Updated on 2022/11/25

写真b

 
IWAKAWA Hirooki
 
*Items subject to periodic update by Rikkyo University (The rest are reprinted from information registered on researchmap.)
Affiliation*
College of Science Department of Life Science
Graduate School of Science Doctoral Program in Life Science
Graduate School of Science Master's Program in Life Science
Title*
Associate Professor
Degree
Doctor of Agriculture ( 3 2010   Kyoto University )
Research Theme*
  • 20–30塩基長の小分子RNAを介したRNAサイレンシングは、配列特異的な遺伝子発現抑制機構であり、分化、発生、ストレス応答などを制御するだけでなく、ウイルスやトランスポゾンなどの非自己核酸の抑制においても中心的な役割を果たす。私達は、生化学、分子生物学、生命情報科学を組み合わせたアプローチで「小分子RNAが働くしくみ」を研究している。

  • Research Interests
  • RNA

  • RNAサイレンシング

  • 翻訳

  • RNAi

  • microRNA

  • 生化学

  • 植物生理学

  • 植物病理学

  • ウイルス学

  • Campus Career*
    • 4 2022 - Present 
      College of Science   Department of Life Science   Associate Professor
    • 4 2022 - Present 
      Graduate School of Science   Master's Program in Life Science   Associate Professor
    • 4 2022 - Present 
      Graduate School of Science   Doctoral Program in Life Science   Associate Professor
     

    Research Areas

    • Life Science / Molecular biology  / RNA

    • Life Science / Molecular biology  / 遺伝子発現制御

    • Life Science / Plant molecular biology and physiology

    • Environmental Science/Agriculture Science / Plant protection science  / 植物病理学

    Research History

    • 4 2022 - Present 
      Rikkyo University   Graduate School of Science Field of Study: Life Science   Associate Professor

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    • 4 2022 - Present 
      Rikkyo University   Graduate School of Science Field of Study: Life Science   Associate Professor

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    • 4 2022 - Present 
      Rikkyo University   College of Science Department of Life Science   Associate Professor

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      Country:Japan

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    • 4 2019 - 3 2022 
      The University of Tokyo   Institute for Quantitative Biosciences (IQB)   Lecturer

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    • 10 2018 - 3 2022 
      JST   PRESTO researcher

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    • 4 2018 - 3 2019 
      The University of Tokyo   Institute for Quantitative Biosciences (IQB)   Research assistant professor

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    • 4 2015 - 3 2019 
      The University of Tokyo   Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences   Research assistant professor

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    • 4 2013 - 3 2018 
      The University of Tokyo   IMCB   Research Assistant Professor

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    • 4 2010 - 3 2013 
      The University of Tokyo   Institute of Molecular and Cellular Biosciences

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    • 4 2007 - 3 2010 
      Kyoto University

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    Education

    • 4 2007 - 3 2010 
      京都大学大学院   農学研究科   応用生物科学専攻・博士課程

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    • 4 2005 - 3 2007 
      京都大学大学院   農学研究科   応用生物科学専攻・修士課程

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    • 4 2001 - 3 2005 
      Kyoto University   Faculty of Agriculture   Faculty of Agriculture

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    Awards

    • 2018  
      Ministry of Education, Culture, Sports, Science and Technology  The Young Scientists’ Prize 
       
      IWAKAWA HIROOKI

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    • 2013  
      日本生化学会  鈴木紘一メモリアル賞 
       
      岩川 弘宙

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    • 2006  
      日本植物病理学会  第一回(平成18年度)学生優秀発表賞 
       
      岩川 弘宙

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    Papers

    • The mechanisms of siRNA selection by plant Argonaute proteins triggering DNA methylation

      Wei Liu, Keisuke Shoji, Masahiro Naganuma, *Yukihide Tomari, *Hiro-oki Iwakawa

      bioRxiv   1 6 2022

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      Authorship:Last author, Corresponding author   Language:English  

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    • Functional specialization of monocot DCL3 and DCL5 proteins through the evolution of the PAZ domain Peer-reviewed

      Shirui Chen, Wei Liu, Masahiro Naganuma, *Yukihide Tomari, *Hiro-oki Iwakawa

      Nucleic Acids Research   5 4 2022

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      Authorship:Last author, Corresponding author   Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press (OUP)  

