Tertiary structural elements determine the extent and specificity of messenger RNA editing

The specificity and extent of RNA editing by ADAR enzymes is determined largely by local primary sequence and secondary structural imperfections in duplex RNA. Here we surgically alter conserved cis elements associated with a cluster of ADAR modification sites within the endogenous Drosophila paralytic transcript. In addition to the local requirement for a central imperfect RNA duplex containing the modified adenosines, we demonstrate that a secondary RNA duplex containing splicing signals strongly modulates RNA editing. A subtle non-coding mutation, extending base pairing of this accessory helix, confers significant phenotypic consequences via effects on splicing. Through mutation/counter-mutation, we also uncover and functionally replace a highly conserved intronic long-range tertiary pseudoknot that is absolutely required for deamination of one particular adenosine in the central duplex. Our results demonstrate that complex RNA tertiary structures, which may be difficult to predict computationally, form in vivo and can regulate RNA-editing events.