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Visualizing adenosine-to-inosine RNA editing in the Drosophila nervous system
npg 2012 Nature America, Inc. All rights reserved.
James E C Jepson1,2, Yiannis A Savva1, Kyle A Jay1,3 & Robert A Reenan1
amber codon to a downstream output such as -galactosidase911.
However, to our knowledge no reporter system for editing activity has been validated in vivo. Here we reverse-engineered a de novo editing site in the context of an artificial UAG stop codon in Aequorea victoria GFP mRNA, which results in full-length GFP only after splicing and A-to-I editing, thus yielding a fluorescent output dependent on dADAR activity. We used this reporter system to detect biologically relevant spatial and temporal endogenous dADAR activity in the fly nervous system, responsiveness to an autoregulatory feedback loop in which dADAR edits its own transcript to fine-tune enzyme function12,13 and inter-individual variation in neuronal dADAR activity.
RESULTSEngineering a fluorescent reporter of ADAR activityTo generate an in vivo reporter for dADAR activity, we used structural information gleaned from comparative genomics8. The Drosophila synaptotagmin-1 (Syt1) mRNA is edited at four positions (AD), of which editing at site D is most robust2. The structure that directs editing at sites C and D has been partially solved and consists of a complex dsRNA pseudoknot formed between the edited exon and two editing site complementary sequence elements (E1 and E2) located in the downstream intron8. The duplex structure that directs editing at site D consists largely of intronic sequence (Fig. 1a).
Using this fact, we generated a chimeric gene construct that would express full-length GFP only when acted upon both by RNA splicing and dADAR modification. First, we inserted the entire intron 9 of Syt1, containing all splicing signals and the E2 cis element that directs editing at site D, directly into the GFP coding sequence, artificially generating two GFP exons (Fig. 1b). We arranged the GFP sequences such that the second position guanosine of a conserved GFP tryptophan codon (UGG) would be positioned at the normally edited third position adenosine of the isoleucine codon (AUA) in the RNA structure of Syt1. We mutated this tryptophan to an amber stop codon (UGG to UAG), recreating a potentially editable adenosine at the analogous position to Syt1 site D. Finally, we engineered element E2 with structurally...