI AYAHO, NAKANISHI AYAKO, SATO RYOSUKE, FUJIWARA TOSHINOBU, ITO YUTAKA, SUGIURA REIKO, MISHIMA MASAKI, Abstr Annu Meet NMR Soc Jpn, 53rd, 216, 217,   2014 11 04 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201502261166309333
  • cap‐poly(A) mRNAの翻訳におけるHuDとPABPの相関関係, OTSUKA HIROSHI, FUKAO AKIRA, FUJIWARA TOSHINOBU, 日本RNA学会年会要旨集, 16th, 182,   2014 07 23 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201402254231545518
  • ポリオウイルスの細胞種特異的なIRES依存的翻訳の解析, SADAHIRO AKITOSHI, SATO RYOSUKE, FUKAO AKIRA, NOMOTO AKIO, FUJIWARA TOSHINOBU, 日本RNA学会年会要旨集, 16th, 204,   2014 07 23 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201402263307005770
  • HuDとeIF3の相互作用による翻訳制御の解析, TAKATSUJI YOSHIFUMI, FUKAO AKIRA, FUJIWARA TOSHINOBU, 日本RNA学会年会要旨集, 16th, 233,   2014 07 23 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201402267939929990
  • Tissue-specific splicing regulator Fox-1 induces exon skipping by interfering E complex formation on the downstream intron of human F1 gamma gene, Kazuhiro Fukumura, Ayako Kato, Yui Jin, Takashi Ideue, Tetsuro Hirose, Naoyuki Kataoka, Toshinobu Fujiwara, Hiroshi Sakamoto, Kunio Inoue, NUCLEIC ACIDS RESEARCH, 35, 16, 5303, 5311,   2007 08 , 10.1093/nar/gkm569
    Summary:Fox-1 is a regulator of tissue-specific splicing, via binding to the element (U) GCAUG in mRNA precursors, in muscles and neuronal cells. Fox-1 can regulate splicing positively or negatively, most likely depending on where it binds relative to the regulated exon. In cases where the (U) GCAUG element lies in an intron upstream of the alternative exon, Fox-1 protein functions as a splicing repressor to induce exon skipping. Here we report the mechanism of exon skipping regulated by Fox-1, using the hF1 gamma gene as a model system. We found that Fox-1 induces exon 9 skipping by repressing splicing of the downstream intron 9 via binding to the GCAUG repressor elements located in the upstream intron 8. In vitro splicing analyses showed that Fox-1 prevents formation of the pre-spliceosomal early (E) complex on intron 9. In addition, we located a region of the Fox-1 protein that is required for inducing exon skipping. Taken together, our data show a novel mechanism of how RNA-binding proteins regulate alternative splicing.
  • Tissue-specific splicing regulator Fox-1 induces exon skipping by interfering E complex formation on the downstream intron of human F1 gamma gene, Kazuhiro Fukumura, Ayako Kato, Yui Jin, Takashi Ideue, Tetsuro Hirose, Naoyuki Kataoka, Toshinobu Fujiwara, Hiroshi Sakamoto, Kunio Inoue, NUCLEIC ACIDS RESEARCH, 35, 16, 5303, 5311,   2007 08 , 10.1093/nar/gkm569
    Summary:Fox-1 is a regulator of tissue-specific splicing, via binding to the element (U) GCAUG in mRNA precursors, in muscles and neuronal cells. Fox-1 can regulate splicing positively or negatively, most likely depending on where it binds relative to the regulated exon. In cases where the (U) GCAUG element lies in an intron upstream of the alternative exon, Fox-1 protein functions as a splicing repressor to induce exon skipping. Here we report the mechanism of exon skipping regulated by Fox-1, using the hF1 gamma gene as a model system. We found that Fox-1 induces exon 9 skipping by repressing splicing of the downstream intron 9 via binding to the GCAUG repressor elements located in the upstream intron 8. In vitro splicing analyses showed that Fox-1 prevents formation of the pre-spliceosomal early (E) complex on intron 9. In addition, we located a region of the Fox-1 protein that is required for inducing exon skipping. Taken together, our data show a novel mechanism of how RNA-binding proteins regulate alternative splicing.
  • Distribution of U3 small nucleolar RNA and fibrillarin during early embryogenesis in Caenorhabditis elegans, Yumi Sasano, Yusuke Hokii, Kunio Inoue, Hiroshi Sakamoto, Chisato Ushida, Toshinobu Fujiwara, BIOCHIMIE, 90, 6, 898, 907,   2008 06 , 10.1016/j.biochi.2008.02.001
    Summary:U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis. (C) 2008 Published by Elsevier Masson SAS.
