IUBMB
Life , 61(6): 572–578, June 2009
Institute of Biochemistry, College of Life Sciences, Zhejiang University (Zijingang Campus),
Hangzhou, Zhejiang, People’s Republic of China
Summary
Adenosine deaminases acting on RNA (ADARs) convert adenosines to inosines in double-stranded RNA in animals.
Identification of more ADAR targets and genome sequences of diverse eukaryotes present an opportunity to elucidate the origin and evolution of ADAR-mediated RNA editing. Comparative analysis of the adenosine deaminase family indicates that the first ADAR might have evolved from adenosine deaminases acting on tRNAs after the split of protozoa and metazoa.
ADAR1 and ADAR2 arose by gene duplications in early metazoan evolution, 700 million years ago, while ADAR3 and
TENR might originate after Urochordata–Vertebrata divergence. More ADAR or ADAR-like genes emerged in some animals ( e.g
., fish). Considering the constrained structure, ADAR targets are proposed to have evolved from transposable elements and repeats, random selection, and fixation, and intermolecular pairs of sense and antisense RNA. In some degree, increased ADAR-mediated gene regulation should substantially contribute to the emergence and evolution of complex metazoans, particularly the nervous system.
Ó 2009 IUBMB
IUBMB
Life , 61(6): 572–578, 2009
Keywords RNA editing; ADAR; ARAR target; evolution.
INTRODUCTION
RNA editing is a molecular process in which the RNA sequences are post-transcriptionally altered by base substitutions, insertions and deletions. RNA editing occurs in the nucleus, as well as in mitochondria and plastids, which are thought to have evolved from prokaryotic-like endosymbionts
( 1 ). However, most of the RNA editing processes appear to be
Received 19 February 2009; accepted 13 March 2009
Address correspondence to: Yongfeng Jin; Institute of Biochemistry,
College of Life Sciences, Zhejiang University(Zijingang Campus),
Hangzhou, Zhejiang, ZJ310058, People’s Republic of China. Tel:
1 0086-571-88206479. Fax: 0086-571-88206478.
E-mail: jinyf@zju.edu.cn
ISSN 1521-6543 print/ISSN 1521-6551 online
DOI: 10.1002/iub.207
evolutionarily recent acquisitions that arose independently ( 1 ).
Of these different kinds of nuclear-encoded RNA editing, the most universal type is adenosine to inosine transition by adenosine deamination in metazoans ( 2 ). Recent reviews on RNA editing have been elaborated on the ADAR gene organization, molecular mechanism, biological significance, and evolution in chordate and invertebrate genomes ( 2–7 ). Here, we focus on the origin and evolution of ADAR-mediated RNA editing.
ORIGIN AND EVOLUTION OF ADAR
All ADARs share a common modular domain organization: dsRNA-binding domains (dsRBDs) and catalytic deaminase domain ( 2 ). Both phylogenetic and biochemical considerations support the idea that the ancestral ADAR may have evolved from an ADAT1-like protein by acquisition of dsRBDs and residue changes in the adenosine-deaminase domain ( 4, 8 ). We searched for ADAR genes in the genome sequences in eukaryotes. ADARs were found only in multicellular animals from sea anemones to human (Fig. 1), and are clearly absent from yeast and plants. They have also not been found in all protozoa examined, including Babesia bovis, Cryptosporidium hominis,
Cryptosporidium parvum, Dictyostelium discoideum, Eimeria tenella, Giardia intestinalis, Leishmania braziliensis, Leishmania infantum, Leishmania major, Paramecium tetraurelia, Plasmodium berghei, Plasmodium chabaudi, Plasmodium falciparum, Plasmodium yoelii, Tetrahymena thermophila, Theileria parva, Trichomonas vaginalis, Trypanosoma brucei,Actinophrys sol, Amoeba proteus, Cyclidium glaucoma, Didinium nasutum,
Eimeria arloingi, Euglena deses, Giardia caprae, Gonium pectorale, Paramecium bursaria, Phacus pleuronectes, Uronema marina, Vorticella campanula . We also failed to find an ADAR gene from the genome of the unicellular choanoflagellate Monosiga brevicollis , the closest known relative of metazoans. Therefore, the first ADAR might appear after the split of protozoa and metazoa.
