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

项目摘要/摘要 RNA编辑是产生时空转录多样性的关键过程，即 在大脑中尤为重要。A-to-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，由 作用于RNA的一组称为腺苷脱氨酶(ADARs)的酶。A-to-I编辑 当双链RNA(DsRNA)被结合和编辑时发生共转录 ADAR酶，在神经系统中出现的频率很高。RNA的改变 编辑水平与许多神经疾病有关。ADAR的丢失可能会导致 神经表型，如癫痫发作、运动改变和昼夜节律。上一首 研究ADAR突变的工作证明了几种关键氨基酸的重要性 适当的编辑活动，包括少数已知会导致人类疾病的编辑活动。然而，我们 目前尚缺乏对ADAR蛋白功能的全面了解。我们对此所知更少 RNA编辑的其他反式调控者，尽管有证据表明它们的存在。在这 在工作中，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。 使用我们最近开发的基于CRISPR的技术，我们将执行饱和 ADAR1和ADAR2的突变在人类细胞中引入点突变。我们会 确定编辑活性降低或增加的功能性ADAR突变体，并进一步 描述这些突变如何影响体内的ADAR编辑活动。第二，我们将 通过生物化学和基因筛选确定RNA编辑的新调节者。我们会 在诱导人神经元中鉴定ADAR相互作用蛋白。我们还将开展一项 在诱导的人神经元中进行全基因组CRISPR/Cas9筛选以寻找改变的候选基因 编辑级别。顶级候选基因在中国接受CRISPR/Cas9二次筛选 小鼠原代神经元以及成对组合中的双基因敲除分析其 基因的相互作用。第三，我们将机械地确定编辑监管机构如何改变 转录组广泛的RNA编辑版图。我们将扰乱监管机构，研究如何 它们影响人类细胞、小鼠原代神经元和 果蝇的大脑。我们将测试调节器是否与ADAR1/2或 以及它们是否以及如何与ADAR RNA底物相互作用。这项工作将提供 对神经元中A-to-I RNA编辑的反式调节的前所未有的理解， 揭示了这种在很大程度上未被探索的机械背后的新机制。

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.