  • Distribution of U3 small nucleolar RNA and fibrillarin during early embryogenesis in Caenorhabditis elegans, Yumi Sasano, Yusuke Hokii, Kunio Inoue, Hiroshi Sakamoto, Chisato Ushida, Toshinobu Fujiwara, BIOCHIMIE, 90, 6, 898, 907,   2008 06 , 10.1016/j.biochi.2008.02.001
    Summary:U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis. (C) 2008 Published by Elsevier Masson SAS.
  • Neuronal RNA binding protein HuD interact with protein kinase B,   2007
  • Movement of Caenorhabditis elegans nucleolar small RNAs in eggs before and after the fertilization,   2007
  • Mechanism of pre-ribosomal RNA processing in Caenorhabditis elegans,   2007
  • Neuronal RNA binding protein HuD interact with protein kinase B,   2007
  • Differential regulation of germline mRNAs in soma and germ cells by zebrafish miR-430, Yuichlro Mishima, Antonio J. Giraldez, Yasuaki Takeda, Toshinobu Fujiwara, Hiroshi Sakamoto, Alexander F. Schier, Kunio Inoue, CURRENT BIOLOGY, 16, 21, 2135, 2142,   2006 11 , 10.1016/j.cub.2006.08.086
    Summary:Early in development, primordial germ cells (PGCs) are set aside from somatic cells and acquire a unique gene-expression program [1].The mechanisms underlying germline-specific gene expression are largely unknown. Nanos expression is required during germline development [2-5] and is posttranscriptionally restricted to PGCs [4,6-8]. Here we report that the microRNA miR-430 targets the 3' untranslated region (UTR) of nanos1 during zebrafish embryogenesis. A miR430 target site within the nanos1 3' UTR reduces poly(A) tail length, mRNA stability, and translation. Repression is disrupted in maternal-zygotic dicer mutants (MZdicer), which lack mature miRNAs [9], and is restored by injection of processed miR-430. Although miR-430 represses other genes equally in germline and soma, specific regions in the nanos1 31 UTR compensate for microRNA-mediated repression in PGCs and allow germline-specific expression. We show that the 3' UTR of an additional PGC-specific gene, TDRD7, is also targeted by miR-430. These results indicate that miR-430 targets the 3' UTRs of germline genes and suggest that differential susceptibility to microRNAs contributes to tissue-specific gene expression.
  • Differential regulation of germline mRNAs in soma and germ cells by zebrafish miR-430, Yuichlro Mishima, Antonio J. Giraldez, Yasuaki Takeda, Toshinobu Fujiwara, Hiroshi Sakamoto, Alexander F. Schier, Kunio Inoue, CURRENT BIOLOGY, 16, 21, 2135, 2142,   2006 11 , 10.1016/j.cub.2006.08.086
    Summary:Early in development, primordial germ cells (PGCs) are set aside from somatic cells and acquire a unique gene-expression program [1].The mechanisms underlying germline-specific gene expression are largely unknown. Nanos expression is required during germline development [2-5] and is posttranscriptionally restricted to PGCs [4,6-8]. Here we report that the microRNA miR-430 targets the 3' untranslated region (UTR) of nanos1 during zebrafish embryogenesis. A miR430 target site within the nanos1 3' UTR reduces poly(A) tail length, mRNA stability, and translation. Repression is disrupted in maternal-zygotic dicer mutants (MZdicer), which lack mature miRNAs [9], and is restored by injection of processed miR-430. Although miR-430 represses other genes equally in germline and soma, specific regions in the nanos1 31 UTR compensate for microRNA-mediated repression in PGCs and allow germline-specific expression. We show that the 3' UTR of an additional PGC-specific gene, TDRD7, is also targeted by miR-430. These results indicate that miR-430 targets the 3' UTRs of germline genes and suggest that differential susceptibility to microRNAs contributes to tissue-specific gene expression.
  • Regulation of 18S ribosomal RNA processing in Caenorhabditis elegans.,   2005
  • Regulation of alternative splicing by Fox-1 via binding to GCAUG sequence upstream of alternative exon.,   2005
  • MRG-1, A C. ELEGANS CHROMODOMAIN PROTEIN IS ESSENTIAL FOR PROPER GENE EXPRESSION IN THE PRIMORDIAL GERM CELLS.,   2005
  • TAP/NXF1, the primary mRNA export receptor, specifically interacts with a neuronal RNA-binding protein HuD, K Saito, T Fujiwara, J Katahira, K Inoue, H Sakamoto, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 321, 2, 291, 297,   2004 08 , 10.1016/j.bbrc.2004.06.140
    Summary:Hu proteins are RNA-binding proteins that are implicated in the control of stabilization, nuclear export, and/or translation of specific mRNAs with AU-rich elements (AREs) in the 3'-untranslated region. Three neuron-specific Hu proteins (HuD, HuB, and HuC), but not a ubiquitously expressed Hu protein HuR, have an activity to induce neurite outgrowth when they are overexpressed in a rat neuronal cell line PC12. Here we show that TAP/NXF1, the primary export receptor for the bulk mRNA, is a specific binding partner for HuD. In vitro binding experiments using recombinant proteins revealed that the interaction between TAP and HuD is direct and that HuD can form a ternary complex together with both TAP and RNA. Interestingly, HuR does not interact with TAP. These results suggest that HuD acts as a novel adaptor protein to recruit TAP for efficient export of ARE-containing mRNAs in neuronal cells. (C) 2004 Elsevier Inc. All rights reserved.
