InteractiveFly: GeneBrief

Adenosine deaminase acting on RNA: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Adenosine deaminase acting on RNA

Synonyms -

Cytological map position - 2B9--10

Function - RNA binding protein

Keywords - RNA editing adenosine deaminase, CNS

Symbol - Adar

FlyBase ID: FBgn0026086

Genetic map position -

Classification - double-stranded RNA adenosine deaminase

Cellular location - presumably nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Ramaswami, G., Deng, P., Zhang, R., Anna Carbone, M., Mackay, T. F. and Billy Li, J. (2015). Genetic mapping uncovers cis-regulatory landscape of RNA editing. Nat Commun 6: 8194. PubMed ID: 26373807
Summary:
Adenosine-to-inosine (A-to-I) RNA editing, catalysed by ADAR enzymes, plays an important role in neurological functions. This study applied a quantitative trait loci (QTL) mapping approach to identify genetic variants associated with variability in RNA editing. With very accurate measurement of RNA editing levels at 789 sites in 131 Drosophila melanogaster strains, this study identified 545 editing QTLs (edQTLs) associated with differences in RNA editing. Many edQTLs can act through changes in the local secondary structure for edited dsRNAs. Furthermore, edQTLs located outside of the edited dsRNA duplex were found to be enriched in secondary structure, suggesting that distal dsRNA structure beyond the editing site duplex affects RNA editing efficiency. Thus work will facilitate the understanding of the cis-regulatory code of RNA editing.

Robinson, J.E., Paluch, J., Dickman, D.K. and Joiner, W.J. (2016). ADAR-mediated RNA editing suppresses sleep by acting as a brake on glutamatergic synaptic plasticity. Nat Commun 7: 10512. PubMed ID: 26813350
Summary:
It has been postulated that synaptic potentiation during waking is offset by a homoeostatic reduction in net synaptic strength during sleep. However, molecular mechanisms to support such a process are lacking. This study demonstrates that deficiencies in the RNA-editing gene Adar increase sleep due to synaptic dysfunction in glutamatergic neurons in Drosophila. Specifically, the vesicular glutamate transporter is upregulated, leading to over-activation of NMDA receptors, and the reserve pool of glutamatergic synaptic vesicles is selectively expanded in Adar mutants. Collectively these changes lead to sustained neurotransmitter release under conditions that would otherwise result in synaptic depression. The study proposes that a shift in the balance from synaptic depression towards synaptic potentiation in sleep-promoting neurons underlies the increased sleep pressure of Adar-deficient animals. These findings provide a plausible molecular mechanism linking sleep and synaptic plasticity. 

Piontkivska, H., Matos, L. F., Paul, S., Scharfenberg, B., Farmerie, W. G., Miyamoto, M. M. and Wayne, M. L. (2016). Role of host-driven mutagenesis in determining genome evolution of sigma virus (DMelSV; Rhabdoviridae) in Drosophila melanogaster. Genome Biol Evol. PubMed ID: 27614234
Summary:
Sigma virus (DMelSV) is ubiquitous in natural populations of Drosophila melanogaster Host-mediated, selective RNA editing of adenosines to inosines (ADAR) may contribute to control of viral infection by preventing transcripts from being transported into the cytoplasm or being translated accurately; or by increasing the viral genomic mutation rate. Previous PCR-based studies showed that ADAR mutations occur in DMelSV at low frequency. This study used SOLiDTM deep sequencing of flies from a single host population from Athens, GA, USA to comprehensively evaluate patterns of sequence variation in DMelSV with respect to ADAR. GA dinucleotides, which are weak targets of ADAR, are strongly overrepresented in the positive strand of the virus, consistent with selection to generate ADAR resistance on this complement of the transient, double-stranded, RNA intermediate in replication and transcription. Potential ADAR sites in a worldwide sample of viruses are more likely to be "resistant" if the sites do not vary among samples. Either variable sites are less constrained and hence are subject to weaker selection than conserved sites, or the variation is driven by ADAR. Evidence was also found of mutations segregating within hosts, hereafter referred to as hypervariable sites. Some of these sites were variable only in one or two flies (i.e. rare); others were shared by four or even all five of the flies (i.e. common). Rare and common hypervariable sites were indistinguishable with respect to susceptibility to ADAR; however, polymorphism in rare sites were more likely to be consistent with the action of ADAR than in common ones, again suggesting that ADAR is deleterious to the virus. Thus, in DMelSV, host mutagenesis is constraining viral evolution both within and between hosts.
Duan, Y., Dou, S., Luo, S., Zhang, H. and Lu, J. (2017). Adaptation of A-to-I RNA editing in Drosophila. PLoS Genet 13(3): e1006648. PubMed ID: 28282384
Summary:
Adenosine-to-inosine (A-to-I) editing, catalyzed by adenosine deaminase acting on RNA (ADAR), is hypothesized to facilitate adaptive evolution by expanding proteomic diversity through an epigenetic approach. However, it is challenging to provide evidences to support this hypothesis at the whole editome level. This study systematically characterized 2,114 A-to-I RNA editing sites in female and male brains of D. melanogaster, and nearly half of these sites had events evolutionarily conserved across Drosophila species. Strong signatures were detected of positive selection on the nonsynonymous editing sites in Drosophila brains, and the beneficial editing sites were significantly enriched in genes related to chemical and electrical neurotransmission. The signal of adaptation was even more pronounced for the editing sites located in X chromosome or for those commonly observed across Drosophila species. A set of gene candidates (termed "PSEB" genes) was identified that had nonsynonymous editing events favored by natural selection. Evidence is presented that editing preferentially increased mutation sequence space of evolutionarily conserved genes, which supported the adaptive evolution hypothesis of editing. Prevalent nonsynonymous editing sites were found that were favored by natural selection in female and male adults from five strains of D. melanogaster. Temperature played a more important role than gender effect in shaping the editing levels, although the effect of temperature is relatively weaker compared to that of species effect. The relevant factors were explored that shape the selective patterns of the global editomes. Altogether this study demonstrated that abundant nonsynonymous editing sites in Drosophila brains were adaptive and maintained by natural selection during evolution. These results shed new light on the evolutionary principles and functional consequences of RNA editing.
Zhang, R., Deng, P., Jacobson, D. and Li, J. B. (2017). Evolutionary analysis reveals regulatory and functional landscape of coding and non-coding RNA editing. PLoS Genet 13(2): e1006563. PubMed ID: 28166241
Summary:
Adenosine-to-inosine RNA editing, catalyzed by adenosine deaminase acting on RNA (ADAR), diversifies the transcriptome and promotes functional diversity, particularly in the brain. A plethora of editing sites has been recently identified; however, how they are selected and regulated and which are functionally important are largely unknown. This study shows the cis-regulation and stepwise selection of RNA editing during Drosophila evolution and pinpoints a large number of functional editing sites. Establishment of editing and variation in editing levels across Drosophila species were found to be largely explained and predicted by cis-regulatory elements. Furthermore, editing events that arose early in the species tree tend to be more highly edited in clusters and enriched in slowly-evolved neuronal genes, thus suggesting that the main role of RNA editing is for fine-tuning neurological functions. While nonsynonymous editing events have been long recognized as playing a functional role, in addition to nonsynonymous editing sites, a large fraction of 3'UTR editing sites is evolutionarily constrained, highly edited, and thus likely functional. These 3'UTR editing events can alter mRNA stability and affect miRNA binding and thus highlight the functional roles of noncoding RNA editing. This work, through evolutionary analyses of RNA editing in Drosophila, uncovers novel insights of RNA editing regulation as well as its functions in both coding and non-coding regions.
Zhang, R., Deng, P., Jacobson, D. and Li, J.B. (2017). Evolutionary analysis reveals regulatory and functional landscape of coding and non-coding RNA editing. PLoS Genet [Epub ahead of print]. PubMed ID: 28166241
Summary:
Adenosine-to-inosine RNA editing (see Adar) diversifies the transcriptome and promotes functional diversity, particularly in the brain. A plethora of editing sites has been recently identified; however, how they are selected and regulated and which are functionally important are largely unknown. This study shows the cis-regulation and stepwise selection of RNA editing during Drosophila evolution and pinpoints a large number of functional editing sites. It was found that the establishment of editing and variation in editing levels across Drosophila species are largely explained and predicted by cis-regulatory elements. Furthermore, editing events that arise early in the species tree tend to be more highly edited in clusters and enriched in slowly-evolved neuronal genes, thus suggesting that the main role of RNA editing is for fine-tuning neurological functions. While nonsynonymous editing events have been long recognized as playing a functional role, in addition to nonsynonymous editing sites, a large fraction of 3'UTR editing sites is evolutionarily constrained, highly edited, and thus likely functional. These 3'UTR editing events can alter mRNA stability and affect miRNA binding and thus highlight the functional roles of noncoding RNA editing.

Buchumenski, I., Bartok, O., Ashwal-Fluss, R., Pandey, V., Porath, H. T., Levanon, E. Y. and Kadener, S. (2017). Dynamic hyper-editing underlies temperature adaptation in Drosophila. PLoS Genet 13(7): e1006931. PubMed ID: 28746393
Summary:
In Drosophila, A-to-I editing is prevalent in the brain, and mutations in the editing enzyme ADAR correlate with specific behavioral defects. This study demonstrates a role for ADAR in behavioral temperature adaptation in Drosophila. Although there is a higher level of editing at lower temperatures, at 29 ° C more sites are edited. These sites are less evolutionarily conserved, more disperse, less likely to be involved in secondary structures, and more likely to be located in exons. Interestingly, hypomorph mutants for ADAR display a weaker transcriptional response to temperature changes than wild-type flies and a highly abnormal behavioral response upon temperature increase. In sum, these data shows that ADAR is essential for proper temperature adaptation, a key behavior trait that is essential for survival of flies in the wild. Moreover, the results suggest a more general role of ADAR in regulating RNA secondary structures in vivo.
BIOLOGICAL OVERVIEW

The central dogma of molecular biology maintains that there is a one-to-one correspondence between the genetic code held in the DNA and the encoded sequence found in protein. RNA is the mediator of this correspondence. The phenomenon of RNA editing was first reported in trypanosomes 14 years ago with evidence of the insertion of four ribonucleotides into a frameshifted mitochondrial transcript (Benne, 1986). The insertion restored an open reading frame that was not encoded by the trypanosome mitochondrial genome. Since then, several distinct classes of RNA editing have been found in both plants and animals (reviewed by Simpson, 1999). ADARs (adenosine deaminase acting on RNA) are capable of the site-specific conversion of A-to-I in precursor messenger RNAs (pre-mRNAs). The examples of site-specific editing are limited to transcripts found in the nervous system of vertebrate and invertebrate animals. Most of these transcripts encode ligand- or voltage-gated ion channels and G protein-coupled receptors (reviewed in O'Connell, 1997; Rueter, 1998; Keller, 1999). Because inosine has basepairing properties like that of guanosine (G), the translation machinery interprets I as G. Thus, A-to-I conversion in mRNA has the potential to recode genomic information and alter protein function (Palladino, 2000b and references therein).

Despite advances in understanding of the mechanisms that underlie RNA editing, a very basic question remains; what is the biological function of this process? To address this question, null mutations have been generated in a gene responsible for an entire class of RNA editing in Drosophila, which possesses a single Adar gene. Correspondingly, all known Drosophila site-specific RNA editing (25 sites in three ion channel transcripts) is abolished. Adult flies lacking Adar are morphologically wild-type but exhibit extreme behavioral deficits including temperature-sensitive paralysis, locomotor uncoordination, and tremors that increase in severity with age. Neurodegeneration accompanies the increase in phenotypic severity. Surprisingly, Adar mutants are not short-lived. Thus, A-to-I editing of pre-mRNAs in Drosophila acts predominantly through nervous system targets to affect adult nervous system function, integrity, and behavior (Palladino, 2000b).

ADAR enzymes convert adenosine (A) to inosine (I) in double-strand (ds)RNA and were first discovered in Xenopus oocytes (Bass, 1988). ADARs have been purified and cloned from many diverse sources and act via hydrolytic deamination of A-to-I. In mammals, there are three known ADAR enzymes; ADAR1, ADAR2, and RED2. They contain two or three dsRNA binding domains and a catalytic deaminase domain. The deaminase domain is distantly related to cytidine deaminases, and in particular to another editing enzyme, APOBEC-1, that catalyzes the conversion of C to U in transcripts of mammalian apolipoprotein B. The deaminase domain is also related to the ADATs (adenosine deaminases acting on tRNA) that convert A-to-I in tRNA (Palladino, 2000b and references therein).

ADARs are defined by their ability to convert A-to-I nonselectively in extended perfect dsRNA duplexes. Such activities have been detected in extracts from lower invertebrates through vertebrates, and hence, the enzymes for converting A-to-I in mRNA are of ancient origin. The exact biological role for this promiscuous activity is not known but the activity has been proposed to play roles in viral defense, viral life cycles, and gene regulation. Recently, edited mRNAs were identified in C. elegans on the basis that they contain inosine (Morse, 1999). These mRNAs have been shown to possess extensive modification of 3' untranslated regions within extended hairpin structures, and candidate ADARs have been identified in C. elegans (Hough, 1999). Likewise, a Drosophila Adar has been identified and shown to possess promiscuous A-to-I activity (Palladino, 2000a).

The best characterized examples of A-to-I RNA editing are found in the glutamate receptor subunit genes (GluRs) of the mammalian nervous system (reviewed by Seeburg, 1998). One site in GluR-B transcripts, the glutamine (Q)/arginine (R) site, undergoes editing that changes a genomically encoded Q codon (CAG) to an R codon (CGG) (Sommer, 1991). The functional consequences of editing at this site are striking: Ca2+ permeability of GluR channels containing GluR-B subunits is determined largely by the editing status of this site (Verdoorn, 1991; Kohler, 1993). Mechanistically, RNA editing of the Q/R site is dependent upon cis-acting sequences in the intron downstream of and complementary to the editing site (Higuchi, 1993). This editing site complementary sequence (ECS) has been shown to base-pair with the region surrounding the Q/R site and form a double-stranded (ds) RNA secondary structure. Transgenic mice missing the ECS fail to edit the Q/R site. Mice expressing such GluR-B Q/R editing-incompetent alleles in various heterozygous combinations exhibit seizures and early death (Brusa, 1995; Feldmeyer, 1999). Thus, site-specific A-to-I RNA editing profoundly affects protein function in vivo (Palladino, 2000b and references therein).

Further examples of specific A-to-I editing in mammals include other GluR editing sites, several sites in the serotonin receptor (5HT2CR) (Burns, 1997; Niswender, 1999), and the ADAR2 gene (Rueter, 1999). Although the functional significance has been investigated for some of these sites, their in vivo significance remains largely unknown. Interestingly, while the best studied example of mammalian A-to-I editing, the GluR-B Q/R site, is edited at a very high frequency in vivo (>99% of transcripts are edited), all of the remaining examples of editing occur at lower frequencies. Such intermediate levels of editing suggest a mixture of edited and unedited proteins in neurons or tissue specificity of editing. This is particularly intriguing considering a recent proposal (Seeburg, 2000) that RNA editing in the nervous system evolved to fine-tune the function of the nervous system through subtle functional effects on ion channels (Palladino, 2000b and references therein).

In support of such a proposal, A-to-I RNA editing as a posttranscriptional regulatory mechanism for generating protein diversity in the nervous system appears to have ancient origins, as evidenced by several invertebrate examples. For instance, the squid voltage-gated potassium channel sqKv2 has been shown to undergo RNA editing at numerous positions (Patton, 1997). Transcripts of the major action-potential Na+ channel in Drosophila, the product of the paralytic (para) locus, have been shown to undergo RNA editing proceeding through a mechanism similar to that of the mammalian GluR Q/R sites (Hanrahan, 2000; Reenan, 2000). Other reported substrates for editing in Drosophila also encode nervous system signaling components, namely, the cacophony (cac) voltage-gated Ca2+ channel (Peixoto, 1997; Smith, 1998b) and the glutamate-gated Cl- channel, DrosGluCl-alpha (Semenov, 1999) (Palladino, 2000b and references therein).

Mutant Adar1F1 larvae appear normal for locomotion, response to stimuli, and are also normal in a specific behavioral assay. The phototactic behavior of Adar1F1 versus WT larvae was examined and no measurable difference was observed. Adar mutant adults, however, exhibit severe neurobehavioral phenotypes. Motor deficits, manifesting as slow uncoordinated locomotion, occasional tremors and varying degrees of abnormal body posture are observed immediately upon eclosion. Also apparent soon after eclosion, Adar- animals spend an inordinate amount of time grooming (5 ± 0.5% for WT, 27 ± 6% for Adar1F1). This obsessive cleaning is apparent throughout the lifetime of mutant animals. Adar mutants are capable of flying and jumping but do so only when repeatedly provoked and then only rarely. Not unexpectedly, flight in Adar mutants is erratic. Adar- males exhibit an extreme mating defect. When presented with WT or Adar- virgin females, males were not observed to initiate any displays of courtship (Palladino, 2000b).

Since the targets of Adar activity are ion channel transcripts of the nervous system, two paradigms that have been found to be extremely successful in detecting ion channel mutations in Adar mutants are temperature-sensitive (ts) paralysis and ether-induced leg shaking. While Adar mutants recover more slowly from ether anesthesia, they do not exhibit the leg-shaking characteristic of mutations that increase membrane excitability. However, Adar mutant flies exhibit a strong temperature-dependent enhancement of behavioral defects at the restrictive temperature (37.5°C) resulting in bouts of paralysis and extreme motor uncoordination. Mutations in a number of genes affecting ion channels or the process of neurotransmission confer similar phenotypes. The lack of a significant developmental phenotype, outwardly normal adult morphology, varied behavioral deficits, and ts phenotype are all consistent with the primary role of Adar being the editing of transcripts encoding signaling components of the adult nervous system (Palladino, 2000b).