      Abstract

      Monocot DICER-LIKE3 (DCL3) and DCL5 produce distinct 24-nt small interfering RNAs (siRNAs), heterochromatic siRNAs (hc-siRNAs) and phased secondary siRNAs (phasiRNAs), respectively. The former small RNAs are linked to silencing of transposable elements and heterochromatic repeats, and the latter to reproductive processes. It is assumed that these DCLs evolved from an ancient ‘eudicot-type’ DCL3 ancestor, which may have produced both types of siRNAs. However, how functional differentiation was achieved after gene duplication remains elusive. Here, we find that monocot DCL3 and DCL5 exhibit biochemically distinct preferences for 5′ phosphates and 3′ overhangs, consistent with the structural properties of their in vivo double-stranded RNA substrates. Importantly, these distinct substrate specificities are determined by the PAZ domains of DCL3 and DCL5, which have accumulated mutations during the course of evolution. These data explain the mechanism by which these DCLs cleave their cognate substrates from a fixed end, ensuring the production of functional siRNAs. Our study also indicates how plants have diversified and optimized RNA silencing mechanisms during evolution.

      DOI: 10.1093/nar/gkac223

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    • Life of RISC: Formation, action, and degradation of RNA-induced silencing complex Invited Peer-reviewed

      *Hiro-oki Iwakawa, *Yukihide Tomari

      Molecular Cell82 ( 1 ) 30 - 43   1 2022

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      Authorship:Lead author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

      DOI: 10.1016/j.molcel.2021.11.026

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    • Cell-free reconstitution reveals the molecular mechanisms for the initiation of secondary siRNA biogenesis in plants Peer-reviewed International journal

      Yuriki Sakurai, Kyungmin Baeg, Andy Y. W. Lam, Keisuke Shoji, *Yukihide Tomari, *Hiro-oki Iwakawa

      Proceedings of the National Academy of Sciences118 ( 31 ) e2102889118 - e2102889118   3 8 2021

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      Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Proceedings of the National Academy of Sciences  

      Secondary small interfering RNA (siRNA) production, triggered by primary small RNA targeting, is critical for proper development and antiviral defense in many organisms. RNA-dependent RNA polymerase (RDR) is a key factor in this pathway. However, how RDR specifically converts the targets of primary small RNAs into double-stranded RNA (dsRNA) intermediates remains unclear. Here, we develop an in vitro system that allows for dissection of the molecular mechanisms underlying the production of trans-acting siRNAs, a class of plant secondary siRNAs that play roles in organ development and stress responses. We find that a combination of the dsRNA-binding protein, SUPPRESSOR OF GENE SILENCING3; the putative nuclear RNA export factor, SILENCING DEFECTIVE5, primary small RNA, and Argonaute is required for physical recruitment of RDR6 to target RNAs. dsRNA synthesis by RDR6 is greatly enhanced by the removal of the poly(A) tail, which can be achieved by the cleavage at a second small RNA-binding site bearing appropriate mismatches. Importantly, when the complementarity of the base pairing at the second target site is too strong, the small RNA–Argonaute complex remains at the cleavage site, thereby blocking the initiation of dsRNA synthesis by RDR6. Our data highlight the light and dark sides of double small RNA targeting in the secondary siRNA biogenesis.

      DOI: 10.1073/pnas.2102889118

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      Other Link: https://syndication.highwire.org/content/doi/10.1073/pnas.2102889118

    • Ribosome stalling caused by the Argonaute-microRNA-SGS3 complex regulates the production of secondary siRNAs in plants Peer-reviewed International journal

      *Hiro-oki Iwakawa, Andy Y.W. Lam, Akira Mine, Tomoya Fujita, Kaori Kiyokawa, Manabu Yoshikawa, Atsushi Takeda, Shintaro Iwasaki, Yukihide Tomari

      Cell Reports35 ( 13 ) 109300 - 109300   29 6 2021

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      Authorship:Lead author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

      <title>Abstract</title>The path of ribosomes on mRNAs can be impeded by various obstacles. One such example is halting of ribosome movement by microRNAs, though the exact mechanism and physiological role remain unclear. Here, we find that ribosome stalling caused by the Argonaute-miRNA-SGS3 complex regulates production of secondary siRNA biogenesis in plants. We show that the double-stranded RNA-binding protein, SGS3, directly interacts with the 3′ end of the microRNA-Argonaute complex, resulting in ribosome stalling. Strikingly, microRNA-mediated ribosome stalling enhances production of secondary small interfering RNAs (siRNAs) from target mRNAs. Our results uncover a previously uncharacterized role for paused ribosomes in regulation of small RNA function that may have broad biological implications across the plant kingdom.