  • TAP/NXF1, the primary mRNA export receptor, specifically interacts with a neuronal RNA-binding protein HuD, K Saito, T Fujiwara, J Katahira, K Inoue, H Sakamoto, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 321, 2, 291, 297,   2004 08 , 10.1016/j.bbrc.2004.06.140
    Summary:Hu proteins are RNA-binding proteins that are implicated in the control of stabilization, nuclear export, and/or translation of specific mRNAs with AU-rich elements (AREs) in the 3'-untranslated region. Three neuron-specific Hu proteins (HuD, HuB, and HuC), but not a ubiquitously expressed Hu protein HuR, have an activity to induce neurite outgrowth when they are overexpressed in a rat neuronal cell line PC12. Here we show that TAP/NXF1, the primary export receptor for the bulk mRNA, is a specific binding partner for HuD. In vitro binding experiments using recombinant proteins revealed that the interaction between TAP and HuD is direct and that HuD can form a ternary complex together with both TAP and RNA. Interestingly, HuR does not interact with TAP. These results suggest that HuD acts as a novel adaptor protein to recruit TAP for efficient export of ARE-containing mRNAs in neuronal cells. (C) 2004 Elsevier Inc. All rights reserved.
  • RBD-1, a nucleolar RNA-binding protein, is essential for Caenorhabditis elegans early development through 18S ribosomal RNA processing, E Saijou, T Fujiwara, T Suzaki, K Inoue, H Sakamoto, NUCLEIC ACIDS RESEARCH, 32, 3, 1028, 1036,   2004 02 , 10.1093/nar/gkh264
    Summary:RBD-1 is the Caenorhabditis elegans homolog of Mrd1p, which was recently shown to be required for 18S ribosomal RNA (rRNA) processing in yeast. To gain insights into the relationship between ribosome biogenesis and the development of multicellular organisms, we examined the expression and function of RBD-1. Maternal RBD-1 in the fertilized egg disappears immediately after cleavage starts, whereas zygotic RBD-1 first appears in late embryos and is localized in the nucleolus in most cells, although zygotic transcription of pre-rRNA is known to be initiated as early as the one-cell stage. RNA interference of the rbd-1gene severely inhibits the processing of 18S rRNA in association with various developmental abnormalities, indicating its essential role in pre-rRNA processing and development in C. elegans. These results provide evidence for the linkage between ribosome biogenesis and the control of development and imply unexpected uncoupling of transcription and processing of pre-rRNA in early C. elegans embryos.
  • RBD-1, a nucleolar RNA-binding protein, is essential for Caenorhabditis elegans early development through 18S ribosomal RNA processing, E Saijou, T Fujiwara, T Suzaki, K Inoue, H Sakamoto, NUCLEIC ACIDS RESEARCH, 32, 3, 1028, 1036,   2004 02 , 10.1093/nar/gkh264
    Summary:RBD-1 is the Caenorhabditis elegans homolog of Mrd1p, which was recently shown to be required for 18S ribosomal RNA (rRNA) processing in yeast. To gain insights into the relationship between ribosome biogenesis and the development of multicellular organisms, we examined the expression and function of RBD-1. Maternal RBD-1 in the fertilized egg disappears immediately after cleavage starts, whereas zygotic RBD-1 first appears in late embryos and is localized in the nucleolus in most cells, although zygotic transcription of pre-rRNA is known to be initiated as early as the one-cell stage. RNA interference of the rbd-1gene severely inhibits the processing of 18S rRNA in association with various developmental abnormalities, indicating its essential role in pre-rRNA processing and development in C. elegans. These results provide evidence for the linkage between ribosome biogenesis and the control of development and imply unexpected uncoupling of transcription and processing of pre-rRNA in early C. elegans embryos.
  • Microubule association of a neuronal RNA-binding protein HuD through its binding to the light chain of MAP1B.,   2003
  • Functional basis for promotion of neuronal differentiation by the ELAV protein HuD.,   2003