Novel Assay System and Analysis Algorithm in Detecting RNA Editing Events and Linked Splicing Isoforms

摘要 RNA编辑是基因组编码的信息在RNA中发生改变的过程。RNA编辑是一种 增加RNA复杂性的有效方法，从而微调基因功能和剂量。腺苷至 肌苷(A-to-I)编辑是动物已知的最常见的RNA编辑类型。蜂窝机械 将肌苷识别为鸟苷，因此密码子的A-to-I编辑和剪接信号直接修饰蛋白质- 编码基因功能，而编辑microRNAs及其结合部位会改变基因表达。浩瀚无边 然而，大多数A-to-I编辑是在非编码区检测到的(例如，内含子和3‘或5’内的Alu-重复 非翻译区)，强烈表明这一细胞机制的调节作用尚不清楚。 信使核糖核酸编辑的失调与几种神经系统疾病有关。此外，我们和其他人 研究小组发现，其中一种5-羟色胺受体(5-羟色胺2C受体)的mRNA编辑发生了变化 与完全自杀、严重抑郁症和可能的物质使用障碍(SUD)有关。目前， 短读Sanger或Illumina测序是RNA编辑研究的主流工具。然而，这些 工具无法检测到编辑事件的“阶段”；即，它们无法检测到同时在两个或两个或 位于同一个mRNA分子上的多个相互距离超过50-100个碱基的位点。在……里面 此外，目前的工具集中在给定编辑站点附近的小区域，不允许我们 在剪接的背景下学习RNA编辑。在提议的项目中，我们的目标是开发一种最先进的工具 它可以被整个科学界所使用。如果我们成功了，这个工具将同时实现 检测和量化大脑中的RNA编辑和剪接异构体，从而使研究人员能够 在剪接的背景下学习RNA编辑。此外，这一工具将能够确定单倍型 具有多个编辑位点的mRNA分子。我们预计该工具将具有巨大的商业潜力 并将促进RNA编辑作为与SUD有关的分子机制之一的研究。

Abstract RNA editing is a process in which the genome-encoded information is altered in RNA. RNA editing is an efficient way to increase RNA complexity, thereby fine-tuning both gene function and dosage. Adenosine to Inosine (A-to-I) editing is the most common type of RNA editing known in animals. The cellular machinery recognizes inosine as guanosine, so A-to-I editing of codons and splicing signals directly modifies protein- coding gene function, whereas editing of microRNAs and their binding sites alter gene expression. The vast majority of A-to-I editing, however, is detected in non-coding regions (e.g. Alu-repeats within introns and 3' or 5' untranslated regions), strongly suggesting a still unknown regulatory role of this cellular mechanism. Dysregulation of mRNA editing was implicated in several neurological diseases. In addition, we and other groups showed that alterations in mRNA editing of one of the serotonin receptors (serotonin 2C receptor) is associated with completed suicide, major depression and possibly substance use disorder (SUD). Currently, short read Sanger or Illumina sequencing are the mainstream tools in RNA editing studies. However, these tools cannot detect the “phase” of editing events; i.e., they cannot detect simultaneous editing events at two or more sites which are situated further than 50-100bp from one another on the same mRNA molecule. In addition, the current tools, which focus on a small region in the vicinity of a given editing site, do not allow us to study RNA editing in the context of splicing. In the proposed project we aim to develop a state-of-the-art tool that can be used by the entire scientific community. If we are successful, this tool will enable a simultaneous detection and quantification of RNA editing and splicing isoforms in the brain hence allowing researchers to study RNA editing in the context of splicing. Moreover, this tool will enable determination of the haplotypes of mRNA molecules with multiple editing sites. We anticipate that this tool will have a great commercial potential and will facilitate research on RNA editing as one of the molecular mechanisms that is implicated in SUD.