Live-cell imaging of endogenous mRNAs with a small molecule

• Project/Area Number: 26440005
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Molecular biology
• Research Institution: Kyoto University
• Principal Investigator: SATO SHINICHI 京都大学, 化学研究所, 准教授 (70534478)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2016: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2015: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2014: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: RNA生細胞内イメージング / 蛍光プローブ / ケミカルバイオロジー / RNAアプタマー / RNA性細胞内イメージング

Outline of Final Research Achievements

Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. We developed a new convenient and versatile method that permits spatiotemporal imaging of specific non-engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene-specific RNA aptamer, combined with a cell-permeable synthetic small molecule whose fluorescence is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live-cell imaging of the endogenous mRNA of β-actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including ARFIP2, CTTN, and CYFIP2. The RNA-imaging technology and its further optimization might permit live-cell imaging of any RNA molecules.

Research Products

• [Journal Article] Vitamin D metabolite, 25-Hydroxyvitamin D, regulates lipid metabolism by inducing degradation of SREBP/SCAP. (2017)
  • Author(s): Asano, L., Watanabe, M., Ryoden, Y., Usuda, K., Yamaguchi, T., Khambu, B., Takashima, M., Sato, S., Sakai, J., Nagasawa, K., Uesugi, M.
  • Journal Title: Cell Chem Biol. Volume: 24 Issue: 2 Pages: 207-217
  • DOI: 10.1016/j.chembiol.2016.12.017
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] A synthetic hybrid molecule for the selective removal of human pluripotent stem cells from cell mixtures. (2017)
  • Author(s): Mao, D., Ando, S., Sato, S., Qin, Y., Hirata, N., Katsuda, Y., Kawase, E., Kuo, T.F., Minami, I., Shiba, Y., Ueda, K., Nakatsuji, N., Uesugi, M.
  • Journal Title: Angew. Chem. Int. Ed. Volume: 56 Issue: 7 Pages: 1765-1770
  • DOI: 10.1002/anie.201610284
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] A small molecule that represses translation of G-quadruplex-containing mRNA. (2016)
  • Author(s): Katsuda, Y., Sato, S., Asano, L., Morimura, Y., Furuta, T., Sugiyama, H., Hagihara, M., Uesugi, M.
  • Journal Title: J. Am. Chem. Soc. Volume: 138 Issue: 29 Pages: 9037-9040
  • DOI: 10.1021/jacs.6b04506
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Intracellular generation of a diterpene-peptide conjugate that inhibits 14-3-3-mediated interactions. (2015)
  • Author(s): Parvatkar P., Kato N., Uesugi M., Sato S., Ohkanda J.
  • Journal Title: J. Am. Chem. Soc. Volume: 137 Issue: 50 Pages: 15624-15627
  • DOI: 10.1021/jacs.5b09817
  • Peer Reviewed
• [Journal Article] Live-cell Imaging of Endogenous mRNAs with a Small Molecule (2015)
  • Author(s): Sato, S.*, Watanabe, M., Katsuda, Y., Murata, A., Wang, D. O., Uesugi M.
  • Journal Title: Angew. Chem. Int. Ed. Volume: 54 Issue: 6 Pages: 1855-1858
  • DOI: 10.1002/anie.201410339
  • NAID: 120005619801
  • Peer Reviewed
• [Journal Article] Synthetic Molecules That Protect Cells from Anoikis and Their Use in Cell Transplantation (2014)
  • Author(s): Frisco-Cabanos, H.L., Watanabe, M., Okumura, N., Kusamori, K., Takemoto, N., Takaya, J., Sato, S., Yamazoe, S., Takakura, Y., Kinoshita, S., Nishikawa, M., Koizumi, M., Uesugi, M
  • Journal Title: Angew. Chem. Int. Ed. Volume: 53 Issue: 42 Pages: 11208-11213
  • DOI: 10.1002/anie.201405829
  • Peer Reviewed
• [Journal Article] A chemical probe that labels human pluripotent stem cells. (2014)
  • Author(s): Hirata N., Nakagawa M., Fujibayashi Y., Yamauchi K., Murata A., Minami I., Tomioka M., Kondo T., Kuo T. F., Endo H., Inoue H., Sato S., Ando S., Kawazoe Y., Aiba K., Nagata K., Kawase E., Chang Y. T., Suemori H., Eto K., Nakauchi H., Yamanaka S., Nakatsuji N., Ueda K., Uesugi M.
