| 研究課題/領域番号 |
19J10101
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| 研究種目 |
特別研究員奨励費
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| 配分区分 | 補助金 |
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| 応募区分 | 国内 |
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| 審査区分 |
小区分43010:分子生物学関連
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| 研究機関 | 筑波大学 |
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研究代表者 |
IGNATOCHKINA ANNA 筑波大学, 人間総合科学研究科, 特別研究員(DC2)
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| 研究期間 (年度) |
2019-04-25 – 2021-03-31
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| 研究課題ステータス |
完了 (2020年度)
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| 配分額 *注記 |
2,100千円 (直接経費: 2,100千円)
2020年度: 1,000千円 (直接経費: 1,000千円)
2019年度: 1,100千円 (直接経費: 1,100千円)
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| キーワード | mRNA 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 |
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| 研究開始時の研究の概要 |
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.
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| 研究実績の概要 |
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.
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| 現在までの達成度 (段落) |
令和2年度が最終年度であるため、記入しない。
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| 今後の研究の推進方策 |
令和2年度が最終年度であるため、記入しない。
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