A-Z junction formation drives recognition of Alu RNAs by ADAR1 and supports viral infectivity and replication

A-Z 连接的形成驱动 ADAR1 对 Alu RNA 的识别，并支持病毒的感染性和复制

基本信息

批准号：
10385045
负责人：
Parker J Nichols
金额：
$ 3.53万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-12-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10385045
关键词：
Adenosine Adopted Alu Elements Amplifiers Binding Binding Sites Biochemical Biological Process Biophysics Cells Characteristics DNA Deaminase Disease Double-Stranded RNA Binding Domain Event Foundations Genetic Transcription Growth Helix-Turn-Helix Motifs Human Human Genome Immune response Infection Inflammatory Response Innate Immune Response Innate Immune System Inosine Interferons Kinetics Knowledge Methods Molecular Molecular Conformation Mutation N-terminal NMR Spectroscopy Nature Pathway interactions Play Population Primates Proliferating Protein Isoforms Proteins RNA RNA Binding RNA Conformation RNA Editing RNA Recognition Motif Research Resolution Retrotransposon Role Sequencing Biochemistry Side Signal Transduction Specificity Structure TLR3 gene Techniques Testing Tissue-Specific Gene Expression Viral Virus Virus Diseases West Nile virus Work X-Ray Crystallography Z-Form DNA alpha helix biological adaptation to stress biological research conformational conversion cost design dsRNA adenosine deaminase experimental study fighting in vivo mutant prevent response structural biology synergism tool transcriptome transcriptome sequencing treatment strategy

项目摘要

Project Summary Self and non-self RNA must be distinguished by the cell in order to avoid triggering the innate immune response when not needed. In humans, self RNAs are edited by adenosine deaminase that acts on RNA (ADAR1), which modifies adenosines to inosines. The vast majority of A-to-I editing events occur in primate- specific Alu elements, which are the most prolific retrotransposon found within the human genome. Alu elements have been shown to be the primary drivers of RIG-I, MDA-5, and TLR3 signaling suggesting that the primary function of ADAR1-dependent editing of Alu elements is to suppress the immune response. Editing is augmented upon infection by viruses primarily through the activity of the longer, interferon-induced, isoform of ADAR1 (ADAR1p150), which is unique from the short isoform in that it has a N-terminal Z-RNA binding domain (Zα) and that it is pro-viral. This suggests that the Z-RNA binding function of Zα plays a critical role in targeting ADAR1p150 to Z-RNA-forming regions within Alu elements along with a nearby structurally homologous Zβ domain, however, the RNA binding mechanisms and specificities of ADAR1p150 are poorly characterized. My hypothesis is that the N-terminal Zα domain and the closely related Zβ domain augment A-to-I editing by targeting ADAR1p150 to Z-RNA-forming regions within Alu elements during the interferon response. This increased editing in-turn “blunts” the interferon response and allows many types of viruses to proliferate unchallenged. In this proposal, I present a strategy which integrates structural biology, RNA-sequencing, and biochemistry techniques to uncover specific Z-RNA-forming sequences within Alu elements and during infection and answer basic questions about A-to-Z RNA transitions in Alu elements by the Zα and Zβ domains of ADAR1p150. My specific aims are (Aim 1) to characterize the transition from A- to Z-RNA in Alu RNAs through biochemical and structural techniques. I also plan (Aim 2) to investigate how the Z-RNA recognizing ability of Zα and Zβ contributes to recognition and editing of Alu elements in vivo and how it correlates to West Nile infection of HEK293T cells by RNA-sequencing techniques. Due to our lack of knowledge about the Z- DNA/RNA binding domain of ADAR1, we have likely vastly overlooked the repertoire of RNA segments able to adopt Z-conformations within the transcriptome. The results of my proposal will help to fill this gap in knowledge, laying the foundations for further research on the importance of Z-RNA in biological processes and help guide studies attempting to manipulate A-to-I editing as a tool for treating many types of diseases or inhibit the pro-viral characteristics of ADAR1p150.

项目概要细胞必须区分自身和非自身RNA，以避免触发先天免疫在人类中，自身 RNA 被作用于 RNA 的腺苷脱氨酶编辑。 (ADAR1)，它将腺苷修饰为肌苷绝大多数 A 到 I 编辑事件发生在灵长类动物中。特定的 Alu 元件，这是人类基因组中发现的最多产的逆转录转座子。元素已被证明是 RIG-I、MDA-5 和 TLR3 信号传导的主要驱动因素，表明 ADAR1 依赖性 Alu 元件编辑的主要功能是抑制免疫反应。主要通过干扰素诱导的较长亚型的活性在病毒感染后增强 ADAR1 (ADAR1p150)，与短亚型不同，它具有 N 端 Z-RNA 结合域 (Zα) 并且它是促病毒的，这表明 Zα 的 Z-RNA 结合功能在靶向中发挥着关键作用。 ADAR1p150 到 Alu 元件内的 Z-RNA 形成区域以及附近的结构同源 Zβ 然而，ADAR1p150 的 RNA 结合机制和特异性尚不清楚。假设 N 端 Zα 结构域和密切相关的 Zβ 结构域增强了 A 到 I 编辑在干扰素反应期间，将 ADAR1p150 靶向 Alu 元件内的 Z-RNA 形成区域。这种增加的编辑反过来“削弱”了干扰素反应，并允许多种类型的病毒增殖在这个提案中，我提出了一个整合了结构生物学、RNA 测序和技术的策略。生物化学技术揭示 Alu 元素内特定的 Z-RNA 形成序列感染并回答有关 Zα 和 Zβ 结构域 Alu 元件中 A 到 Z RNA 转换的基本问题我的具体目标是（目标 1）表征 Alu RNA 中从 A-RNA 到 Z-RNA 的转变。我还计划（目标 2）通过生物化学和结构技术研究 Z-RNA 是如何识别的。 Zα 和 Zβ 的能力有助于体内 Alu 元素的识别和编辑及其与 West 的关联由于我们缺乏对 Z-的了解，通过 RNA 测序技术对 HEK293T 细胞进行 Nile 感染。 ADAR1 的 DNA/RNA 结合域，我们可能极大地忽略了能够在转录组中采用 Z 构象我的提案的结果将有助于填补这一空白。知识，为进一步研究 Z-RNA 在生物过程中的重要性奠定基础帮助指导尝试将 A-to-I 编辑作为治疗多种疾病的工具的研究，或抑制 ADAR1p150 的促病毒特性。