Functional conservation in human and Drosophila of Metazoan ADAR2 involved in RNA editing: loss of ADAR1 in insects

Abstract

Flies with mutations in the single Drosophila Adar gene encoding an RNA editing enzyme involved in editing 4% of all transcripts have severe locomotion defects and develop age-dependent neurodegeneration. Vertebrates have two ADAR-editing enzymes that are catalytically active; ADAR1 and ADAR2. We show that human ADAR2 rescues Drosophila Adar mutant phenotypes. Neither the short nuclear ADAR1p110 isoform nor the longer interferon-inducible cytoplasmic ADAR1p150 isoform rescue walking defects efficiently, nor do they correctly edit specific sites in Drosophila transcripts. Surprisingly, human ADAR1p110 does suppress age-dependent neurodegeneration in Drosophila Adar mutants whereas ADAR1p150 does not. The single Drosophila Adar gene was previously assumed to represent an evolutionary ancestor of the multiple vertebrate ADARs. The strong functional similarity of human ADAR2 and Drosophila Adar suggests rather that these are true orthologs. By a combination of direct cloning and searching new invertebrate genome sequences we show that distinct ADAR1 and ADAR2 genes were present very early in the Metazoan lineage, both occurring before the split between the Bilateria and Cnidarians. The ADAR1 gene has been lost several times, including during the evolution of insects and crustacea. These data complement our rescue results, supporting the idea that ADAR1 and ADAR2 have evolved highly conserved, distinct functions.

Solution structure of the N-terminal dsRBD of Drosophila ADAR and interaction studies with RNA

“…The domain would fail to be fixed at a single position and might slide back and forth between different sites, resulting in a broadening of the resonances at the protein-RNA interface. Additionally, this might also account for the looser specificity of dADAR as compared with ADAR1 and ADAR2 [22]. Nevertheless, the affinity of binding between dADAR dsRBD1 and GluR-2 USL is strong (Kd = 0.40 ± 0.05 μM) and is very close to the affinity that have been determined for ADAR2 dsRBD1 binding to the same RNA substrate (Kd = 0.33 ± 0.03 μM) [7].…”

“…This question was of primary interest in order to support our structural NMR characterization, and has of course important implications for the understanding of the molecular determinant at the origin of the distinct selectivity of the different ADAR enzymes. Indeed, we recently demonstrated that Drosophila ADAR edits other sites in mammalian GluR-2 pre-mRNA, namely the Q/R site which is a natural substrate of ADAR2 and an intronic hotspot 1 site which is preferentially modified by ADAR1 [22], showing that dADAR is less selective than mammalian ADARs. Additionally, mammalian ADAR2 can modify highly specific sites of editing in Drosophila substrates [22], indicating an overlap in the editing specificity of dADAR and ADAR2.…”

“…Interestingly, such a mutation M67A (dADAR residue numbering) has been introduced in ADAR2 and results in a five fold decrease in editing efficiency for the GluR-2 R/G site [7]. It would be interesting to test whether such a mutant with less editing efficiency on the R/G site would also start editing other adenosines with a higher efficiency, and especially some ADAR1 specific sites that are efficiently edited by the Drosophila enzyme [22].…”

“…The role of the dsRBDs in ADAR proteins is to recognize and bind to dsRNA thereby bringing the deaminase catalytic domain to its substrate adenosine at specific editing sites. Whereas two functional enzymes are present in vertebrates (ADAR1 and ADAR2), Drosophila has a single ADAR protein (dADAR) related to vertebrates ADAR2 [22,23]. The structure of mammalian ADAR2 deaminase domain [24] and ADAR2 dsRBDs have been determined in their free state [25].…”

The emerging role of RNA editing in plasticity

“…Finally, multiple editing sites within a transcript create exponential diversity. All true metazoans express ADARs so they probably also edit their RNAs (Keegan et al, 2011). Vertebrates have two functional isoforms (ADAR1 and ADAR2), Drosophila has one (an ADAR2 ortholog), and most other invertebrates have at least two.…”