      DOI: 10.1016/j.celrep.2021.109300

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    • Plant 22-nt siRNAs mediate translational repression and stress adaptation Peer-reviewed International coauthorship International journal

      Huihui Wu, Bosheng Li, Hiro-oki Iwakawa, Yajie Pan, Xianli Tang, Qianyan Ling-hu, Yuelin Liu, Shixin Sheng, Li Feng, Hong Zhang, Xinyan, Zhang, Zhonghua Tang, Xinli Xia, Jixian Zhai, Hongwei Guo

      Nature581   89 - 93   5 2020

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      DOI: 10.1038/s41586-020-2231-y

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    • In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6 Invited Peer-reviewed International journal

      Kyungmin Baeg, Yukihide Tomari, *Hiro-oki Iwakawa.

      Bio-protocol8 ( 1 )   1 2018

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      Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

      DOI: 10.21769/BioProtoc.2673

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    • Silencing messages in a unique way Invited Peer-reviewed

      *Hiro-oki Iwakawa and *Yukihide Tomari.

      Nature Plants3 ( 10 ) 769 - 770   10 2017

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      Authorship:Lead author, Corresponding author   Language:English   Publisher:NATURE PUBLISHING GROUP  

      DOI: 10.1038/s41477-017-0028-2

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    • Biochemical and single-molecule analyses of the RNA silencing suppressing activity of CrPV-1A Peer-reviewed

      Mariko Watanabe, Hiro-oki Iwakawa, Hisashi Tadakuma, Yukihide Tomari

      Nucleic Acids Research45 ( 18 ) 10837 - 10844   10 2017

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

      Viruses often encode viral silencing suppressors (VSSs) to counteract the hosts' RNA silencing activity. The cricket paralysis virus 1A protein (CrPV-1A) is a unique VSS that binds to a specific Argonaute protein (Ago)-the core of the RNA-induced silencing complex (RISC)-in insects to suppress its target cleavage reaction. However, the precise molecular mechanism of CrPV-1A action remains unclear. Here we utilized biochemical and single-molecule imaging approaches to analyze the effect of CrPV-1A during target recognition and cleavage by Drosophila Ago2-RISC. Our results suggest that CrPV-1A obstructs the initial target searching by Ago2-RISC via base pairing in the seed region. The combination of biochemistry and single-molecule imaging may help to pave the way for mechanistic understanding of VSSs with diverse functions.

      DOI: 10.1093/nar/gkx748

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    • Requirement for eukaryotic translation initiation factors in cap-independent translation differs between bipartite genomic RNAs of red clover necrotic mosaic virus Peer-reviewed

      Yuri Tajima, Hiro-oki Iwakawa, Kiwamu Hyodo, Masanori Kaido, Kazuyuki Mise, Tetsuro Okuno

      Virology509   152 - 158   9 2017

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

      The bipartite genomic RNAs of red clover necrotic mosaic virus (RCNMV) lack a 5' cap and a 3' poly(A) tail. RNA1 encodes viral replication proteins, and RNA2 encodes a movement protein (MP). These proteins are translated in a cap-independent manner. We previously identified two cis-acting RNA elements that cooperatively recruit eukaryotic translation initiation factor (eIF) complex eIF4F or eIFiso4F to RNA1. Such cis-acting RNA elements and host factors have not been identified in RNA2. Here we found that translation of RNA1 was significantly compromised in Arabidopsis thaliana carrying eif4f mutation. RNA1 replicated efficiently in eifiso4fl mutants, suggesting vigorous translation of the replication proteins from RNA1 in the plants. In contrast, MP accumulation was decreased in eifiso4f1 mutants but not in eif4f mutants. Collectively, these results suggest that RCNMV uses different eIF complexes for translation of its bipartite genomic RNAs, which may contribute to fine-tuning viral gene expression during infection.

      DOI: 10.1016/j.virol.2017.06.015

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    • In Vitro Analysis of ARGONAUTE-Mediated Target Cleavage and Translational Repression in Plants Invited

      Yukihide Tomari and *Hiro-oki Iwakawa.

      Methods in Molecular Biology1640   55 - 71   6 2017

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      Authorship:Last author, Corresponding author   Language:English   Publishing type:Part of collection (book)   Publisher:Humana Press Inc.  

      MicroRNAs (miRNAs) are endogenous small RNAs, which negatively regulate expression of complementary target genes at the post-transcriptional level. In plants, miRNAs are mainly loaded onto ARGONAUTE1 to form RNA-induced silencing complexes (RISCs), which mediate target mRNA cleavage as well as translational repression. The cell-free system derived from tobacco BY-2 protoplasts has become a powerful tool not only for the analysis of RISC assembly mechanism but also for mechanistic dissection of plant RISC functions. Here we describe the detailed protocols for the preparation of BY-2 cell lysate and the procedure to analyze the dual function of plant RISC—target cleavage and translational repression—in vitro.