Although Adar mutants are severely compromised for many aspects of adult behavior, they perform the behaviors and functions necessary to sustain life (eating, respiration, metabolism). It was of interest to determine whether Adar mutants are short-lived as a result of their neurobehavioral enfeeblement. Life span analysis was performed on Adar1F1 male populations under optimal environmental conditions (low population density). As controls, the life spans of populations of Adar1F1 males bearing Dup(X;Y)901 and FM7G males were determined. The life span of Adar1F1 males under these ideal conditions parallels that of control animals. Importantly, the maximal life span of Adar1F1 is not different from controls. The oldest flies obtained from Adar1F1 and the corresponding Dup(X;Y)901 rescued male controls were 112 and 115 days, respectively. Similar results were obtained for Adar1F4 and controls. Therefore, most Adar- individuals, though extraordinarily compromised neurologically, can survive as long as WT animals (Palladino, 2000b).

A stark contrast to the apparent vitality of Adar mutants is their genesis in a screen for lethal mutations. Given the normalcy of development and lack of inviability during larval development and morphogenesis, one question was whether Adar mutants would be compromised under conditions of competition with WT animals. Simply, populations were composed of flies of a given mixture of genotypes. Females in the populations were phenotypically WT and heterozygous for Adar1F1 and FM7G. Males were of two genotypes; half were Adar1F1 and the remaining half were Adar1F1 bearing Dup(X;Y)901. Survivorship for each of the genotypes in these populations was determined for one generation (14 days). That is, no progeny from the populations in question were allowed to eclose and thus alter the size of the population. Even under relatively low population density, Adar mutants exhibit a high mortality rate with respect to WT animals. Thus, Adar mutants are at an extreme selective disadvantage even early in their life span, and this disadvantage is rescued by a translocation that provides Adar+ function and behavioral rescue (Palladino, 2000b).

The behavioral phenotypes of Adar- animals become more severe with age and some new phenotypes appear. Tremors increase dramatically such that locomotion is severely compromised in animals beyond day 50. Animals fall over and become increasingly inefficient at righting themselves; sometimes leading to periods of inactivity resting on their backs. Many animals beyond day 30 exhibit circling behavior that varies from wide circling to circling while standing in place. A majority of animals beyond day 50 exhibit a persistent upheld wing phenotype. Marked asymmetries appear in animals, manifesting as one upheld wing or leg, extension of one or both back legs, and more severe asymmetries in posture. The progressive nature of these defects prompted a determination of the gross nervous system morphology of Adar mutants. Frontal sections through adult heads of male Adar1F1 and Adar1F4 were stained with hematoxylin and eosin. One gross difference between Adar mutants and controls is the organization of the retina. The photoreceptors in Adar- animals appear disorganized and extend longitudinally projecting further to reach the laminar layer in mutants versus the more compact, organized retinal structure seen in WT controls. This retinal abnormality is seen in all sections through mutant heads of all ages tested (Palladino, 2000b).

Other than structural abnormalities in the retina, young Adar mutant brains (1–3 day) appear grossly normal. However, by day 30, lesions appear in the brains of Adar mutants and are distributed randomly in the central brain, optic lobes, and retina. The lesions appear as vacuolated regions, are distributed throughout the sectioned brains, and are most prevalent in the retina, lamina, and optic lobes. Vacuoles appear to increase in size and number with age and, by day 50, animals can be found with extensive brain degeneration. Control animals do not demonstrate brain degeneration and seldom have even one small vacuole per head (Palladino, 2000b).

What are the global roles of ADARs in gene regulation and organismal function? A hint at the answer may be the prevalence of nervous system targets of ADARs in both invertebrates and mammals. From data presented here, it is concluded that the process of A-to-I RNA editing in Drosophila is, primarily, a process subserving the execution of adult behaviors through effects on numerous and varied nervous system targets of ADAR activity. Moreover, it is proposed that the process of A-to-I editing is, by its very nature, ideally suited for modifying activity in the nervous system (Palladino, 2000b).

The developmental time course of Adar- animals is essentially the same as that of WT controls. Thus Adar activity does not appear to be necessary maternally or zygotically for normal morphology of adult animals. Additionally, lack of Adar does not shorten the life span of mutants under optimal conditions. The mutant phenotypes associated with loss of Adar activity are entirely consistent with perturbations of electrical and chemical signaling mechanisms in the nervous system and proximal effects on behavior. In fact, mutations have been generated in some of the ion channel targets of Adar (described in this paper) which confer behavioral phenotypes. For instance, cac mutants exhibit specific defects in male courtship song, visual defects, and temperature-dependent convulsions and uncoordination. Mutations of para also confer a wide range of behavioral phenotypes including recessive and dominant ts-paralysis, defects in learning and olfaction, cold-sensitive lethality, genetic suppression of certain mutations in the Shaker K+ channel gene, and resistance to insecticides. Glutamate-gated Cl- channels, like DrosGluCl-alpha, have only been reported in invertebrates. They are a target of the avermectin class of insecticides and are found in several neuronal and muscle preparations. Though no mutations in DrosGluCl-alpha have been reported, the presence of glutamate-gated Cl- channels on the soma of neurons and their similarity to the mammalian inhibitory glycine receptor, mutations that cause inherited startle disorders to unexpected stimuli, are also consistent with the idea that RNA editing of DrosGluCl-alpha modulates neuronal membrane excitability (Palladino, 2000b).

The extensive and varied behavioral defects in Adar mutants are consistent with affects on target genes such as para, cac, and DrosGluCl-alpha; ion channels of the nervous system. Like all other examples of site-specific A-to-I editing though, these 25 sites in three Drosophila genes were discovered serendipitously and other unidentified targets of Adar activity surely exist. Thus, it appears unlikely that the nervous system impairment seen in Adar mutants is due solely to any one particular Adar target site. A more likely scenario, given the potential for hundreds of Adar editing sites in scores of nervous system genes, is that the Adar- phenotype is complex and results from effects on the posttranscriptional processing of many transcripts in the nervous system (Palladino, 2000b).

Since most A-to-I editing sites have been discovered in signaling components of the nervous system, one role of editing that has been proposed is in the fine-tuning of neurophysiological processes (Seeburg, 2000). Site-specific mRNA editing can have profound biological significance for individuals. The only examples, and these are extreme, are the in vivo consequences of failure to edit the GluR-B Q/R site in mice. Editing at this site occurs at high frequency (>99%) and mutant mice expressing an editing-incompetent allele of GluR-B die postnatally from neurological dysfunction (Brusa, 1995). However, most editing sites are not edited at such high levels and must confer altered signaling properties in a cell-specific manner or alter neuronal signaling properties through dominant effects on protein function (Palladino, 2000b and references therein).

The singular role of Adar in Drosophila pre-mRNA editing along with the adult neurological phenotypes of Adar null mutants suggest a novel evolutionary role for Adar in supplying genetic diversity. Genetic variation in large, diploid, randomly mating populations appears through mutation and is discrete in nature. That is, an individual animal can have zero, one or two alleles for a given nucleotide position and stochastic or selective processes act to determine the final frequency of that allele in a population over time. Cells can usually express, at most, two alleles differing at a single amino acid in a 1:1 ratio (Palladino, 2000b).

An important example of selection acting on variation in nature exists for the para gene. In natural insect pest populations, such as Heliothis (tobacco horn worm) or Blatella (cockroach), resistance to pyrethroid insecticides is prevalent and confers a strong selective advantage on animals carrying knock-down resistant (Kdr) alleles in para orthologs. In the laboratory as well, certain Drosophila para mutations that are recessive ts-paralytic mutations confer dominant resistance to pyrethroid insecticides and DDT in heterozygotes. In fact, certain doubly heterozygous mutant combinations confer even higher levels of resistance. Thus, the advantage of modifying certain amino acid positions in ion channels is not without precedence and demonstrates the selective value of possessing two different alleles of a particular locus (Palladino, 2000b and references therein).

A-to-I RNA editing via ADARs is different from the usual mutational gene-altering mutational paradigm. Changes introduced by editing are not discrete, such as naturally occurring genetic variation, but appear as a continuum. Within the 25 examples given in this report, the level of editing at particular sites ranges from ~5% to 90%. Though little is known about what determines the efficiency of deamination of pre-mRNAs in vivo, the substrate requirements of ADARs must play a large role. Specific pre-mRNA editing via ADARs has been shown, in mammals, to proceed through dsRNA intermediates in which intronic editing-site complementary sequences (ECSs) base-pair with regions around the edited adenosine (Higuchi, 1993; Herb, 1996). This mechanism has been conserved in Drosophila para editing (Reenan, 2000). Thus, the evolution of such RNA editing sites likely proceeds through the mutation of noncoding sequences. Many mutational changes to the genome may be required in intronic sequences to generate an initial, weakly complementary ECS. This nascent ECS would then direct modification of a particular nucleotide position at a low efficiency in nearby coding sequences resulting in an amino acid change in a small percent of messages. Certain de novo editing sites, though edited at low levels, would be selectively advantageous through dominant effects on protein function and resultant changes in behavior. Such a model provides an ideal substrate for evolution, the intron, for which mutation can provide a large sequence space of variants most of which would be selectively neutral. Larger introns would perforce generate more sequence variants through time. Editing introduced at a low level at a particular site within a protein would introduce subtle variants within a population that could benefit from a selective advantage in a niche-specific manner altering behavior to suit environment. Future enhancements of editing, through improvements of the ECS-editing site interaction to generate a better ADAR substrate, could titrate in the edited form of the protein to maximally advantageous levels -- all via changes to noncoding sequences. In effect, RNA editing provides the possibility to make use of amino acid changes that would be selectively disadvantageous at 50% (as a mutation appearing in coding sequence) offering a continuum of expression of two residues at a given amino acid position. Further diversity could be obtained in cases where the process of RNA editing became spatially regulated (Palladino, 2000b).

A-to-I RNA editing, while providing the potential for tailored mixtures of proteins differing at a single amino acid position, seems to have supplied this diversity primarily to the nervous system -- a tissue whose hierarchical function, complex structure and direct influence on organismal behavior ideally suit it to take advantage of such continuous and subtle genetic variation. The future challenge of studying Adar mutants will be in addressing the potential multitude of unknown pre-mRNA targets, the role of different Adar isoforms in the editing of targets, the association of specific editing sites with specific modifications of behavior, and the natural history of ADARs and their substrates in the course of evolution (Palladino, 2000b).

RNA editing regulates transposon-mediated heterochromatic gene silencing

Heterochromatin formation drives epigenetic mechanisms associated with silenced gene expression. Repressive heterochromatin is established through the RNA interference pathway, triggered by double-stranded RNAs (dsRNAs) that can be modified via RNA editing. However, the biological consequences of such modifications remain enigmatic. This study shows that RNA editing regulates heterochromatic gene silencing in Drosophila. The binding activity of an RNA-editing enzyme was used to visualize the in vivo production of a long dsRNA trigger mediated by Hoppel transposable elements. Using homologous recombination, this trigger was deleted, dramatically altering heterochromatic gene silencing and chromatin architecture. Furthermore, it was shown that the trigger RNA is edited and that dADAR serves as a key regulator of chromatin state. Additionally, dADAR auto-editing generates a natural suppressor of gene silencing. Lastly, systemic differences in RNA editing activity generates interindividual variation in silencing state within a population. These data reveal a global role for RNA editing in regulating gene expression (Savva, 2013).

This study pursued an observation of the in vivo localization of the RNA-editing enzyme, dADAR, to the proof of its action on an endogenously expressed inverted repeat of the TE, Hoppel. The results explicitly demonstrate a functional intersection between the processes of RNA editing and RNA silencing. Previous studies in Drosophila implicate Hoppel and the RNAi pathway in determining the global silencing state of chromosome 4, although no dsRNA trigger had been experimentally identified. This study showed that the inverted repeat acts as a genetic element, Hok, and regulates PEV, the global architecture of chromosome 4, and silences the Hoppel transposase. As a general mechanism, ADAR's action on dsRNA should oppose RNAi. It was shown that deficiency for ADAR acts as a global enhancer of silencing state, and dADAR hypomorphism even extends lifespan. In Drosophila, gene silencing decreases with age and has been implicated in the aging process (Wood, 2010). Thus, substantial decreases in ADAR activity may lead to lifespan extension through increased silencing. Interestingly, polymorphisms within a human ADAR gene have been associated with extreme longevity, indicating that interventions involving ADAR activity may be capable of affecting lifespan. Importantly, mutations in human ADAR1 cause Aicardi–Goutières syndrome in which it is hypothesized that ADAR has a role in regulating dsRNA metabolism from repeated elements in the human genome. Thus, the current data are consistent with a conserved role in the regulation of dsRNA levels in animals through RNA editing or RNA binding (Savva, 2013).

Mechanistically, evidence is provided that dADAR auto-editing has evolved as a natural inhibitor of RNAi, generating dADARG. In dAdar null or dAdarS genetic backgrounds, no dADARG can be produced. Thus, both backgrounds effectively act as enhancers of PEV (E(var)). In the wild-type background, PEV occurs to the extent that each animal expresses dADAR (and the corresponding amount of dADARG). In the extreme, the dAdarG background acts as a strong suppressor of PEV (Su(var)). How can a single amino acid change in dADAR protein affect such a silencing switch? It is speculated that dADARG may interfere indirectly with Dicer activity on dsRNA, simply by blocking access via binding irrespective of editing activity, analogous to the FHV-B2 protein. Alternately, a recent study showed a direct functional interaction between mammalian ADAR and Dicer that is necessary for the processing of small RNAs (Ota, 2013). If dADAR has a similar interaction, it could also mediate all of the effects in the model via dominant-negative interactions of dADARG with Dicer, whereas dADARS (which encodes the conserved amino acid) would function in a similar manner described in mammals to promote small RNA biogenesis. Further biochemical experiments will be necessary to determine whether this phenomenon is conserved across species and the exact molecular mechanisms through which dADARG exerts its effects (Savva, 2013).

The most engaging aspect of these results lay in their implications for somatic regulation of heterochromatin functioning as a safeguard of transposon activity, especially in the nervous system. The RNA-induced silencing complex isolated from Drosophila tissue-culture cells was shown to be programmed with esiRNAs, largely derived from transposon sequences, a significant portion of which bears the signature of a single dADAR modification (Kawamura, 2008). Likewise, in C. elegans, ADAR activity has a profound effect on the abundance and identity of small RNA profiles. Further experiments in this system using deep sequencing technologies will be necessary to shed light on the effects of ADAR on endo-siRNA abundances and functionality. It is envisioned that such RNA-editing-mediated effects may be quite specific to the nature of individual dsRNA triggers. Studies in both mammals and Drosophila have shown that TEs are mobile in the nervous system, revealing an intriguing mechanism for the generation of somatic mutations potentially conferring adaptive value in individuals (Li, 2013; Muotri, 2005; Perrat, 2013). This study demonstrates a mechanistic link between RNA editing and the regulation of transposon silencing, particularly in the nervous system, which may have domesticated uses as diversifiers of neuronal genomes on a neuron-to-neuron and an individual-to-individual basis. The implications of these results, given the universal prevalence of dsRNAs as a component of transcriptomes, are that ADAR activity has an evolved role in determining the fate of RNAs entering silencing pathways, thus globally influencing somatic genomic integrity, gene expression and downstream organismal phenotypes (Savva, 2013).


REGULATION

Biochemical Characterization of Adar activity in wild-type and mutant flies

The mammalian ADARs (ADAR1 and ADAR2), which are capable of specifically editing transcripts of the glutamate receptor subunit genes and the 5-HT2C receptor gene, also possess additional nonspecific A-to-I conversion activity on synthetic duplex RNA. Such a nonspecific ADAR activity has been directly detected in embryonic extracts of Drosophila and Adar has been shown to possess A-to-I activity (Palladino, 2000). In order to address the contribution of Adar to total Adar activity in flies, nonspecific A-to-I conversion activity present in crude protein extracts from WT and Adar1F4 adults were examined. In brief, [alpha-32P]ATP labeled synthetic dsRNA was transcribed and incubated with crude head extract. Modified dsRNA was digested with P1 ribonuclease and the resulting mononucleotide mixtures separated by thin-layer chromatography to resolve adenosine from inosine. While dsRNA-dependent adenosine deaminase activity was detectable in WT male head extracts, no A-to-I conversion activity was detected in extracts from the heads of Adar1F4. Quantitation of the amount of inosine converted in extracts reveals an average overall decrease in promiscuous A-to-I conversion activity of at least 12-fold in Adar1F4 (n = 4). This is a conservative estimate of the decrease in activity, since the level of ADAR activity was frequently lower in the mutant flies than the negative control (no extract). Extracts from WT versus Adar1F4 whole animals gives similar results (Palladino, 2000b).

Drosophila has been shown to possess at least one adenosine deaminase acting on tRNA, dADAT1 (Keegan, 1999). To ensure that the mutant and WT are equivalent with regard to a related enzymatic activity, the same extracts were assayed for the conversion of A-to-I at positions 34 and 37 of Bombyx mori tRNAAla. The mutant extract is indistinguishable from WT extract as regards specific conversion of A-to-I in the tRNA substrate. The adenosine deaminases acting on tRNAs (ADATs) have adenosine deaminase catalytic domains homologous to those of ADARs but lack dsRNA binding domains and are active on tRNAs (Gerber, 1998; Gerber, 1999 ; Keegan, 1999). Thus, Adar mutants abolish nonspecific A-to-I editing activity without affecting tRNA-specific editing activity. From this and the transcriptional analysis of Adar mutants, it is concluded that Adar encodes the major dsRNA-specific adenosine deaminase in Drosophila.

