A comparative analysis among ADAR mutant mice reveals site-specific regulation of RNA editing

Abstract

Adenosine-to-inosine RNA editing is an essential posttranscriptional modification catalyzed by adenosine deaminase acting on RNA (ADAR)1 and ADAR2 in mammals. For numerous sites in coding sequences (CDS) and microRNAs (miRNAs), editing is highly conserved and has significant biological consequences, for example, by altering amino acid residues and target recognition. However, technical limitations have prevented a comprehensive and quantitative study to determine how specific ADARs contribute to each site. Here, we developed a simple method in which each RNA region with an editing site was amplified separately and combined for deep sequencing. Using this method, we compared the editing ratios of all sites that were either definitely or possibly conserved in CDS and miRNAs in the cerebral cortex and spleen of wild-type mice, Adar1 E861A/E861A Ifih -/mice expressing inactive ADAR1 (Adar1 KI) and Adar2 -/-Gria2 R/R (Adar2 KO) mice. We found that the editing ratio was frequently upregulated in either Adar mutant mouse strain. In contrast, we found that the presence of both ADAR1 and ADAR2 was required for the efficient editing of specific sites.In addition, some sites, such as miR-3099-3p, showed no preference for either ADAR. We further created double mutant Adar1 KI Adar2 KO mice and observed viable and fertile animals with complete absence of editing, suggesting that ADAR1 and ADAR2 are the sole enzymes responsible for all editing sites in vivo. Collectively, these findings indicate that editing is regulated in a site-specific manner by the different interplay between ADAR1 and ADAR2.

Quantification of AMPA receptor subunits and RNA editing-related proteins in the J20 mouse model of Alzheimer’s disease by capillary western blotting

“…Changes in ADAR1 and 2 have different but overlapping roles in RNA editing ADAR1 has been identified as the principal factor responsible for the majority of editing within non-coding regions and repeat elements in mRNAs. It does not exhibit the specificity that ADAR2 has for protein-recoding genes, particularly in regards to A-I editing events that alter codons such as the GluA2 Q/R site (Tan et al, 2017;Chalk et al, 2019;Costa Cruz et al, 2020). Nevertheless, both proteins possess the capability to edit numerous identical sites and are essential for the preservation of A-to-I editing in the epitranscriptome (Wong et al, 2001).…”

Harnessing ADAR-Mediated Site-Specific RNA Editing in Immune-Related Disease: Prediction and Therapeutic Implications

“…The ADAR enzymes exhibit a regional preference to catalyze specific sites by recognizing nearby sequences, as confirmed in Drosophila and humans [ 46 , 47 , 48 ]. ADAR1 and ADAR2 catalyze their preferred characteristic dsRNAs [ 45 , 49 , 50 ]. There is no significant difference between ADAR1 and ADAR2 in the global editing sites or regional preference [ 50 ].…”