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

摘要 RNA编辑是一个改变RNA中基因组编码信息的过程。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。腺苷与 肌苷（A-to-I）编辑是动物中最常见的RNA编辑类型。的细胞机器 将肌苷识别为鸟苷，因此密码子和剪接信号的A到I编辑直接修饰蛋白质- 编码基因的功能，而编辑microRNA及其结合位点改变基因表达。绝 然而，大多数A-to-I编辑在非编码区（例如内含子内的重复序列和3'或5'内含子内的重复序列）中检测到。 非翻译区），强烈表明这种细胞机制的调节作用仍然未知。 mRNA编辑的失调与几种神经系统疾病有关。此外，我们和其他 研究表明，其中一种5-羟色胺受体（5-羟色胺2C受体）的mRNA编辑的改变是 与自杀未遂、重度抑郁症和可能的物质使用障碍（SUD）有关。目前， 短读段桑格或Illumina测序是RNA编辑研究中的主流工具。但这些 工具不能检测编辑事件的“阶段”;即，它们无法检测两个或两个以上的同时编辑事件， 在相同的mRNA分子上彼此相距50- 100 bp的更多位点。在 此外，目前的工具，集中在一个小区域附近的一个给定的编辑网站，不允许我们 在剪接的背景下研究RNA编辑。在拟议的项目中，我们的目标是开发一种最先进的工具 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 检测和定量大脑中的RNA编辑和剪接异构体，从而使研究人员能够 在剪接的背景下研究RNA编辑。此外，该工具将能够确定 具有多个编辑位点的mRNA分子。我们预计这一工具将具有巨大的商业潜力 并将促进RNA编辑作为SUD中涉及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.