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: 9365748
• 负责人: Jin Billy Li
• 金额: $ 41.86万
• 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/9365748
• 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; 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; Process; 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-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

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.

鉴于后生动物中的基因比以前预测的要少得多 生物学的几种机制在转录和翻译上产生多样性 等级。一种这样的机制是RNA编辑，其中RNA中的基部通过酶修饰 形成不同的基础。最常见的RNA编辑类型是腺苷对肌苷（A- to-i），由作用于RNA（ADAR）酶家族的腺苷脱氨酶催化。阿达 结合双链RNA并去除腺苷形成肌苷，然后是 通过细胞机械读取为鸟氨酸。因此，RNA编辑可以有助于 转录组的多样性通过改变蛋白质的氨基酸序列，改变 开始或停止密码子的位置，影响替代剪接模式，并影响 miRNA与目标位点结合的能力。通过RNA编辑戏剧的严格调节导入 许多与疾病相关的病例所示的角色。我们最近的结果表明 CIS调节在RNA编辑调节中起主要作用。但是，RNA编辑是如何的 由顺式调节元件调节，在很大程度上尚未探索。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 开发系统的方法来解密RNA编辑CIS的调节守则 规定。首先，我们将绘制与RNA相关的CIS定量性状基因座（QTL） 跨人的编辑水平。其次，我们将研究涉及的编辑QTL 在其他可能与RNA编辑功能相关的细胞过程中。第三，我们会的 应用合成生物学方法在DSRNA底物中引入突变 测量体外和人类在每个碱基的变体的编辑特异性和效率 细胞，并通过实验确定ADAR结合亲和力和RNA二级结构 体外。综上所述，这个拟议项目的目标将提供前所未有的 理解主导CI的主要序列和二级结构特征 调节A到I RNA编辑，并揭示RNA编辑之间的功能关系 和其他细胞过程。