      DOI: 10.1007/978-1-4939-7165-7_4

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    • The poly(A) tail blocks RDR6 from converting self mRNAs into substrates for gene silencing Peer-reviewed

      Kyungmin Baeg, *Hiro-oki Iwakawa and *Yukihide Tomari.

      Nature Plants3 ( 4 ) Article number: 17036   4 2017

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      Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

      It remains unclear how post-transcriptional gene silencing (PTGS) in plants discriminates aberrant RNAs from canonical messenger RNAs (mRNAs). The key step of plant PTGS is the conversion of aberrant RNAs into double-stranded RNAs by RNA-DEPENDENT RNA POLYMERASE6 (RDR6). Here, we show that RDR6 itself selects aberrant poly(A)-less mRNAs over canonical polyadenylated mRNAs as templates at the initiation step of complementary strand synthesis. This mechanism can be viewed as an innate safeguard against 'self-attack' by PTGS.

      DOI: 10.1038/nplants.2017.36

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    • The Functions of MicroRNAs: mRNA Decay and Translational Repression. Invited Peer-reviewed

      Hiro-oki Iwakawa, Yukihide Tomari

      Trends in cell biology25 ( 11 ) 651 - 665   11 2015

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      Authorship:Lead author   Language:English   Publisher:ELSEVIER SCIENCE LONDON  

      MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs, which regulate complementary mRNAs by inducing translational repression and mRNA decay. Although this dual repression system seems to operate in both animals and plants, genetic and biochemical studies suggest that the mechanism underlying the miRNA-mediated silencing is different in the two kingdoms. Here, we review the recent progress in our understanding of how miRNAs mediate translational repression and mRNA decay, and discuss the contributions of the two silencing modes to the overall silencing effect in both kingdoms.

      DOI: 10.1016/j.tcb.2015.07.011

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    • MicroRNAs Block Assembly of eIF4F Translation Initiation Complex in Drosophila Peer-reviewed

      #Takashi Fukaya, #Hiro-oki Iwakawa, Yukihide Tomari.

      Molecular Cell56 ( 1 ) 67 - 78   10 2014

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      Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:CELL PRESS  

      miRNAs silence their complementary target mRNAs by translational repression as well as by poly(A) shortening and mRNA decay. In Drosophila, miRNAs are typically incorporated into Argonaute1 (Ago1) to form the effector complex called RNA-induced silencing complex (RISC). Ago1-RISC associates with a scaffold protein GW182, which recruits additional silencing factors. We have previously shown that miRNAs repress translation initiation by blocking formation of the 48S and 80S ribosomal complexes. However, it remains unclear how ribosome recruitment is impeded. Here, we examined the assembly of translation initiation factors on the target mRNA under repression. We show that Ago1-RISC induces dissociation of eIF4A, a DEAD-box RNA helicase, from the target mRNA without affecting 50 cap recognition by eIF4E in a manner independent of GW182. In contrast, direct tethering of GW182 promotes dissociation of both eIF4E and eIF4A. We propose that miRNAs act to block the assembly of the eIF4F complex during translation initiation.

      DOI: 10.1016/j.molcel.2014.09.004

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    • Molecular insights into microRNA-mediated translational repression in plants. Peer-reviewed

      Hiro-oki Iwakawa and Yukihide Tomari.

      Molecular cell52 ( 4 ) 591 - 601   11 2013

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      Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:CELL PRESS  

      microRNAs (miRNAs) bind Argonaute proteins in order to form RNA-induced silencing complexes (RISCs) that can silence the expression of complementary mRNAs. Plant miRNAs can mediate the cleavage of their target mRNAs as well as the repression of their translation. Here, by using an in vitro system prepared from plant culture cells, we biochemically dissect the mechanisms by which Arabidopsis thaliana ARGONAUTE1 RISC (AtAGO1-RISC) silences its mRNA targets. We find that AtAGO1 RISC has the ability to repress translation initiation without promoting deadenylation or mRNA decay. Strikingly, AtAGO1-RISC bound in the 5' untranslated region or the open reading frame can sterically block the recruitment or movement of ribosonnes. These silencing effects require more extensive base pairing to the target site in comparison to typical animal miRNAs. Our data provide mechanistic insights into miRNA-mediated translational repression in plants.

