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

项目摘要 为了避免触发先天免疫，必须按细胞区分自体和非自体RNA 在不需要的时候做出反应。在人类，自身RNA是由作用于RNA的腺苷脱氨酶编辑的 (ADAR1)，它将腺苷修饰为肌苷。绝大多数A-to-I编辑事件发生在灵长类动物- 特定的Alu元件，这是在人类基因组中发现的最多的反转录转座子。Alu 已证明这些元件是RIG-I、MDA-5和TLR3信号的主要驱动因素，这表明 依赖ADAR1的Alu元件编辑的主要功能是抑制免疫反应。正在编辑 在病毒感染时主要通过干扰素诱导的较长的亚型 ADAR1(ADAR1p150)，它不同于短的异构体，因为它有一个N-末端的Z-RNA结合域 (Zα)，而且它是亲病毒的。这表明Z-α的Z-RNA结合功能在靶向中起着关键作用 Alu元素中的ADAR1p150到Z-RNA形成区以及附近结构上同源的Zβ 然而，ADAR1p150的RNA结合机制和特异性尚不清楚。我的 假设N端的Zα结构域和密切相关的Zβ结构域增强了A-to-I编辑 在干扰素应答期间，通过靶向ALU元件内的ADAR1p150到Z-RNA形成区域。 这种增加的编辑反过来会使干扰素反应变得迟钝，并允许多种类型的病毒扩散 无懈可击。在这个提案中，我提出了一种策略，它将结构生物学、RNA测序和 生物化学技术在铝元素内和过程中发现特定的Z-RNA形成序列 通过Zα和Zβ结构域感染并回答有关AlU元素A到Z核糖核酸转变的基本问题 ADAR1p150。我的具体目标是(目标1)描述Alu RNA中从A-RNA到Z-RNA的转变 通过生化和结构技术。我还计划(目标2)研究Z-RNA识别 Zα和Zβ的能力有助于体内铝元素的识别和编辑及其与西方的关联 RNA测序技术检测HEK293T细胞的尼罗河病毒感染由于我们对Z-缺乏了解- ADAR1的DNA/RNA结合域，我们可能极大地忽视了能够 转录组采用Z-构象。我的提议的结果将有助于填补这一空白 知识，为进一步研究Z-RNA在生物过程和 帮助指导试图将A-to-I编辑作为治疗多种疾病或 抑制ADAR1p150的亲病毒特性。

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.