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

摘要

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.