      DOI: 10.1016/j.molcel.2013.10.033

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    • Arabidopsis ARGONAUTE7 selects miR390 through multiple checkpoints during RISC assembly. Peer-reviewed

      Yayoi Endo, *Hiro-oki Iwakawa and *Yukihide Tomari.

      EMBO reports14 ( 7 ) 652 - 658   7 2013

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      Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

      Plant ARGONAUTE7 (AGO7) assembles RNA-induced silencing complex (RISC) specifically with miR390 and regulates the auxin-signalling pathway via production of TAS3 trans-acting siRNAs (tasiRNAs). However, how AGO7 discerns miR390 among other miRNAs remains unclear. Here, we show that the 5' adenosine of miR390 and the central region of miR390/miR390* duplex are critical for the specific interaction with AGO7. Furthermore, despite the existence of mismatches in the seed and central regions of the duplex, cleavage of the miR390* strand is required for maturation of AGO7-RISC. These findings suggest that AGO7 uses multiple checkpoints to select miR390, thereby circumventing promiscuous tasiRNA production.

      DOI: 10.1038/embor.2013.73

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    • Poly(A)-Binding Protein Facilitates Translation of an Uncapped/Nonpolyadenylated Viral RNA by Binding to the 3 ' Untranslated Region Peer-reviewed

      Hiro-oki Iwakawa, Yuri Tajima, Takako Taniguchi, Masanori Kaido, Kazuyuki Mise, Yukihide Tomari, Hisaaki Taniguchi and Tetsuro Okuno.

      Journal of Virology86 ( 15 ) 7836 - 7849   8 2012

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      Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC MICROBIOLOGY  

      Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5' cap and a 3' poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3' untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3' CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3' CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3' CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3' UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3' CITE-mediated translation of RCNMV RNAl. We also found that both the ARS and 3' CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3 ' UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3' CITE as substitutes for the 3' poly(A) tail and the 5' cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA.

      DOI: 10.1128/JVI.00538-12

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    • A long-distance RNA-RNA interaction plays an important role in programmed-1 ribosomal frameshifting in the translation of p88 replicase protein of Red clover necrotic mosaic virus Peer-reviewed

      Yuri Tajima, Hiro-oki Iwakawa, Masanori Kaido, Kazuyuki Mise, Tetsuro Okuno

      Virology417 ( 1 ) 169 - 178   8 2011

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

      Programmed - 1 ribosomal frameshifting (- 1 PRF) is one viral translation strategy to express overlapping genes in positive-strand RNA viruses. Red clover necrotic mosaic virus ( RCNMV) uses this strategy to express its replicase component protein p88. In this study, we used a cell-free translation system to map cis-acting RNA elements required for 1 PRF. Our results show that a small stem-loop structure adjacent to the cap-independent translation element in the 3&apos; untranslated region (UTR) of RCNMV RNA1 is required for - 1 PRF. Site-directed mutagenesis experiments suggested that this stem-loop regulates - 1 PRF via base-pairing with complementary sequences in a bulged stem-loop adjacent to the shifty site. The existence of RNA elements responsible for - 1 PRF and the cap-independent translation of replicase proteins in the 3&apos; UTR of RNA1 might be important for switching translation to replication and for regulating the ratio of p88 to p27. (C) 2011 Published by Elsevier

      DOI: 10.1016/j.virol.2011.05.012

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    • Identification of amino acids in auxiliary replicase protein p27 critical for its RNA-binding activity and the assembly of the replicase complex in Red clover necrotic mosaic virus. Peer-reviewed

      Kiwamu Hyodo, Akira Mine, Hiro-oki Iwakawa, Masanori Kaido, Kazuyuki Mise and Tetsuro Okuno.

      Virology413 ( 2 ) 300 - 309   5 2011

    • Template Recognition Mechanisms by Replicase Proteins Differ between Bipartite Positive-Strand Genomic RNAs of a Plant Virus Peer-reviewed

      Hiro-oki Iwakawa, Akira Mine, Kiwamu Hyodo, Mengnan An, Masanori Kaido, Kazuyuki Mise, Tetsuro Okuno

      Journal of Virology85 ( 1 ) 497 - 509   1 2011

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      Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC MICROBIOLOGY  

