Analysis of mRNP remodeling during NMD

• Project/Area Number: 19770151
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Single-year Grants
• Research Field: Molecular biology
• Research Institution: Yokohama City University
• Principal Investigator: 山下 暁朗 Yokohama City University, 医学研究科, 客員准教授 (20405020)
• Project Period (FY): 2007 – 2008
• Project Status: Completed (Fiscal Year 2008)
• Budget Amount: ¥3,820,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥420,000); Fiscal Year 2008: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2007: ¥2,000,000 (Direct Cost: ¥2,000,000)
• Keywords: 遺伝情報複製 / 転写装置 / 再編 / 制御

Research Abstract

本研究により、動物細胞内からの高精度mRNP複合体精製システムを新たに構築することに成功した。具体的には、mRNP精製のためのタグであるBoxB配列を挿入したbeta-globin遺伝子由来のmRNA(翻訳型mRNA)とその5'非翻訳領域に翻訳開始を阻害する強固なステムループを形成する配列を挿入した遺伝子由来のmRNA(非翻訳型mRNA)をmRNP精製アフィニティータグを用いて精製した。mRNP精製アフィニティータグとして、BoxB結合ペプチドであるバクテリオファージNタンパク質(Nペプチド)にStreptavidin binding peptide(SBP)とGSTとFlag epitope tagを融合した物を使用した。この解析により、動物細胞内で翻訳中のリボソームが結合したmRNP(リボソーム、翻訳伸張因子を含む、スプライシング依存的mRNA結合タンパク質複合体であるEJCを含まない)と翻訳されていないmRNP(EJCをふくむ、リボソーム翻訳伸張因子を含まない)の構成因子が大きく異なることを生化学的に明らかとした。この結果は、動物細胞内で翻訳依存的にmRNPの大きな再構成が起こるということを初めて示した物である。

