Analysis of the molecular mechanisms of RNA-directed DNA methylation

• Project/Area Number: 16H06159
• Research Category: Grant-in-Aid for Young Scientists (A)
• Allocation Type: Single-year Grants
• Research Field: Molecular biology
• Research Institution: The University of Tokyo
• Principal Investigator: Iwakawa Hiro-oki 東京大学, 定量生命科学研究所, 助教 (60710415)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥24,310,000 (Direct Cost: ¥18,700,000、Indirect Cost: ¥5,610,000); Fiscal Year 2018: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000); Fiscal Year 2017: ¥7,800,000 (Direct Cost: ¥6,000,000、Indirect Cost: ¥1,800,000); Fiscal Year 2016: ¥10,660,000 (Direct Cost: ¥8,200,000、Indirect Cost: ¥2,460,000)
• Keywords: RNAサイレンシング / siRNA / DNA-RNAハイブリッド / RNA-DNAハイブリッド / RNA-DNA ハイブリッド

Outline of Final Research Achievements

We conducted our research by focusing on how RNA-induced silencing complexes (RISCs) mediate sequence specific DNA methylation in nuclei. Through in vitro binding assays, we found that the plant cytoplasmic RISCs interact more tightly with RNAs than with DNAs, conversely, the plant nuclear RISCs bind to DNAs with much higher affinity than to RNAs. Moreover, we obtained novel insights into nuclear RISC assembly and template specificity of RNA-dependent RNA polymerase 6 (RDR6), which is known to be involved in de novo DNA methylation.

Academic Significance and Societal Importance of the Research Achievements

植物の核内RISCがRNAよりもDNAと強く結合することを世界で初めて生化学的に示したことで、「核内RISCは新生RNAとの結合を介して標的DNAに接近しメチル化を促進する」と信じられてきた従来のモデルを書き換える可能性がある。さらに核内RISC形成機構やRDR6の鋳型特異性の生化学的な理解は、未だ多くの謎に包まれている核内RNAサイレンシング機構を理解する上で重要な基盤的知見となる。