To address the status of specific A-to-I RNA editing in Adar mutants, RNA editing site in cac voltage-gated Ca2+ channel transcripts was examined. cac cDNAs were generated via RT-PCR from WT and Adar- flies. In the case of the cac N/S-1 site, editing abolishes cleavage by a restriction enzyme. RT-PCR products from behaviorally normal animals heterozygous for Adar mutations have WT levels of RNA editing while editing in two different Adar mutants (1F1 and 1F4) was undetectable. All of the mutants were tested in complementation tests by Adar1F1 or Adar1F4. Females doubly heterozygous for either of these alleles and any of the other 11 Adar mutations display behavioral phenotypes that are indistinguishable from females homozygous for Adar1F1 or Adar1F4. Correspondingly, all double heterozygote Adar- combinations show failure of complementation at the molecular level, revealing no detectable editing at the cac N/S-1 site. Since Adar5G1 removes the entire Adar transcription unit and is indistinguishable in behavioral and molecular tests from all of the other Adar alleles, it is concluded that all 13 Adar mutations generated in this study are null (Palladino, 2000b).

Extending this analysis of specific editing, the editing status of all reported editing sites in transcripts of the cac, para (voltage-gated Na+), and DrosGluCl-alpha (glutamate-gated chloride ion channel) genes was determined. Where editing events generate or abolish restriction enzyme cleavage sites, RT-PCR products were obtained from whole fly RNA and digested with the appropriate restriction enzyme. Otherwise, RT-PCR products were subjected to direct sequence analysis and analyzed for the presence of a mixed A/G signal in the chromatographic data. Editing was undetectable at all 25 sites analyzed. Control experiments demonstrate that the level of detection of editing using the direct sequence method is about 5% (Palladino, 2000b).

Although no editing was seen at any site using restriction enzyme assays or sequence data in Adar mutants, it was of interest to determine a lower limit to the detection of specific RNA editing in Adar mutants. To this end, more than 100 partial para cDNAs spanning three RNA editing sites from both WT and Adar1F1 mutants were isolated and their editing status was assessed. In wild type, where 112 cDNAs were analyzed, 116 out of a potential 336 adenosine residues were modified. In Adar1F1 males, analysis of 103 partial cDNAs from para failed to detect a single modified adenosine residue in 309 potential sites. Thus, this method for detection of site-specific editing would be sufficient to detect less than 1% residual activity remaining in Adar1F1 mutants. From this, it is again concluded that all Adar mutant alleles described here are null mutations and that Adar is the site-specific A-to-I RNA editing enzyme of all reported pre-mRNA editing sites in Drosophila (Palladino, 2000b).

Dissecting the splicing mechanism of the Drosophila editing enzyme; dADAR

In Drosophila, the expression of adenosine deaminase acting on RNA is regulated by transcription and alternative splicing so that at least four different isoforms are generated that have a tissue-specific splicing pattern. Even though dAdar has been extensively studied, the complete adult expression pattern has yet to be elucidated. The present study investigated mature transcripts of dAdar arising from different promoters. Two predominant isoforms of dAdar are expressed in gonads and dAdar is transcribed from both the embryonic and the adult promoters. Furthermore, full-length transcripts containing the alternatively spliced exon-1 are expressed in a tissue-specific manner. The splicing factor B52/SRp55 binds within the alternative spliced exon 3a and plays a role in this alternative splicing event (Marcucci, 2009; full text of article).

Targets of Activity

Messenger RNA editing of transcripts encoding voltage-sensitive ion channels has not been extensively analyzed--least of all in Drosophila, for which several channel-encoding genes are known. Previous sequence studies of D. melanogaster's cacophony gene, which encodes an alpha 1 calcium-channel subunit called Dmca1A, suggested that several nucleotides within the ORF of the primary transcript are edited such that 'A-to-G' substitutions occur (these two nucleotides being the adenine that is found at the relevant sites in the sense strand of genomic DNA or the primary transcript, compared to the substitution of guanine that is detected at the level of cDNA analysis). Such A-to-G changes are the same kind of post-transcriptional variations originally discovered (in a neurobiological context) for a ligand-sensitive channel in vertebrates. RNA was extracted from adult flies and it has been revealed, by RT-PCR and restriction-enzyme analyses, that transcript heterogeneity exists in vivo for three distinct edited sites within the cac-encoded RNA. Each such nucleotide would lead to channel variability at the level of the Dmca1A polypeptide. Owing to cacophony being originally identified as a 'behavioral gene', the possible significance of Dmca1A RNA editing for influencing the relevant neuro-functional phenotypes is discussed (Smith, 1998).

A Drosophila melanogaster gene has been identified and analyzed that encodes a chloride channel subunit (DrosGluCl-alpha) previously shown to function as a glutamate-gated chloride channel in an in vitro expression system. Sequence analysis of several cDNAs corresponding to the gene reveals sequence diversity in their open reading frames at seven specific sites. Site-specific A-to-G variations between cDNA and genomic sequences, consistent with RNA editing, were detected at five nucleotide positions. In addition, sequence variations among cDNA clones consistent with alternative splicing of mRNA were found at two different sites. In the 5' region, two small adjacent exons, containing similar but distinct modular sequences, are alternatively incorporated into the mature mRNA. In the 3' region, alternative splicing generates a variant encoding a protein with four additional amino acids just upstream of the fourth transmembrane domain. Combinations of RNA editing and alternative splicing can lead to extensive diversification of transcripts. These results give the first example of RNA editing in neurotransmitter-gated chloride channel genes or of alternative splicing in a glutamate-gated chloride channel gene of Drosophila (Semenov, 1999).

RNA editing occurs at three sites in the para transcript, which encodes the major voltage-activated Na+ channel polypeptide in Drosophila. The occurrence of RNA editing at the three sites was found to be developmentally regulated. Editing at two of these sites is also conserved across species between the D. melanogaster and D. virilis. In each case, a highly conserved region was found in the intron downstream of the editing site and this region was shown to be complementary to the region of the exonic editing site. Thus, editing at these sites would appear to involve a mechanism whereby the edited exon forms a base-paired secondary structure with the distant conserved noncoding sequences located in adjacent downstream introns, similar to the mechanism shown for A-to-I RNA editing of mammalian glutamate receptor subunits (GluRs). For the third site, neither RNA editing nor the predicted RNA secondary structures are evolutionarily conserved. Transcripts from transgenic Drosophila expressing a minimal editing site construct for this site were shown to faithfully undergo RNA editing. These results demonstrate that Na+ channel diversity in Drosophila is increased by RNA editing via a mechanism analogous to that described for transcripts encoding mammalian GluRs (Hanrahan, 2000).

A total of four A-to-I RNA-editing sites have been identified within Drosophila para transcripts, three of which are described in detail in this study. The editing sites are named for restriction enzyme recognition sequences that are either generated or abolished by RNA editing. For instance, the SfcI site contains the sequence ctataa in genomic DNA but edited cDNAs have the sequence ctatag, which generates a SfcI site. These sites were originally discovered through sequence analysis of cDNAs that were subsequently compared with genomic DNA from D. melanogaster and D. simulans. It is estimated that these two sibling species are separated by 2.5 million years of divergence. In each case, adenosine (A) was observed in the genomic sequence with guanosine (G) at the corresponding position in numerous cDNAs (Hanrahan, 2000).

It was postulated that if editing of para transcripts is biologically important for Na+ channel function in Drosophila, it should be evolutionarily conserved among distant relatives of D. melanogaster. The frequency of editing at each site in adult D. melanogaster was determined for a number of independent cDNAs via restriction enzyme analysis. The frequency of editing varies among the three sites: the frequency of editing is 68 ± 3% at the Fsp site; 43 ± 5% at the Sfc site, and 21 ± 2% at the Ssp site. Editing at these sites in D. melanogaster was compared with the corresponding regions in D. virilis. It is estimated that these two species diverged from 61 to 65 million years ago. The frequency of editing in adult D. virilis is increased slightly at the Ssp site whereas the Sfc site shows a slight decrease with respect to D. melanogaster. In contrast, editing at the Fsp site is not detectable at all in D. virilis. This suggests that editing has been conserved for only two of the three sites (Hanrahan, 2000).

Two of the editing sites (Ssp and Sfc) are separated by 2000 bp and two introns in pre-mRNA. It was hypothesized that editing of these sites might not occur independently. That is, the editing of the two sites might be mutually exclusive or interdependent. To examine this, RT-PCR products that encompass both the Ssp and Sfc sites were cloned and the editing status for each was determined. A total of 131 cDNAs were analyzed for editing at both sites. Using Fisher's exact test, editing at the Ssp and Sfc sites was determined to be independent despite their proximity. It is concluded that editing at the Ssp or Sfc sites occurs independently of the editing status of the other site (Hanrahan, 2000).

Alternatively spliced exons are present at several different locations throughout the para transcript resulting in the potential to encode at least 192 Na+ channel isoforms in D. melanogaster and at least 128 in D. virilis. RNA editing further increases the transcript diversity of para. However, since both processes may require conserved intronic elements, alternative splicing decisions may influence editing or vice versa. It was hypothesized that certain splice forms might be preferentially edited at the Fsp site, which lies 200 bp upstream of an intron involved in the alternative splicing of exons i and a. For example, exon-i-containing splice forms may be edited significantly more than splice forms lacking exon i. No preference for editing of any splice form was found near the Fsp editing site regardless of splice form abundance. These results suggest that specific editing occurs independently of alternative splicing at the Fsp site (Hanrahan, 2000).

To determine whether editing of the para transcript is developmentally regulated as is editing of GluR transcripts, para cDNAs representing transcripts from each stage of development were analyzed. The Ssp and Sfc sites show only minimal editing (1%-2% of cDNAs) at all stages from embryos through the third larval instar. However, a dramatic 20- to 40-fold increase in editing of the Ssp and Sfc sites occurs at pupation, with the frequency of editing approaching adult levels. The Fsp site shows a different pattern of editing during development, exhibiting higher starting levels in the embryo (15%), and increasing less than fourfold during the pupal stage to adult levels. Thus, specific editing at the three sites in the para transcript examined here is developmentally regulated (Hanrahan, 2000).

If A-to-I editing were occurring in para transcripts via a mechanism analogous to that for mammalian GluR transcripts, it could be hypothesized that each editing site has a corresponding editing site complementary sequence (ECS) within the downstream intron. Exonic regions of para are highly conserved between D. melanogaster and D. virilis, whereas intronic regions are divergent. Therefore, if an intron contains within it an ECS complementary to a conserved editing site, this should be apparent as a conserved sequence surrounded by a highly divergent flanking intronic sequence (Hanrahan, 2000).

Because editing at the Ssp and Sfc sites is conserved between D. melanogaster and D. virilis, intronic regions surrounding these sites were cloned and sequenced in these two species. An identity profile was generated based on aligned sequences. As expected, exonic regions are nearly identical, whereas intronic sequences vary considerably, generally exhibiting <50% identity. However, regions of high sequence identity between D. melanogaster and D. virilis were discovered within the introns downstream from each editing site. For the Ssp site, a 40-bp region located 240 bp downstream of the exon-intron boundary is identical in the two species. For the Sfc site, a stretch of 62 bp found 1036 bp downstream of the exon-intron boundary is nearly identical (61/62 nucleotides) between D. melanogaster and D. virilis. On the basis of their conservation and location, these two intronic regions appear to be good candidates for ECSs. Moreover, a preliminary analysis reveals that these regions of conservation are complementary to the sequences surrounding the exonic editing sites and are thus capable of forming base-paired duplexes in this region, the essential feature of an ECS (Hanrahan, 2000).

In addition to an ECS, editing of mammalian GluR transcripts requires formation of an extended RNA secondary structure that aligns the distant ECS with the region encompassing the edited adenosine. To determine whether the putative ECSs would be capable of base pairing with their respective exonic counterparts in the context of a larger RNA secondary structure, a computer program was used to generate secondary structures in these regions for the para pre-mRNA sequence. For the Ssp site, the predicted secondary structures for both species appear strikingly similar despite the divergence of intron sequences. For instance, they each have an extended repeat capable of forming a hairpin of duplex RNA <24 bp long. Both structures also contain a large A-rich loop (60%-70% adenosines over 53 or 73 nucleotides in D. melanogaster and D. virilis, respectively). However, the most striking aspect of these structures involves the specifically edited adenosine at the Ssp site. It is contained within a relatively long region of duplex RNA in which the editing site base pairs with the 40-bp conserved intronic segment, confirming its identity as an ECS. For the Sfc site, the predicted structure in D. virilis also juxtaposes the editing site with the predicted ECS. However, a similar structure was not predicted when the D. melanogaster sequence was used in this analysis. Nonetheless, when base-paired structures are generated by manually aligning the putative ECS and editing site, identical local structures are predicted for both D. virilis and D. melanogaster. Moreover, these local structures resemble those formed in edited mammalian transcripts. In addition, it is clear that the sequences outside of the ECS/exon base-pairing regions are under less constraint. It is concluded that the conserved intronic elements are ECSs and that they are necessary to direct editing by forming duplex RNA substrates for dsRNA editases within larger energetically stable RNA secondary structures (Hanrahan, 2000).

In contrast with editing at the Ssp and Sfc sites, which is conserved between D. melanogaster and D. virilis, editing at the Fsp site appears to occur only in D. melanogaster. To examine this region in more detail, genomic DNA from both species that is found flanking this editing site, including a nearby upstream intron, was cloned and sequenced. No intronic regions were found that were highly conserved between the two species. Because the Fsp site is edited in D. melanogaster, it was predicted that this would require an ECS as for the other edited sites. The upstream intron is only 58 bp from the Fsp site and the possibility that it or the exon itself contains the corresponding ECS was examined. Computer-generated RNA secondary structures using the D. melanogaster sequence predicts formation of a stable RNA duplex in the Fsp region. Surprisingly, the adenosine to be edited in the D. melanogaster sequence is predicted to lie within an extended hairpin that forms entirely from exonic sequences. Nonetheless, the predicted local RNA secondary structure for the Fsp site compares favorably with other known editing sites. If editing at the Fsp site also occurs in D. virilis, an RNA secondary structure analogous to that in D. melanogaster would be expected for the corresponding virilis sequence. To test this possibility, attempts were made to generate the analogous structure in D. virilis and a structure that differs markedly from the structure observed in D. melanogaster was obtained, consistent with the failure to find editing of the Fsp site in D. virilis. The computed free energies for the two structures differ (D. melanogaster = -1620 kcal/mol vs. D. virilis = -538 kcal/mol), with the D. melanogaster structure predicted to form a much more stable structure. Furthermore, local structures were generated using a pairwise alignment of the sequence surrounding the edited adenosine. Even using these local comparisons, the D. virilis structure is less stable compared with the melanogaster structure. These RNA secondary structure predictions are consistent with the lack of conservation of RNA editing at the Fsp site (Hanrahan, 2000).

A valid test of RNA secondary structure predictions would be to assay RNA editing on substrates removed from the context of the entire para primary transcript. To this end, transgenic flies capable of expressing an FSP editing site minimal construct were generated via germline transformation. The region of the FSP site genomic DNA was subcloned into a heat-shock-inducible transformation vector and numerous stable transgenic fly lines were obtained. Since the transgene contains the intron upstream of the edited exon as well as part of the upstream exon, processing of the transgene by the splicing could be monitored. The FSP transgene produces transcripts in which the intron is removed efficiently under noninduced conditions. By restriction analysis of RT-PCR product from numerous transgenic lines, efficient RNA editing of the FSP transgene was observed in all lines. Direct sequence analysis of RT-PCR products of both cognate para and transgene transcripts revealed that the transgene is edited faithfully and specifically; only the single adenosine is edited in both para and transgene products. Thus, all the sequences necessary to direct RNA editing at the FSP editing site are contained within the transgene, which includes all the sequences shown in the predicted structure (Hanrahan, 2000).

It is concluded that flanking sequences near the para editing sites support editing by an ADAR-based mechanism. The 5' nucleotide neighboring each edited adenosine is consistent with known editase preferences described by (A = T > C > G). That is, two of the sites (Ssp and Sfc) have the most preferred 5' neighbor, an adenosine, while the third site (Fsp) has a 5' cytosine. None of the sites have the least preferred 5' neighbor, a guanosine. Also, duplex formation surrounding the editing sites conforms to editing structures described for other known A-to-I RNA-editing sites. The Ssp and Sfc editing site adenosines base pair with uracil, which occurs with the GluR-B Q/R site. However, there is no absolute requirement that the edited adenosine be base paired; in fact, the adenosines edited in GluR-5 and GluR-6 Q/R sites are present within a small bubble. In vitro experiments have demonstrated that the local duplex affects the efficiency of editing. Introduction of a cytosine opposite the edited adenosine in the GluR Q/R sites increases the efficiency of modification by recombinant ADAR1, while introduction of guanosine opposite the GluR-B Q/R editing site does not. Interestingly, the Fsp adenosine, which is edited at the highest frequency of any para editing site, forms a mismatch with cytosine similar to the GluR R/G and HDV sites. These observations are consistent with a Drosophila ADAR activity mediating editing of these sites (Hanrahan, 2000).

Initially, the location of one editing site suggested experiments that might reveal a correlation between editing and alternative splicing. The Fsp site is upstream of alternatively spliced exons. It was suspected that the Fsp site might be edited more frequently in one splice form than another. This hypothesis was unsubstantiated by experimental evidence (Hanrahan, 2000).

Although the Ssp and Sfc sites are 2000 bp apart in pre-mRNAs, analysis of a large number of cDNAs (n = 131) spanning both of these editing sites revealed that editing occurs independently. One interpretation of this result is that the editing activity is present in all tissues in which para is expressed and the sites themselves determine the intrinsic level of modification independently of one another. Another interpretation is that these sites are edited in a spatially regulated manner. In this model, some tissues would perform one edit, while some perform both in a manner that mimics expected ratios. This could be accomplished by multiple editases or isoforms, each editing a different site in a spatially distinct manner (Hanrahan, 2000).

The presence of three unique editing sites has allowed a comparison of the regulation of editing during development. Two of the sites (Ssp and Sfc) appear to be tightly regulated in a similar manner, while the third site (Fsp) has a different editing profile. There are several possible interpretations of these observations. (1) There could be different accessory factors involved in the editing of each site. In the case of the Fsp site, which is edited throughout development, either no additional regulatory factors would be required or they would be constitutively expressed. In contrast, the Ssp and Sfc sites would require accessory factors or the accessory factor expression would be induced during pupation. Alternatively, a repressor of RNA editing that acts at these sites specifically may be expressed early in development. (2) Different enzymes or isoforms may recognize different secondary structures. In this case, editing at the Fsp site involves an enzyme that recognizes its limited secondary structure, while the Ssp and Sfc sites might utilize an enzyme whose binding and activity requires more extensive secondary structures. The different enzymes may have tissue-specific distributions that would add further diversification to the expression of edited proteins (Hanrahan, 2000).

