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.

蛋白质和两种RNA靶标已经成功产生，并且复合物的形成已经使用EMSA用生物化学表征进行了验证。ADAR 2：GLI 1 - 61 bp的EMSA表征显示，在蛋白质与RNA的摩尔比为2：1时，复合物在由一个、两个或三个ADAR 2单体结合的RNA之间平衡存在，其中大多数处于两个或三个ADAR 2状态。选择2：1的比例进行cryoEM数据收集。在Titan Krios冷冻电子显微镜上收集了超过15，000张ADAR 2：GLI 1 - 61 bp复合物的显微照片。在分离出良好的颗粒图像和3D重建之后，发现冰中颗粒的角分布不足以进行精确的3D重建。为了增加角分布，在开始数据收集之前将样品倾斜至30度角。在收集了大约3500张显微照片后，该技术导致了ADAR 2：GLI 1 - 61 bp在5埃处的3D重建，其中可以清楚地看到蛋白质二级结构。二级结构的纯化允许将结构模型准确地分配给重建，包括RNA、脱氨酶结构域和dsRBDADAR 2：HT 2cR-89聚体复合物的EMSA表征显示，在蛋白质与RNA的摩尔比为4：1时，复合物仅作为由两个ADAR 2分子结合的RNA存在。4：1的比例用于制备cryoEM网格，但冷冻和数据收集条件仍在优化中。

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