Research Products

• [Journal Article] In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6 (2018)
  • Author(s): Kyungmin Baeg, Yukihide Tomari, Hiro-oki Iwakawa
  • Journal Title: Bio-protocol Volume: 8 Issue: 1
  • DOI: 10.21769/bioprotoc.2673
  • Peer Reviewed / Open Access
• [Journal Article] ilencing messages in a unique way (2017)
  • Author(s): Hiro-oki Iwakawa and Yukihide Tomari,
  • Journal Title: Nature Plants, Volume: 3 Issue: 10 Pages: 769-770
  • DOI: 10.1038/s41477-017-0028-2
  • Peer Reviewed
• [Journal Article] In Vitro Analysis of ARGONAUTE-Mediated Target Cleavage and Translational Repression in Plants (2017)
  • Author(s): Tomari Yukihide、Iwakawa Hiro-oki
  • Journal Title: Methods in Molecular Biology Volume: 1640 Pages: 55-71
  • DOI: 10.1007/978-1-4939-7165-7_4
  • ISBN: 9781493971640, 9781493971657
  • Peer Reviewed
• [Journal Article] 植物のRNAサイレンシング機構が自己のmRNAを攻撃しないメカニズム (2017)
  • Author(s): 岩川 弘宙
  • Journal Title: BSJ-Review Volume: 8 Issue: B Pages: 58-70
  • DOI: 10.24480/bsj-review.8b4.00114
  • ISSN: 2432-9819
  • Peer Reviewed / Open Access
• [Journal Article] The poly(A) tail blocks RDR6 from converting self mRNAs into substrates for gene silencing. (2017)
  • Author(s): Baeg K, *Iwakawa HO, *Tomari Y.
  • Journal Title: Nature Plants Volume: 3 Issue: 4 Pages: 17036-17036
  • DOI: 10.1038/nplants.2017.36
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] In vitro recapitulation of the secondary siRNA biogenesis in plants (2019)
  • Author(s): Hiro-oki Iwakawa, Kyungmin Baeg, Yuriki Sakurai, Manabu Yoshikawa, Yukihide Tomari
  • Organizer: 第20回武田科学振興財団生命科学シンポジウム RNAネオバイオロジー
  • Int'l Joint Research
• [Presentation] In vitro recapitulation of the secondary siRNA biogenesis in plants (2019)
  • Author(s): Kyungmin Baeg, Yuriki Sakurai, Andy Lam, Keisuke Shoji, Manabu Yoshikawa, Yukihide Tomari, Hiro-oki Iwakawa
  • Organizer: POST-TRANSCRIPTIONAL GENE REGULATION IN PLANTS (PGRP) 2019 Nara
  • Int'l Joint Research
• [Presentation] The PIWI domain of RdDM Argonautes in plants plays a critical role for sensing the identity of the 5′-base of small RNAs (2019)
  • Author(s): Wei Liu, Yukihide Tomari, Hiro-oki Iwakawa
  • Organizer: POST-TRANSCRIPTIONAL GENE REGULATION IN PLANTS (PGRP) 2019 Nara
• [Presentation] 植物のRNAサイレンシング機構 (2018)
  • Author(s): 岩川弘宙
  • Organizer: 第53回 植物感染生理談話会
  • Invited
• [Presentation] 植物における二次的小分子RNA生成の試験管内再現 (2018)
  • Author(s): Baeg Kyungmin、櫻井 友理希、吉川 学、泊 幸秀、岩川 弘宙
  • Organizer: 第20回日本RNA学会年会
• [Presentation] In vitro analysis of small RNA preference of ARGONAUTE 4 in Arabidopsis thaliana (2018)
  • Author(s): Wei Liu, Yukihide Tomari, Hiro-oki Iwakawa
  • Organizer: 第59回日本植物生理学会年会
• [Presentation] Plant ARGONAUTE4 family proteins prefer to bind to DNA targets in vitro (2017)
  • Author(s): Iwakawa HO, Tomari Y
  • Organizer: The 22nd Annual Meeting of the RNA Society
  • Int'l Joint Research
• [Presentation] The poly(A) tail blocks RDR6 from converting self mRNAs into the substrates for gene silencing (2017)
  • Author(s): Baeg K, Iwakawa HO, Tomari Y
  • Organizer: The 22nd Annual Meeting of the RNA Society
  • Int'l Joint Research
• [Presentation] Plant ARGONAUTE4 family proteins prefer to bind to DNA targets in vitro (2017)
  • Author(s): Iwakawa HO, Tomari Y
  • Organizer: 12th Microsymposium on small RNA biology
  • Int'l Joint Research
• [Presentation] The poly(A) tail blocks RDR6 from converting self mRNAs into the substrates for gene silencing (2017)
  • Author(s): Baeg K, Iwakawa HO, Tomari Y
  • Organizer: 12th Microsymposium on small RNA biology
  • Int'l Joint Research
• [Presentation] Plant ARGONAUTE4 family proteins prefer to bind to DNA targets in vitro (2016)
  • Author(s): Hiro-oki Iwakawa, Yukihide Tomari
  • Organizer: The 2016 joint annual meeting of the RNA Society and the RNA Society of Japan
  • Int'l Joint Research
• [Presentation] 植物のRNAサイレンシング機構 (2016)
  • Author(s): 岩川 弘宙
  • Organizer: 第39回日本分子生物学会
  • Invited
• [Presentation] 生化学で迫る植物の RNA サイレンシング (2016)
  • Author(s): 岩川 弘宙
  • Organizer: 日本植物学会第80回大会
  • Invited
• [Remarks] researchmap
  • URL: https://researchmap.jp/Hiro_Iwa/
• [Remarks] researchmap 岩川 弘宙
  • URL: https://researchmap.jp/Hiro_Iwa/