Repurposing Endonuclease V for the detection and engineering of adenosine-to-inosine editing

重新利用核酸内切酶 V 进行腺苷至肌苷编辑的检测和改造

基本信息

批准号：
10322142
负责人：
Jennifer Margaret Heemstra
金额：
$ 19.67万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2022-09-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10322142
关键词：
Adenosine Affinity Amino Acid Sequence Antibodies Autoimmune Diseases Base Pairing Binding Biological Biosensing Techniques Brain Diseases Catalytic RNA Cell Line Cell physiology Cells Cellular Immunity Communities Cytosine Data Deaminase Deamination Detection Development Directed Molecular Evolution Disease Embryonic Development Endonuclease V Engineering Enzymes Event Frequencies Gene Expression Genetic Transcription Guanine High-Throughput RNA Sequencing Human Hybrids Hydrogen Bonding Immune response Immunoprecipitation In Vitro Information Storage Inosine Knowledge Link Location Malignant neoplasm of brain Maps Measures Mediating Messenger RNA Methods Modification Nervous System Physiology Nucleic Acids Nucleotides Pattern Play Prevalence Process Property Proteins RNA RNA Editing RNA-Binding Proteins Reagent Reporter Genes Research Research Personnel Role Sampling Site Site-Directed Mutagenesis Structure Techniques Technology Tissue Sample Transcript adenosine deaminase aged cancer type endonucleaseV epitranscriptomics improved method development nervous system disorder next generation novel diagnostics novel therapeutic intervention nucleobase overexpression preference recruit repair enzyme stem cell differentiation tool transcriptome transcriptome sequencing

项目摘要

Project Summary/Abstract RNA editing is a widespread strategy employed by cells to post-transcriptionally alter protein sequence and gene expression levels. Adenosine-to-inosine (A-to-I) editing is among the most common and impactful of these RNA modifications and is catalyzed by adenosine deaminases acting on RNA (ADARs). Deamination changes the structure and hydrogen bonding pattern of the nucleobase, and resulting inosines hybridize with cytosine to effectively recode these sites as guanine. Editing is essential for a number of processes including embryogene- sis, neurological function, and innate cellular immunity, and dysfunctional editing is also linked to autoimmune diseases, neurological disorders, and several types of cancer. Despite the critical role of A-to-I editing in cellular function, our understanding of the locations and frequency of this modification are confined by the inherent limi- tations in the currently available methods for mapping and quantifying inosine in the transcriptome. Additionally, the ability to site-selectively induce A-to-I editing would be extremely valuable for both the study of this modifi- cation and the development of new therapeutic approaches, yet current methods are hampered by their reliance on the substrate binding preferences of native or modified ADAR enzymes. A central challenge that has hindered the development of methods for studying A-to-I editing is the lack of availability of anti-inosine antibodies or other affinity reagents capable of selective binding to this modified nucleotide. We have overcome this challenge by repurposing the naturally occurring EndoV protein from an RNA-cleaving enzyme into an RNA-binding protein and have used this to develop a workflow to enrich inosine-containing RNAs from total cellular RNA. We have shown that this increases the fraction of reads in RNA-seq data that contain inosine and facilitates the discovery of new A-to-I editing sites in the transcriptome. The proposed research will leverage our EndoV method to de- velop a toolbox of technologies to advance the study and engineering of A-to-I editing. Together, these new methods will enable researchers to more accurately map editing sites in the transcriptome, quantify changes in overall editing prevalence rapidly and in high-throughput, and direct editing at specific target sites in living cells. Additionally, the methods developed here can be applied to other epitranscriptomic modifications beyond ino- sine, providing a set of technologies that are of broad utility to the RNA editing community.

项目概要/摘要 RNA编辑是细胞广泛采用的策略，用于转录后改变蛋白质序列和基因表达水平。腺苷到肌苷 (A-to-I) 编辑是这些 RNA 中最常见和最有影响力的编辑之一修饰并由作用于 RNA 的腺苷脱氨酶 (ADAR) 催化。脱氨基改变了核碱基的结构和氢键模式，以及产生的肌苷与胞嘧啶杂交有效地将这些位点重新编码为鸟嘌呤。编辑对于包括胚胎发生在内的许多过程至关重要 sis、神经功能和先天细胞免疫以及功能失调的编辑也与自身免疫有关疾病、神经系统疾病和几种类型的癌症。尽管 A 到 I 编辑在细胞中发挥着关键作用函数，我们对这种修改的位置和频率的理解受到固有限制的限制目前可用的转录组中肌苷图谱和定量方法中的一些内容。此外，位点选择性诱导 A 到 I 编辑的能力对于这种修饰的研究非常有价值阳离子和新治疗方法的开发，但目前的方法因其依赖性而受到阻碍天然或修饰 ADAR 酶的底物结合偏好。阻碍的主要挑战研究 A 到 I 编辑方法的发展是由于缺乏抗肌苷抗体或其他抗体能够选择性结合该修饰核苷酸的亲和试剂。我们克服了这一挑战将天然存在的 EndoV 蛋白从 RNA 切割酶重新利用为 RNA 结合蛋白并用它开发了一个工作流程，从总细胞 RNA 中富集含肌苷的 RNA。我们有结果表明，这增加了 RNA-seq 数据中含有肌苷的读数比例，并促进了发现转录组中新的 A 到 I 编辑位点。拟议的研究将利用我们的 EndoV 方法来开发一个技术工具箱来推进 A-to-I 编辑的研究和工程。在一起，这些新这些方法将使研究人员能够更准确地绘制转录组中的编辑位点，量化转录组中的变化总体编辑普遍快速且高通量，并在活细胞中的特定靶位点进行直接编辑。此外，这里开发的方法可以应用于除 ino- 之外的其他表观转录组修饰。正弦，提供了一套对 RNA 编辑界有广泛用途的技术。