ADARs are a class of dsRNA binding proteins that catalyze the deamination of adenosines to inosines in double-stranded RNA, disrupting base-pairing at the editing site. A standard approach to characterize ADAR targets involves identifying A-to-G changes in cDNA sequences relative to the reference genome. Several well-characterized ADAR-editing events occur in coding regions and alter the amino acid sequence, including those in the glutamate ion channels, seratonin 2C receptor, and voltage-gated potassium channel (Kv1.1)1–5
. Despite these prominent examples, genome-wide assays for A-to-I editing sites in mammals show that the majority of ADAR targets are disbursed in clusters amongst non-coding regions (e.g. 3′ UTRs) and genomic repeat structures (e.g. SINEs)6–16
. Studies of ADAR activity in vitro
show that ADARs edit perfect hairpins throughout the double-stranded structure17
What is the functional consequence of promiscuous A-to-I editing for individual transcripts? Transcripts with inosine-containing 3′ UTRs have been suggested to undergo degradation, sequestration, additional processing, or enter other dsRNA-binding pathways18–24
. However, several HeLa and C. elegans
transcripts with inosine-containing 3′ UTRs showed no changes in protein levels, mRNA levels, or degree of ribosome association in animals lacking ADARs25
, suggesting that some observed effects may not be direct consequences of loss of editing.
The ADAR class of enzymes is conserved across metazoans26
, with loss of activity in mice and flies leading to a dramatic disruption: loss of ADAR1 in mice results in defective hematopoiesis and embryonic lethality27–30
without dADAR exhibit adult-stage uncoordination and temperature-sensitive paralysis31,32
. C. elegans adr
mutant strains exhibit chemotaxis defects and reduced life-span, but remain viable and fertile33,34
. As C. elegans adr
mutants lack extreme phenotypes, they provide a valuable system for studying the molecular contributions of A-to-I editing in transcriptome regulation.
Transcript levels for C. elegans adr-1
reach peak expression in embryos and the developing vulva. Examination of editing targets in select 3′ UTRs in mutant backgrounds has implicated ADR-2 as the active A-to-I editing enzyme, while ADR-1 appears to control site specificity and editing frequency33
.Physiological chemotaxis dependence has also been characterized, with defects evident in adr-1
mutant strains, more severe in adr-2
, and most severe in adr-1;adr-2
In addition to the physiological phenotypes, C. elegans adr-1;adr-2
mutant animals also exhibit a modulated response to transgene expression. Reporter transgenes introduced into the animal exhibit reduced expression, independent of sequence composition of the transgene35
. This phenomenon has been suggested to derive from unintended duplex structures produced from indiscriminant strand-nonspecific transcription of transgene arrays. The resulting duplex RNAs, which may serve as ADAR substrates in wild-type animals, would then trigger RNAi in the absence of active ADAR. Correspondingly, the modulated transgene effect is also rescued in adr-1;adr-2;rde-1
triple mutants, while transgene transcripts exhibit extensive A-to-I editing in the presence of ADAR35
The competitive relationship between the ADAR and RNAi pathways in C. elegans
appears to extend to regulation of endogenous genes. In particular, the chemotaxis defect of adr-1;adr-2
animals is rescued in strains additionally lacking critical components of the RNAi machinery (adr-1;adr-2;rde-4
. These observations suggest that ADARs may act to unwind endogenous dsRNAs capable of entering the RNAi pathway36
(). Alternatively, it is possible that components of the RNAi pathway act upstream of the ADAR pathway, possibly modulating editing activity or the availability of editing targets. Additionally, ADAR activity may act in a specific or non-specific manner to antagonize or modulate small RNA processing and maturation37–39
, and in such a role may act as a modulator of the miRNA pathway. These possibilities are certainly not mutually exclusive.
Diverse consequences of dsRNA formation
Assignment of ADARs to distinct cellular roles has been constrained by the lack of an identifiable sequence motif at editing sites40
and by challenges in definitive identification of functional ADAR targets41
. Nevertheless, conserved characteristics of binding and activity appear to implicate ADARs in a broad regulation of dsRNA populations42,43
. In mice, for example, the liver failure associated with defective hematopoiesis in ADAR mutants is suggested to arise from dsRNA-triggered interferon response27
. Mutant phenotypes in organisms lacking ADAR could thus arise as a secondary consequence of the deregulation of a much broader class of transcripts. Although the salient phenotype of C. elegans
ADAR mutants is neurological (resulting in defective chemotaxis), the broad role of ADAR editing may or may not be directly related to regulation of transcripts involved neurological development.
Taken in this view, elucidation of ADAR function will entail general characterization of broad classes of ADAR targets. Antagonistic interactions between the ADAR and RNAi pathways in C. elegans suggest that such a population of endogenous ADAR-targets could be evident by examining endogenous small RNA populations in editing-deficient animals. We introduce a genome-wide approach to identify highly edited loci that trigger a dramatic RNAi response in C. elegans in the absence of ADAR, characterizing a population of transcripts that naturally engage the ADAR mechanism.