      Recognition of RNA templates by viral replicase proteins is one of the key steps in the replication process of all RNA viruses. However, the mechanisms underlying this phenomenon, including primary RNA elements that are recognized by the viral replicase proteins, are not well understood. Here, we used aptamer pulldown assays with membrane fractionation and protein-RNA coimmunoprecipitation in a cell-free viral translation/replication system to investigate how viral replicase proteins recognize the bipartite genomic RNAs of the Red clover necrotic mosaic virus (RCNMV). RCNMV replicase proteins bound specifically to a Y-shaped RNA element (YRE) located in the 3&apos; untranslated region (UTR) of RNA2, which also interacted with the 480-kDa replicase complexes that contain viral and host proteins. The replicase-YRE interaction recruited RNA2 to the membrane fraction. Conversely, RNA1 fragments failed to interact with the replicase proteins supplied in trans. The results of protein-RNA coimmunoprecipitation assays suggest that RNA1 interacts with the replicase proteins coupled with their translation. Thus, the initial template recognition mechanisms employed by the replicase differ between RCNMV bipartite genomic RNAs and RNA elements are primary determinants of the differential replication mechanism.

      DOI: 10.1128/JVI.01754-10

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    • A Y-shaped RNA structure in the 3' untranslated region together with the trans-activator and core promoter of Red clover necrotic mosaic virus RNA2 is required for its negative-strand RNA synthesis. Peer-reviewed

      Mengnan An, Hiro-oki Iwakawa, Akira Mine, Masanori Kaido, Kazuyuki Mise and Tetsuro Okuno.

      Virology405 ( 1 ) 100 - 109   9 2010

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      Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA virus with a bipartite genome RNA1 encodes N-terminally overlapping replication proteins, p27 and p88 RNA2 is replicated efficiently by the replication proteins supplied in trans, whereas RNA1 needs p88 preferentially in cis for its replication cis-Acting elements required for RNA2 replication have been mapped to the 3&apos; terminal stem-loop structure conserved between RNA I and RNA2, and to the protein-coding region including the trans-activator Here, we have identified a Y-shaped RNA structure with three-way RNA Junctions predicted m the 3&apos; untranslated region of RNA2 as a novel element required for negative-strand synthesis using an in vitro translation/replication system We also show that, in addition to the 3&apos; terminal core promoter, several RNA elements including the trans-activator are also required for negative-strand synthesis Functional roles and structural requirements of these cis-acting elements in RCNMV RNA replication are discussed (C) 2010 Elsevier Inc All rights reserved

      DOI: 10.1016/j.virol.2010.05.022

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    • Host-dependent roles of the viral 5' untranslated region (UTR) in RNA stabilization and cap-independent translational enhancement mediated by the 3' UTR of Red clover necrotic mosaic virus RNA1. Peer-reviewed

      Siriruk Sarawaneeyaruk, Hiro-oki Iwakawa, Hiroyuki Mizumoto, Hiromi Murakami, Masanori Kaido, Kazuyuki Mise and Tetsuro Okuno.

      Virology391 ( 1 ) 107 - 118   8 2009

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

      The genome of Red clover necrotic mosaic virus (RCNMV) consists of RNA1 and RNA2, both lacking a cap structure and a poly(A)tail. RNA1 has a translational enhancer element (3&apos;TE-DR1) in the 3&apos; untranslated region (UTR). In this study, we analyzed the roles of 5&apos; and 3&apos; UTRs of RNA1 in 3&apos;TE-DR1-mediated cap-independent translation in cowpea and tobacco BY-2 protoplasts using a dual-luciferase (LUC) reporter assay system. Most mutations introduced into RNA15&apos; UTR in reporter LUC mRNA abolished or greatly reduced cap-independent translation in BY-2 promplasts, whereas those mutations had no or Much milder effects if any on translational activity in cowpea protoplasts. Our results suggest that a stern-loop structure predicted in the 5&apos; proximal region of RNAI plays important roles in both translation and RNA stability. We also show that 3&apos;TE-DR1-mediated cap-independent translation relies on a ribosome-scanning mechanism in both protoplasts. (C) 2009 Elsevier Inc. All rights reserved.

      DOI: 10.1016/j.virol.2009.05.037

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    • A viral noncoding RNA generated by cis-element-mediated protection against 5'-&gt;3' RNA decay represses both cap-independent and cap-dependent translation. Peer-reviewed

      Hiro-oki Iwakawa, Hiroyuki Mizumoto, Hideaki Nagano, Yuka Imoto, Kazuma Takigawa, Siriruk Sarawaneeyaruk, Masanori Kaido, Kazuyuki Mise and Tetsuro Okuno.

