Engineered Alterations in RNA Editing Modulate Complex Behavior in Drosophila

Abstract

Select proteins involved in electrical and chemical neurotransmission are re-coded at the RNA level via the deamination of particular adenosines to inosine by adenosine deaminases acting on RNA (ADARs). It has been hypothesized that this process, termed RNA editing, acts to "fine-tune" neurophysiological properties in animals and potentially downstream behavioral outputs. However, the extreme phenotypes resulting from deletions of adar loci have precluded investigations into the relationship between ADAR levels, target transcripts, and complex behaviors. Here, we engineer Drosophila hypomorphic for ADAR expression using homologous recombination. A substantial reduction in ADAR activity (>80%) leads to altered circadian motor patterns and abnormal male courtship, although surprisingly, general locomotor coordination is spared. The altered phenotypic landscape in our adar hypomorph is paralleled by an unexpected dichotomous response of ADAR target transcripts, i.e. certain adenosines are minimally affected by dramatic ADAR reduction, whereas editing of others is severely curtailed. Furthermore, we use a novel reporter to map RNA editing activity across the nervous system, and we demonstrate that knockdown of editing in fruitless-expressing neurons is sufficient to modify the male courtship song. Our data demonstrate that network-wide temporal and spatial regulation of ADAR activity can tune the complex system of RNA-editing sites and modulate multiple ethologically relevant behavioral modalities.Informational recoding of RNA by the catalytic deamination of adenosine to inosine proceeds through the action of ADARs 3 (1). Long double strand RNA duplexes exhibiting perfect complementarity can be modified extensively by promiscuous ADAR activity. However, mRNAs may also serve as site-specific substrates for ADARs via base pairing interactions that generate short imperfect duplexes that generally include the exon destined for editing and a cis-acting complementary sequence, usually found in a neighboring intron (2, 3). Because inosine is recognized by the translation machinery as guanosine (4), A-to-I editing in mRNAs can lead to the incorporation of amino acids differing from those specified by the literal genome.In Drosophila, the spectrum of ADAR substrates is peculiarly specific, consisting primarily of mRNAs encoding an array of voltage-and ligand-gated ion channels, as well as numerous pre-synaptic proteins involved in exo-and endocytosis of synaptic vesicles (5-8). Similarly, several mammalian ion channels and G-protein-coupled receptors are also subject to RNA editing (2, 7, 9 -11). In light of the ontological class and high sequence conservation of ADAR target genes, RNA editing has been invoked as an essential function in controlling synaptic transmission and neurophysiology. Correspondingly, deletion of the single Drosophila adar locus (dAdar) results in severe adult-stage behavioral abnormalities, including extreme uncoordination, seizures and a complete lack of courtship in dAdar null (dAdar 5g1 ) males (1...

Auto-regulatory RNA editing fine-tunes mRNA re-coding and complex behaviour in Drosophila

“…1c). We have previously shown that the HA-tag has no effect on dADAR activity on several known targets 28 , and western blot analyses revealed that modifying auto-editing has no significant effect on levels of dADAR protein expression (Fig. 1d).…”

“…We recently engineered a novel hypomorphic allele of dAdar ( dAdar hyp ), in which dADAR expression is reduced by ~ 80% (ref. 28). In this background, editing at many sites, but not all, is significantly reduced.…”

“…A common theme of ADAR mutations in several higher metazoan model genetic systems is the disruption of normal nervous system function 5–7,28 . We therefore asked whether the relatively subtle changes in editing observed in the dAdar S and dAdar G backgrounds could also confer abnormal adult-stage behaviors.…”

“…7a,b). The anticipation of morning is an output of a subset of the circadian neuronal network, and interestingly is also absent in dAdar hypomorphs 28,31 . To quantify the degree of increase in locomotor activity in the hours preceding the onset of morning, we calculated the total number of beam breaks occurring in the three hours before lights-on divided by the total in the six hours before lights-on (i.e a value of 0.5 indicates no anticipation).…”

Reovirus-mediated induction of ADAR1 (p150) minimally alters RNA editing patterns in discrete brain regions

“…ADAR substrates in Drosophila melanogaster (dADAR) appear to fall into “high” or “low” categories based upon structure and sequence determinants that affect their intrinsic ability to be selectively deaminated (Jepson et al, 2011). Higher levels of dADAR are required for the editing of “low efficiency sites”, whereas low levels of enzyme can effectively edit “high efficiency” sites.…”