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5 prime mRNA cap modifications involved in gene expression control in T. brucei

Research Project

Project/Area Number 19J10101
Research Category

Grant-in-Aid for JSPS Fellows

Allocation TypeSingle-year Grants
Section国内
Review Section Basic Section 43010:Molecular biology-related
Research InstitutionUniversity of Tsukuba

Principal Investigator

IGNATOCHKINA ANNA  筑波大学, 人間総合科学研究科, 特別研究員(DC2)

Project Period (FY) 2019-04-25 – 2021-03-31
Project Status Completed (Fiscal Year 2020)
Budget Amount *help
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2020: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2019: ¥1,100,000 (Direct Cost: ¥1,100,000)
KeywordsmRNA cap / Trypanosome / Trypanosoma brucei / Trypanosoma cruzi / Cap 4 / CRISPR-Cas9 system / RNA methyltransferase / RNA methylation / mRNA capping / N6-N6 base methylation / RNA processing / Inducible CRISPR-Cas9 system
Outline of Research at the Start

RNA modification plays an important role in regulating the gene expression. In trypanosome, the 5′-end of mRNA is modified by hypermethylation to form a cap 4 structure. In this research, I aim to characterize RNA methyltransferase enzymes to determine the role of hypermethylated cap structure in parasite gene expression.

Outline of Annual Research Achievements

RNA methylation is rapidly emerging as a gene expression regulator. In Trypanosoma brucei and related parasites, seven RNA methylations are present at the 5′-end of capped mRNA, which may play a role in post-transcriptional gene expression by regulating protein synthesis. This makes the Trypanosome an outstanding target for mRNA cap methylation studies.
We showed that putative RNA methyltransferase possesses a novel cap-dependent RNA methyltransferase activity. The recombinant enzyme was capable of transferring the methyl group to the capped RNA. The activity was enhanced by the presence of m7G cap and 2′-O ribose methylations. However, it does not appear to methylate the known methylation site found. We plan to determine the position and what kind of RNA modification this enzyme support by analyzing the RNA products using mass spectrometry.
We expect to identify the role of the RNA methyltransferase in parasite gene expression by generating CRISPR knock-out and RNAi knockdown in T. cruzi and T.brucei, respectively. We constructed a targeting plasmid encoding for Cas9-EGFP protein and guide RNA under the control of a tetracycline-inducible T7 RNA polymerase promoter. A stable T. cruzi cell line was obtained to analyze the system's effectiveness. As an alternative and complementary approach, we also prepared targeting plasmid to deplete the RNA methyltransferase in T. brucei. Comparative, genome-wide analyses of RNA isolated from CRISPR knock-out and RNAi knockdown cell lines should provide insight into how RNA methylation plays a role in eukaryotic gene expression.

Research Progress Status

令和2年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

令和2年度が最終年度であるため、記入しない。

Report

(2 results)
  • 2020 Annual Research Report
  • 2019 Annual Research Report
  • Research Products

    (4 results)

All 2021

All Presentation (4 results)

  • [Presentation] Purification of a Putative Cap-dependent RNA Methyltransferase in Trypanosoma brucei2021

    • Author(s)
      Anna Ignatochkina
    • Organizer
      3rd Tsukuba Purification Forum
    • Related Report
      2020 Annual Research Report
  • [Presentation] Development of a Tet-inducible CRISPR-Cas9 system to elucidate the role of cap 4 methylation in Trypanosoma cruzi2021

    • Author(s)
      Ignatochkina Anna, Takagi Yuko, Ho C. Kiong
    • Organizer
      The Joint Meeting of the 90th Annual Meeting of the Japanese Society of Parasitology and the 32th Annual Meeting of the Japanese Society of Clinical Parasitology
    • Related Report
      2020 Annual Research Report
  • [Presentation] Characterization of Trypanosome cap-dependent RNA Methyltransferase2021

    • Author(s)
      Anna Ignatochkina, Yuko Takagi, C. Kiong Ho
    • Organizer
      The 22nd Annual Meeting of the RNA Society of Japan
    • Related Report
      2020 Annual Research Report
  • [Presentation] Characterization of cap methyltransferase in Trypanosoma brucei2021

    • Author(s)
      Anna Ignatochkina
    • Organizer
      Tsukuba Conference 2021
    • Related Report
      2020 Annual Research Report

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Published: 2019-05-29   Modified: 2024-03-26  

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