      Journal of virology82 ( 20 ) 10162 - 10174   10 2008

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      Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC MICROBIOLOGY  

      Positive-strand RNA viruses use diverse mechanisms to regulate viral and host gene expression for ensuring their efficient proliferation or persistence in the host. We found that a small viral noncoding RNA (0.4 kb), named SR1f, accumulated in Red clover necrotic mosaic virus (RCNMV)-infected plants and protoplasts and was packaged into virions. The genome of RCNMV consists of two positive-strand RNAs, RNA1 and RNA2. SR1f was generated from the 3' untranslated region (UTR) of RNA1, which contains RNA elements essential for both cap-independent translation and negative-strand RNA synthesis. A 58-nucleotide sequence in the 3' UTR of RNA1 (Seq1f58) was necessary and sufficient for the generation of SR1f. SR1f was neither a subgenomic RNA nor a defective RNA replicon but a stable degradation product generated by Seq1f58-mediated protection against 5'-&gt; 3' decay. SR1f efficiently suppressed both cap-independent and cap-dependent translation both in vitro and in vivo. SR1f trans inhibited negative-strand RNA synthesis of RCNMV genomic RNAs via repression of replicase protein production but not via competition of replicase proteins in vitro. RCNMV seems to use cellular enzymes to generate SR1f that might play a regulatory role in RCNMV infection. Our results also suggest that Seq1f58 is an RNA element that protects the 3'-side RNA sequences against 5'-&gt; 3' decay in plant cells as reported for the poly(G) tract and stable stem-loop structure in Saccharomyces cerevisiae.

      DOI: 10.1128/JVI.01027-08

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    • cis-Preferential requirement of a -1 frameshift product p88 for the replication of Red clover necrotic mosaic virus RNA1. Peer-reviewed

      Kimiyuki Okamoto, Hideaki Nagano, Hiro-oki Iwakawa, Hiroyuki Mizumoto, Atsushi Takeda, Masanori Kaido, Kazuyuki Mise and Tetsuro Okuno.

      Virology375 ( 1 ) 205 - 212   5 2008

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

      The genome of Red clover necrotic mosaic virus (RCNMV) consists of RNA1 and RNA2. RNA1 encodes N-terminally overlapping replication proteins, p27 and p88, which are translated in a cap-independent manner. The 3' untranslated region of RNA1 contains RNA elements essential for cap-independent translation and negative-strand RNA synthesis. In this study, we investigated how p27 and p88 were engaged in the replication of RCNMV genomic RNAs by using DNA vectors or in vitro transcribed RNAs in protoplasts and in a cell-free extract of evacuolated BY-2 protoplasts. Our results show a cis-preferential requirement of p88, but not of p27, for the replication of RNA1. This mechanism might help to facilitate a switch in the role of RNA1 from mRNA to a replication template. (c) 2008 Elsevier Inc. All rights reserved.

      DOI: 10.1016/j.virol.2008.02.004

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    • cis-Acting core RNA elements required for negative-strand RNA synthesis and cap-independent translation are separated in the 3'-untranslated region of Red clover necrotic mosaic virus RNA1. Peer-reviewed

      Hiro-oki Iwakawa, Masanori Kaido, Kazuyuki Mise and Tetsuro Okuno.

      Virology369 ( 1 ) 168 - 181   12 2007

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      Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)  

      The genome of Red clover necrotic mosaic virus (RCNMV) is positive-sense and divided into RNA1 and RNA2. RNA1 has a translation enhancer element (3′ TE-DR1) in the 3′ untranslated region (UTR) that substitutes for a 5′ cap. In this study, we determined the regions required for cap-independent translation and RNA synthesis in the 3′ UTR of RNA1 using a cell-free extract of evacuolated BY-2 protoplasts (BYL) and by an assay in BY-2 protoplasts. The use of capped viral RNA transcripts in the BYL system allowed us to distinguish the effects of introduced mutations on cap-independent translation and negative-strand RNA synthesis of RNA1. We found that the core RNA element of 3′ TE-DR1 essential for cap-independent translation of RNA1 is dispensable for negative-strand RNA synthesis. Thus, cis-acting RNA elements essential for cap-independent translation are separated from those required for negative-strand RNA synthesis in the 3′ UTR of RCNMV RNA1. © 2007 Elsevier Inc. All rights reserved.

      DOI: 10.1016/j.virol.2007.07.016

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    • Cap-independent translation mechanism of red clover necrotic mosaic virus RNA2 differs from that of RNA1 and is linked to RNA replication. Peer-reviewed

      #Hiroyuki Mizumoto, #Hiro-oki Iwakawa, Masanori Kaido, Kazuyuki Mise and Tetsuro Okuno.

