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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

基本信息

批准号：
10000212
负责人：
Jin Billy Li
金额：
$ 41.8万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10000212
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

项目摘要

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之前预测的要少得多，生物学中产生转录和翻译多样性的几种机制等级。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地。最常见的 RNA 编辑类型是腺苷至肌苷（A- to-I)，由作用于 RNA (ADAR) 酶家族的腺苷脱氨酶催化。阿达与双链 RNA 结合并使腺苷脱氨基形成肌苷，然后被细胞机器读取为鸟苷。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 miRNA 与其靶位点结合的能力。 RNA 编辑的严格监管很重要一些与疾病有关的案例就证明了这一作用。我们最近的结果表明顺式调控在RNA编辑调控中发挥着重要作用。然而，RNA 编辑是如何进行的？顺式监管要素的监管在很大程度上仍未被探索。缺乏系统性、全基因组研究阐明 RNA 编辑的顺式调控。在这项工作中，我们的目标是开发系统方法来破译RNA编辑顺式调控密码规定。首先，我们将绘制与 RNA 相关的顺式数量性状位点 (QTL) 人类个体的编辑水平。其次，我们将检查也涉及的编辑QTL 其他可能与 RNA 编辑功能相关的细胞过程。第三，我们将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每个碱基变异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。总而言之，该拟议项目的目标将提供前所未有的了解控制顺式的一级序列和二级结构特征 A-to I RNA编辑的调控，揭示RNA编辑之间的功能关系和其他细胞过程。