The first ADAR gene ( ADAR1 ) was identified from the vertebrate Xenopus ( 9 ), and later ADAR2 has also been identified

ORIGINS AND EVOLUTION OF ADAR-MEDIATED RNA EDITING 573
Figure 1.
Phylogenetic analysis of ADARs and ADATs. Taxa for which we collected new data are marked with circle. Genomic
DNA sequences and corresponding protein sequences of other homologues were obtained through BLAST, using the sequence of the most closely related organisms. Tool: Insect genomics in http://flybase.org/blast/; Nematostella vectensis, Daphnia magna ,
Ciona intestinalis , Branchiostoma floridae, M. brevicollis , and so forth, in JGI genome Web: http://genome.jgi-psf.org/; Strongylocentrotus purpuratus, Danio rerio, Gallus gallus , and Protozoa ( Schistosoma japonicum , Aureococcus anophagefferens, Batrachochytrium dendrobatidis , Trichoplax adhaerens , Trypanosoma cruzi , Plasmodium falciparum , Tetrahymena thermophila , etc),
Plantae ( Arabidopsis thaliana ; Oryza sativa ; Selaginella moellendroffii and Sorghum bicolor , etc), and so forth in http://www.ncbi.
nlm.nih.gov/mapview/. All protein sequences were aligned with Clustal W (http://www.ebi.ac.uk/Tools/clustalw2/) and a tree based on the alignment was generated using MEGA4.
E. coli TadA was selected as the outgroup.
because ADAR1 failed to efficiently catalyze editing at the
GluR-B Q/R site ( 10, 11 ). ADAR1 and ADAR2 have been found not only from primitive chordates Ciona intestinalis (Urochordata), Branchiostoma floridae (Cephalochordata), and echinodermatan Strongylocentrotus purpuratus , but also from cnidarian Nematostella vectensis (sea anemones) (Fig. 1). This sug-

574 JIN ET AL.
gests that ADAR1 and ADAR2 arise from the parental gene by gene duplications in early metazoan evolution, perhaps 700 million years ago. During subsequent evolution, either ADAR1 or
ADAR2 was lost in some species ( i.e
., insects), while both
ADAR1 and ADAR2 were kept in some species ( i.e
., chordates).
Moreover, two ADAR2 genes were located in the genomes of the cephalochordatan B. floridae and the vertebrate D. rerio
(Fig. 1), perhaps emerging independently by gene duplications.
There are at least two ADAR -like genes in vertebrate genomes, encoding enzymes with unknown function. The
ADAR3 gene, also called Red2 , is more closely related with
ADAR2 ( 12 ) (Fig. 1).
ADAR3 is conserved in all vertebrates from fish to human. However, we failed to find ADAR3 orthologs from primitive chordates C. intestinalis (Urochordata), B.
floridae (Cephalochordata), and S. purpuratus (Echinodermata).
Thus, ADAR3 might arise from ADAR2 by gene duplication within the vertebrate lineage (Fig. 1).
A fourth ADAR -like gene in vertebrates, TENR , is expressed in the male germline ( 12 ). Schumacher et al. has shown that this protein is localized to round and early elongating spermatid cells and suggests that it may be involved in testis-specific nuclear posttranscriptional processes such as alternative splicing, hnRNA packaging, or transport of mRNAs ( 13 ). Thus, TENR may bind to substrates but not have the ability to edit them and may even act as an inhibitor of mRNA editing ( 14 ). In addition to mammalians, we found TENR orthologs in fish and chicken genomes.
Similar to ADAR3 , no TENR orthologs have been found from primitive chordates C. intestinalis (Urochordata), B. floridae
(Cephalochordata), and S. purpuratus (Echinodermata). Thus,
TENR might originate after Urochordata–Vertebrata divergence.
Moreover, two distinct TENR genes were located in the genome of D. rerio . Interestingly, three distinct TENR-like genes have been found in the genome of T. rubripes and D. rerio , one of which encodes interleukin enhancer binding factor 3.
The Drosophila genome encodes one ADAR ( 15 ), which is more similar to vertebrate ADAR2 than to ADAR1 ( 4 ) (Fig. 1).
