Molecular recognition by ADAR1 of Z-RNA within transcriptomes

项目摘要 对细胞RNA的RNA编辑有助于细胞区分自体和非自体RNA。这 腺苷编辑成肌苷通常是由作用于肌苷的腺苷脱氨酶催化的 RNA-1‘蛋白(ADAR1)。A&gt；I编辑在肿瘤和感染时增强，主要是 通过干扰素诱导的包含Z-DNA/Z-RNA结合的更长的ADAR1亚型 在其N端命名为‘Zα’的域。编辑错误与神经系统疾病有关，如 艾卡迪-古蒂埃综合征。以胞嘧啶和鸟苷(CpG)重复形式存在的Z-RNA 在细胞中已经提出了左旋双螺旋构象，但这种构象的流行 结构和它们的确切作用尚不清楚。此外，许多地区--如果不是大多数地区--提议 采用Z构象不像规则的(CpG)n。这些局部Z-RNA构象如何 在A型螺旋中产生，由ADAR1的Zα稳定和调节，以及它们的 这些RNA在功能中的确切作用尚不清楚。我们的假设是， Z-α到Z-RNA的转换在编辑过程中起着至关重要的作用。在此，我们建议 回答以下问题：CpG中Z-RNA的形成机制是什么，而且 非CpG序列？向Z-RNA的转变在转录本中有多普遍？是Z- 是以自由形式采样，还是仅在Zα结合时采用？什么是结构上的 Zα在非CpG序列上识别Z-RNA的特征？我们首先要动态地 表征各种序列上下文采用Z-RNA构象的倾向。 这一目标将使用先进的核磁共振方法来表征导致 从A-型到Z-型的RNA区域。第二，我们将确定无偏3D结构 在溶液中与Z-α结合。最后，我们将标识并本地化Zα绑定 站点和Zα依赖的A&>I编辑事件。这一目标将利用稳健的 在我们的园区和签约的 公司。总体而言，我们作为共同投资促进机构的联合工作将为形成 Z-RNA和由此形成的跨越各种RNA的A-Z连接。我们最终的目标是 为了解释ADAR1的Z-RNA结合域如何增加ADAR1的特异性和活性 ADAR1.我们的发现将有助于超越这一申请，提出一种全面的 ADAR1编辑机制及其RNA介导的转录组调控，以及 有助于了解癌症或自身免疫缺陷等疾病。

Project Summary RNA editing of cellular RNAs helps the cell distinguish between self and non-self RNAs. This editing of adenosines into inosines is generally catalyzed by the `adenosine deaminase acting on RNA-1' protein (ADAR1). A>I editing is augmented in tumors and 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. Misediting is implicated in neurological diseases such as Aicardi-Goutières syndrome. Z-RNA in the form of repeats of cytosine and guanosine (CpG) in a left-handed double-helical conformation has been proposed 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, as well as their exact role in the function of these RNAs, remain unknown. Our hypothesis is that the binding of Zα to Z-RNA plays an essential role during the editing process. Here, we propose to answer the following questions: What is the mechanism for Z-RNA formation at CpG but also non-CpG sequences? How widespread are transitions to Z-RNA across transcriptomes? Is Z- RNA sampled in the free form or only adopted upon binding by Zα? What are the structural features of Z-RNA recognition by Zα at non CpG sequences? We will first dynamically characterize the propensity of various sequence contexts to adopt Z-RNA conformations. This aim will use advanced NMR methods to characterize the sequence of events that lead an RNA region from A-form to Z-form. Second, we will determine the unbiased 3D structure of RNA fragments bound to Zα in solution. Finally, we will identify and localize Zα binding sites and Zα-dependent A>I editing events. This aim will take advantage of the robust expertise and support for next-generation sequencing on our campus and at a contracted company. Overall, our joint work as co-PIs will provide a structural rationale for the formation of Z-RNA and the resulting formation of A-Z junctions across a variety of RNAs. We ultimately aim to explain how the Z-RNA binding domain of ADAR1 increases the specificity and activity of ADAR1. Our findings will help beyond this application with proposing a comprehensive mechanism for ADAR1 editing and its RNA-mediated transcriptome-wide regulation, and contribute to understanding disease such as cancer or auto-immune deficiencies.