2023 Fiscal Year Research-status Report
Enhancing the Site-directed RNA Editing Toolkit with Cryo-EM Structures of Native RNA Editing Complexes
| Project/Area Number |
22K15050
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
Matthews Melissa 沖縄科学技術大学院大学, 生体分子電子顕微鏡解析ユニット, ポストドクトラルスカラー (20866298)
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| Project Period (FY) |
2022-04-01 – 2025-03-31
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| Keywords | cryoEM / RNA |
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| Outline of Annual Research Achievements |
The protein of interest (adenosine deaminase acting on dsRNA 2, ADAR2) has been successfully characterized in complex with two target dsRNA sequences by cryoEM. To accomplish this, roughly 300,000 particles extracted from 9300 micrographs containing ADAR2:GLI-61bp complex or 225,000 particles extracted from 15,000 micrographs were averaged together. Deaminase and double-stranded RNA binding domains were elucidated to 4-3.5-angstrom resolution, while RNA resolution varied from 6-3.5 angstroms. At the current resolution, cryoEM reconstructions are sufficient to elucidate the locations of deaminase domains and dsRBDs. Although density is not observed for all dsRBDs, novel interactions between protein and RNA can be hypothesized.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Several months were spent troubleshooting unforeseen problems with protein purification, but the issue has now been resolved. Quality sample was prepared and analyzed by cryoEM.
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| Strategy for Future Research Activity |
Work is ongoing to prepare accurate molecular models and perform biochemical experiments to validate these models. After that, work will begin on a manuscript.
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| Causes of Carryover |
This work has not yet been published, so award will be carried over to the next year to contribute toward publication fees.
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