We searched for ADAR genes in the complete genome sequences of other insects: Culex pipiens, Bombyx mori, Tribolium castaneum , Apis mellifera , and Pediculus humanus . As a result, only a single ADAR locus was found. Similarly, only one
ADAR gene was found in the genome of Daphnia pulex , a species closely related with insects. In contrast, the genomes of the nematodes Caenorhabditis elegans contain two ADAR genes, adr1 and adr2 ( 16 ), which do not reflect a direct correspondence with vertebrate ADAR1 and ADAR2 proteins ( 4 ) (Fig.
1). Furthermore, adr2 is more similar to vertebrate ADAR1 than to ADAR2, and adr1 is more similar to vertebrate TENR
(Fig. 1).
EVOLUTION OF ADAR TARGETS
ADARs act on RNA that is completely, or largely, doublestranded, and catalyze the deamination of adenosine to produce inosine ( 2 ). Any double-stranded region of at least 15–20 base pairs may potentially be a substrate for ADARs ( 17 ). However, dsRNAs for ADARs can be broadly classified into nonspecific and specific types. In the former long regions of perfectly paired dsRNA may be edited nonspecifically ( 2 ). In contrast, shorter regions of imperfectly paired dsRNA may be precisely edited at particular adenosine in the midst of hundreds. This form of editing is most often observed in the coding regions of neuronal transcripts targeted by ADARs, such as pre-mRNA encoding the vertebrate glutamate receptor subunit GluR-B , the first example of site-specific editing ( 18 ). Later, we discuss several potential mechanisms for the evolution of editing targets.
ORIGIN OF ADAR TARGETS FROM TRANSPOSABLE
ELEMENTS AND REPEATS
Many animal ADAR targets appear to be derived from repeats and transposable elements, such as Alu and LINE.
Alu elements belong to the short interspersed element (SINE) family of retrotransposons, typically 300 nucleotides long, which altogether represent 10% of the whole human genome ( 19 ). Abundant A-to-I editing sites have recently been identified in more than 1,600 different human genes ( 20–23 ). Most of these editing sites reside within closely localized Alu elements inserted in inverse orientations, which would be predicted to form large dsRNA structures upon transcription ( 20–23 ). The extraordinary frequency of RNA editing in human is attributed to the dominance of the primate-specific Alu element in the human transcriptome ( 24 ).
Other mammals have a number of different SINEs, for example, rodent-specific SINEs in the mouse genome is more than human Alu SINEs ( 25, 26 ). The difference in RNA editing can be attributed to the basic requirements from two nearby, oppositely oriented repeats to form a long stable dsRNA ( 26 ).
The overall rate of RNA editing is determined by specific properties of different repeats such as abundance, length, and homology
( 26 ). DNA transposons are also extensively located in invertebrates, such as D. melanogaster and C. elegans . TIR (Terminal inverted repeat) of DNA transposon Tc1 of C. elegans is a target of RNA editing ( 27 ). Although the occurrence of editing was not yet reported in Drosophila transposons, it is conceivable that some of transposons are a target of ADAR. Insertion of transposons into new genomic sites might be one of the driving forces of
ADAR targets. Future studies are required to elucidate its regulatory function.
ADAR TARGETS FROM INTERMOLECULAR PAIRS
OF SENSE AND ANTISENSE RNA
Sense and antisense transcript pairs are RNAs containing sequences that are complementary to each other. They can be transcribed in cis , from opposing DNA strands at the same genomic locus, or in trans , from distinct loci. In humans, between 5 and 10% of all genes were found to have a cis antisense counterpart ( 28–30 ), and similar results were reported in mouse ( 31 ), and Drosophila ( 32 ). It is tantalizing to hypothesize

ORIGINS AND EVOLUTION OF ADAR-MEDIATED RNA EDITING 575
Figure 2.
Origin and evolution of ADAR targets.
(A) Origin of RNA editing from genomic repeats or transposable elements in animals.
(B) Sense and antisense transcript pairs are RNAs containing sequences that are complementary to each other. They can be transcribed in cis , from opposing DNA strands at the same genomic locus. Sense–antisense RNA-transcript pairs create dsRNA duplexes that undergo extensive A-to-I RNA editing.
