| 研究実績の概要 |
Protein and both RNA targets have been successfully produced, and complex formation has been verified with biochemical characterization using EMSA. Data collection is ongoing.
EMSA characterization of ADAR2:GLI1-61bp showed that at a 2:1 molar ratio of protein to RNA, complex exists in equilibrium between RNA bound by one, two, or three ADAR2 monomers, with a majority being in the two- or three-ADAR2 state. The 2:1 ratio was chosen for cryoEM data collection. Over 15,000 micrographs of ADAR2:GLI1-61bp complex were collected on the Titan Krios cryo-electron microscope. After isolation of good particle images and 3D reconstruction, the angular distribution of particles in the ice was found to be insufficient for accurate 3D reconstruction. To increase the angular distribution, the sample was tilted to a 30deg angle before beginning data collection. After collecting about 3500 micrographs, this technique resulted in a 3D reconstruction of ADAR2:GLI1-61bp at 5-angstroms in which protein secondary structures can clearly be seen. The distinguishment of secondary structures allowed for accurate assignment of structural models to the reconstruction, including the RNA, deaminase domain, and dsRBDs.
EMSA characterization of the ADAR2:HT2cR-89mer complex showed that at a 4:1 molar ratio of protein to RNA, complex exists exclusively as RNA bound by two ADAR2 molecules. The 4:1 ratio was used for preparation of cryoEM grids, but freezing and data collection conditions are still being optimized.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
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理由
Although protein production and complex formation of ADAR2:GLI1-61bp is straightforward, complex in the vitrified ice used for cryoEM imaging is preferentially oriented so that certain side views of the complex were not observed, resulting in very poor 3D reconstructions which lacked detail when viewing from particular directions. Many different techniques to resolve this issue were attempted over the past year before the sample tilting technique was successful.
Production of HT2cR-89mer was not completed until March 2023, so analysis of this sample is only just beginning.
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| 今後の研究の推進方策 |
Although 5-angstrom resolution of the ADAR2:GLI1-61bp complex is sufficient for determining the most common location of dsRBDs on the RNA target, additional binding sites may be observed if the number of particles in the dataset is increased. Therefore, more data will be collected on the ADAR2:GLI1-61bp complex. Increased data will also improve resolution, enabling the generation of more accurate models.
The plunging and data collection conditions for the ADAR2:HT2cR-89mer complex will be optimized on the Talos Arctica before collection of high-resolution data on the Titan Krios, processing, and model building. Since these procedures are expected to be similar to those of the GLI1-61bp complex, this is not expected to take more than 3-4 months.
Lastly, by comparing the location of the dsRBDs in both reconstructions, hypotheses concerning their role in selective editing of ADAR2 will be generated. A few simple protein mutation/truncation EMSA experiments, along with an extensive literature review on the editing selectivity of previously published ADAR2 mutants will be sufficient to confirm and expanded upon the conclusions of this study in its resulting publication.
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