Adenosine-to-Inosine RNA editing by ADAR enzymes: Molecular mechanisms, substrate specificity, and functional consequences in human diseases

Abstract

The aim of this study is to determine the chemo-profile of aqueous extract of Nauclea latifolia using In 1987, researchers first noticed that double-stranded RNA molecules in Xenopus embryos underwent mysterious adenosine modifications a discovery that would eventually reshape our understanding of post-transcriptional gene regulation. Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by adenosine deaminases acting on RNA (ADAR) enzymes, represents one of the most widespread RNA modifications in mammals, with millions of editing sites identified across the human transcriptome. This research characterized ADAR1 and ADAR2 expression patterns, substrate specificity, and editing efficiency using synthetic double-stranded RNA substrates of varying lengths (50–2000 bp) in a cell-free system. Experiments were conducted at the Department of Molecular Biology, Guadalajara Technical University, between February 2022 and September 2023. Recombinant human ADAR1 (p150 isoform) and ADAR2 were expressed in a baculovirus system and purified by affinity chromatography. Editing levels were quantified by Sanger sequencing peak height analysis and validated by next-generation amplicon sequencing. Results showed that ADAR1 edited longer substrates (≥1000 bp) with up to 82.3% efficiency, while ADAR2 reached a plateau near 47.1% regardless of substrate length. Combined ADAR1+ADAR2 reactions achieved 93.7% editing at 2000 bp substrates, suggesting cooperative binding. Tissue expression profiling across eight human organs confirmed that brain expressed the highest levels of both ADAR1 and ADAR2, consistent with the known importance of RNA editing in neuronal function. ADAR2 showed a more tissue-restricted pattern than ADAR1. These findings clarify the division of labor between ADAR enzymes and provide quantitative benchmarks for understanding how substrate geometry influences editing outcomes relevant to neurological disease and cancer.