Evolutionary conservation of the Ssp and Sfc sites suggests that A-to-I RNA editing of the para transcript provides a selective advantage to the organism. Evolutionarily conserved intronic elements that maintain significant complementarity to exonic sequences containing the Ssp and Sfc editing sites further support this conclusion. In contrast, the lack of conservation of a cis-element for the Fsp site correlates with the absence of editing in D. virilis. This correlation is further strengthened when the estimated 61–65 million years of divergence between the two species is considered. In addition, the Fsp editing site occurs in one of the least conserved portions of the para protein and thus the absence of RNA editing in this region in D. virilis may simply reflect reduced selective constraints in this region of the protein (Hanrahan, 2000).

An evolutionary comparison of editing frequencies in adult flies is intriguing. At the Ssp site the editing frequency is similar between D. melanogaster and D. virilis, while the editing frequency at the Sfc site is slightly higher in D. melanogaster. These data may reflect intronic sequence differences that would alter the RNA secondary structure of editing site substrates. The similarity of predicted RNA secondary structures, both globally and locally, for the Ssp site is consistent with similar editing frequencies. For the Sfc site, the large size of the intron downstream of the Sfc site, without significant selective pressures, provides ample opportunity for sequence changes that may alter RNA secondary structure or even tertiary interactions, which could affect the efficiency of editing. Despite differences in editing efficiencies, evolutionary conservation of A-to-I RNA editing itself is significant. Conservation of necessary structural elements would have required many compensatory mutations, because the intronic sequences are highly divergent (Hanrahan, 2000).

The length of intronic sequences that compose the conserved ECS in the Sfc (65 bp with one difference) and Ssp (40 bp of identity) sites are notable. Aside from these areas of conservation, intronic sequences were only 40%-60% conserved between D. melanogaster and D. virilis. Several known genes contain conserved intronic sequence corresponding to homeodomain protein binding sites or expression enhancers. However, these 'conserved sequences' include multiple differences, have identical stretches of <30 bp of identity, and often contain repeated sequences. Extensive studies of alternative splicing indicate that conserved intronic regions of >30 bp are rare. Most importantly, the conserved candidate ECSs are capable of forming extended imperfect duplexes with the regions of the edited adenosines and these local structures resemble the structure of known ADAR substrates. It has been shown for vertebrate GluR-R/G sites that the most highly conserved nucleotides in the editing site are those participating in base pairing interactions within the hairpin known to be important for RNA editing. In all cases presented here, the para editing site/ECS pairings predicted are almost completely conserved, including mismatched pairs and bulges. In addition, a recent study of ADAR1 shows that an important determinant of ADAR specificity may be the presence of nearby loops or bulges that act as helix ends. The presence of such helical defects in one defined substrate has been shown to limit ADAR1 activity to certain A residues in a substrate that would otherwise be edited more extensively. Such helical defects are present in all the para substrates predicted in this study and may serve the purpose of positioning a Drosophila ADAR in a manner similar to that seen for ADAR1. These criteria make these conserved sequences excellent candidates for ECSs, not only by definition, and taken together suggest that energetically favorable RNA secondary structures have been predicted that are utilized for A-to-I RNA editing of para (Hanrahan, 2000).

In addition, for the FSP site, RNA editing of a minimal substrate occurs in vivo. The transgene that was constructed in this instance contains the predicted secondary structure and some additional upstream intron and exon sequences. Interestingly, for this editing site, a contrast is seen with all other known editing sites. While all other reported mammalian ion channel editing sites require downstream intronic ECSs, the FSP editing construct contains no downstream intron sequences. In fact, while the predicted ECS for the FSP site is downstream, it lies entirely within the coding region of the same exon as the edited adenosines. Efficient editing of the transgene supports the predicted secondary structure and shows that no downstream intronic sequences are necessary. Moreover, the absence of RNA editing in D. virilis is also supported by this evidence since a similar secondary structure for this species is predicted to differ significantly from the predicted D. melanogaster structure. In particular, changes are predicted in exactly the region of the D. virilis structure near the edited adenosines and similar changes at known sites of RNA editing have been shown to disrupt RNA editing in vitro (Hanrahan, 2000).

The discovery of RNA editing of a Na+ channel transcript follows the trend of A-to-I edited ligand- and voltage-gated ion channels, including the first and most extensively studied GluR subunits, the serotonin receptor 2C, and the squid K+ channel. In contrast with these other edited channels, the functional significance of para editing must be inferred. Evolutionary conservation of para editing suggests that Ssp and Sfc editing sites are biologically important. The Sfc site is edited in both D. melanogaster and D. virilis and has also been observed in Musca domestica. The functional consequences of all three editing sites are intriguing considering the structural properties of the Na+ channel. (1) The Fsp site is contained within the first cytoplasmic domain, which is known to contain several PKA phosphorylation sites. Although the Fsp site itself does not create or abolish a phosphorylation site, the charge change introduced by editing (Q-to-R) may affect regulation by phosphorylation. Histidine is encoded by the D. virilis genome at this position. Thus, in D. virilis, this position may be positively charged depending on local pH. In D. melanogaster, editing changes the coding potential from an uncharged Q to a positively charged R residue, which may be functionally equivalent to the D. virilis encoded histidine. (2) The Sfc site is contained within a short cytoplasmic linker between homology domains III and IV, a region known to be important for inactivation of the Na+ channel. More importantly, the edited Sfc site introduces the serine (N-to-S) of a consensus PKC phosphorylation site. The functional changes introduced by phosphorylation of this region have been studied in rat brain Na+ channels. For example, phosphorylation of a protein kinase C (PKC) site, whose serine is seven amino acids from the Sfc serine in para, is required to slow inactivation of the Na+ channel. In addition, this PKC phosphorylation site is required for reduction in peak sodium currents induced by cAMP. The addition of a second PKC phosphorylation site by RNA editing of the Sfc site may prolong the slow inactivation associated with phosphorylation at the adjacent genomic encoded site (Hanrahan, 2000).

Genome analysis of the fruit fly reveals three new ligand-gated ion channel subunits with the characteristic YXCC motif found only in alpha-type nicotinic acetylcholine receptor subunits. The subunits are designated Dalpha5, Dalpha6, and Dalpha7. Cloning of the Dalpha5 embryonic cDNAs reveals an atypically large N terminus, part of which is without identifiable sequence motifs and is specified by two polymorphic alleles. Embryonic clones from Dalpha6 contain multiple variant transcripts arising from alternative splicing as well as A-to-I pre-mRNA editing. Alternative splicing in Dalpha6 involves exons encoding nAChR functional domains. The Dalpha6 transcript is a target of the Drosophila adenosine deaminase acting on RNA (dADAR). This is the first case for any organism where a nAChR gene is the target of mRNA editing. Seven adenosines could be modified in the extracellular ligand-binding region of Dalpha6, four of which are also edited in the Dalpha6 ortholog in the tobacco budworm Heliothis virescens. The conservation of an editing site between the insect orders Diptera and Lepidoptera makes nAChR editing the most evolutionarily conserved invertebrate RNA editing site so far described. These findings add to understanding of nAChR subunit diversity, which is increased and regulated by mechanisms acting at the genomic and mRNA levels (Grauso, 2002).

In the mutant Drosophila dADAR- that completely lacks ADAR activity, site-specific A-to-I editing of all known pre-mRNA targets in Drosophila is abolished. RT-PCR on dADAR mutant RNA for the Dalpha6 gene showed only adenosine in all the seven editing sites identified, thus demonstrating that Dalpha6 editing is dADAR dependent and is abolished in the ADAR mutant fly. In mammals, editing by ADAR has been shown to occur within the context of predicted RNA secondary structure formed through interactions between exon and intron sequences. RNA secondary structure leading to base pairing between the main group of editing sites in Dalpha6 exon 5 and its downstream or upstream intron were sought. In both cases, the edited region seems to form base pairing within exon 5 itself. A similar result was obtained for the Fsp site in the para channel (Grauso, 2002).

The presence of two additional putative sites of editing, found only in the adult Dalpha6 EST clone (sites 1 and 2), suggests that some sites could be edited in a stage-specific manner. The existence of such developmentally regulated editing has been also demonstrated at two of the four editing sites in the D. melanogaster para channel transcript, the Ssp and Sfc sites. It is speculated that Dalpha6 alternative splicing of multiple exons could also be developmentally regulated, as recently found for the exon 4 region of the Drosophila Dscam pre-mRNA (Grauso, 2002). Most of the Dalpha6 residues changed by both alternative splicing and pre-mRNA editing are localized to key functional domains like the ligand-binding loops and TM2. The dADAR mutant fly, where editing is missing in a number of ligand and voltage-gated ion channels, exhibits various age-dependent behavioral deficits accompanied by neurodegeneration. The dADAR mutant has also been independently discovered in a genetic screen for mutants sensitive to hypoxia conditions. Electrophysiological recordings on primary culture of dADAR mutant neurons show that the para channel conductivity is altered in the mutant fly especially in oxygen deprivation conditions. This result clearly indicates that misediting of channels could result in neuronal activity defects. Thus the not-edited/edited Dalpha6 subunit-containing receptors could play a critical role in nervous system function and integrity. Because editing at some Dalpha6-specific sites occurs also in the homologous positions of the alpha7-2 H. virescens gene (sites 3–6), there is a high degree of evolutionary conservation of the pre-mRNA editing between two distantly related insect groups (moths and flies). This implies modifications introduced by editing are of functional relevance (Grauso, 2002).

Pre-mRNA adenosine deaminase (ADAR) is involved in many physiological processes by either directly converting adenosine to inosine in certain pre-mRNAs or indirectly regulating expression of certain genes. Mutations of Drosophila ADAR (dADAR) result in neuronal dysfunction and hypersensitivity to oxygen deprivation. Mutant flies were very resistant to paraquat, a compound that generates free radicals. In order to further characterize the neuronal role of dADAR and understand the basis for the resistance to the oxidative stress, the effect of dADAR on the expression of genes encoding scavengers of cellular reactive oxygen species (ROS) was investigated in both dADAR mutant and overexpression flies. The data show that the expression of the genes encoding known ROS scavengers [superoxide dismutase (SOD) and catalase] is not regulated by dADAR. However, the transcripts of genes encoding two potential ROS scavengers (dhd and Cyp4g1) are robustly increased in dADAR mutant flies, and conversely both are significantly decreased in dADAR overexpressing flies. Using dhd [encoding a Drosophila homolog of the mammalian protein thioredoxin (Trx)] transgenic flies, it was confirmed that the resistance of dADAR mutant flies to paraquat results, at least partially, from the up-regulation of dhd gene in dADAR mutant flies. The data not only confirm the importance of ADAR in maintenance of neuronal function but also reveal its regulatory role in the expression of genes encoding ROS scavengers (Chen, 2004).

Mutation of ADAR in Drosophila (dADAR) results in pathological and physiological changes, such as sensitivity to hypoxia and neuronal degeneration. To understand the full scope of dADAR function, it is crucial to identify new dADAR targets. A polyclonal antibody against inosine was developed and used to enrich inosine-containing mRNAs. The efficiency of immunoaffinity purification was confirmed for the Q/R editing site of GluR-B pre-mRNA that has been edited by ADAR2 to generate inosines at the editing site. This approach was applied to enrich inosine-containing mRNAs from total mRNAs of wild-type and dADAR mutant flies, respectively. The enriched mRNA portion was then amplified and hybridized with Drosophila cDNA arrays. With this method, over 500 mRNAs were identified as potential dADAR targets by showing a higher amount in the enriched mRNA portion from wild-type flies than from dADAR mutant flies. The occurrence of A-to-G conversion in these mRNAs was further analyzed by comparing over 7,000 Drosophila cDNAs sequences with their genomic sequences. A final list of 62 candidates was generated from the overlap of the two approaches. Twelve genes from the final list were further examined by sequencing the RT-PCR products of these genes from wild-type and dADAR mutant flies. Seven of the 12 genes were proven to have A-to-G changes in the wild-type but not in mutant flies. It is concluded that the combination of immunoaffinity enrichment of inosine-containing mRNA, DNA microarrays, and sequence comparison could facilitate the discovery of new dADAR substrates, which in turn allows a better understanding of the targets of dADAR and the biological function of A-to-I RNA editing in flies (Xia, 2005).

Nervous system targets of RNA editing identified by comparative genomics

An unknown number of precursor messenger RNAs undergo genetic recoding by modification of adenosine to inosine, a reaction catalyzed by the adenosine deaminases acting on RNA (ADARs). Discovery of these edited transcripts has always been serendipitous. Using comparative genomics, a phylogenetic signature of RNA editing has been identified. The identification and experimental verification of 16 previously unknown ADAR target genes is reported in Drosophila and one in humans - more than the sum total previously reported. All of these genes are involved in rapid electrical and chemical neurotransmission, and many of the edited sites recode conserved and functionally important amino acids. These results point to a pivotal role for RNA editing in nervous system function (Hoopengardner, 2003).

One factor frustrating ADAR site discovery is the lack of a signature sequence motif. The pre-mRNA substrate required by an ADAR enzyme is usually an imperfect duplex RNA formed by base-pairing between the exon that contains the adenosine to be edited and an intronic noncoding element, called the editing site complementary sequence (ECS). An ECS can be several hundred to several thousand nucleotides upstream or downstream of the edited adenosine. In short, any given ADAR editing site comprises the adenosine (s) to be modified within a stretch of gene-specific coding sequence, plus a pairing partner whose presence is inferred and whose location must be determined experimentally (Hoopengardner, 2003).

To investigate the substrate requirements of ADAR enzymes, a phylogenetic study of an editing site of the para Na+ channel gene, which is edited in numerous Drosophila species, was undertaken. It was hypothesized that if RNA editing of a particular site were conserved between species, then intronic cis-elements required for editing site/ECS duplex formation would be conserved as well. Unexpectedly, this approach revealed highly conserved exonic sequences neighboring sites of ADAR modification. Mutations, including synonymous changes, were virtually absent near sites of ADAR modification in 18 Drosophila species tested. Sequences within the same exon, distal to the region of ADAR modification, acquired substitutions at a much higher rate. Assuming that RNA editing confers a selective advantage upon organisms, this highly conserved region surrounding an editing site is interpreted as having arisen from a selective constraint against any mutation near a site of ADAR modification. Such mutations would perturb the secondary structure required for editing and have been shown experimentally to disrupt or diminish editing. A relaxation of this constraint would be expected in flanking coding sequences not involved in editing (Hoopengardner, 2003).

Using this high degree of sequence identity between species as a potential signature of ADAR editing sites, new ADAR targets were sought. Several groups of genes in Drosophila melanogaster were compared with their orthologs in Drosophila pseudoobscura. Choice of genes in this study was directed, in part, by the neurological phenotype of dADAR null mutants. 914 genes were examined: those annotated as ion channels, G protein-coupled receptors (GPCRs, n=178), proteins involved in synaptic transmission (n=102), and transcription factors (n=499). Of these, 41 genes contained regions within coding sequences that displayed unusually high sequence conservation compared with surrounding sequences. To determine whether any of these genes are subject to editing, reverse transcription-polymerase chain reaction (RTPCR) was performed followed by sequence analysis of amplification products for the 41 candidate genes in both wild-type and dADAR-deficient genetic backgrounds. Electropherograms from 16 of the 41 genes showed one or more sites with mixed A/G peaks in a wildtype background, indicative of a mixture of edited and unedited transcripts. In the same analysis involving dADAR- animals, all sites yielded pure A signal in sequenced products (Hoopengardner, 2003).

This method of discovering new ADAR targets introduces no a priori assumptions and makes no predictions as to the identity or number of targeted adenosines or the level of editing for a given site. For example, the D. pseudoobscura Drosophila Sodium Channel I (DSCI) gene contains the same editing site identified in the D. melanogaster gene, although the level of editing is higher for the former. The same exon of the D. pseudoobscura gene contained an additional editing site that was absent from the D. melanogaster gene, although both species encode identical exonic sequences (155/155 nucleotides). It is presumed that species-specific variation of the ECS element for this editing site is responsible for the different pattern of ADAR modification observed between species (Hoopengardner, 2003).

All of the ADAR targets identified by this method function in rapid electrical and chemical neurotransmission. The majority of ADAR targets identified were voltage-gated ion channels (VGICs) or ligand-gated ion channels (LGICs). Most VGICs are tetrameric integral membrane proteins consisting of a central ion-specific conduction pathway (S5-pore-S6) surrounded by portions of the protein involved in voltage-dependent gating (S1 to S4). The gating of VGICs upon membrane depolarization results in channel opening and flow of ionic currents. Frequently, current flow is ended by another gated process, channel inactivation. The firing properties of a given neuron are determined largely by the gating characteristics of the spectrum of VGICs expressed there. Both voltage-gated Na+ and Ca2++ channels were found in this screen, channels which serve to depolarize neuronal membranes. The editing sites in these channels occur in transmembrane (TM) domains or in other functionally relevant portions of the protein involved in channel gating or inactivation. For instance, editing of the DSCI Na+ channel occurs within the cytoplasmic linker between HD-III and HD-IV, termed 'the inactivation gate'. The editing site is in the functionally critical IFM motif [in this case, (M3V)FL]. Studies have shown that mutational substitution or deletion of these residues alters the inactivation kinetics or renders the channels noninactivating (Hoopengardner, 2003).