      Journal of virology80 ( 8 ) 3781 - 3791   4 2006

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      Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC MICROBIOLOGY  

      The genome of Red clover necrotic mosaic virus (RCNMV) in the genus Dianthovirus is divided into two RNA molecules of RNA1 and RNA2, which have no cap structure at the 5' end and no poly(A) tail at the 3' end. The 3' untranslated region (3' UTR) of RCNMV RNA1 contains an essential RNA element (3'TE-DR1), which is required for cap-independent translation. In this study, we investigated a cap-independent translational mechanism of RNA2 using a firefly luciferase (Luc) gene expression assay system in cowpea protoplasts and a cell-free lysate (BYL) prepared from evacuolated tobacco BY2 protoplasts. We were unable to detect cis-acting RNA sequences in RNA2 that can replace the function of a cap structure, such as the 3'TE-DR1 of RNA1 However, the uncapped reporter RNA2, RNA2-Luc, in which the Luc open reading frame (ORF) was inserted between the 5' UTR and the movement protein ORF, was effectively translated in the presence of p27 and p88 in protoplasts in which RNA2-Luc was replicated. Time course experiments in protoplasts showed that the translational activity of RNA2-Luc did not reflect the amount of RNA2. Mutations in cis-acting RNA replication elements of RNA2 abolished the cap-independent translational activity of RNA2-Luc, suggesting that the translational activity of RNA2-Luc is coupled to RNA replication. Our results show that the translational mechanism differs between two segmented genomic RNAs of RCNMV. We present a model in which only RNA2 that is generated de novo through the viral RNA replication machinery functions as mRNA for translation.

      DOI: 10.1128/JVI.80.8.3781-3791.2006

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    Misc.

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    Teaching Experience

    • 4 2019 - 3 2021 
      全学自由研究ゼミナール「生命科学の最前線」(東京大学)

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      全学体験ゼミナール ( 東京大学 )

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    Research Projects

    • 植物RNAiの理解と応用:自在な人工ゲノム発現にむけて

      国立研究開発法人科学技術振興機構(JST)  創発的研究支援事業 

      岩川 弘宙

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      4 2022 - 3 2029

      Authorship:Principal investigator 

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    • 小分子RNA依存的リボソーム停滞機構の統合的理解

      日本学術振興会  科学研究費助成事業 学術変革領域研究(A) 

      岩川 弘宙

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      9 2021 - 3 2023

      Grant number:21H05717

      Grant amount:\10400000 ( Direct Cost: \8000000 、 Indirect Cost:\2400000 )

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    • Evading unwanted RNA silencing: Toward large-scale genome reconstitution

      Japan Science and Technology Agency (JST)  PRESTO 

      IWAKAWA HIROOKI

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      10 2018 - 3 2022

      Authorship:Principal investigator  Grant type:Competitive

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    • 核内小分子RNAの作用メカニズムの解明

      独立行政法人日本学術振興会(JSPS)  若手研究(A) 

      岩川 弘宙

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      4 2016 - 3 2019

      Authorship:Principal investigator  Grant type:Competitive

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    • mRNAとタンパク質の品質管理機構における新生鎖の新規機能の解明

      独立行政法人日本学術振興会(JSPS)  新学術領域研究(研究領域提案型) 

      稲田 利文

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      7 2014 - 3 2019

      Grant type:Competitive

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    • 二次的小分子RNA生成における反応モジュール間連動機構の解明

      独立行政法人日本学術振興会(JSPS)  挑戦的萌芽研究 

      岩川 弘宙

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      4 2015 - 3 2018

      Authorship:Principal investigator  Grant type:Competitive

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    • 植物microRNAによる翻訳抑制機構の解明

      独立行政法人日本学術振興会(JSPS)  研究活動スタート支援 

      岩川 弘宙

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      4 2013 - 3 2015

      Authorship:Principal investigator  Grant type:Competitive

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    • 植物におけるsmall RNA依存的翻訳抑制機構の生化学的解析

      独立行政法人日本学術振興会(JSPS)  特別研究員奨励費 

      岩川 弘宙

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      2010 - 2012

      Authorship:Principal investigator  Grant type:Competitive

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    • ダイアンソウイルスの遺伝子発現制御と複製機構の解明

      独立行政法人日本学術振興会(JSPS)  特別研究員奨励費 

      岩川 弘宙

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      2007 - 2009

      Authorship:Principal investigator  Grant type:Competitive

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