| Project/Area Number |
17K15080
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Multi-year Fund |
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| Research Field |
Structural biochemistry
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| Research Institution | The University of Tokyo (2018)
Okinawa Institute of Science and Technology Graduate University (2017) |
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Principal Investigator |
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| Research Collaborator |
Ohi Melanie D.
Wolf Matthias
KURUMIZAKA Hitoshi
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| Project Period (FY) |
2017-04-01 – 2019-03-31
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| Project Status |
Completed (Fiscal Year 2018)
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| Budget Amount *help |
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2018: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2017: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
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| Keywords | スプライソソーム / クライオ電子顕微鏡 / 単粒子解析 |
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| Outline of Final Research Achievements |
The spliceosome is a dynamic macromolecular machine that catalyzes the excision of introns from pre-mRNA to mature mRNA. Although we know the composition of the spliceosome, we still lack a mechanistic understanding of how this dynamic machine assemble and recycle. To elucidate the snRNP biogenesis, I attempted to determine the three-dimensional structure of pre-U5 snRNP, which is known as a catalytic snRNP of the spliceosome, from fission yeast using cryo-electron microscopy. First, I purified the pre-U5 snRNP using a pre-U5 snRNP specific protein, Aar2 by Tandem affinity purification. I then successfully obtained the pre-U5 snRNP containing the Aar2, and observed the oval shape complex of the pre-U5 snRNP by the negative stain EM. I next tried to prepare the sample for cryo-electron microscopy. Unfortunately, I haven’t obtained the suitable sample for cryo-EM yet. I need further experiments for cryo-EM to analyze the structure of pre-U5 snRNP.
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| Academic Significance and Societal Importance of the Research Achievements |
RNA-タンパク質複合体 (snRNP)であるスプライソソームによるpre-mRNAのスプライシング反応は、真核生物に普遍的な触媒反応である。本研究の中心であるU5 RNA-タンパク質複合体 (U5 snRNP)を精製し、成熟過程における構造をネガティブ染色電子顕微鏡法により明らかにした。これにより、真核生物に普遍的なスプライソソームの生合成過程の一端を電子顕微鏡負染色法により観察することができ、クライオ電子顕微鏡解析への足がかりを作ることができた。
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