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 细胞必须区分自身和非自身RNA，以避免触发先天免疫。 在不需要的时候回应。在人类中，自身RNA由作用于RNA的腺苷脱氨酶编辑 ADAR 1基因，它将腺苷修饰为肌苷。绝大多数的A到I编辑事件发生在灵长类动物中- 特定的Alu元件，其是在人类基因组中发现的最多产的反转录转座子。Alu 元件已被证明是RIG-I、MDA-5和TLR 3信号传导的主要驱动因子，这表明， ADAR 1依赖性编辑Alu元件的主要功能是抑制免疫应答。编辑是 在病毒感染后，主要通过干扰素诱导的较长的 ADAR 1（ADAR 1 p150），其与短同种型不同之处在于其具有N-末端Z-RNA结合结构域 （Zα），它是前病毒。这表明Zα的Z-RNA结合功能在靶向中起着关键作用。 ADAR 1 p150与Alu元件内的Z-RNA形成区域沿着，附近有结构同源的Zβ 然而，ADAR 1 p150的RNA结合机制和特异性尚不清楚。我 一种假设是N-末端Zα结构域和密切相关的Zβ结构域增强了A-to-I编辑 通过在干扰素应答期间将ADAR 1 p150靶向到Alu元件内的Z-RNA形成区域。 这种增加的编辑反过来“钝化”干扰素反应，并允许许多类型的病毒增殖 无人质疑在这个建议中，我提出了一个整合结构生物学，RNA测序， 生物化学技术，以揭示特定的Z-RNA形成序列内的Alu元件和过程中， Zα和Zβ结构域对Alu元件中A-to-ZRNA转换的影响，并回答有关Alu元件中A-to-ZRNA转换的基本问题 ADAR 1 p150的。我的具体目标是（目标1）描述Alu RNA中从A-到Z-RNA的转变 通过生物化学和结构技术。我还计划（目的2）研究Z-RNA如何识别 Zα和Zβ的能力有助于在体内识别和编辑Alu元件，以及它如何与West 通过RNA测序技术对HEK 293 T细胞的Nile感染。由于我们对Z的了解不够， 由于ADAR 1的DNA/RNA结合结构域，我们可能极大地忽略了能够 在转录组中采用Z构象。我的建议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了基础， 帮助指导试图操纵A到I编辑作为治疗多种疾病的工具的研究， 抑制ADAR 1 p150的前病毒特征。