Potassium channels in the nervous system serve many roles, such as maintenance of resting membrane potential, membrane repolarization, and tuning the firing properties of a given neuron. Multiple editing sites have been found in three K+ channel genes -- Shaker (Sh), ether-a-go-go (eag), and slowpoke (slo). The design of the conducting pore of K+ channels is ancient, as can be seen by the similarity in crystal structures of the non-voltage-gated KcsA and voltage-gated KvAP channel pores, which share sequence homology with all voltage-gated ion channels. slo site A, eag site A, and Sh site C can all be placed into the crystal structure of the KcsA channel and lie in the extracellular vestibule of the channel or within the conducting pore. Other editing sites in these K+ channels are found in highly conserved regions involved in channel gating and modulation (Hoopengardner, 2003).

Electrical impulses in neurons terminate with an increase in intracellular Ca2 and neurotransmitter release at a synapse. Unexpectedly, several components of the synaptic release machinery were found to undergo RNA editing. The best-studied of the identified targets is synaptotagmin (syt), whose C2 domains serve as the Ca2++ sensor for neurotransmitter vesicle fusion. Editing at several positions in the syt C2B domain was observed in regions essential for Syt self-association, binding to Ca2++ channels, and association with accessory proteins involved in vesicle reuptake. Editing was also seen for other proteins of the core-complex synaptic release machinery in C2 domains or other interaction surfaces within the core-complex (Hoopengardner, 2003).

Rapid responses of a neuron to vesicle fusion and neurotransmitter release into the synaptic cleft are accomplished through the action of LGICs that either excite or inhibit postsynaptic neuronal output. Both kinds of LGICs were found in this screen: nicotinic acetylcholine receptor (nAChR) and alpha and beta subunits and a gamma-aminobutyric acid (GABA) receptor, Resistance to dieldrin (Rdl). Editing of these receptors in Drosophila occurs in several conserved regions including the ligand- binding domain, TM domains involved in gating and agonist sensitivity and, importantly, the lining of the conducting pore, TM2. Editing sites found in TM2 in these channels are in hydrophilic or charged residues that have been shown by mutagenesis studies to affect ion selectivity and channel conductance (Hoopengardner, 2003).

The screening strategy was applied to mammalian Shaker genes (Kv1.1-5). Orthologs of the Kv1.1 gene in mouse, rat, and human showed a region of unusually high sequence conservation not seen in the other family members. Subsequent analysis revealed RNA editing of a site within this conserved region in all three species. RNA editing of the human Kv1.1 ortholog (KCNA1) was spatially regulated. Levels of editing ranged from 17% in the caudate nucleus to 77% in the medulla. High levels of editing were also observed in spinal cord RNA (68%). A comparison of Kv1.1 editing in different regions of mouse and rat brain revealed spatial differences consistent with those seen in humans. Thus, spatial regulation of RNA editing for Kv1.1 genes is evolutionarily conserved (Hoopengardner, 2003).

The location of this editing site within the Kv1.1 protein is especially provocative. The isoleucine (I) to valine (V) change introduced by editing is in a highly conserved position (I400 in human KCNA1) within the pore-lining S6 domain of Shaker-type K+ channels (Hoopengardner, 2003).

Structural work indicates that the inactivation particle of a Shaker-type channel or its accessory beta subunit enters the channel and binds to residues in the pore itself, blocking conduction. Hydrophobic residues at the N-terminus of the particle enter the inner vestibule and interact strongly with the I400 residue, as do quaternary amine inhibitors such as tetrabutylammonium (TBA). Directed substitution of this residue by the smaller alanine residue reduces the affinity of the pore for the blocking particle by more than 400-fold, resulting in channels that either fail to inactivate or inactivate incompletely, and that consequently pass more current than the wild-type channel. In the case of Kv1.1 channel editing, the I3V change also reduces the amino acid sidechain volume at this position that conceivably would have functional consequences (Hoopengardner, 2003).

The genes identified as targets of genetic recoding by ADARs all serve prominent roles in rapid signaling in the nervous system. This is despite the fact that the screen included a majority of genes not directly involved in fast electrical or chemical signaling. The preponderance of ADAR-mediated codon reassignment in the nervous system is supported by the neurological phenotypes of animals lacking ADARs. Given the spatially and temporally hierarchical events that occur in neurons on the time scale of milliseconds, it is proposed that the advantage of regulating VGICs, LGICs, and the synaptic release machinery by RNA editing lies in a finer level of control than discrete genetic change permits. Clearly, different species have chosen different subsets of these targets to edit in order to modify signaling in their nervous systems. Yet, a commonality does exist, in that both fruit flies and mammals edit their Shaker channels (Hoopengardner, 2003).

Certain dominant neurological disorders such as episodic ataxia-1 (EA-1) and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) result from heterozygosity for a missense mutation in some of the analogous positions or regions to editing sites identified in this study. The implications of this for human genetic disease are that mutations of the recoding process of RNA editing, particularly when it targets functionally critical residues in proteins, may be a source of variation or disease. Moreover, certain mutations in noncoding regions (for example, the ECS) may affect particular A-to-I RNA editing sites while being far removed from the editing site itself. As the number of human ADAR targets increases, such mutations will need to be considered as another potential source of inherited behavioral differences as well as neurological disorders (Hoopengardner, 2003).

Tuning of RNA editing by ADAR is required in Drosophila

RNA editing increases during development in more than 20 transcripts encoding proteins involved in rapid synaptic neurotransmission in Drosophila central nervous system and muscle. Adar (adenosine deaminase acting on RNA) mutant flies that express only genome-encoded, unedited isoforms of ion-channel subunits are viable, but show severe locomotion defects. The Adar transcript itself is edited in adult wild-type flies to generate an isoform with a serine to glycine substitution close to the ADAR active site. Editing restricts ADAR function since the edited isoform of ADAR is less active in vitro and in vivo than the genome-encoded, unedited isoform. Ubiquitous expression in embryos and larvae of an Adar transcript that is resistant to editing is lethal. Expression of this transcript in embryonic muscle is also lethal, with above-normal, adult-like levels of editing at sites in a transcript encoding a muscle voltage-gated calcium channel (Keegan, 2005).

In adult Drosophila, ADAR edits its mRNA with 40% efficiency to encode a less active isoform. It is proposed that this own-transcript editing is a form of negative autoregulation. When this editing is prevented in vivo so that only the genome-encoded ADAR is expressed, then levels of total ADAR protein otherwise sufficient for phenotypic rescue become lethal. This early lethality is associated with hyperediting of sites in at least one target transcript (Keegan, 2005).

It has been shown that mammalian ADAR2, which is the orthologue of Drosophila ADAR, edits a 3' splice site in its own pre-mRNA so that an additional 47 nucleotides are included in the mRNA. This editing is also considered to be a negative autoregulatory mechanism due to frameshifts and inefficient translation initiation from internal methionines that lead to a reduction in protein levels from the edited transcript. Therefore, both Drosophila ADAR and ADAR2 appear to have evolved completely different own-transcript RNA editing sites to control ADAR activity. In another example of convergent evolution of RNA editing sites, the same codon change is introduced by editing at the same position in Drosophila and vertebrate voltage-gated potassium channels in different channel families (Keegan, 2005).

ADAR editing requires only a short RNA duplex to target it; nevertheless, the evolution of an RNA duplex within Drosophila Adar exon 7 is surprising and suggests that editing of the mature Adar mRNA may be required. Adar exon 7 probably folds into a very stable secondary structure, as editing of this minisubstrate in vitro to 70% is higher than for any other transcript so far tested in vitro. The Adar exon 7 structure that supports RNA editing is conserved in other Drosophila species but not in the mosquito. If transient increases or decreases in ADAR activity occur in response to unknown regulatory signals in Drosophila, then Adar editing of mature Adar mRNA could respond immediately to restore the correct level of RNA editing. In vertebrates, modulation of ADAR2 activity in response to changes in neurotransmitter levels has been suggested (Keegan, 2005).

The conversion of serine to glycine has an unexplained dramatic effect on RNA editing activity and this requires further study. This amino acid is conserved in all ADAR2-type enzymes, whereas ADAR1-type enzymes have a conserved aspartic acid at the same position; one possibility is that the serine is phosphorylated but this could not be detected. When the serine is mutated to aspartic acid to introduce a negative charge as found in ADAR1, dADAR is completely inactive. Defining the function of this serine residue in the deaminase domain awaits the crystallisation of ADAR (Keegan, 2005).

Since alternative splicing is critical for generating proteomic diversity, it will be important in the future to determine the functional differences between protein isoforms in an animal model. Drosophila is an excellent animal model to study proteomic diversity. An excellent correlation was observed between the activities of the different ADAR isoforms that have been overexpressed in yeast and assayed in vitro and their in vivo activities as tested in flies. Even though ADAR 3/4 S is very active in vitro, it is not intuitive that this would be toxic in Drosophila when it is ubiquitously expressed, considering that ADAR 3/4 G, which differs by one amino acid, has no toxic effects. It is improbable that many isoforms of other proteins will have such dramatic effects on activity in vivo; however, some will. Drosophila may be the proteomics choice of model organism in future, considering the resource costs of generating equivalent numbers of transgenic mice. While the present work required a great deal of effort to overcome the variations due to position effects on transgene insertions, these limitations arising from the Drosophila transformation method may be relieved by using new genetic techniques that reduce position effects on transgene expression or that express variants from a common site in the chromosome (Keegan, 2005).

In Drosophila, the number of transcripts known to be edited to cause recoding events is much greater than in vertebrates; the list of vertebrate targets may now be nearly complete and includes a large amount of editing of Alu elements embedded in transcripts. The number of recoding sites per target transcript is also higher in Drosophila, suggesting that flies make considerable use of RNA editing as an easy and efficient method of generating proteomic diversity. RNA editing contributes to an enormous potential diversity of transcripts from the cacophony gene encoding the large, pore-forming alpha subunit of the main, non-L-type, voltage-gated calcium channel in the CNS. This protein has a critical role at synapses on axon termini where it responds to depolarising signals by allowing calcium entry to induce vesicle fusion and neurotransmitter release. Pairwise choices due to alternative splicing events, predict 25 splice forms. Pairwise choices of edited or unedited isoforms at each editing site where editing changes codon meaning could multiplex this with potentially 211 edited isoforms. Transcript diversity in cacophony and other RNA editing targets may not reach the full possible maximum level as is the case for the Adar transcript itself where the edited form of the Adar 3a transcript is not observed. It is not surprising that the misregulation of RNA editing that was induced experimentally in this study, leading to this proteomic complexity being expressed at the wrong developmental stage, causes lethality (Keegan, 2005).

The highest levels of editing are seen in adult flies, consistent with the Adar mutant phenotype that includes adult locomotion defects and age-dependent neurodegeneration. ADAR mutant phenotypes in mice and Caenorhabditis elegans also demonstrate that editing is essential for viability and for proper functioning of the nervous system. Editing also normally occurs outside the nervous system in Drosophila. Loss of RNA editing in muscles may contribute to the locomotion defects in Adar mutant flies. RNA editing outside the nervous system is also necessary in vertebrates; ADAR1 mutant mouse embryos undergo widespread apoptosis due to loss of editing in unknown target. The primary requirement for RNA editing in Drosophila may be in the abundant cholinergic neurons in the brain, consistent with efficient rescue using a Cha-GAL4 driver, with patterns of neurodegeneration seen in Adar mutants, and with electrophysiological measurements linking slow recovery from anoxic stupor to defects in brain interneurons. Also, several transcripts encoding acetylcholine receptor alpha and beta subunits are edited in Drosophila. Adar expression is much lower in muscle than in CNS in embryos and it is possible that toxicity arising from ADAR overexpression in embryos could be due to hyperediting of the Ca-alpha 1D transcript encoding an L-type voltage-gated calcium channel. In vertebrates, this channel class is found in muscles and has a critical role on the postsynaptic side of the neuro muscular junction where it responds to depolarisation by allowing calcium entry into muscle cells and subsequent release of calcium from intracellular stores for muscle contraction. It is not, however, know what the effects of editing on most of the channels are nor is it known which channels are critical for either rescue or hyperediting toxicity (Keegan, 2005).

This study describes some of the RNA editing events that were found to be almost 100% efficient in adult flies. In mice, the GluR-B Q/R site is edited with virtually 100% efficiency. An edited GluR-B construct is able to rescue all the defects in a mouse ADAR2 mutant, and no functional requirement has been found for the unedited GluR-B isoform, which is not found even early in development. For the sites that are 100% edited in adult Drosophila, the situation is somewhat different, that is, the unedited transcripts are expressed significantly earlier in development and the results suggest that these forms are required at this stage. Lethality due to increased ADAR activity is associated with increasing editing at some sites in an embryonic ion channel transcript close to levels seen in adults. In contrast to the case of the GluR-B Q/R site, it is suggested that flies require unedited and edited isoforms at different stages. It may be possible to rescue the Adar mutant phenotype in flies by expressing cDNA constructs encoding edited versions of target ion channel transcripts; however, the corresponding unedited transcripts may also be required (Keegan, 2005).

The fruit fly undergoes complete metamorphosis to build two very different-looking complex organisms using a gene set that is small and relatively nonredundant compared to that of vertebrates. Many fly genes show complex patterns of embryonic and adult expression, with different enhancers, promoters, RNA splicing patterns and, finally, different RNA editing patterns in adult transcripts. Customising fast neurotransmission to the requirements of the different life-cycle stages by RNA editing is the most baroque of the gene-sparing strategies so far described in Drosophila (Keegan, 2005).

Protein Interactions

Drosophila ADAR (adenosine deaminase acting on RNA) forms a dimer on double-stranded (ds) RNA, a process essential for editing activity. The minimum region required for dimerization is the N-terminus and dsRNA-binding domain 1 (dsRBD1). Single point mutations within dsRBD1 abolish RNA-binding activity and dimer formation. These mutations and glycerol gradient analysis indicate that binding to dsRNA is important for dimerization. However, dimerization can be uncoupled from dsRNA-binding activity, as evidenced by the observation that a deletion of the N-terminus (amino acids 1-46) yields a monomeric ADAR that retains the ability to bind dsRNA but is inactive in an editing assay, demonstrating that ADAR is only active as a dimer. Different isoforms of ADAR with different editing activities can form heterodimers and this can have a significant effect on editing in vitro as well as in vivo. A model is proposed for ADAR dimerization whereby ADAR monomers first contact dsRNA; however, it is only when the second monomer binds and a dimer is formed that deamination occurs (Gallo, 2003).

Sequence comparison of more thatn 100 dsRBDs has identified clusters of highly conserved amino acids, and NMR and X-ray crystallography studies have revealed that the domain has an alpha1-ß1-ß2-ß3-alpha2 secondary structure. The most conserved region of the dsRBD is the second alpha helix that makes contacts with the major groove of the dsRNA, and mutations within this helix can completely destroy RNA binding. In the present study, the amino acids A106 and A110 of ADAR were independently mutated to glutamate in the alpha2 helix in dsRBD1. These ADAR mutants are unable to bind dsRNA as assayed by filter binding, do not form dimers with full-length ADAR in a yeast two-hybrid assay and do not inhibit RNA editing in mixing experiments with active ADAR isoforms. These experiments suggest that dsRNA is important for dimer formation and that dsRBD1 from two different monomers must bind dsRNA. However, the N-terminus (amino acids 1-53) or dsRBD1 (amino acids 55-133) are unable to interact with the full-length ADAR when expressed as independent domains, indicating that dsRNA and a single dsRBD1 are not sufficient to form a stable ADAR dimer (Gallo, 2003).

The N-terminus of ADAR is also important for dimerization, as evidenced by the observation a truncated ADAR lacking the first 46 amino acids failed to interact with the full-length ADAR in vivo despite the presence of an intact dsRBD1. However, this mutant retained the ability to bind dsRNA better than wild-type ADAR. More importantly, this mutant was inactive in an in vitro editing assay, demonstrating that ADAR is only active as a dimer. Apparently, dimerization does not increase the affinity of ADAR for RNA, since the DeltaN-ADAR not only binds as well as wild-type protein but even competes efficiently with wild-type ADAR in RNA-editing assays. This mutant also demonstrates that dimerization and binding to dsRNA can be separated. The results do not rule out the possibility that there are also contacts between deaminase domains in ADAR dimers (Gallo, 2003).

How can the evidence for ADAR dimerization be interpreted in relation to what is known about editing targets? Most ADAR substrates may be edited on only one side of the putative ADAR dimer, and the complex is probably pseudo-symmetric, as proposed for substrate recognition by APOBEC. A dimeric model for ADAR predicts that in some cases symmetrical editing may be seen if there are target adenosine residues on opposite strands at a fixed distance apart. In fact, a possible symmetric editing event has been reported in which five adenosine residues are edited in a substrate containing the top strand sequence AA-(N15)-TTT. It is proposed that the ADAR dimer has active sites positioned 16 base pairs apart and that each active site flips out and edits a number of adenosines in succession. Hybrid proteins made between vertebrate ADAR1 and ADAR2 show that ADAR deaminase domains contribute to the different target site specificities of those proteins. The dimer model predicts that deaminase domain recognition of RNA sequence or structure 16 bases away from the edited adenosine may contribute to specifying the editing site (Gallo, 2003).

Thus the dimerization of ADAR enzymes is essential for deamination and is a key step in regulating RNA editing. These findings are important in understanding the enzymatic reaction of ADARs. The RNA structures formed at editing sites must now be re-examined for evidence of symmetry. dADAR is a good model for mammalian ADARs, since it is very homologous in domain structure and sequence to hADARs, particularly to hADAR2. hADAR2 is able to rescue locomotion defects in Adar mutant flies, indicating that mechanisms of site recognition in editing of ion channel transcripts are conserved (Gallo, 2003).

Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein

Loss of FMR1 gene function results in fragile X syndrome, the most common heritable form of intellectual disability. The protein encoded by this locus (FMRP) is an RNA-binding protein that is thought to primarily act as a translational regulator; however, recent studies have implicated FMRP in other mechanisms of gene regulation. This study found that the Drosophila fragile X homolog (dFMR1) biochemically interacts with the adenosine-to-inosine RNA-editing enzyme dADAR. Adar and Fmr1 mutant larvae exhibit distinct morphological neuromuscular junction (NMJ) defects. Epistasis experiments based on these phenotypic differences revealed that Adar acts downstream of Fmr1 and that dFMR1 modulates dADAR activity. Furthermore, sequence analyses revealed that a loss or overexpression of dFMR1 affects editing efficiency on certain dADAR targets with defined roles in synaptic transmission. These results link dFMR1 with the RNA-editing pathway and suggest that proper NMJ synaptic architecture requires modulation of dADAR activity by dFMR1 (Bhogal, 2011).

