Enhancing the Site-directed RNA Editing Toolkit with Cryo-EM Structures of Native RNA Editing Complexes

利用天然 RNA 编辑复合物的冷冻电镜结构增强定点 RNA 编辑工具包

• 批准号: 22K15050
• 负责人: Matthews Melissa
• 金额: $ 1.41万
• 依托单位: Okinawa Institute of Science and Technology Graduate University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Early-Career Scientists
• 财政年份: 2022
• 资助国家: 日本
• 起止时间: 2022-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-22K15050/
• 关键词: ADAR; cryoEM; RNA editing; RNA; structural biology; cryo-electron microscopy; adenosine deaminase; dsRNA binding domain; autoimmune disease

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.

蛋白质和两个RNA靶标已经成功产生，并且使用EMSA通过生化表征验证了复合形成。数据收集正在进行中。EMSA的ADAR2：GLI1-61BP的表征表明，在蛋白质与RNA的2：1摩尔比时，复数在RNA之间以一个，两个或三个ADAR2单体结合的RNA之间存在平衡，其中大多数在两个或三个ADAR2状态中。为冷冻数据收集选择了2：1的比率。在Titan Krios Cryo-Electron显微镜上收集了超过15,000个ADAR2：GLI1-61BP复合物。分离出良好的粒子图像和3D重建后，发现冰中颗粒的角度分布不足以准确3D重建。为了增加角度分布，在开始数据收集之前，将样品倾斜至30度角。在收集了约3500个显微照片之后，该技术在5角处进行了3D重建：gli1-61bp的3D重建，其中可以清楚地看到蛋白质二级结构。二级结构的区分允许将结构模型准确地分配到重建中，包括RNA，Deaminase romain和DSRBDS.EMSA的ADAR2：HT2CR-89MER络合物的表征，表明，在蛋白质与RNA的4：1摩尔比时，Complex complex complece都会被独立地限制为RNA，因为RNA被两种Adar2 morecules by boldus boldus boldus boldus boldus boldus boldus bocles by by by the Adar2 metar2 morecules。 4：1的比率用于制备冷冻电网，但仍在优化冷冻和数据收集条件。

项目成果

Structural Understanding of A-to-I RNA Editing: Adenosine Deaminase Acting on RNA (ADAR2) Complexed with dsRNA

A 到 I RNA 编辑的结构理解：作用于与 dsRNA 复合的 RNA (ADAR2) 的腺苷脱氨酶

• 发表时间: 2022
• 作者: Melissa Matthews