Research Products

• [Journal Article] SMG-8 and SMG-9, two novel subunits of the SMG-1 complex, regulate remodeling of the mRNA surveillance complex during nonsense-mediated mRNA decay. (2009)
  • Author(s): Yamashita A, Izumi N, Kashima I, Ohnishi T, Saari B, Katsuhata Y, Muramatsu R, Morita T, Iwamatsu A, Kurata R, Hachiya T, Hirano H, Anderson P, Ohno S
  • Journal Title: Genes Dev 23(9)(in press)
• [Journal Article] SMG-8 and SMG-9, two novel subunits of the SMG-1 complex, regulate remodeling ofthe mRNA surveillance complex during nonsense-mediated mRNA decay (2009)
  • Author(s): Akio Yamashita, Natsuko Izumi, Isao Kashima, Tetsuo Ohnishi, Bonnie Saari, Yukiko Katsuhata, Reiko Muramatsu, Tomoko Morita, Akihiro Iwamatsu, Rie Kurata, Takahisa Hachiya, Hisashi Hirano, Philip Anderson. and Shigeo Ohno
  • Journal Title: Genes and development 23
  • Peer Reviewed
• [Journal Article] Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells (2007)
  • Author(s): Chen CY, Yamashita Y, Chang TC, Yamashita A, Zhu W, Zhong Z, Shyu AB
  • Journal Title: RNA (10) Pages: 1775-1786
• [Journal Article] Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation. (2007)
  • Author(s): Ezzeddine N, Chang TC, Zhu W, Yamashita A, Chen CY, Zhong Z, Yamashita Y, Zheng D, Shyu AB
  • Journal Title: Mol. Cell. Biol. 27(22) Pages: 7791-7801
• [Journal Article] Distant N- and C-terminal domains are required for intrinsic kinase activity of SMG-1, a critical component of nonsense-mediated mRNA decay. (2007)
  • Author(s): Morita T, Yamashita A, Kashima I, Ogata K, Ishiura S, Ohno S.
  • Journal Title: J Biol Chem. 16 ; 282(11) Pages: 7799-7808
• [Journal Article] Human TOB,an antiproliferative transcription factor,is apoly(A)-binding protein-dependent positive regulator of cyto plasmic mRNA deadenylation. (2007)
  • Author(s): Ezzeddine N, Chang TC, Zhu W, Yamashita A, Chen CY, Zhong Z, Yamashita Y, Zheng D, Shyu AB
  • Journal Title: Mol.Cell.Biol 27 Pages: 7791-7801
  • Peer Reviewed
• [Journal Article] Versatile applications of transcriptional pulsing to study m RNA turnover in mammalian cells (2007)
  • Author(s): Chen CY, Yamashita Y, Chang TC, Yamashita A, Zhu W, Zhong Z, Shyu AB
  • Journal Title: RNA 13 Pages: 1775-1786
  • Peer Reviewed
• [Journal Article] Distant N-andC-terminal domatns are required fbr intrinsic kinase activity of SMG-1,a critical component of nonsense-mediated mRNA decay (2007)
  • Author(s): Morita T, Yamashita A, Kashima I, Ogata K, Ishiura S, Ohno S
  • Journal Title: J.Biol., Chem 282 Pages: 7799-7808
  • Peer Reviewed
• [Presentation] TRANSLATION TERMINATION COMPLEX DURING NONS ENSE- MEDIATED MRNA DECAY (2008)
  • Author(s): Yamashita A, Izumi N, Kashima I, Katsuhata Y, Muramatsu R. Ohno S
  • Organizer: 2008 Cold spring harver labatory meeting on translational control
  • Place of Presentation: New York, USA
  • Year and Date: 2008-09-03
• [Presentation] The formation of SMG-1 : Upf1 : eRF1 : eRF3 complex (SURF) on ribosomes precedes translation dependent Upf1 phosphorylation during nonsense-mediated mRNA decay (2008)
  • Author(s): Yamashita A, Kashima I, Izumi N, Katsuhata Y, Muramatsu R, Ohno S
  • Organizer: THE 21st NAITO CONFERENCE Nuclear Dynamics and RNA [I]
  • Place of Presentation: Yamanashi
  • Year and Date: 2008-06-26
• [Presentation] The formation of SMG-1 : Upfl : eRF1 : eRF3 complex (SURF) on ribosomes precedes translation-dependent-Upfl phosphorylatio n during nonsense-mediated mRNA decay (2008)
  • Author(s): Yamashita A, Kashima I, Izumi N, Katsuhata Y, Muramatsu R, Ohno S
  • Organizer: THE 21st NAITO CONFERENCE "Nuclea Dynamics and RNA [I]
  • Place of Presentation: Yamanashi, Japan
  • Year and Date: 2008-06-26
• [Presentation] TRANSLATION TERMINATION COMPLEX DURING NONSENSE- MEDIATED MRNA DECAY. (2008)
  • Author(s): Yamashita A, Izumi N, Kashima I, Katsuhata Y, Muramatsu R, Ohno S.
  • Organizer: Cold spring harbor laboratory meeting 2008 Translational control
  • Place of Presentation: New York
• [Presentation] 翻訳依存的なmRNA監視複合体形成および再構成がUpf1リン酸化を誘導する (2007)
  • Author(s): 山下 暁朗, 泉 奈津子, 鹿島 勲, 森田 智子, 村松 玲子
  • Organizer: 第30回日本分子生物学会年会〜第80回日本生化学会大会合同大会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2007-12-15
• [Presentation] The SMG-1 : SMG-8 : p60 Complex Regulated Phosphorylation Of Upf1 Is Required For The Dissociation Of eRFs From mRNA Surveillance Complex During Nonsense-mediated mRNA decay. (2007)
  • Author(s): Yamashita A, Izumi N, Kashima I, Ohnishi T, Anderson P, Ohno S
  • Organizer: 第9回日本RNA学会年会
  • Place of Presentation: 名古屋国際会議場
  • Year and Date: 2007-07-31
• [Presentation] The SMG-1:SMG-8:p60 Complex Regulated Phosphorylation Of Upfl Is Required For The Dissociation Of eRFs From mRNA Surveillance Complex During Nonsense-mediated mRNA decay (2007)
  • Author(s): Yamashita A, Izumi N, Kashima I, Ohnishi T, Anderson P, Ohno S
  • Organizer: 第9回日本RNA学会年会
  • Place of Presentation: 名古屋国際会議場
  • Year and Date: 2007-07-31
• [Presentation] The SMG-1:SMG-8:p60 Complex Regulated Phosphorylation Of Upfl Is Required For The Dissociation Of eRFs From mRNA Surveillance Comp lex During Nonsense-mediated mRNA decay (2007)
  • Author(s): Yamashita A, Kashima I, Izumi N, Ohnishi T, Anderson P, Ohno S
  • Organizer: 第12回国際RNA学会年会
  • Place of Presentation: University of Wisconsin,Madison
  • Year and Date: 2007-06-03
• [Presentation] 翻訳依存的なmRNA監視複合体形成および再構成がUpf1リン酸化を誘導する (2007)
  • Author(s): 山下暁朗,泉奈津子,鹿島勲,森田智子,村松玲子,大野茂男
  • Organizer: BMB2007-第30回日本分子生物学会年会・第80回日本生化学会大会合同大会
  • Place of Presentation: 横浜
• [Presentation] The SMG-1 : SMG-8 : p60 Complex Regulated Phosphorylation Of Upf1 Is Required For The Dissociation Of eRFs From mRNA Surveillance Complex During Nonsense-mediated mRNA decay. (2007)
  • Author(s): Yamashita A, Kashima I, Izumi N, Ohnishi T, Anderson P, Ohno S.
  • Organizer: 第12回国際RNA学会年会
  • Place of Presentation: Wisconsin