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

• 批准号: 10540225
• 负责人: Parker J Nichols
• 金额: $ 3.62万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 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/10540225
• 关键词: Adenosine; Adopted; Alu Elements; Amplifiers; Binding; Binding Sites; Biochemical; Biochemistry; 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; Side; Signal Transduction; Specificity; Structure; TLR3 gene; Techniques; Testing; Tissue-Specific Gene Expression; Viral; Virus; Virus Diseases; West Nile; 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; novel therapeutic intervention; prevent; response; structural biology; synergism; theories; tool; transcriptome; transcriptome sequencing

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.

项目摘要 自我和非自我RNA必须通过细胞区分，以避免触发先天免疫 不需要的响应。在人类中，自我RNA由作用于RNA的腺苷脱氨酶编辑 （ADAR1），将腺苷修饰为插入。绝大多数A到I编辑事件发生在初级 - 特定的ALU元素，这是人类基因组中发现的最多产的逆转录子。阿鲁 元素已被证明是RIG-I，MDA-5和TLR3信号的主要驱动因素，表明 ADAR1依赖性编辑ALU元素的主要功能是抑制免疫反应。编辑是 病毒感染后，主要是通过较长的，干扰素引起的，同工型的活性 ADAR1（ADAR1P150），它在短同工型中是独特的，因为它具有N末端Z-RNA结合域 （Zα），它是促病毒的。这表明Zα的Z-RNA结合函数在靶向中起关键作用 ADAR1P150至ALU元素内的Z-RNA形成区域以及附近的结构同源Zβ 但是，域，ADAR1P150的RNA结合机理和规格的特征很差。我的 假设是N末端Zα结构域和密切相关的Zβ结构域增强A-至I编辑 通过将ADAR1P150靶向干扰素反应期间Alu元素内的Z-RNA形成区域。 这增加了编辑的内部“钝”干扰素响应，并允许许多类型的病毒增殖 不受挑战。在此提案中，我提出了一项策略，该策略将结构生物学，RNA测序和 生物化学技术可在Alu元素和期间发现特定的Z-RNA形成序列 Zα和Zβ域中有关A-t-Z RNA转变的感染并回答有关A-to-Z RNA转变的基本问题 ADAR1P150。我的具体目的是（AIM 1）表征从Alu RNA中从A-到Z-RNA的过渡 通过生化和结构技术。我还计划（AIM 2）来研究Z-RNA如何识别 Zα和Zβ的能力有助于识别和编辑体内Alu元素及其与西方的相关性 通过RNA测序技术，HEK293T细胞的尼罗河感染。由于我们对Z-缺乏了解 ADAR1的DNA/RNA结合域，我们可能非常忽略了RNA段的曲目 在转录组中采用Z形成。我的建议的结果将有助于填补这一空白 知识，为Z-RNA在生物过程中的重要性和 帮助指导试图操纵A到I编辑的研究，以作为治疗多种疾病或 抑制ADAR1P150的促病毒特征。