ADAR genes in chordate and invertebrate genomes

The role of ADARs is to deaminate adenosine to inosine non-specifically in long double-stranded RNA (dsRNA) or site-specifically in transcripts. Non-specific adenosine deamination has a role in defence against viruses that have dsRNA stages in their life-cycle [1], as hypermutation of the viral genome by adenosine-to-inosine conversion disrupts viral open reading frames. The second role of ADARs, site-specific deamination of individual adenosines in pre-mRNAs, can change individual codons in open reading frames, including start or stop codons, or it may affect splicing or untranslated regions [2].

The first ADAR gene to be identified was the vertebrate ADAR1. The protein was purified from Xenopus, bovine liver and calf thymus using non-specific deamination of dsRNA as an assay [3-5], and peptide-sequence information was used to design PCR primers to clone the gene [6,7]. The ADAR1 gene was found to encode three double-stranded-RNA binding domains (dsRBDs) and a deaminase domain with zinc-binding motifs (Figure ​(Figure1),1), similar to those of zinc-dependent cytosine deaminases and to the cytosine-to-uracil RNA-editing enzyme APOBEC1. The first example of site-specific editing, in a pre-mRNA encoding the vertebrate glutamate-receptor subunit GluR-B, was found at the same time; editing converts a glutamine codon to an arginine codon (Q to R in the single-letter amino-acid code) [8]. ADAR1 was unable to catalyze site-specific editing of the Q/R editing site in the GluR-B transcript in vitro, however. This observation led to the idsentification of ADAR2, which edits the GluR-B Q/R site specifically [9,10]. ADAR2 contains two dsRBDs and an adenosine-deaminase domain (Figure ​(Figure1).1). Other ADAR genes in vertebrates and ADAR genes in other organisms were identified either by PCR using primers derived from the deaminase motifs or by sequence homology.

Open in a separate window

Figure 1

Primary structures of ADARs and ADATs (adenosine deaminases acting on tRNAs), showing arrangements of dsRNA-binding domains and deaminase domains. The deaminase motifs (I-III) consist of four or five highly conserved amino acids surrounding each of the cytosines and histidines that bind zinc. Proteins have been drawn to scale and are aligned at the catalytic glutamate residue (E) in the active site in adenosine deaminase motif I. The deaminase domains of ADAR and ADAT1 proteins are larger than the core deaminase domain (which we define as the region of homology between Tad2/ADAT2 and Tad3/ADAT3) because of the insertion of sequence between the second and third zinc-chelating residues, separating deaminase motifs II and III, and probably also because of fusion with a carboxy-terminal subdomain of unknown origin. Abbreviation: aa, amino acids.

There are two ADAR-like genes in vertebrate genomes, encoding enzymes with unknown function. The ADAR3 gene, also called Red2, is very similar to ADAR2 (Figure ​(Figure2)2) [11]. It has all the key conserved residues that are known to be required for catalysis, but it has been shown to lack non-specific and site-specific dsRNA adenosine-deaminase activity [12]. ADAR3 may have arisen from ADAR2 by gene duplication within the chordate lineage. ADAR3 is very well conserved in fish genomes, even though no function has been ascribed to it, suggesting that it is active on an unknown substrate. A fourth ADAR-like gene in vertebrates, TENR, is expressed in the male germline and has only one dsRBD [13]. TENR lacks a key catalytic glutamate residue in the deaminase domain and also lacks zinc-chelating residues; the protein has not been characterized. We have been able to identify TENR genes only in mammalian and not in fish genomes.

Open in a separate window

Figure 2

A phylogenetic tree of core deaminase sequences. Protein sequences that are predicted from genomic sequences rather than from full cDNA sequences are indicated by asterisks. The vertebrate genes listed include one from the crab-eating monkey Macacus fascicularis. The fish genomes examined are from the zebrafish Danio rerio and pufferfish, Takifugu rubripes and Tetraodon fluviatilis. Two tunicate genomes have been examined, from Ciona intestinalis and Ciona savigni. One squid (Loligo pealeii) is included (J. Rosenthal, personal communication). The insect genomes are of the two Drosophila species, Drosophila melanogaster and Drosophila pseudoobscura, and the malaria mosquito Anopheles gambiae. Alignments were made using T-COFFEE, the boundaries of the core deaminase domain were specified and a tree based on the alignment was generated using MEGA. Bootstrap values are given on all branches.

The presence of four ADAR genes in vertebrates represents a degree of gene-family expansion in the chordate lineage similar to that found in many other gene families, notably including the cytosine deaminases acting on RNA (CDARs) [14]. The vertebrate ADAR genes are unlinked in the genome, but some chordate genomes contain multiple copies of particular ADAR genes: for example, the Xenopus genome, which is large and not completely sequenced, contains two ADAR1 genes, and Takifugu rubripes has two ADAR2 genes.

The Drosophila genome encodes one ADAR, which has two dsRBDs and a deaminase domain and is more similar to vertebrate ADAR2 than to ADAR1 [15]. We searched for ADAR genes in the complete genome sequences of the primitive chordates Ciona intestinalis and Ciona savigni (our unpublished work); predicted deaminase domains were found that seem to correspond to ADAR1 and ADAR2 as well as a separate ADAT1 gene (see below; Figure ​Figure2)2) [16]. We have not proven that these are full ADAR1 genes, because there is no cDNA sequence available for them and because the full genes with dsRBDs are difficult to predict from the genome sequence. ADAR1-like deaminase domains are clearly present in basal chordates, however. This is a little surprising and argues against the idea that ADAR1 and ADAR2 diverged from a parental gene during the period of widespread gene duplications in early vertebrate evolution; it suggests, instead, that the ADAR duplication occurred earlier (our unpublished work). An ADAR2 gene has been cloned from the squid Loligo pealeii, but it is not clear whether squid also have an ADAR1 gene.