Genetic and molecular findings lead to a proposal that modulation of dADAR activity by dFMR1 is important for NMJ synaptic architecture. The epistatic relationship of these two genes, the requirement of RNA editing by dADAR for normal NMJ morphology, and the genetic suppression of the Fmr1 loss-of-function NMJ defects all support a model in which dFMR1 affects the editing activity of dADAR. Molecular analyses of dADAR substrates support this prediction, since both loss and overexpression of dFMR1 was found to result in changes in editing efficiency in several dADAR-dependent editing sites. Although the changes in editing observed in Fmr1 mutant whole larvae were not large (for example, a ~15% change in editing was observed for lap), they are statistically significant, and it is proposed that these changes would likely be larger if analyses could be performed using mRNA prepared from isolated neurons or synapses rather than whole larvae. In addition, despite a few transcripts that were highly edited throughout development, dADAR function during developmental stages is relatively low compared with its high editing activity in pupal and adult stages. Thus, a larger effect of dFMR1 levels on dADAR substrates with lower efficiency editing sites cannot be ruled out (Bhogal, 2011).

In considering how dFMR1 affects editing, an important clue might come from the fact that both dFMR1 and dADAR are RNA-binding proteins that associate with secondary and higher order RNA structures. FMRP can bind to two separate complex RNA structures that are believed to allow for specificity of FMRP-associated transcripts: the RGG domain in the C terminus of FMRP protein interacts with an intramolecular G quartet stem loop RNA structure, whereas the KH2 domain associates with a complex tertiary kissing complex RNA structure. Similarly, the dADAR family of proteins contains several double-stranded RNA-binding domains and requires duplex RNA structures to identify, bind to and function on its target RNAs. The RNA structure required for dADAR activity, however, can vary from a simple hairpin to complex pseudoknot structures (Bhogal, 2011).

In addition, immunoprecipitation experiments indicate that dFMR1 and dADAR associate on common RNA targets. The dADAR-dFMR1 biochemical interaction was reduced through both a decrease in the amount of RNA in lysates using RNase A as well as by mutating the KH domains of dFMR1, suggesting that the ability for dFMR1 to bind to RNA is important for its association with dADAR. Molecular analyses of lap<.i> and <>Caα1D in the dFMR1 RNA-binding mutants further support this theory, although differential effects were observed with respect to the two transcripts analyzed. It is possible that dFMR1 associates with these two particular transcripts via different RNA-binding motifs. Although the analogous I307N mutation in mammals reduces FMRP's ability to associate with both poly(U)-rich sequences and large RNP complexes, FMRP can still bind to RNA, including transcripts containing G-quartet structures, through an intact RGG RNA-binding motif. Thus, it is proposed that the I244N and I307N mutations in dFMR1 reduce particular dFMR1-dADAR complexes associating with certain edited transcripts while concurrently enriching for dFMR1-dADAR complexes associating with the dFMR1 RGG box. Further studies delving into the importance of each RNA-binding domain in both dFMR1 and dADAR will give more insight into the biochemical and functional interaction between these two proteins (Bhogal, 2011).

On the basis of these results, it is predicted that dFMR1 and dADAR can associate in a common complex and converge on similar RNA substrates. Because the effect that dFMR1 has on the editing efficiency is context dependent, it is proposed that the association of dFMR1 with dADAR has no net positive or negative effect on the editing activity of dADAR, but instead maintains a balance of dADAR activity. At sites that demonstrate enhanced editing in the presence of dFMR1, dFMR1 could promote editing by either recruiting dADAR to the site via its own RNA-binding activity, or it could help form and/or stabilize RNA structures that create a site for editing by dADAR. At sites that are negatively affected by the presence of dFMR1, it is proposed that the RNA binding activity of dFMR1 interferes with the formation of a substrate for dADAR (Bhogal, 2011).

This analyses revealed several transcripts whose level of editing is regulated by the interaction between dFMR1 and dADAR; however, at this time, it is not known how many such transcripts are important for the proper formation of the NMJ. Although many identified dADAR targets encode for proteins that function in synaptic transmission at the NMJ34 and mutations in several dADAR substrates (for example, syt1, lap and unc-13) affect NMJ synaptic architecture and/or function, how editing is affecting the function of most of these gene products remains unknown. It is also important to note that a role for dFMR1 in translational regulation is already proposed to be important for proper NMJ development through its genetic interaction with the microtuble-associated protein homolog Futsch. Collectively, these studies suggest that both dADAR and dFMR1 have multifaceted roles at the NMJ (Bhogal, 2011).

In summary, this study found that dFMR1 physically and genetically interacts with dADAR-dependent RNA editing. This is the first report of a disease-associated protein that associates with and modulates A-to-I RNA editing. In addition, these findings reveal a previously unknown function for FMRP with respect to neuronal architecture and expand FMRP's predicted role as a translational regulator. Understanding all of the mechanisms by which FMRP functions to regulate synaptic development and function is essential to better understand the pathogenesis of the FXS symptoms, and consequently can lead to effective therapeutic treatments for people afflicted with this disease (Bhogal, 2011).


DEVELOPMENTAL BIOLOGY

Embryonic

Adar is expressed in the developing nervous system, making it a candidate for the editase that acts on para voltage-gated Na+ channel transcripts in the central nervous system. Surprisingly, Adar itself undergoes developmentally regulated RNA editing that changes a conserved residue in the catalytic domain. These findings show that both transcription and processing of Adar transcripts are under strict developmental control and suggest that the process of RNA editing in Drosophila is dynamically regulated (Palladino. 2000a).

A molecular, phylogenetic and functional study of the mRNA truncated isoform during embryonic development reveals an editing-independent function

Adenosine Deaminases Acting on RNA (ADARs) have been studied in many animal phyla, where they have been shown to deaminate specific adenosines into inosines in duplex mRNA regions. In Drosophila, two isoform classes are encoded, designated full-length (contains the editase domain) and truncated (lacks this domain). Much is known about the full-length isoform, which plays a major role in regulating functions of voltage-gated ion channel proteins in the adult brain. In contrast, almost nothing is known about the functional significance of the truncated isoform. In situ hybridization shows that both isoform mRNA classes are maternally derived and transcripts for both localize primarily to the developing central nervous system. Quantitative RT-PCR shows that about 35% of all dADAR mRNA transcripts belong to the truncated class in embryos. 3'-RACE results show that abundance of the truncated isoform class is developmentally regulated, with a longer transcript appearing after the mid-blastula transition. 3'-UTR sequences for the truncated isoform have been determined from diverse Drosophila species and important regulatory regions including stop codons have been mapped. Western analysis shows that both mRNA isoform classes are translated into protein during embryonic development, as full-length variant levels gradually diminish. The truncated protein isoform is present in every Drosophila species studied, extending over a period spanning about 40 x 106 years, implying a conserved function. Previous work has shown that a dADAR protein isoform binds to the evolutionarily conserved rnp-4f pre-mRNA stem-loop located in the 5'-UTR to regulate splicing, while no RNA editing was observed, suggesting the hypothesis that it is the non-catalytic truncated isoform which regulates splicing. To test this hypothesis, RNAi technology was used, the results of which support the hypothesis. These results demonstrate a novel, non-catalytic function for the truncated dADAR protein isoform in Drosophila embryonic development, which is very likely evolutionarily conserved (Ghosh, 2014: PubMed).


EFFECTS OF MUTATION

A search for available mutations near the Adar locus revealed no breakpoints or useful transposon insertions. Insertions in the Adar locus, on the X chromosome, were obtained through site-selected P element mutagenesis. In a screen of 20,000 lines, three insertions into the 5' regulatory region of the Adar gene, near the two predicted promoters of Adar, were generated. Molecular analysis reveals that all 13 mutations harbor deletions of the Adar locus and comprise several classes. One class deletes DNA unidirectionally in the 5' direction removing exon -4a and upstream 5' regulatory regions (alleles 2B3 and 5A1). Another, the largest class, consists of bi-directional deletions that remove all of exons -4a and -4b and the predicted promoter regions (alleles 1F1, 1F4, 1G3, 2J3, 4A1, 4F1, 5I2, 6A2, and 6K1). Lastly, two deletions were recovered that are unidirectional in the 3' direction; allele 6C1 deletes from the P element insertion site to a region between alternative exons -1 and 0, and allele 5G1 completely deletes the Adar locus including all coding sequences (Palladino, 2000b).

All of the Adar mutants obtained from the P-element screen are conditionally lethal. That is, under ideal growth conditions Adar mutants developed into morphologically normal adults that display profound behavioral deficits and these phenotypes are recessive. All Adar alleles are phenotypically indistinguishable, including the 5G1 allele that deletes the entire Adar locus (Palladino, 2000b).

One behavioral defect observed in Adar mutant males is an extreme defect in mating. Adar- males rarely successfully mate with wild-type (WT) females. Nevertheless, a rare successful mating between females heterozygous for Adar2B3 and Adar2B3 males was able to produce viable progeny amongst which were females homozygous for Adar2B3. Mutant females display behavioral defects similar to hemizygous males. Females homozygous for all alleles of Adar display mutant behavioral phenotypes similar to hemizygous Adar- males. In contrast, homozygous Adar- females can be mated by WT males, are fertile, and give rise to morphologically normal Adar- male progeny, which exhibit the Adar- adult behavioral defects. Since Adar- male progeny of mothers lacking Adar are indistinguishable from those generated by mothers heterozygous for Adar-, it is concluded that there is no significant requirement for a maternal contribution of Adar activity. Thus, Adar would seem not to be required during early development (Palladino, 2000b).

Since it has been shown that maternal Adar is dispensable, it was of interest to determine whether Adar- animals display normal development in the absence of a zygotic contribution of Adar. The time course of development from egg-laying to pupariation of WT and Adar1F1 was examined. No significant difference was observed. As well, no significant difference between WT and mutant animals in the time course from onset of pupariation to eclosion into morphologically normal adults was observed. Adar mutant animals show a slight reduction in viability versus WT (Palladino, 2000b).

Engineered alterations in RNA editing modulate complex behavior in Drosophila: regulatory diversity of adenosine deaminase acting on RNA (ADAR) targets

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. This study engineered 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 the 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, a novel reporter was used to map RNA editing activity across the nervous system, and it was demonstrated that knockdown of editing in fruitless-expressing neurons is sufficient to modify the male courtship song. These 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 (Jepson, 2011).

Using a novel hypomorphic allele of dAdar generated through homologous recombination coupled with cell-specific dADAR knockdown, this study has demonstrated that RNA editing serves a modulatory role in multiple adaptive behaviors in Drosophila. In short, this study provides linkage between the loss of conserved and taxa-specific amino acid re-coding sites and alterations in wild-type ethological outputs that directly impinge on organismal fitness. Importantly, the behavioral defects observed in dAdarhyp males correlate with the severe loss of a particular subset of edited adenosines, namely those that are preferentially edited at the adult stage (Jepson, 2011).

This molecular analysis of dAdar revealed a striking diversity in the response of edited adenosines to changes in endogenous dADAR levels. Both the local sequence surrounding edited adenosines and their predicted secondary structures vary widely between dADAR substrates, providing a potential mechanism to generate differential affinities for dADAR binding and deamination. This finding has important implications as follows. First, it provides a explanatory basis for the developmental regulation of a select population of editing sites, a phenomenon common to both Drosophila and mammals. Second, cell-specific variation in dADAR expression may allow spatial control of low efficiency (LE) sites while simultaneously maintaining robust network-wide editing of HE sites, thus providing a means to fine-tune neuronal physiology through the diversification of a constrained population of proteins (Jepson, 2011).

Pan-neuronal expression of the two hairpin RNAi constructs used in this study reduces locomotor activity by ∼90%, and this effect could not be phenocopied by dADAR knockdown in any particular neuronal subset tested. Furthermore, dADAR knockdown under these conditions was robust enough to strongly reduce editing even at high efficiency (HE) sites such as syt-1 site 4. Although knockdown is subject to the level of hairpin expression and efficiency of RNAi in particular neurons, the abrogation of ADAR expression by transgenic knockdown was clearly effective. In contrast, editing at HE sites is still maintained in a purely genetic model using dAdarhyp mutant males and females, as is coordinated locomotion (albeit at lower levels). Indeed, even dAdarhyp/dAdar5g1 trans-heterozygote females, predicted to express dADAR at ∼ 10% of wild-type levels, do not appear uncoordinated. Collectively, these data imply that network-wide editing of HE sites is sufficient to provide motor tone and prevent the extreme uncoordination observed in dAdar null flies (Jepson, 2011).

Conversely, it is tempting to speculate that developmentally regulated LE sites modulate adult-specific behaviors. Indeed, two ethologically relevant behaviors were examined in dAdarhyp males, which show a severe disruption of developmentally regulated editing. dAdarhyp males did not show the circadian anticipation of lights-on seen in wild-type Drosophila, and multiple aspects of courtship behavior were abnormal in dAdarhyp males, including the time required to initiate courtship and the waveform of the mating song. It should be stressed that defects in both of the above parameters are likely to be severely detrimental to reproductive fitness under competitive conditions in the wild (Jepson, 2011).

Although these data lead to a hypothesis that loss of adult-stage LE sites may underlie the locomotor and courtship defects exhibited by dAdar , the loss of particular editing sites cannot be currently linked to the behavioral defects seen in dAdarhyp males due to the large number of characterized dADAR substrates. Over 100 editing sites in 24 mRNAs have been identified either serendipitously or through comparative genomics approaches, although a recent bioinformatic screen identified a further potential 27 mRNAs subject to re-coding. The existence of functionally epistastic interactions between editing sites also makes it unlikely that any particular phenotype observed in dAdarhyp males can be fully mapped to the loss of a single editing site. Rather, the relationship between re-coding and behavior can instead be viewed through the prism of the pleiotropic actions of dADAR on a wide range of RNA substrates, with many edited proteins simultaneously contributing to the total phenotype of interests (Jepson, 2011).

The cellular foci for behavioral abnormalities associated with stringent loss of dADAR expression were mapped using transgenic RNAi. Knockdown of dADAR specifically in fruitless-expressing neurons partially recapitulated the polycyclic songs observed in dAdarhyp males but did not phenocopy alterations in other song properties or mating behavior, suggesting that these highly specific phenotypic components are influenced by editing in other fru-negative neurons and/or muscle tissue. Surprisingly, targeted expression of a molecular reporter for editing activity suggests that male and female fru neurons within both the brain and thoracic ganglion may differ in terms of dADAR activity. Because only small subpopulations of fru neurons exhibit morphological sexual dimorphism, it has been hypothesized that expression of the male-specific isoform of Fruitless (FruM) may modify the physiological properties of fru neurons. Given the large number of transcripts re-coded by A-to-I editing, an alteration of dADAR expression or activity by FruM could hypothetically provide a means of enabling functional modulation of a wide range of ion channels and synaptic release proteins. Further experiments will be required to test whether the alterations in editing observed between male and female fru neurons represent large differences in a subset of fru neurons, subtle alterations across the fru neuron network, or are due to numerical sexual dimorphism in the fru neuron populations (Jepson, 2011).

That RNA editing can modulate song properties is particularly intriguing, because editing sites are not static throughout insect evolution. Indeed, even within the Drosophila lineage, species-specific changes are observed in the magnitude of editing at orthologous adenosines in several ion channels. Therefore, these data open the possibility that alterations in RNA editing may contribute to species-specific song waveforms, a key mechanism implicated in the reproductive isolation between Drosophilids. More broadly, the data suggest that, in principle, evolutionary divergences in RNA editing may contribute to the generation of adult-stage species-specific behavioral patternss (Jepson, 2011).

Adar is essential for optimal presynaptic function

RNA editing is a powerful way to recode genetic information. Because it potentially affects RNA targets that are predominantly present in neurons, it is widely hypothesized to affect neuronal structure and physiology. Across phyla, loss of the enzyme responsible for RNA editing, Adar, leads to behavioral changes, impaired locomotion, neurodegeneration and death. However, the consequences of a loss of Adar activity on neuronal structure and function have not been studied in detail. In particular, the role of RNA editing on synaptic development and physiology has not been investigated. This study tested the physiological and morphological consequences of the lack of Adar activity on the Drosophila neuromuscular junction (NMJ). Detailed examination of synaptic transmission showed that loss of Adar increases quantal size, reduces the number of quanta of neurotransmitter released and perturbs the calcium dependence of synaptic release. In addition, it was found that staining for several synaptic vesicle proteins is abnormally intense at Adar deficient synapses. Consistent with this finding, Adar mutants showed a major alteration in synaptic ultrastructure. Finally, evidence is presented of compensatory changes in muscle membrane properties in response to the changes in presynaptic activity within the Adar mutant NMJs (Maldonado, 2013).

The ADAR RNA editing enzyme controls neuronal excitability in Drosophila melanogaster

RNA editing by deamination of specific adenosine bases to inosines during pre-mRNA processing generates edited isoforms of proteins. Recoding RNA editing is more widespread in Drosophila than in vertebrates. Editing levels rise strongly at metamorphosis, and Adar5G1 null mutant flies lack editing events in hundreds of CNS transcripts; mutant flies have reduced viability, severely defective locomotion and age-dependent neurodegeneration. On the other hand, overexpressing an adult dADAR isoform with high enzymatic activity ubiquitously during larval and pupal stages is lethal. Advantage was taken of this to screen for genetic modifiers; Adar overexpression lethality is rescued by reduced dosage of the Rdl (Resistant to dieldrin), gene encoding a subunit of inhibitory GABA receptors. Reduced dosage of the Gad1 gene encoding the GABA synthetase also rescues Adar overexpression lethality. Drosophila Adar5G1 mutant phenotypes are ameliorated by feeding GABA modulators. This study demonstrates that neuronal excitability is linked to dADAR expression levels in individual neurons; Adar-overexpressing larval motor neurons show reduced excitability whereas Adar5G1 null mutant or targeted Adar knockdown motor neurons exhibit increased excitability. GABA inhibitory signalling is impaired in human epileptic and autistic conditions, and vertebrate ADARs may have a relevant evolutionarily conserved control over neuronal excitability (Li, 2013).


