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Development of luminescence indicator of transcript abundance using RNA recognition module

Research Project

Project/Area Number 20K15395
Research Category

Grant-in-Aid for Early-Career Scientists

Allocation TypeMulti-year Fund
Review Section Basic Section 37010:Bio-related chemistry
Research InstitutionThe University of Tokyo

Principal Investigator

Kawamura Genki  東京大学, 大学院理学系研究科(理学部), 特任研究員 (10852791)

Project Period (FY) 2020-04-01 – 2023-03-31
Project Status Completed (Fiscal Year 2022)
Budget Amount *help
¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
Fiscal Year 2021: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2020: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Keywords発光 / ルシフェラーゼ / RNA / Cas13 / スプライシング / 転写量 / 発光タンパク質 / RNA認識 / 転写物 / 遺伝子工学
Outline of Research at the Start

次世代シーケンスによる大規模な転写物解析は、様々な動態を持つ転写物の同定を可能にした。今後は同定された転写物に対してより詳細な動態解析が必要となる。しかし既存の手法には、転写物量変動に対して時間分解能が十分でない、という課題がある。本研究では転写物を認識するタンパク質と発光タンパク質ルシフェラーゼを組み合わせることで高時間分解能な転写物量の分析手法の開発を目指す。開発する手法では認識する転写物は20残基程度の短いRNAにより変更可能であり、様々な種類の転写物に応用することができる。これら特性は未知の転写制御現象の解明へ寄与することが期待される。

Outline of Final Research Achievements

In this study, an attempt to develop a probe that detects the intracellular abundance of target transcript based on a new measurement principle using luciferase. To this end, we constructed a probe that converts the structural changes associated with transcript recognition into changes in the luminescent activity of luciferase, using the RNA-cleaving enzyme Cas13 and luciferase. When perturbation of transcript levels was applied to cells expressing the developed probe, changes in the luminescence values were observed. Furthermore, we attempted to detect splicing variant-specific transcript levels using this probe. As a result, the probe showed a fluctuation in luminescence intensity when the splicing product ratio was artificially perturbed. These results indicate that the developed probe is capable of transcript-selective detection in living cells.

Academic Significance and Societal Importance of the Research Achievements

本研究では、既存の手法とは異なる原理に基づいて細胞内転写物量の経時変化を測定する技術を開発した。近年では次世代シーケンスなどによる大規模な転写物関連のデータが蓄積されており、特定の転写物の時空間的動態を調べるニーズが高まっている。開発したプローブは比較的容易に標的転写物を変更できること、また、実際の転写物量変化に対する時間的な遅延が小さいこと、などの特徴を持つ。これらの特徴は様々な転写物の動態を解析する際に有益であるため、転写物の動態解析において新たな選択肢となる技術として確立することが期待される。

Report

(4 results)
  • 2022 Annual Research Report   Final Research Report ( PDF )
  • 2021 Research-status Report
  • 2020 Research-status Report
  • Research Products

    (9 results)

All 2023 2022 2021

All Journal Article (2 results) (of which Peer Reviewed: 2 results,  Open Access: 1 results) Presentation (7 results) (of which Int'l Joint Research: 1 results)

  • [Journal Article] Optogenetic decoding of Akt2-regulated metabolic signaling pathways in skeletal muscle cells using transomics analysis2023

    • Author(s)
      Kawamura Genki、Kokaji Toshiya、Kawata Kentaro、Sekine Yuka、Suzuki Yutaka、Soga Tomoyoshi、Ueda Yoshibumi、Endo Mizuki、Kuroda Shinya、Ozawa Takeaki
    • Journal Title

      Science Signaling

      Volume: 16 Issue: 773 Pages: 773-773

    • DOI

      10.1126/scisignal.abn0782

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Mechanistic insights into cancer drug resistance through optogenetic PI3K signaling hyperactivation2022

    • Author(s)
      Ueda Yoshibumi、Miura Yuri、Tomishige Nario、Sugimoto Naotoshi、Murase Megumi、Kawamura Genki、Sasaki Norihiko、Ishiwata Toshiyuki、Ozawa Takeaki
    • Journal Title

      Cell Chemical Biology

      Volume: 29 Issue: 11 Pages: 1576-1587.e5

    • DOI

      10.1016/j.chembiol.2022.10.002

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed
  • [Presentation] Dissecting the role of Akt2 in cellular metabolism by optogeneticsaided trans-omics analysis2023

    • Author(s)
      Genki Kawamura
    • Organizer
      日本化学会第102春季年会
    • Related Report
      2022 Annual Research Report
  • [Presentation] Trans-omics analysis of Akt-mediated metabolic pathways by an optogenetics approach2022

    • Author(s)
      Genki Kawamura
    • Organizer
      第82回分析化学討論会
    • Related Report
      2022 Annual Research Report
  • [Presentation] Reconstruction of Akt2-mediated cellular signaling pathways by integrating multiple omics data.2022

    • Author(s)
      Genki Kawamura
    • Organizer
      日本分析化学会第71年会
    • Related Report
      2022 Annual Research Report
  • [Presentation] Optogenetic approach to analyze Akt2-regulated cellular metabolic signaling pathways2022

    • Author(s)
      Genki Kawamura
    • Organizer
      第45回日本分子生物学会年会
    • Related Report
      2022 Annual Research Report
  • [Presentation] Optical control of Akt to analyze cellular metabolic signalling pathways2022

    • Author(s)
      Genki Kawamura
    • Organizer
      日本化学会第102春季年会
    • Related Report
      2021 Research-status Report
  • [Presentation] Analysis of Akt signaling pathway using a molecule-specific Akt activation tool by an optogenetic approach2021

    • Author(s)
      Genki Kawamura
    • Organizer
      日本分析化学会第69年会
    • Related Report
      2021 Research-status Report
  • [Presentation] Single-cell analysis of circadian synchronization process using luciferase probes.2021

    • Author(s)
      Genki Kawamura
    • Organizer
      Pacifichem2021
    • Related Report
      2021 Research-status Report
    • Int'l Joint Research

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Published: 2020-04-28   Modified: 2024-01-30  

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