The genomes of the nematodes Caenorhabditis elegans and Caenorhabditis briggsae contain two ADAR genes, adr1 and adr2 [17]. The deaminase domains of ADR1 and ADR2 are not closely related to each other, and their names do not reflect a direct correspondence with vertebrate ADAR1 and ADAR2 proteins; the sequences differ so much from vertebrate ADARs that it is impossible to make such links (Figure ​(Figure2).2). Both adr1 and adr2 are required for editing activity in vivo, and the proteins may form heterodimers. ADR1 has two dsRBDs; ADR2 has only one and lacks the active-site glutamate. The C. elegans adr1 gene structure shows some resemblance to the Drosophila Adar gene, however. In addition to encoding an ADAR with two dsRBDs, C. elegans adr1, like Drosophila Adar, produces several isoforms by alternative 3' splice-site usage affecting the sequence between the dsRBDs.

Human ADAR1 has two transcription start sites and two promoters, leading to production of a shorter isoform that lacks the first 296 residues of the long form (Figure ​(Figure1).1). The first 530 amino acids of human ADAR1, up to the middle of the first dsRBD, is encoded by a single exon. Interestingly, each of the regions encoding the three dsRBDs in ADAR1 is interrupted in the middle by an intron in a conserved position, suggesting that they could have arisen by duplication of a pair of exons. The deaminase motifs are encoded on separate exons. Human ADAR2 has both dsRBDs on one exon, as does ADAR3. As in human ADAR1 the deaminase motifs are encoded on separate exons but intron positions in the deaminase domain are not well conserved between human ADAR1 and ADAR2. Human ADAR2 produces two isoforms one of which (ADAR-2L) has an exonized Alu sequence that inserts an additional 40 amino acids between the second and third deaminase motifs (the ADAR2-S form lacking the Alu sequence is shown in Figures ​Figures11 and ​and3).3). These splice variants should not be confused with the two distinct ADAR2a and ADAR2b genes found in the genome of T. rubripes. Alternative splice forms of ADAR3 or TENR have not been described.

Open in a separate window

Figure 3

An alignment of core deaminase domains from vertebrate ADAR1 and ADAR2 proteins, showing residues characteristic for each. Asterisks indicate cysteines and histidines that chelate zinc, as in Figure ​Figure1,1, and the three active-site motifs are bracketed.

The adenosine deaminase domain

Experimental exchanges of the deaminase domains of ADAR1 and ADAR2 have shown that the deaminase domain determines the difference in specificity between the two proteins [23]. With the availability of more genomic sequences, we have aligned 14 different sequences of ADAR1 and ADAR2 deaminase domains and found a high degree of conservation in a region of 179 amino acids (Figure ​(Figure3).3). The amino acids that are downstream of the second deaminase motif appear to specify the enzyme that is encoded. The sequence differences between ADAR1 and ADAR2 that are highlighted (Figure ​(Figure3)3) make it possible to assign newly sequenced vertebrate ADAR genes as ADAR1 or ADAR2 orthologs, even in cases in which there in no full-length cDNA sequence and the number of dsRNA-binding domains is not known. With this obvious difference close to the catalytic site, it is not surprising that ADAR1 and ADAR2 show very little overlap in the specific sites they edit in transcripts, despite the fact that they both perform the same enzymatic activity. The three active site sequence motifs are particularly characteristic of ADARs. Each motif contains a cysteine or histidine that is thought to chelate zinc at the active site (asterisks in Figure ​Figure3);3); the first deaminase motif (CHAE) includes a glutamate (E) that is required for catalysis.

Structural information is available for one E. coli free-nucleotide cytosine deaminase [24]. This protein has deaminase-domain motifs similar to ADAT2 and tadA and dimerizes so that each monomer contributes to form the active site; it has very little homology to ADARs apart from the zinc-binding motifs, and it is not clear whether the ADARs dimerize in an equivalent way around the active site. The additional sequence between motif II and motif III in ADARs may be incompatible with this mechanism of dimerization. Structural information from this cytosine deaminase cannot be used reliably to model ADAR deaminase domains but may be more useful for modeling the deaminase domains of ADAT2 and ADAT3.

We do not know how the deaminase domain interacts with RNA. The ADAR2 deaminase domain without dsRBDs is capable of site-specific editing in vivo [25], and the same is true of the Drosophila ADAR deaminase domain (G. Ring and M.O'C., unpublished observations). The ADAR deaminase domain was therefore a dsRNA-binding domain even before it acquired dsRBDs. It clearly evolved from ADAT1 [19], but the residues responsible for binding to tRNA in ADAT1 and in the heterodimer ADAT2/ADAT3 are as yet unknown. Considering the sequence homology between the ADARs and ADAT1, the intriguing possibility arises that recognition of pre-mRNA and tRNA substrates by these proteins follows a common structural model. Both ADARs and ADAT1 probably flip the target adenosine out of the DNA and into the enzyme's active site; studies with a fluorescent adenine analog at a second GluR-B DNA-editing site (the R/G site) are consistent with base-flipping by ADAR2 [26,27]. (The R/G site is separated from the Q/R site and is more experimentally accessible.) Consistent with this idea, some similarity to DNA methyltransferases that flip out the target base has been found in the deaminase domain of ADARs [13].