Structure and functional analysis of a molecular switch that regulated the mRNA synthesis rate.

• Project/Area Number: 15510171
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Living organism molecular science
• Research Institution: Tokyo Institute of Technology
• Principal Investigator: WADA Tadashi Tokyo Institute of Technology, Department of Bioscience and Biotechnology, Lecturer, 大学院生命理工学研究科, 講師 (60262309)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,900,000); Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000); Fiscal Year 2003: ¥2,000,000 (Direct Cost: ¥2,000,000)
• Keywords: transcription elongation / DSIF / RNA polymerase II / antisense / zebrafish / NELF / mRNA synthesis / 転写 / 転写阻害剤 / mRNA合成速度 / リン酸化 / キャッピング酵素 / 競合 / mRNA / 変異体 / トランスフェクション / レポーターアッセイ / Spt5 / Gene Chip

Research Abstract

The transcription elongation factor DSIF, which is comprised of human Spt4 and Spt5, is unique in its ability to regulate RNA polymerase II processivity. Experiments with cultured HeLa cells and in vitro transcription assays revealed that stimulation of transcription by the DNA-binding transcription activator Ga14VP16 is dependent on human Spt5, indicating that actively transcribed genes require DSIF activity. Microarray analysis revealed that approximately 7.5% of genes decreased their expression by a specific morpholino antisense-mediated knockdown of zebrafish Spt5. Further investigation of the down-regulated genes showed that the genes most intensely repressed by the knockdown were strongly activated during early development in untreated embryos, supporting above results. On the other hand, cooperative action of DSIF and the transcription elongation factor NELF negatively regulates transcription elongation by RNA polymerase II. In this study, I show that DSIF and NELF contribute to the regulation of junB gene expression not only by pausing RNA polymerase II elongation at promoter proximal region before cytokine IL-6 induction, but also by attenuating mRNA induction level after induction. In addition, two deletion mutants of human Spt5 have been isolated to elucidate the regulatory mechanisms of the mRNA synthesis rate.

Research Products

• [Journal Article] GABP, HCF-1 and YY1 are involved in Rb gene expression during myogenesis. (2005)
  • Author(s): Delehouzee, S. et al.
  • Journal Title: Genes Cells 10 Pages: 717-731
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] NF-Y is essential for the recruitment of RNA polymerase II and inducible transcription of several CCAAT box-containing genes. (2005)
  • Author(s): Kabe, Y. et al.
  • Journal Title: Mol. Cell. Biol. 25 Pages: 512-522
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Poly(ADP-ribose) polymerase-1 is a negative regulator of HIV-1 transcription through competitive binding to TAR RNA with Tat.positive transcriptionelongation factor b (p-TEFb) complex. (2005)
  • Author(s): Parent, M. et al.
  • Journal Title: J. Biol. Chem. 280 Pages: 448-457
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] GABP, HCF-1 and YY1 are involved in Rb gene expression during myogenesis. (2005)
  • Author(s): Delehouzee, S., Yoshikawa, T., Sawa, C., Sawada, J., Ito, T., Omori, M., Wada, T., Yamaguchi, Y., Kabe, Y., Handa, H.
  • Journal Title: Genes Cells 10 Pages: 717-731
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] NF-Y is essential for the recruitment of RNA polymerase II and inducible transcription of several CCAAT box-containing genes. (2005)
  • Author(s): Kabe, Y., Yamada, J., Uga, H., Yamaguchi, Y., Wada, T., Handa, H.
  • Journal Title: Mol.Cell.Biol. 25 Pages: 512-522
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Poly(ADP-ribose) polymerase-1 is a negative regulator of HIV-1 transcription through competitive binding to TAR RNA with Tat positive transcription elongation factor b (p-TEFb) complex. (2005)
  • Author(s): Parent, M., Yung, T.M., Rancourt, A., Ho, E.L., Vispe, S., Suzuki-Matsuda, F., Uehara, A., Wada, T., Handa, H., Satoh, M.S.
  • Journal Title: J.Biol.Chem. 280 Pages: 448-457
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] NF-Y is Essential for the Recruitment of RNA Polymerase II and Inducible Transcription of Several CCAAT Box-Containing Genes. (2005)
  • Author(s): Kabe, Y., et al.
  • Journal Title: Mol.Cell.Biol. 25・1 Pages: 512-522
• [Journal Article] Bromodomain factor 1 (Bdf1) is phosphorylated by protein kinase CK2. (2004)
  • Author(s): Sawa, C. et al.
  • Journal Title: Mol. Cell. Biol. 24 Pages: 4734-4742
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II. (2004)
  • Author(s): Mandal, S.S. et al.
  • Journal Title: Proc. Natl. Acad. Sci. USA 101 Pages: 7572-7577
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Human Spt6 stimulates transcription elongation by RNA polymerase II in vitro. (2004)
  • Author(s): Endoh, M. et al.
  • Journal Title: Mol. Cell. Biol. 24 Pages: 3324-3336
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Bromodomain factor 1 (Bdf1) is phosphorylated by protein kinase CK2. (2004)
  • Author(s): Sawa C, Nedea E, Krogan N, Wada T, Handa H, Greenblatt J, Buratowski S.
  • Journal Title: Mol.Cell.Biol. gaw24 Pages: 4734-4742
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II. (2004)
  • Author(s): Mandal, S.S., Chu, C., Wada, T., Handa, H., Shatkin, A.J., Reinberg, D.
  • Journal Title: Proc.Natl.Acad.Sci.USA. 101 Pages: 7572-7577
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Human Spt6 stimulates transcription elongation by RNA polymerase II in vitro. (2004)
  • Author(s): Endoh, M., Zhu, W., Hasegawa, J., Watanabe, H., Kim, D.K., Aida, M., Inukai, N., Narita, T., Yamada, T., Furuya, A., Sato, H., Yamaguchi, Y., Mandal, S.S., Reinberg, D., Wada, T., Handa, H.
  • Journal Title: Mol.Cell.Biol. 24 Pages: 3324-3336
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Human Spt6 stimulates transcription elongation by RNA polymerase II in vitro. (2004)
  • Author(s): Endoh, M., et al.
  • Journal Title: Mol.Cell.Biol. 24・8 Pages: 3324-3336
• [Journal Article] Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II. (2004)
  • Author(s): Mandal, S.S., et al.
  • Journal Title: Proc.Natl.Acad.Sci.USA 101・20 Pages: 7572-7577
• [Journal Article] Bromodomain factor 1 (Bdft) is phosphorylated by protein kinase CK2. (2004)
  • Author(s): Sawa, C., et al.
  • Journal Title: Mol.Cell.Biol. 24・11 Pages: 4734-4742
• [Publications] M.Endoh, et al.: "Human Spt6 Stimulates transcription elongation by RNA polymerase II in vitro"Mol.Cell.Biol.. (in press).
• [Publications] T.Narita, et al.: "Human transcription elongation factor NELF : identification of novel subunits and reconstitution of the functionally active complex"Mol.Cell.Biol.. 23. 1863-1873 (2003)