EVOLUTIONARY HOMOLOGS

A second Drosophila RNA editing enzyme

Pre-mRNA editing involving the conversion of adenosine to inosine is mediated by adenosine deaminases that act on RNA (ADAR1 and ADAR2). ADARs contain multiple double-stranded RNA(dsRNA)-binding domains in addition to an adenosine deaminase domain. An adenosine deaminase acting on tRNAs, scTad1p (also known as scADAT1), cloned from Saccharomyces cerevisiae has a deaminase domain related to the ADARs but lacks dsRNA-binding domains. A gene homologous to scADAT1 has been identified in the region of Drosophila Adh chromosome II. Recombinant Drosophila ADAT1 (dADAT1) has been expressed in the yeast Pichia pastoris and purified. The enzyme has no activity on dsRNA substrates but is a tRNA deaminase with specificity for adenosine 37 of insect alanine tRNA. dADAT1 shows greater similarity to vertebrate ADARs than to yeast Tad1p, supporting the hypothesis of a common evolutionary origin for ADARs and ADATs. dAdat1 transcripts are maternally supplied in the egg. Zygotic expression is widespread initially and later concentrates in the central nervous system (Keegan, 1999).

Mechanism of RNA editing

A functionally critical position (Q/R site) of the AMPA receptor subunit GluR-B is controlled by RNA editing that operates in the nucleus, since in brain and clonal cell lines of neural origin, unspliced GluR-B transcripts occur edited in the Q/R site CAG codon and, additionally, in intronic adenosines. Transfection of GluR-B gene constructs into PC12 cells reveals that the proximal part of the intron downstream of the unedited exonic site is required for Q/R site editing. This intron portion contains an imperfect inverted repeat preceding a 10 nt sequence with exact complementarity to the exon centered on the unedited codon. Single nucleotide substitutions in this short intronic sequence or its exonic complement curtails Q/R site editing, which was recovered by restoring complementarity in the respective partner strand. Base conversion in the channel-coding region of GluR-B directed by base paired sequences may be executed by a ubiquitous nuclear adenosine deaminase specific for double-stranded RNA (Higuchi, 1993).

RNA editing by adenosine deamination in brain-expressed pre-mRNAs for glutamate receptor (GluR) subunits alters gene-specified codons for functionally critical positions, such as the channel's Q/R site. Transcript analysis of minigenes transiently expressed in PC-12 cells shows that, in contrast to GluR-B pre-mRNA, where the two editing sites (Q/R and R/G) require base pairing with nearby intronic editing site complementary sequences (ECSs), editing in GluR5 and GluR6 pre-mRNAs recruits an ECS located as far as 1900 nucleotides distal to the Q/R site. The exon-intron duplex structure of the GluR5 and GluR6 pre-mRNAs appears to be a substrate of double-stranded RNA-specific adenosine deaminase. This enzyme when coexpressed in HEK 293 cells preferentially targets the adenosine of the Q/R site and of an unpaired position in the ECS which is highly edited in brain (Herb, 1996).

Adenosine deaminases that act on RNA (ADARs) are a family of RNA editing enzymes that convert adenosines to inosines within double-stranded RNA (dsRNA). Although ADARs promiscuously deaminate perfectly base-paired dsRNA, deamination is limited to a few, selected adenosines within dsRNA containing mismatches, bulges and internal loops. As a first step in understanding how RNA structural features promote selectivity, the role of internal loops within ADAR substrates has been investigated. A dsRNA helix is deaminated at the same sites whether it exists as a free molecule or is flanked by internal loops. Thus, internal loops delineate helix ends for ADAR1. Since ADAR1 deaminates short RNAs at fewer adenosines than long RNAs, loops decrease the number of deaminations within an RNA by dividing a long RNA into shorter substrates. For a series of symmetric internal loops related in sequence, larger loops (greater than or equal to six nucleotides) act as helix ends, whereas smaller loops (less than or equal to four nucleotides) do not. This work provides the first information about how secondary structure within ADAR substrates dictates selectivity, and suggests a rational approach for delineating minimal substrates for RNAs deaminated by ADARs in vivo (Lehmann, 1999).

The interferon-inducible RNA-specific adenosine deaminase (ADAR1) is an RNA editing enzyme implicated in the site-selective deamination of adenosine to inosine in cellular pre-mRNAs. The pre-mRNA for the rat serotonin-2C receptor (5-HT2CR) possesses four editing sites (A, B, C, and D), which undergo A-to-I nucleotide conversions that alter the signaling function of the encoded G-protein-coupled receptor. Measurements of 5-HT2CR pre-mRNA editing in vitro revealed site-specific deamination catalyzed by ADAR1. Three splice site variants, ADAR1-a, -b, and -c, all efficiently edit the A site of 5-HT2CR pre-mRNA, but the D site does not serve as an efficient substrate for any of the ADAR1 variants. Mutational analysis of the three double-stranded (ds) RNA binding motifs present in ADAR1 reveal a different relative importance of the individual dsRNA binding motifs for deamination of the A site of 5-HT2CR and synthetic dsRNA substrates. Quantitative reverse transcription-polymerase chain reaction analyses demonstrate that the 5-HT2CR pre-mRNA is most highly expressed in the choroid plexus of rat brain. However, ADAR1 and the related deaminase ADAR2 shows significant expression in all regions of the brain examined, including cortex, hippocampus, olfactory bulb, and striatum, where the 5-HT2CR pre-mRNA is extensively edited (Liu, 1999a).

The interferon-inducible RNA-specific adenosine deaminase (ADAR1) is an RNA-editing enzyme that catalyzes the deamination of adenosine in double-stranded RNA structures. Three alternative splice-site variants of ADAR1 (ADAR1-a, -b, and -c) occur that possess functionally distinct double-stranded RNA-binding motifs, as measured with synthetic double-stranded RNA substrates. The pre-mRNA transcript encoding the B subunit of glutamate receptor (GluR-B) has two functionally important editing sites (Q/R and R/G sites) that undergo selective A-to-I conversions. The ability of the three ADAR1 splice-site variants to catalyze the editing of GluR-B pre-mRNA at the Q/R and R/G sites as well as an intron hotspot (+60) of unknown function has been examined. Measurement of GluR-B pre-mRNA editing in vitro reveals different site-specific deamination catalyzed by the three ADAR1 variants. The ADAR1-a, -b, and -c splice variants all efficiently edit the R/G site and the intron +60 hotspot but exhibit little editing activity at the Q/R site. ADAR1-b and -c show higher editing activity than ADAR1-a for the R/G site, whereas the intron +60 site is edited with comparable efficiency by all three ADAR1 splice variants. Mutational analysis reveals that the functional importance of each of the three RNA-binding motifs of ADAR1 varies with the specific target editing site in GluR-B RNA. Quantitative reverse transcription-polymerase chain reaction analyses of GluR-B RNA from dissected regions of rat brain show significant expression and editing at the R/G site in all brain regions examined except the choroid plexus. The relative levels of the alternatively spliced flip and flop isoforms of GluR-B RNA vary among the choroid plexus, cortex, hippocampus, olfactory bulb, and striatum, but in all regions of rat brain the editing of the flip isoform is greater than that of the flop isoform (Liu, 1999b).

The ADAR family of RNA-editing enzymes deaminates adenosines within RNA that are completely or largely double stranded. In mammals, most of the characterized substrates encode receptors involved in neurotransmission, and these substrates are thought to be targeted by the mammalian enzymes ADAR1 and ADAR2. Although some ADAR substrates are deaminated very promiscuously, mammalian glutamate receptor B (gluR-B) pre-mRNA is deaminated at a few specific adenosines. Like most double-stranded RNA (dsRNA) binding proteins, ADARs bind to many different sequences, but few studies have directly measured and compared binding affinities. Attempts have been made to determine if ADAR deamination specificity occurs because the enzymes bind to targeted regions with higher affinities. To explore this question, an examination was made of the binding of rat ADAR2 to a region of rat gluR-B pre-mRNA that contains the R/G editing site, and a wild-type molecule was compared with one containing mutations that decreased R/G site editing. Although binding affinity to the two sequences is almost identical, footprinting studies indicate ADAR2 binds to the wild-type RNA at a discrete region surrounding the editing site, whereas binding to the mutant appears to be nonspecific (Ohman, 2000).

Structural analogs of a natural RNA editing substrate have been synthesized and editing reactions of these substrates by recombinant ADAR-2, an RNA-editing adenosine deaminase, were compared. Deamination rates were shown to be sensitive to structural changes at the 2[prime]-carbon of the edited adenosine. Methylation of the 2[prime]-OH causes a large decrease in deamination rate, whereas 2[prime]-deoxyadenosine and 2[prime]-deoxy-2[prime]-fluoroadenosine are deaminated at a rate similar to adenosine. In addition, a duplex containing as few as 19 bp of the stem structure adjacent to the R/G editing site of the GluR-B pre-mRNA supports deamination of the R/G adenosine by ADAR-2. This identification and initial characterization of synthetic RNA editing substrate analogs, further defines structural elements in the RNA that are important for the deamination reaction and sets the stage for additional detailed structural, thermodynamic and kinetic studies of the ADAR-2 reaction (Yi-Brunozzi, 1999).

RNA editing changes the read-out of genetic information, increasing the number of different protein products that can be made from a single gene. One form involves the deamination of adenosine to form inosine, which is subsequently translated as guanosine. The reaction requires a double-stranded RNA (dsRNA) substrate and is catalyzed by the adenosine deaminase that act on dsRNA (ADAR) family of enzymes. These enzymes possess dsRNA-binding domains (DRBM) and a catalytic domain. ADAR1 so far has been found only in vertebrates and is characterized by two Z-DNA-binding motifs, the biological function of which remains unknown. The role of the various functional domains of ADAR1 in determining the editing efficiency and specificity of ADAR1 was examined in cell-based assays. A variety of dsRNA substrates was tested. It was found that a 15-bp dsRNA stem with a single base mismatch was sufficient for editing. The particular adenosine that was be modified could be varied by changing the position of the mismatch. Editing efficiency could be increased by placing multiple pyrimidines 5' to the edited adenosine. With longer substrates, editing efficiency also increased and was partly due to the use of DRBMs. Additional editing sites were also observed that clustered on the complementary strand 11-15 bp from the first. An unexpected finding was that the DRBMs are not necessary for the editing of the shorter 15-bp substrates. However, mutation of the Z-DNA-binding domains of ADAR1 decreased the efficiency with which such a substrate was edited (Herbert, 2001).

ADAR enzymes, adenosine deaminases that act on RNA, form a family of RNA editing enzymes that convert adenosine to inosine within RNA that is completely or largely double-stranded. Site-selective A-->I editing has been detected at specific sites within a few structured pre-mRNAs of metazoans.The editing selectivity of ADAR enzymes was examined, and the naturally edited R/G site in the pre-mRNA of the glutamate receptor subunit B (GluR-B) was chosen. A comparison of editing by ADAR1 and ADAR2 revealed differences in the specificity of editing. These results show that ADAR2 selectively edits the R/G site, while ADAR1 edits more promiscuously at several other adenosines in the double-stranded stem. To further understand the mechanism of selective ADAR2 editing, the importance of internal loops in the RNA substrate was investigated. The immediate structure surrounding the editing site was found to be important. A purine opposite the editing site has a negative effect on both the selectivity and efficiency of editing. More distant internal loops in the substrate were found to have minor effects on site selectivity, while efficiency of editing was found to be influenced. Finally, changes in the RNA structure that affect editing did not alter the binding abilities of ADAR2. Overall these findings suggest that binding and catalysis are independent events (Kallman, 2003).

Evolutionary conservation of intronic sites regulating editing

Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosines to inosines within cellular and viral RNAs. Certain glutamate receptor (gluR) pre-mRNAs are substrates for the enzymes in vivo. For example, at the R/G editing site of gluR-B, -C, and -D RNAs, ADARs change an arginine codon (AGA) to a glycine codon (IGA) so that two protein isoforms can be synthesized from a single encoded mRNA; the highly related gluR-A sequence is not edited at this site. To gain insight into what features of an RNA substrate are important for accurate and efficient editing by an ADAR, phylogenetic analysis of sequences required for editing at the R/G site was performed. Highly conserved sequences were found that are shared by gluR-B, -C, and -D, but absent from gluR-A. Surprisingly, in contrast to results obtained in phylogenetic analyses of tRNA and rRNA, it is the bases in paired, helical regions whose identity is conserved, whereas bases in nonhelical regions vary, but maintain their nonhelical state. It is speculated that this pattern in part reflects constraints imposed by ADAR's unique specificity and support for these hypotheses was obtained with mutagenesis studies. Unexpectedly, some of the gluR introns are conserved beyond the sequences required for editing. The approximately 600-nt intron 13 of gluR-C is particularly remarkable, showing greater than 94% nucleotide identity between human and chicken, organisms estimated to have diverged 310 million years ago (Aruscavage, 2000).

Subcellular localization of ADAR

The adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine (A to I) in primary mRNA transcripts, thereby affecting the splicing pattern or coding potential of mature mRNAs. Although the subnuclear localization of A-to-I editing has not been precisely defined, ADARs have been shown to act before splicing, suggesting that they function near nucleoplasmic sites of transcription. ADAR2, a member of the vertebrate ADAR family, is concentrated in the nucleolus, a subnuclear domain disparate from the sites of mRNA transcription. Selective inhibition of ribosomal RNA synthesis or the introduction of mutations in the double-stranded RNA-binding domains within ADAR2 results in translocation of the protein to the nucleoplasm, suggesting that nucleolar association of ADAR2 depends on its ability to bind to ribosomal RNA. Fluorescence recovery after photobleaching reveals that ADAR2 can shuttle rapidly between subnuclear compartments. Enhanced translocation of endogenous ADAR2 from the nucleolus to the nucleoplasm results in increased editing of endogenous ADAR2 substrates. These observations indicate that the nucleolar localization of ADAR2 represents an important mechanism by which RNA editing can be modulated by the sequestration of enzymatic activity from potential RNA substrates in the nucleoplasm (Sansam, 2003).

Function of mRNA editing

The arginine residue at position 586 of the GluR-B subunit renders heteromeric AMPA-sensitive glutamate receptor channels impermeable to calcium. The codon for this arginine is introduced at the precursor messenger RNA (pre-mRNA) stage by site-selective adenosine editing of a glutamine codon. Heterozygous mice engineered by gene targeting to harbor an editing-incompetent GluR-B allele, synthesize unedited GluR-B subunits and, in principal neurons and interneurons, express AMPA receptors with increased calcium permeability. These mice develop seizures and die by 3 weeks of age, showing that GluR-B pre-mRNA editing is essential for brain function (Brusa, 1995).

Mouse mutants were generated with targeted AMPA receptor (AMPAR) GluR-B subunit alleles, functionally expressed at different levels and deficient in Q/R-site editing. All mutant lines have increased AMPAR calcium permeabilities in pyramidal neurons, and one of these shows elevated macroscopic conductances of these channels. The AMPAR-mediated calcium influx induces NMDA-receptor-independent long-term potentiation (LTP) in hippocampal pyramidal cell connections. Calcium-triggered neuronal death was not observed, but mutants have mild to severe neurological dysfunctions, including epilepsy and deficits in dendritic architecture. The seizure-prone phenotype correlates with an increase in the macroscopic conductance, as independently revealed by the effect of a transgene for a Q/R-site-altered GluR-B subunit. Thus, changes in GluR-B gene expression and Q/R site editing can affect critical architectural and functional aspects of excitatory principal neurons (Feldmeyer, 1999).

Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that convert adenosine to inosine within double-stranded RNA. In the 12 years since the discovery of ADARs only a few natural substrates have been identified. These substrates were found by chance, when genomically encoded adenosines were identified as guanosines in cDNAs. To advance an understanding of the biological roles of ADARs, a method was developed for systematically identifying ADAR substrates. In the first application of the method, five additional substrates in Caenorhabditis elegans were identified. Four of those substrates are mRNAs edited in untranslated regions, and one is a noncoding RNA edited throughout its length. The edited regions are predicted to form long hairpin structures, and one of the RNAs encodes POP-1, a protein involved in cell fate decisions (Morse, 1999).

RNA transcripts encoding the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor (5-HT2CR) undergo adenosine-to-inosine RNA editing events at up to five specific sites. Compared with rat brain, human brain samples express higher levels of RNA transcripts encoding the amino acids valine-serine-valine (5-HT2C-VSV) and valine-glycine-valine (5-HT2C-VGV) at positions 156, 158, and 160, respectively. Agonist stimulation of the nonedited human receptor (5-HT2C-INI) and the edited 5-HT2C-VSV and 5-HT2C-VGV receptor variants stably expressed in NIH-3T3 fibroblasts demonstrate that serotonergic agonists are less potent at the edited receptors. Competition binding experiments reveal a guanine nucleotide-sensitive serotonin high affinity state only for the 5-HT2C-INI receptor; the loss of high affinity agonist binding to the edited receptor demonstrates that RNA editing generates unique 5-HT2CRs that couple less efficiently to G proteins. This reduced G protein coupling for the edited isoforms is primarily due to silencing of the constitutive activity of the nonedited 5-HT2CR. The distinctions in agonist potency and constitutive activity suggest that different edited 5-HT2CRs exhibit distinct responses to serotonergic ligands and further imply that RNA editing represents a novel mechanism for controlling physiological signaling at serotonergic synapses (Niswender, 1999).