(C) Random selection model of origin of ADAR targets. A newly evolved editing site arises by chance from hairpins encoded in the genome. Random targeting of transcripts by potential RNA editing could be deleterious, with only a few targets being selectively neutral or advantageous. If the new editing is physiologically important, selectively advantageous editing sites would be improved by mutations during subsequent evolution. Purifying selection would purge deleterious editing sites off. Thus, physiologically beneficial ADAR targets are fixed. Exons are represented by red boxes (in
DNA) and lines (in pre-mRNA) and introns are represented by blue lines.
that sense–antisense RNA-transcript pairs create dsRNA duplexes that undergo extensive A-to-I RNA editing. Indeed, there are a few cases of sense-antisense RNA transcript pairs creating dsRNA duplexes that undergo extensive A-to-I RNA editing ( 22, 33, 34 ). For example, A-to-I editing occurs within untranslated regions of eri-6 and eri-7 pre-mRNAs, whereas editing of the double-stranded intermediate does not affect eri-
6/7 trans -splicing ( 34 ). In addition, dsRNA duplexes from sense–antisense transcripts undergo extensive A-to-I RNA editing in some RNA viruses ( 2 ).
Although complementary RNA oligonucleotides could be synthesized to mimic RNA editing of intermolecular duplexes
( 35 ), the editing level in these human and mouse genomic antisense regions was found to be negligible ( 36, 37 ). These results might contribute to the facts that sense–antisense RNA pairings rarely form intermolecular duplexes after transcription in the nucleus ( 37 ). Alternatively, pairing might actually occur within the cell, but the edited duplexes might be retained in the nucleus ( 38, 39 ).
RANDOM SELECTION AND FIXATION
MODEL FOR THE EMERGENCE OF NOVEL
ADAR TARGETS
However, many more editing sites might exist outside repeats. For example, most of recoding by RNA editing occurs due to formation of dsRNA duplexes between nonrepetitive exonic and intronic sequences or within the coding sequences
(40-42) . Likewise, noncoding and intronic parts of the gene may also be edited out of known repeats ( 43 ). Therefore, additional ADAR targets are from a pool from which they are randomly drawn to potential hairpin. Indeed, recent genome-wide bioinformatic screen showed that the human genome encoded millions of potential hairpins (excluding Alu elements) mostly located in the transcribed part of the genome ( 44 ). Therefore, the number of functional editing sites could be much smaller than the number of potential editing sites that could be randomly produced by ADAR.
How are random ADAR targets fixed in the lineage? Three steps in the model are as follows (Fig. 2C). First, a newly evolved

576 JIN ET AL.
Figure 3.
Origins and evolution of ADAR-mediated RNA editing. This phylogenetic tree illustrates the relative position of the organisms examined here and is provided as a quick reference for taxonomic relationships. Possible biological pathways related with RNA editing are shown. ‘‘ n ’’ indicates the number of ADARs and ‘asterisk’ indicates a likely gene duplication event. Only those branches for organisms containing a novel ADAR ortholog are shown.
editing site would arise by chance when unconstrained sequences changed enough to form a partial duplex surrounding the edited nucleotide. This idea could be supported by the fact that site-specific A-to-I editing could occur after in vitro transcribed Arabidopsis mRNA was injected into Xenopus oocyte nuclei (data not shown). This initial editing level is postulated to be low to avoid an adverse effect on the fitness of the organism. Second, with time, provided that the regulation elicited by the new editing is physiologically important, selectively advantageous editing sites would be improved by additional changes to noncoding sequences or synonymous nucleotides during subsequent evolution. Purifying selection would purge deleterious editing sites off. Thus, physiologically beneficial ADAR targets are fixed by purifying selection. A few examples about convergent evolution of ADAR targets supported this scenario ( 18 , 42 , 45–46 ). In addition to recoding, a few beneficial ADAR targets in noncoding RNA are fixed (Fig. 2C), such as miRNA ( 47 ). Third, these fixed ADAR targets will also be lost via mutation. For example, two nAChR alpha6 editing sites, conserved in different insects spanning 300 million years of evolution— D. melanogaster, B. mori, T. castaneum , and A. mellifera ( 48–50 ), were lost in Anopheles ( 51 ). This loss of editing might correlate with the lack of downstream intronic sequences, which were necessary to direct editing by forming duplex RNA substrates for ADARs ( 50 ).
