Structural characterization of the mechanism leading to recognition of Alu elements by the Z-RNA-binding domain of ADAR1

DNA代表了我们基因组成的储存库，但它在RNA中的许多拷贝构成了生命的实际蓝图。这些RNA拷贝开始时是精确的DNA转录本，但它们后来以精确的方式被改变，最终决定了细胞的命运。一个非常常见的RNA组成部分（腺苷或a）与一个不太常见的RNA组成部分（肌苷或I）的交换。这种对细胞rna的编辑有助于细胞区分自我rna和非自我rna。如果这种机制被破坏，免疫系统就会受到损害，或者可能出现自身免疫性疾病，在癌细胞中也会观察到错误编辑。导致这种修饰的生物途径是复杂的，因为并不是每个A都需要或应该转化为I。一种名为ADAR1的蛋白质酶负责选择修饰位点，以及催化导致“将A编辑为I”的化学反应。催化部分已经被理解，但ADAR1如何识别RNA分子中的特定位点仍然是一个谜。该项目基于一个假设，即ADAR1中的一个区域是识别RNA上应该在哪里进行精确编辑的关键。已知该区域选择性地与一种罕见的左旋RNA结合。本研究将使用先进的核磁共振技术来监测从a型到z型RNA的转变，并结合等温量热法和圆二色性等技术来定位长ADAR1在天然RNA上的结合位置。然后，该项目将通过精确定位导致完全组装复合物的步骤，描绘ADAR1识别的签名。该项目将把企业研究纳入课程模块，并培训来自代表性不足社区的研究生、本科生和高中生。细胞RNA的RNA编辑有助于细胞区分自身和非自身RNA。将腺苷编辑为肌苷（A-to-I）通常由作用于灵长类动物特异性Alu逆转录转座子上的RNA-1蛋白（ADAR1）的“腺苷脱氨酶”催化。a -to- i编辑在感染后增强，主要是通过干扰素诱导的ADAR1的较长异构体，该异构体在其n端包含一个名为“Zα”的Z-DNA/Z-RNA结合结构域。在细胞中发现了左旋双螺旋结构的胞嘧啶和鸟苷（CpG）重复形式的Z-RNA，但这种结构的普遍程度及其确切作用尚不清楚。此外，许多（如果不是大多数的话）被提议采用Z构象的区域并不像规则的（CpG）n。这些局部Z-RNA构象是如何在a型螺旋内产生、稳定和受ADAR1的Zα（与下游Zβ结构域协同作用）调节的，以及它们在这些rna功能中的确切作用尚不清楚。该项目将验证Zα与Alu元件的结合在编辑过程中起重要作用的假设，其总体目标是实现A-Z RNA连接形成及其在转录组中被Zα(-Zβ)识别的结构和动态表征。本项目由分子与细胞生物科学部分子生物物理组支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

DNA represents the repository of our genetic makeup, but its many copies into RNA constitute the actual blueprint for life. These RNA copies start off as exact DNA transcripts, but they later get altered in precise ways that end up determining the fate of the cell. The swap of a very common RNA building block (adenosine or A) to a less common one (inosine or I). Such editing of cellular RNAs helps a cell distinguish between self and non-self RNAs. If that mechanism is corrupted, the immune system can be compromised, or auto-immune diseases may arise and misediting is also observed in cancerous cells. The biological pathway leading to such modifications is complex because not every A needs to or should be turned into an I. A single protein enzyme called ADAR1 is responsible for selecting the modification site, as well as for catalyzing the chemical reaction that leads to ‘editing of A to I’. The catalysis part is understood, but how ADAR1 recognizes a particular site in an RNA molecule remains a mystery. This project is grounded in the hypothesis that a region in ADAR1 is key for recognizing where exactly editing should occur on RNA. That region is known to selectively bind to a rare left-handed form of RNA. This proposal will use advanced NMR methods to monitor the transition from A-form to Z-form RNA, combined with techniques such as isothermal calorimetry and circular dichroism to localize where on natural RNA this part of long ADAR1 binds. The project will then delineate the signature for recognition by ADAR1, through pinpointing the steps leading to a fully assembled complex. This project will in corporate research into course modules and train graduate, undergraduate, and high school students from underrepresented communities.RNA editing of cellular RNAs helps a cell distinguish between self and non-self RNAs. Editing of adenosines into inosines (A-to-I) is generally catalyzed by the ‘adenosine deaminase acting on RNA-1’ protein (ADAR1) at primate-specific Alu retrotransposons. A-to-I editing is augmented 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. Z-RNA in the form of repeats of cytosine and guanosine (CpG) in a left-handed double-helical conformation has been found 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 (acting in synergy with the downstream Zβ domain), as well as their exact role in the function of these RNAs, remain unknown. This project will test the hypothesis that the binding of Zα to Alu elements plays an essential role during the editing process with the overall goal of achieving structural and dynamic characterization of the formation of A-Z RNA junctions and their recognition by Zα(-Zβ) across transcriptomes. This project is supported by the Molecular Biophysics Cluster of the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.