Systematic characterization of trans regulation of A-to-I RNA editing in neurons

项目概要/摘要 RNA编辑是产生时空转录组多样性的关键过程， 在大脑中尤为重要。A到I（腺苷到肌苷，被认为是 鸟苷）编辑是后生动物中最常见的RNA编辑形式， 一种称为腺苷脱氨酶的酶家族，作用于RNA（ADAR）。A-to-I编辑 当双链RNA（dsRNA）被结合并编辑时， 阿达尔酶，其在神经系统中以高频率发生。RNA的改变 编辑水平与许多神经系统疾病有关。失去阿达尔可导致 神经学表型，如癫痫发作、改变的运动和昼夜节律。先前 研究阿达尔突变的工作证明了一些关键氨基酸的重要性 进行适当的编辑活动，包括少数已知会导致人类疾病的基因。但我们 仍然缺乏对阿达尔蛋白功能全面了解。我们甚至更不了解 其他RNA编辑的反式调节因子，尽管有证据表明它们的存在。在这 工作，我们的目标是开发系统的方法来破译A到I的反式调节 RNA编辑。首先，我们将在人类细胞中鉴定ADAR1和ADAR2的功能突变体。 使用我们最近开发的一种基于CRISPR的技术， ADAR 1和ADAR 2的突变以在人细胞中引入点突变。我们将 鉴定编辑活性降低或增加的功能性阿达尔突变体，并进一步 表征这些突变如何影响体内阿达尔编辑活性。二是 通过生物化学和遗传筛选鉴定RNA编辑的新型调节剂。我们将 在诱导的人类神经元中鉴定ADAR相互作用蛋白。我们亦会进行一项 在诱导的人类神经元中进行全基因组CRISPR/Cas9筛选，以寻找改变人类神经元的候选者。 编辑级别。热门候选基因将接受CRISPR/Cas9二次筛选， 小鼠原代神经元以及成对组合的双敲除，以分析它们的 基因相互作用第三，我们将机械地确定编辑调节器如何改变 转录组范围的RNA编辑景观。我们将扰乱监管机构， 它们影响人类细胞、小鼠原代神经元和 果蝇的大脑我们将测试调节子是否与ADAR 1/2或 以及它们是否以及如何与阿达尔RNA底物相互作用。这项工作将提供 对神经元中A-to-IRNA编辑的反式调节有了前所未有的理解， 揭示了这种基本上未被探索的机制背后的新机制。

Project Summary/Abstract RNA editing is a critical process for generating spatiotemporal transcriptomic diversity that is particularly important in the brain. A-to-I (adenosine to inosine, which is recognized as guanosine) editing is the most common form of RNA editing in metazoans and is catalyzed by a family of enzymes called adenosine deaminases acting on RNA (ADARs). A-to-I editing occurs co-transcriptionally when double-stranded RNA (dsRNA) is bound and edited by ADAR enzymes, which occurs at high frequency in the nervous system. Alteration of RNA editing levels is implicated in a number of neurological disorders. Loss of ADAR can lead to neurological phenotypes such as seizure, altered locomotion and circadian rhythm. Previous work studying mutations in ADAR demonstrates the importance of a few amino acids critical for proper editing activity, including a handful known to cause human diseases. However, we still lack a comprehensive understanding of ADAR protein function. We know even less about other trans regulators of RNA editing despite the evidence suggesting their existence. In this work, we aim to develop systematic approaches to deciphering the trans regulation of A-to-I RNA editing. First, we will identify functional mutants of ADAR1 and ADAR2 in human cells. Using a CRISPR-based technology we recently developed, we will perform saturation mutagenesis of ADAR1 and ADAR2 to introduce point mutations in human cells. We will identify functional ADAR mutants with decreased or increased editing activity and further characterize how these mutations affect ADAR editing activity in vivo. Second, we will identify novel regulators of RNA editing through biochemical and genetic screens. We will identify ADAR-interacting proteins in induced human neurons. We will also carry out a genome-wide CRISPR/Cas9 screen in induced human neurons to find candidates that alter editing levels. Top candidate genes are subject to secondary CRISPR/Cas9 screening in mouse primary neurons as well as double knockout in pairwise combinations to analyze their genetic interactions. Third, we will determine mechanistically how editing regulators alter the transcriptome-wide landscape of RNA editing. We will perturb the regulators to examine how they affect editing levels transcriptome-wide in human cells, mouse primary neurons, and Drosophila brains. We will test whether the regulators physically interact with ADAR1/2 or each other, and if and how they interact with ADAR RNA substrates. This work will provide an unprecedented understanding of trans regulation of A-to-I RNA editing in neurons, revealing novel mechanisms underlying this largely unexplored machinery.