A-to-I RNA editing remodels 5′-UTR initiation codons to tune translational output

Abstract

Abstract A-to-I RNA editing is a prevalent post-transcriptional modification in higher eukaryotes that converts adenosine to inosine within RNA molecules. Because inosine is interpreted as guanosine during translation, editing can alter codon identity and potentially influence translation initiation signals. Here, we examined whether A-to-I editing within the 5′ untranslated region (5′-UTR) can remodel upstream initiation codons and thereby tune downstream translation. Using luciferase-based reporter systems, we show that AUA-to-AUI editing generates an initiation-competent inosine-containing codon, whereas AUG-to-IUG editing markedly attenuates initiation and can relieve uORF-mediated repression. Quantitative in vitro and cellular assays establish the initiation hierarchy AUA < AUI < AUG, with IUG exhibiting strongly reduced initiation efficiency. Importantly, AUI-mediated upstream initiation did not behave like a canonical AUG-initiated uORF in the tested contexts; its effect on downstream ORF translation was modest and context-dependent. Transcriptome-wide bioinformatic analysis identified endogenous human transcripts whose 5′-UTRs harbor editing sites compatible with initiation-codon gain or attenuation. Reporter validation using native 5′-UTR sequences supports the possibility that editing-dependent initiation-codon remodeling can tune translational output in living cells, particularly through AUG-to-IUG-mediated derepression. Together, these findings establish a reporter-based framework in which A-to-I editing can remodel 5′-UTR initiation codons, while highlighting the need for endogenous protein-level and native-locus validation to determine physiological relevance.