CONVERGENT EVOLUTION OF ADAR TARGETS
There are several examples about convergence of ADAR-mediated RNA editing. The precedent for convergence of RNA editing was firstly discovered in the glutamate receptor subunit genes encoding the AMPA and kainate receptor subtypes in mammals. In this case, the GluR2 (AMPA), GluR5, and GluR6 (kainate) subunit mRNAs are edited at the same critical Q/R site through different intron-directed RNA secondary structures ( 18, 45 ). The same I/V editing site occurs in both mammalian Kv1.1 and D. melanogaster and Loligo pealei Kv2 channels, evolutionarily far-removed members of different Kv subfamilies, further supporting evolutionary convergence ( 42 ). Fruit flies, mosquitoes, and butterflies have shared and species-specific syt I editing sites, while beetles, honeybees, and roaches do not edit syt I ( 41 ). Verification of these two A-to-I editing sites in squid indicates that RNA editing events might occur independently in insects and squid ( 46 ). These editing events occur independently in ion-channel families, suggesting that there is an adaptive benefit to regulate physiologically ion channels in this way.
Because RNA editing occurs at the posttranscriptional stage, the properties of the channel can be modified on a faster timescale and in a more precise manner.
RNA EDITING AND GENETIC VARIATION
Comparative analysis indicated that A-to-I editing usually occurred in highly conserved coding regions, but usually recoded less-conserved coding positions of these regions ( 46,
52 ). Our analyses indicate that edited sites accumulate mutations much more rapidly than unedited sites, and thus evolution of sites undergoing mRNA editing is accelerated to enhance protein diversity ( 46, 52 ). Furthermore, more than half of these edited amino acids are genomically encoded in the orthologs of other species ( 46 ). Therefore, ADAR-mediated RNA editing not only extends the sequence diversity at the RNA and protein level, but also is a novel source of genetic variation at least in some genome sequence ( 52 ).

ORIGINS AND EVOLUTION OF ADAR-MEDIATED RNA EDITING 577
RNA editing mechanisms appear to have evolved much later to compensate for gene sequences gone awry or to increase evolutionary variation ( 1 ). Although ADAR-mediated RNA editing is mechanistically very different from other types of editing in mitochondria and chloroplasts, they should have some parallel evolutionary rationale. In plants, it is suggested that RNA editing helps repair otherwise-deleterious genomic mutations ( 53–57 ). The
ADAR-mediated RNA editing system is driven by genetic variation to maintain phylogenetic conservation, similar to plant mitochondrial RNA editing. Unlike most cases of plant RNA editing, however, animal genes are seldom fully edited ( 2–7 ).
PERSPECTIVES AND PROBLEMS
There are crucial questions that remain to be answered regarding the origin and evolution of ADAR-mediated RNA editing.
Did ADAR-mediated RNA editing play a role in early metazoan evolution? In particular, what were the structure and mechanism of biogenesis of ancestral ADAR targets, if any such existed, and what is the predominant path(s) to generate new ADAR targets during the further course of eukaryotic evolution? A comprehensive search for ADAR targets in early metazoans (such as sea anemones) is indispensable to address these questions.
The emergence of ADARs in metazoans suggests that increased ADAR-mediated gene regulation accompanied and, probably, substantially contributed to the emergence of complex, organ-containing animal body plans, particularly the nervous system (Fig. 3). New ADAR targets seem to contribute to functional innovation at early and later stages of animal evolution. An example is the majority of editing sites in coding regions identified in the nervous system ( 58 ), indicating that RNA editing might play a critical role in nervous system function. However, very little is known about how ADARs interface with other biological pathways. Another fact is the occurrence of abundant ADAR targets in primates but not in other mammals ( 24 ). Did abundant ADARmediated RNA editing play a role in primate evolution? Editing of some exonic and intronic Alu elements could alter alternative splicing, and thus expanding the transcriptomic repertoire ( 59–
61 ). Sequence comparisons of representatives of all primate infraorders revealed the critical evolutionary steps leading to this editing-mediated exonization ( 61 ). The consequence of widespread editing in Alu elements remains to be elucidated.
ACKNOWLEDGEMENTS
The authors are grateful to Cao Jun for helping with sequence analysis. We also acknowledge James E. Jepson for help in commenting on the manuscript. This work was partly supported by research grants from the National Natural Science Foundation of China (90508007, 30770469), and 863 Program
(2006AA10A119) and the Program for New Century Excellent
Talents in University (NCET-04-0531).
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