| Project/Area Number |
22K15050
|
|---|
| Research Category |
Grant-in-Aid for Early-Career Scientists
|
| Allocation Type | Multi-year Fund |
|---|
| Review Section |
Basic Section 43020:Structural biochemistry-related
|
|---|
| Research Institution | Okinawa Institute of Science and Technology Graduate University |
|---|
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)
|
|---|
| Project Period (FY) |
2022-04-01 – 2025-03-31
|
| Project Status |
Granted (Fiscal Year 2023)
|
| Budget Amount *help |
¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2023: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2022: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
|
|---|
| Keywords | cryoEM / RNA / ADAR / RNA editing / structural biology / cryo-electron microscopy / adenosine deaminase / dsRNA binding domain / autoimmune disease |
|---|
| Outline of Research at the Start |
ADARs are enzymes responsible for conversion of adenosine to inosine in double-stranded RNA (dsRNA) in humans. Double-stranded RNA-binding domains (dsRBDs) are present in all ADARs and play a key role in determining which RNAs get edited.
An unhealthy level of editing by ADARs leads to autoimmune and neurological diseases. This project will determine 3D structures of ADAR:dsRNA complexes using cryo-electron microscopy. The structures from this work will provide molecular frameworks for RNA editing technologies which include dsRBDs, enabling the creation of new, versatile therapeutic techniques.
|
| 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.
|
| 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.
|
| 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.
|
