Repurposing Endonuclease V for the detection and engineering of adenosine-to-inosine editing

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Project Summary/Abstract RNA editing is a widespread strategy employed by cells to post-transcriptionally alter protein sequence and gene expression levels. Adenosine-to-inosine (A-to-I) editing is among the most common and impactful of these RNA modifications and is catalyzed by adenosine deaminases acting on RNA (ADARs). Deamination changes the structure and hydrogen bonding pattern of the nucleobase, and resulting inosines hybridize with cytosine to effectively recode these sites as guanine. Editing is essential for a number of processes including embryogene- sis, neurological function, and innate cellular immunity, and dysfunctional editing is also linked to autoimmune diseases, neurological disorders, and several types of cancer. Despite the critical role of A-to-I editing in cellular function, our understanding of the locations and frequency of this modification are confined by the inherent limi- tations in the currently available methods for mapping and quantifying inosine in the transcriptome. Additionally, the ability to site-selectively induce A-to-I editing would be extremely valuable for both the study of this modifi- cation and the development of new therapeutic approaches, yet current methods are hampered by their reliance on the substrate binding preferences of native or modified ADAR enzymes. A central challenge that has hindered the development of methods for studying A-to-I editing is the lack of availability of anti-inosine antibodies or other affinity reagents capable of selective binding to this modified nucleotide. We have overcome this challenge by repurposing the naturally occurring EndoV protein from an RNA-cleaving enzyme into an RNA-binding protein and have used this to develop a workflow to enrich inosine-containing RNAs from total cellular RNA. We have shown that this increases the fraction of reads in RNA-seq data that contain inosine and facilitates the discovery of new A-to-I editing sites in the transcriptome. The proposed research will leverage our EndoV method to de- velop a toolbox of technologies to advance the study and engineering of A-to-I editing. Together, these new methods will enable researchers to more accurately map editing sites in the transcriptome, quantify changes in overall editing prevalence rapidly and in high-throughput, and direct editing at specific target sites in living cells. Additionally, the methods developed here can be applied to other epitranscriptomic modifications beyond ino- sine, providing a set of technologies that are of broad utility to the RNA editing community.