Systematic approaches to deciphering cis regulation of A-to-I RNA editing

破译 A-to-I RNA 编辑顺式调控的系统方法

• 批准号: 9554985
• 负责人: Jin Billy Li
• 金额: $ 41.84万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9554985
• 关键词: Adenosine; Affect; Affinity; Alternative Splicing; Amino Acid Sequence; Binding; Biological Assay; Biology; Case Study; Catalytic RNA; Cell Nucleus; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; Computational Technique; DNA Methylation; DNA Sequence Alteration; Data; Data Set; Disease; Double-Stranded RNA; Drosophila genus; Engineering; Enzymes; Event; Family; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome engineering; Genomics; Genotype; Genotype-Tissue Expression Project; Goals; Guanosine; Human; Human Genome; In Vitro; Individual; Initiator Codon; Inosine; Lead; Libraries; Link; Location; Lower Organism; Maps; Measurement; Measures; MicroRNAs; Microfluidics; Mutagenesis; Mutation; Nucleotides; Oligonucleotides; Pattern; Play; Population; Quantitative Trait Loci; RNA; RNA Binding; RNA Decay; RNA Editing; RNA Processing; RNA Splicing; Regulation; Regulatory Element; Role; Sampling; Site; Specificity; Structure; Techniques; Technology; Terminator Codon; Tissues; Translations; Triplet Multiple Birth; Variant; Work; adenosine deaminase; base; computerized tools; genetic variant; genome sequencing; genome-wide; genome-wide analysis; histone modification; mRNA Precursor; malignant neurologic neoplasms; nervous system disorder; protein expression; repaired; synthetic biology; transcriptome; transcriptome sequencing; transcriptomics; tumor initiation; tumor progression

Given that there are much fewer genes in metazoans than previously predicted, there are several mechanisms in biology to generate diversity at the transcriptional and translational level. One such mechanism is RNA editing, in which a base in RNA is modified by an enzyme to form a different base. The most common type of RNA editing is Adenosine-to-Inosine (A- to-I), catalyzed by the adenosine deaminase acting on RNA (ADAR) family of enzymes. ADAR binds to double-stranded RNA and de-aminates Adenosine to form Inosine, which is then read as Guanosine by the cellular machinery. Thus, RNA editing can contribute to the diversity of the transcriptome by changing the amino acid sequences of proteins, altering the locations of start or stop codons, influencing alternative splicing patterns, and affecting the ability of miRNAs to bind to their target sites. Tight regulation by RNA editing plays import roles as exemplified by a number of cases linked to diseases. Our recent results suggest that cis regulation plays a major role in RNA editing regulation. However, how RNA editing is regulated by cis regulatory elements remains largely unexplored. There is a lack of systematic, genome-wide studies to elucidate the cis regulation of RNA editing. In this work, we aim to develop systematic approaches to deciphering the regulatory code of RNA editing cis regulation. First, we will map cis quantitative trait loci (QTLs) that are associated with RNA editing levels across human individuals. Second, we will examine editing QTLs also involved in other cellular processes that may be functionally related to RNA editing. Third, we will apply synthetic biology approaches to introduce mutations in the dsRNA substrates to measure editing specificity and efficiency for variants at each base in vitro and in human cells, and experimentally determine ADAR binding affinity and RNA secondary structure in vitro. Taken together, the goals of this proposed project will provide an unprecedented understanding of primary sequence and secondary structure features that govern the cis regulation of A-to-I RNA editing, and reveal the functional relationship between RNA editing and other cellular processes.

考虑到后生动物中的基因比以前预测的要少得多， 生物学中的几种机制在转录和翻译上产生多样性， 水平其中一种机制是RNA编辑，其中RNA中的碱基被酶修饰 形成不同的基础。最常见的RNA编辑类型是腺苷到肌苷（A- to-I），由作用于RNA的腺苷脱氨酶（阿达尔）家族酶催化。阿达尔 与双链RNA结合，使腺苷脱氨形成肌苷，然后 被细胞机器解读为鸟苷。因此，RNA编辑可以有助于 通过改变蛋白质的氨基酸序列，改变转录组的多样性， 起始或终止密码子的位置，影响选择性剪接模式，并影响 miRNAs与其靶位点结合的能力。RNA编辑的严格调控起着重要作用 一些与疾病有关的案例说明了这一点。我们最近的研究结果表明， 顺式调节在RNA编辑调节中起主要作用。然而，RNA编辑是如何 由顺式调节元件调节的蛋白质在很大程度上仍未被探索。缺乏系统的、 全基因组研究，以阐明RNA编辑的顺式调节。在这项工作中，我们的目标是 开发系统的方法来破译RNA编辑顺式的调控密码 调控首先，我们将定位与RNA相关的顺式数量性状基因座（QTL）， 编辑人类个体之间的水平。其次，我们将研究编辑QTL也涉及 在其他可能与RNA编辑功能相关的细胞过程中。三是 应用合成生物学方法在dsRNA底物中引入突变， 在体外和人体中测量每个碱基处变体的编辑特异性和效率 细胞，并通过实验确定阿达尔结合亲和力和RNA二级结构， 体外总的来说，这个拟议项目的目标将提供前所未有的 了解控制顺式异构体的一级序列和二级结构特征 A-to-I RNA编辑的调控，并揭示RNA编辑之间的功能关系 和其他细胞过程。