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

DNA代表着我们基因组成的仓库，但它的许多复制品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，但后来它们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构建单元（腺苷或A）与一个不太常见的（肌苷或I）的交换。细胞RNA的这种编辑有助于细胞区分自身和非自身RNA。如果这种机制被破坏，免疫系统可能会受到损害，或者可能出现自身免疫疾病，并且在癌细胞中也观察到错误。导致这种修饰的生物学途径是复杂的，因为不是每个A都需要或应该变成I。一种名为ADAR 1的蛋白酶负责选择修饰位点，并催化导致“将A编辑为I”的化学反应。催化部分已经被理解，但ADAR 1如何识别RNA分子中的特定位点仍然是一个谜。该项目基于这样的假设，即ADAR 1中的一个区域是识别RNA上确切编辑位置的关键。已知该区域选择性地结合一种罕见的左手形式的RNA。 该提案将使用先进的NMR方法来监测从A型到Z型RNA的转变，结合等温量热法和圆二色性等技术来定位天然RNA上这部分长ADAR 1结合的位置。然后，该项目将通过精确定位导致完全组装复杂的步骤，描绘ADAR 1识别的签名。该项目将在企业研究中纳入课程模块，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我和非自我RNA。腺苷编辑成肌苷（A至I）通常由灵长类特异性Alu逆转录转座子上的“腺苷脱氨酶作用于RNA-1”蛋白（ADAR 1）催化。A到I的编辑在感染后增强，主要是通过干扰素诱导的ADAR 1的较长同种型，其在其N末端包含名为“Zα”的Z-DNA/Z-RNA结合结构域。在细胞中发现了左旋双螺旋构象的胞嘧啶和鸟苷（CpG）重复形式的Z-RNA，但此类结构的普遍性及其确切作用尚不清楚。此外，许多（如果不是大多数）建议采用Z构象的区域不类似于常规（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ADAR 1的Zα（与下游Zβ结构域协同作用）调节的，以及它们在这些RNA功能中的确切作用仍然未知。该项目将测试Zα与Alu元件的结合在编辑过程中起重要作用的假设，总体目标是实现A-Z RNA连接形成的结构和动力学表征以及Zα（-Zβ）跨转录组的识别。该项目由分子和细胞生物科学部的分子生物物理学小组支持。该奖项反映了NSF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.