RNA modifications, alternative splicing and circular RNA landscape in the mouse brain: inosine and beyond [circRNA-seq]

RNA modifications, including adenosine-to-inosine (A-to-I) editing, are fundamental for normal brain physiology, as they participate in multiple biological processes as well as in pathological conditions. These modifications can influence RNA secondary structure and alter affinity for RNA-binding proteins, thereby modulating RNA processing mechanisms such as backsplicing, which gives rise to circular RNAs (circRNAs). In this study, we investigated the impact of A-to-I editing on circRNAs in the mouse brain using Adar enzyme–deficient mice, as ADARs catalyze A-to-I RNA editing. We applied a self-developed protocol to sequence full-length circRNAs using Oxford Nanopore Technologies (ONT) and performed a comprehensive characterization of these molecules. In addition, Illumina sequencing was used to conduct a robust differential expression analysis of circRNAs upon ADAR depletion. Overall design: Total RNA from whole brains of adult 3–4-month-old male mice was used for circRNA sequencing. Total RNA was enriched for circular RNAs and sequecing libraries for ONT and Illumina were prepared as described in the Methods section of this publication. Three biological replicates per genotype were sequenced: ADAR2 knockout (Adar2-/-Gria2R/R), double ADAR1 catalytically inactive/ADAR2 knockout (Adar1E861A/E861A Ifih-/- Adar2-/-Gria2R/R), and wild-type controls. All mice were on a C57BL/6J background.