Structural characterization of the mechanism leading to recognition of Alu elements by the Z-RNA-binding domain of ADAR1

ADAR1 的 Z-RNA 结合域识别 Alu 元件的机制的结构表征

基本信息

批准号：
2153787
负责人：
Beat Vogeli
金额：
$ 100万
依托单位：
University of Colorado at Denver
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-15 至 2026-06-30
项目状态：
未结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=2153787&HistoricalAwards=false
关键词：
Structural characterization mechanism leading recognition

项目摘要

DNA represents the repository of our genetic makeup, but its many copies into RNA constitute the actual blueprint for life. These RNA copies start off as exact DNA transcripts, but they later get altered in precise ways that end up determining the fate of the cell. The swap of a very common RNA building block (adenosine or A) to a less common one (inosine or I). Such editing of cellular RNAs helps a cell distinguish between self and non-self RNAs. If that mechanism is corrupted, the immune system can be compromised, or auto-immune diseases may arise and misediting is also observed in cancerous cells. The biological pathway leading to such modifications is complex because not every A needs to or should be turned into an I. A single protein enzyme called ADAR1 is responsible for selecting the modification site, as well as for catalyzing the chemical reaction that leads to ‘editing of A to I’. The catalysis part is understood, but how ADAR1 recognizes a particular site in an RNA molecule remains a mystery. This project is grounded in the hypothesis that a region in ADAR1 is key for recognizing where exactly editing should occur on RNA. That region is known to selectively bind to a rare left-handed form of RNA. This proposal will use advanced NMR methods to monitor the transition from A-form to Z-form RNA, combined with techniques such as isothermal calorimetry and circular dichroism to localize where on natural RNA this part of long ADAR1 binds. The project will then delineate the signature for recognition by ADAR1, through pinpointing the steps leading to a fully assembled complex. This project will in corporate research into course modules and train graduate, undergraduate, and high school students from underrepresented communities.RNA editing of cellular RNAs helps a cell distinguish between self and non-self RNAs. Editing of adenosines into inosines (A-to-I) is generally catalyzed by the ‘adenosine deaminase acting on RNA-1’ protein (ADAR1) at primate-specific Alu retrotransposons. A-to-I editing is augmented upon infection, primarily through the interferon-induced longer isoform of ADAR1 that comprises a Z-DNA/Z-RNA binding domain named ‘Zα’ at its N-terminus. Z-RNA in the form of repeats of cytosine and guanosine (CpG) in a left-handed double-helical conformation has been found in cells, but the prevalence of such structures and their exact role are unknown. In addition, many —if not most— regions proposed to adopt a Z conformation do not resemble regular (CpG)n. How these local Z-RNA conformations are generated within A-form helices, stabilized, and regulated by Zα of ADAR1 (acting in synergy with the downstream Zβ domain), as well as their exact role in the function of these RNAs, remain unknown. This project will test the hypothesis that the binding of Zα to Alu elements plays an essential role during the editing process with the overall goal of achieving structural and dynamic characterization of the formation of A-Z RNA junctions and their recognition by Zα(-Zβ) across transcriptomes. This project is supported by the Molecular Biophysics Cluster of the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

DNA 代表了我们基因组成的储存库，但它的许多 RNA 副本构成了生命的实际蓝图。这些 RNA 副本最初是精确的 DNA 转录物，但后来以精确的方式发生改变，最终决定了细胞的命运。将一种非常常见的 RNA 构建模块（腺苷或 A）替换为不太常见的 RNA 构建模块（肌苷或 I），可以帮助细胞区分自身 RNA 和非自身 RNA。系统可能受到损害，导致这种修饰的生物途径很复杂，因为并非每个 A 都需要或应该转化为 I。一种称为 ADAR1 的单一蛋白酶负责选择 A。催化部分已被了解，但 ADAR1 如何识别 RNA 分子中的特定位点仍然是一个谜。假设 ADAR1 中的一个区域是识别 RNA 上应该发生编辑的关键区域，该区域已知选择性地结合罕见的左手形式的 RNA。该提案将使用先进的 NMR 方法来监测从 A 型的转变。然后，该项目将通过精确定位导致 ADAR1 识别的步骤，描绘出 ADAR1 识别的签名，并结合等温量热法和圆二色性等技术来定位长 ADAR1 的这部分在天然 RNA 上的结合位置。该项目将在课程模块中进行企业研究，并培训来自代表性不足的社区的研究生、本科生和高中生。细胞 RNA 的 RNA 编辑有助于细胞区分自身和非自身 RNA 将腺苷编辑为肌苷。 (A-to-I) 通常由“作用于 RNA-1”蛋白 (ADAR1) 的腺苷脱氨酶催化，在灵长类动物特异性 Alu 逆转录转座子上进行增强的 A-to-I 编辑。感染后，主要通过干扰素诱导的更长的 ADAR1 亚型，其 N 末端包含一个名为“Zα”的 Z-DNA/Z-RNA 结合域，以胞嘧啶和鸟苷 (CpG) 重复形式存在。已在细胞中发现了左手双螺旋构象，但这种结构的普遍性及其确切作用尚不清楚。此外，许多（如果不是大多数）提议采用 Z 构象的区域并未发现。类似于常规 (CpG)n。这些局部 Z-RNA 构象如何在 A 型螺旋内生成、稳定并受 ADAR1 的 Zα 调节（与下游 Zβ 结构域协同作用），以及它们在功能中的确切作用。该项目将测试 Zα 与 Alu 元件的结合在编辑过程中发挥重要作用的假设，总体目标是实现 A-Z RNA 连接形成的结构和动态表征。该项目得到了分子和细胞生物科学部分子生物物理学集群的支持。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力优势和评估进行评估，被认为值得支持。更广泛的影响审查标准。