  • Journal Title: Cell Rep. Volume: 6 Pages: 1165-1174
  • NAID: 120005466668
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] In vitro selection of RNA aptamers for a small-molecule dye. (2014)
  • Author(s): Murata, A., Sato, S.
  • Journal Title: Methods Mol. Biol. Volume: 1111 Pages: 17-28
  • DOI: 10.1007/978-1-62703-755-6_2
  • ISBN: 9781627037549, 9781627037556
  • Peer Reviewed
• [Journal Article] Affinity-Based Isolation of Molecular Targets of Clinically Used Drugs (2014)
  • Author(s): Sato, S., Uesugi, M.
  • Journal Title: Concepts and Case Studies in Chemical Biology Volume: 15 Pages: 221-230
  • DOI: 10.1002/9783527687503.ch15
  • Peer Reviewed
• [Presentation] Exploration of an RNA G-quadruplex using a small molecule (2017)
  • Author(s): Yousuke Katsuda, Shin-ichi Sato, Kenji Yatsuzuka, Motonari Uesugi
  • Organizer: 日本化学会第97春季年会
  • Place of Presentation: 横浜
  • Year and Date: 2017-03-16
• [Presentation] Live-Cell Imaging of Bioorganic Molecules with a Small Molecule and a Short RNA (2017)
  • Author(s): Kenji Yatsuzuka, Shin-ichi Sato, Yousuke Katsuda, Kathleen Beverly Pe, Motonari Uesugi
  • Organizer: 日本化学会第97春季年会
  • Place of Presentation: 横浜
  • Year and Date: 2017-03-16
• [Presentation] RNA G-quadruplex選択的化合物を用いた網羅的なRNA G-quadruplexの探索 (2016)
  • Author(s): 勝田 陽介・佐藤 慎一・萩原 正規・八塚 研治・上杉 志 成
  • Organizer: 第10回バイオ関連化学シンポジウム
  • Place of Presentation: 金沢
  • Year and Date: 2016-09-07
• [Presentation] 蛍光プローブを用いた内在性mRNAイメージング法の改 良 (2016)
  • Author(s): 八塚 研治・佐藤 慎一・勝田 陽介・上杉 志成
  • Organizer: 第10回バイオ関連化学シンポジウム
  • Place of Presentation: 金沢
  • Year and Date: 2016-09-07
• [Presentation] Chemical and RNA tools for investigating cell biology (2016)
  • Author(s): Sato S.
  • Organizer: Leading Edge Chemical Biology Seminar
  • Place of Presentation: 京都
  • Year and Date: 2016-06-14
  • Invited
• [Presentation] Live-Cell Imaging of Endogenous mRNAs with a Small Molecules (2016)
  • Author(s): Sato, S
  • Organizer: 2016Queenstown Molecular Biology Meetings in Shanghai
  • Place of Presentation: Shanghai
  • Year and Date: 2016-03-17
  • Int'l Joint Research / Invited
• [Presentation] A small-molecule based approach for live-cell imaging of endogenous RNAs (2015)
  • Author(s): Sato S., Watanabe M., Katsuda Y., Murata A., Wang D. O., Uesugi M
  • Organizer: The International Chemical Congress Of Pacific Basin Societies
  • Place of Presentation: Honolulu
  • Year and Date: 2015-12-15
  • Int'l Joint Research
• [Presentation] RNA tools for cell biology (2015)
  • Author(s): Sato, S.
  • Organizer: The Tohoku Branch of the Chemical Society of Japan
  • Place of Presentation: Hirosaki
  • Year and Date: 2015-09-10
  • Invited
• [Presentation] A fluorescent probe for imaging of energy metabolism in cells. (2015)
  • Author(s): Sato S., Watanabe M., Katsuda Y., Tamura T., Nakata E., Morii T.
  • Organizer: The 6th International Symposium of Advanced Energy Science
  • Place of Presentation: Kyoto
  • Year and Date: 2015-09-01
  • Int'l Joint Research
• [Presentation] 内在性mRNA細胞内動態の新規イメージング法 (2015)
  • Author(s): 佐藤 慎一・勝田 陽介・渡邊 瑞貴・上杉 志成
  • Organizer: 日本化学会 第95春季年会
  • Place of Presentation: 千葉
  • Year and Date: 2015-03-28
• [Presentation] 内在性mRNA細胞内動態の新規イメージング法の開発 (2014)
  • Author(s): 佐藤 慎一・渡邉 瑞貴・勝田 陽介・王 丹・上杉 志成
  • Organizer: 第 8回バイオ関連化学シンポジウム
  • Place of Presentation: 岡山
  • Year and Date: 2014-09-12
• [Presentation] A small molecule-based method for live-cell imaging of endogenous mRNAs (2014)
  • Author(s): Sato S., Watanabe M., Katsuda Y., Wang D. O., Uesugi M.
  • Organizer: The 37th Naito Conference: Bioimaging—a Paradigm Shift for the Life Sciences
  • Place of Presentation: Niseko
  • Year and Date: 2014-07-18
• [Book] ビオチン化体を利用した生化学的標的タンパク質精製法 (2015)
  • Author(s): 佐藤慎一，勝田陽介，上杉志成
  • Total Pages: 5
  • Publisher: 化学同人