RNA editing by site-selective deamination of adenosine to inosine alters codons and splicing in nuclear transcripts, and therefore protein function. ADAR2 is a candidate mammalian editing enzyme that is widely expressed in brain and other tissues, but its RNA substrates are unknown. ADAR2-mediated RNA editing has been studied by generating mice that are homozygous for a targeted functional null allele. Editing in ADAR2-/- mice is substantially reduced at most of 25 positions in diverse transcripts; the mutant mice become prone to seizures and die young. The impaired phenotype appears to result entirely from a single underedited position, as it reverts to normal when both alleles for the underedited transcript are substituted exonically, with alleles encoding the edited version. The critical position specifies an ion channel determinant, the Q/R site, in AMPAreceptor GluR-B pre-messenger RNA. It is concluded that this transcript is the physiologically most important substrate of ADAR2 (Higuchi, 2000).

Mammalian transcripts that are known to be edited by site-selective adenosine deamination are expressed largely in brain: most encode subunits of ionotropic glutamate receptors (GluRs) that mediate fast excitatory neurotransmission. The only position edited to nearly 100% is the Q/R site of GluR-B, for which the mRNA contains an arginine (R) codon (CIG) in place of the genomic glutamine (Q) codon (CAG). The physiological importance of this codon substitution wrought by RNA editing has been revealed by early onset epilepsy and premature death of mice heterozygous for an intron-11-modified GluR-BECS allele with Q/R site-uneditable transcripts (Higuchi, 2000).

Heterozygous ADAR2+/- mice are phenotypically normal, but ADAR2-/- mice die between P0 and P20 and become progressively seizure-prone after P12, akin to GluR-B+/delta ECS mice. Therefore, this investigation focussed on the effect of ADAR2 deficiency on Q/R site editing of GluR-B pre-mRNA, the substrate for a nuclear RNA-dependent adenosine deaminase activity. As determined from cloned polymerase chain reaction with reverse transcription (RT-PCR) products from brain RNA6, Q/R site editing in primary GluR-B transcripts is tenfold lower in ADAR2-/- than in wild-type mice (10% compared with 98%). This identifies ADAR2 as the principal RNA-editing enzyme at the Q/R site. The remaining low level of Q/R site editing in GluR-B pre-mRNA cannot be mediated by the residual, enzymatically inactive, truncated ADAR2 protein, but is mediated by another ADAR, perhaps ADAR1, for which gene expression appeared unchanged in ADAR2 -/- mice (Higuchi, 2000).

The low extent of Q/R site editing of GluR-B pre-mRNA led to nuclear accumulation of incompletely processed primary GluR-B transcripts and to a fivefold reduction in GluR-B mRNA, as assessed by RNase protection and quantitative RT-PCR. The increased level of intron 11-containing GluR-B transcripts and the decrease in GluR-B mRNA are easily visualized by in situ hybridization. Editing is thus a prerequisite for efficient splicing and processing of the pre-mRNA. The edited GluR-B transcripts are preferentially spliced, as revealed by a shift in Q/R site editing from 10% to 40% when comparing intron-11-containing transcripts with GluR-B mRNA. A defect in transcript processing caused by the interaction of the residual truncated ADAR2 protein with RNA can be excluded because GluR-B pre-mRNA accumulation is also observed in ADAR2+/+ mice expressing the Q/R site-uneditable GluR-BdeltaECS allele (Higuchi, 2000).

Strains are described containing homozygous deletions in each, or both, of the two C. elegans ADAR genes that have been characterized: adr-1 and adr-2. adr-1 is expressed in most, if not all, cells of the C. elegans nervous system and also in the developing vulva. Using chemotaxis assays, it has been shown that both ADARs are important for normal behavior. Biochemical, molecular and phenotypic analyses indicate that ADR-1 and ADR-2 have distinct roles in C. elegans, but sometimes act together (Tonkin, 2002).

The observation that animals with a deletion in adr-1 have vulva defects, as well as the strong expression of the adr-1::GFP construct in the developing vulva, implicates the ADR-1 protein in vulva morphogenesis. However, because the protruding-vulva defects are subtle, future studies will be needed to confirm this. ADARs could act in any of the signaling steps involved in vulval development. Since animals with a deletion in adr-2 have no detectable editing, it is perplexing that these animals do not have vulva defects. Possibly, adr-1 has functions beyond RNA editing or, alternatively, some adr-1-specific editing may exist in adr-2(gv42) animals but is beyond the limits of detection. The latter is consistent with the observation that the RNAi defects of the adr mutants are less severe in adr-2(gv42) animals compared with the double mutants. Although all mutant animals were back-crossed to wild-type animals eight times, since it has not been possible to rescue the Pvl defect, in theory the defect could derive from a mutation in a very closely linked gene. However, the strong vulva expression of the adr-1::GFP construct in multiple transgenic lines argues against this possibility. Finally, while there are no other genes with obvious sequence similarity to ADARs in the C. elegans genome, ADR-1 activity in the vulva could be mediated by interaction with an as yet unknown factor (Tonkin, 2002).

C. elegans ADARs are required for normal chemotaxis. Once an odorant is detected by a sensory neuron, a particular behavioral response is elicited through specific connections to interneurons, other sensory neurons and motor neurons. The data collected so far are not sufficient to indicate where in the chemosensation pathway ADARs are acting. The adr-1::GFP construct is expressed in the sensory neurons and cilia, but also in the ventral nerve cord, motor neurons and interneurons; at present, it is possible that ADARs are acting in any or all of these cells. RNA editing could affect chemosensation by targeting RNAs that encode receptors or signaling molecules within the AWA or AWC neurons, or affect molecules in the downstream cells that mediate the response of these neurons. Two of the C. elegans ADAR substrates analyzed in this study, unc-64 syntaxin and laminin-gamma mRNAs, are important for proper function of the nervous system. Editing sites in the 3'-UTRs of both of these substrates are altered in the adr deletion mutants and, in theory, either of these substrates could be involved in the chemotaxis defects observed. However, since there are probably hundreds of ADAR substrates in the worm nervous system to choose from, future studies will be required to determine this (Tonkin, 2002).

Calcium-permeable AMPA receptors containing Q/R-unedited GluR2 direct human neural progenitor cell differentiation to neurons

Calcium-permeable AMPA receptors have been identified on human neural progenitor cells (NPCs) and present a physiological role in neurogenesis. RNA editing of the GluR2 subunit at the Q/R site is responsible for making most AMPA receptors impermeable to calcium. Because a single-point mutation could eliminate the need for editing at the Q/R site and Q/R-unedited GluR2 exists during embryogenesis, the Q/R-unedited GluR2 subunit presumably has some important actions early in development. Using calcium imaging, this study found that NPCs contain calcium-permeable AMPA receptors, whereas NPCs differentiated to neurons and astrocytes express calcium-impermeable AMPA receptors. RTPCR and BbvI digestion were used to demonstrate that NPCs contain Q/R-unedited GluR2, and differentiated cells contain Q/R-edited GluR2 subunits. This is consistent with the observation that the nuclear enzyme responsible for Q/R-editing, adenosine deaminase (ADAR2), is increased during differentiation. Activation of calcium-permeable AMPA receptors induces NPCs to differentiate to the neuronal lineage and increases dendritic arbor formation in NPCs differentiated to neurons. AMPA-induced differentiation of NPCs to neurons is abrogated by overexpression of ADAR2 in NPCs. This elucidates the role of AMPA receptors as inductors of neurogenesis and provides a possible explanation for why the Q/R editing process exists (Whitney, 2008).

Pin1 and WWP2 regulate GluR2 Q/R site RNA editing by ADAR2 with opposing effects

ADAR2 catalyses the deamination of adenosine to inosine at the GluR2 Q/R site in the pre-mRNA encoding the critical subunit of AMPA receptors. Among ADAR2 substrates this is the vital one as editing at this position is indispensable for normal brain function. However, the regulation of ADAR2 post-translationally remains to be elucidated. This study demonstrates that the phosphorylation-dependent prolyl-isomerase Pin1 interacts with ADAR2 and is a positive regulator required for the nuclear localization and stability of ADAR2. Pin1(-/-) mouse embryonic fibroblasts show mislocalization of ADAR2 in the cytoplasm and reduced editing at the GluR2 Q/R and R/G sites. The E3 ubiquitin ligase WWP2 plays a negative role by binding to ADAR2 and catalysing its ubiquitination and subsequent degradation. Therefore, ADAR2 protein levels and catalytic activity are coordinately regulated in a positive manner by Pin1 and negatively by WWP2 and this may have downstream effects on the function of GluR2. Pin1 and WWP2 also regulate the large subunit of RNA Pol II, so these proteins may also coordinately regulate other key cellular proteins (Marcucci, 2011).

Mutation of ADAR genes

One type of RNA editing involves the conversion of adenosine residues into inosine in double-stranded RNA through the action of adenosine deaminases acting on RNA (ADAR). A-to-I RNA editing of the coding sequence could result in synthesis of proteins not directly encoded in the genome. ADAR also edits non-coding sequences of target RNAs, such as introns and 3'-untranslated regions, which may affect splicing, translation, and mRNA stability. Three mammalian ADAR gene family members (ADAR1-3) have been identified. Phenotypes of mice homozygous for ADAR1 null mutation have been investigated. Although live ADAR1-/- embryos with normal gross appearance can be recovered up to E11.5, widespread apoptosis was detected in many tissues. Fibroblasts derived from ADAR1-/- embryos are also prone to apoptosis induced by serum deprivation. These results demonstrate an essential requirement for ADAR1 in embryogenesis and suggest that it functions to promote survival of numerous tissues by editing one or more double-stranded RNAs required for protection against stress-induced apoptosis (Wang, 2004).

Characterization of ADAR genes

The C. elegans T20H4.4 open reading frame predicted by Genefinder encodes a 367 amino acid protein that is 32%-35% identical to the C-terminal domain of adenosine deaminases that act on RNA T20H4.4 cDNAs encode a larger 495 amino acid protein that is extended at its N-terminus to include a single double-stranded RNA-binding motif, and T20H4.4 has been shown to occupy the second position in a six-gene operon. Ten different spliced-leader (SL) sequences were found attached to T20H4.4 mRNAs, including SL1, SL2 and eight SL2-like leaders that include two new variants. Characterization of cDNAs derived from all six genes confirms the essential features of C. elegans operons: intercistronic distances in the range of 104-257 nt between the upstream polyadenylation sites and the downstream trans-splice sites; SL2, or SL2-like leaders, attached to the downstream mRNAs. Polycistronic mRNA fragments reveal a 5'-untranslated region (5'-UTR) greater than 705 nt. The 5'-UTR is removed in mature mRNAs from the first gene (T20H4.5) and replaced primarily by SL1, and to a lesser extent by SL2. This study provides new information regarding operons and how they are processed (Hough, 1999).

The double-stranded RNA-specific adenosine deaminase (ADAR) is an interferon-inducible RNA-editing enzyme implicated in the site-selective deamination of adenosine to inosine in viral RNAs and cellular pre-mRNAs. Human genomic clones of the ADAR gene and cDNA clones encoding splice site variants of the ADAR protein have been isolated and characterized. Southern blot and sequence analyses reveal that the gene spans about 30 kilobase pairs and consists of 15 exons. The codon phasing of the splice site junctions of exons 3, 5, and 7 that encode the three copies of the highly conserved RNA-binding R-motif (RI, RII, and RIII) is exactly conserved and identical to those R-motif exons of the interferon-inducible RNA-dependent protein kinase. Alternative splice site variants of the 1226-amino acid ADAR-a protein, designated b and c, were identified that differ in exons 6 and 7. ADAR-b is a 5'-splice site variant that possesses a 26-amino acid deletion within exon 7; ADAR-c is a 3'-splice site variant that possesses an additional 19-amino acid deletion within exon 6. The wild-type ADAR-a, -b, and -c proteins all possess comparable double-stranded RNA-specific adenosine deaminase activity. However, mutational analysis of the R-motifs reveals that the exon 6 and 7 deletions of ADAR-b and -c variants alter the functional importance of each of the three R-motifs (Liu, 1997).

Members of the double-stranded RNA- (dsRNA) specific adenosine deaminase gene family convert adenosine residues into inosines in dsRNA and are involved in A-to-I RNA editing of transcripts of glutamate receptor (GluR) subunits and serotonin receptor subtype 2C [5-HT(2C)R]. hADAR3, the third member of this class of human enzyme has been isolated and its editing site has been selectivity investigated using in vitro RNA editing assay systems. Purified ADAR3 proteins could not edit GluR-B RNA at the 'Q/R' site, the 'R/G' site, and the intronic 'hot spot' site. In addition, ADAR3 did not edit any of five sites discovered recently within the intracellular loop II region of 5-HT(2C)R RNAs, confirming its total lack of editing activity for currently known substrate RNAs. Filter-binding analyses reveal that ADAR3 is capable of binding not only to dsRNA but also to single-stranded RNA (ssRNA). Deletion mutagenesis has identified a region rich in arginine residues located in the N-terminus that is responsible for binding of ADAR3 to ssRNA. The presence of this ssRNA-binding domain as well as its expression in restricted brain regions and postmitotic neurons make ADAR3 distinct from the other two ADAR gene family members, editing competent ADAR1 and ADAR2. ADAR3 inhibits in vitro the activities of RNA editing enzymes of the ADAR gene family, raising the possibility of a regulatory role in RNA editing (Chen, 2000).

RNA-specific adenosine deaminase (ADAR1) catalyzes the deamination of adenosine to inosine in viral and cellular RNAs. Two size forms of the ADAR1 editing enzyme are known, an IFN-inducible approximately 150-kDa protein and a constitutively expressed N-terminally truncated approximately 110-kDa protein. Alternative exon 1 structures of human ADAR1 transcripts have been identified that initiate from unique promoters, one constitutively expressed and the other IFN inducible. Cloning and sequence analyses of 5'-rapid amplification of cDNA ends (RACE) cDNAs from human placenta have established a linkage between exon 2 of ADAR1 and two alternative exon 1 structures, designated herein as exon 1A and exon 1B. Analysis of RNA isolated from untreated and IFN-treated human amnion cells demonstrates that exon 1B-exon 2 transcripts are synthesized in the absence of IFN and are not significantly altered in amount by IFN treatment. By contrast, exon 1A-exon 2 transcripts are IFN inducible. Transient transfection analysis with reporter constructs has identified two functional promoters, designated PC and PI. Exon 1B transcripts are initiated from the PC promoter, whose activity in transient transfection reporter assays is not increased by IFN treatment. The 107-nt exon 1B maps 14.5 kb upstream of exon 2. The 201-nt exon 1A that maps 5.4 kb upstream of exon 2 is initiated from the interferon-inducible PI promoter. These results suggest that two promoters, one IFN inducible and the other not, initiate transcription of the ADAR1 gene, and that alternative splicing of unique exon 1 structures to a common exon 2 junction generates RNA transcripts with the deduced coding capacity for either the constitutively expressed approximately 110-kDa ADAR1 protein (exon 1B) or the interferon-induced approximately 150-kDa ADAR1 protein (exon 1A) (George, 1999).

ADAR1 forms a complex with Dicer to promote microRNA processing and RNA-induced gene silencing

Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosine residues to inosine specifically in double-stranded RNAs. This study investigated the interaction of the RNA editing mechanism with the RNA interference (RNAi) machinery and found that ADAR1 forms a complex with Dicer through direct protein-protein interaction. Most importantly, ADAR1 increases the maximum rate (Vmax) of pre-microRNA (miRNA) cleavage by Dicer and facilitates loading of miRNA onto RNA-induced silencing complexes, identifying a new role of ADAR1 in miRNA processing and RNAi mechanisms. ADAR1 differentiates its functions in RNA editing and RNAi by the formation of either ADAR1/ADAR1 homodimer or Dicer/ADAR1 heterodimer complexes, respectively. As expected, the expression of miRNAs is globally inhibited in ADAR1(-/-) mouse embryos, which, in turn, alters the expression of their target genes and might contribute to their embryonic lethal phenotype (Ota, 2013).

Extent of mRNA modification by ADARs

The general view that mRNA does not contain inosine has been challenged by the discovery of adenosine deaminases that act on RNA (ADARs). Although inosine monophosphate (IMP) cannot be detected in crude preparations of nucleotides derived from poly(A)+ RNA, it is readily detectable and quantifiable once it is purified away from the Watson-Crick nucleotides. IMP is present in mRNA at tissue-specific levels that correlate with the levels of ADAR mRNA expression. The amount of IMP present in poly(A)+ RNA isolated from various mammalian tissues suggests adenosine deamination may play an important role in regulating gene expression, particularly in brain, where one IMP is present for every 17,000 ribonucleotides (Paul, 1998).

ADAR substrates

Adenosine deaminases that act on RNA (ADARs) constitute a family of RNA-editing enzymes that convert adenosine to inosine within double-stranded regions of RNA. A method to identify inosine-containing RNAs has been developed and has been used to identify ADAR substrates in Caenorhabditis elegans. All 10 of the C. elegans substrates identified by this method are edited in long stem-loop structures located in noncoding regions, and thus contrast with previously identified substrates of other organisms, in which ADARs target codons. To determine whether editing in noncoding regions is a conserved ADAR function, the method was applied to poly(A)+ RNA of human brain and 19 previously unknown ADAR substrates were identified. The substrates were strikingly similar to those observed in C. elegans, since editing was confined to 3' untranslated regions, introns, and a noncoding RNA. Also similar to what was found in C. elegans, 15 of the 19 substrates were edited in repetitive elements. The identities of the newly identified ADAR substrates suggest that RNA editing may influence many biologically important processes, and that for many metazoa, A-to-I conversion in coding regions may be the exception rather than the rule (Morse, 2002).

ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine. ADAR2 can modify its own pre-mRNA, leading to the creation of a proximal 3'-splice junction containing a non-canonical adenosine-inosine (A-I) dinucleotide. Alternative splicing to this proximal acceptor shifts the reading frame of the mature mRNA transcript, resulting in the loss of functional ADAR2 expression. Both evolutionary sequence conservation and mutational analysis support the existence of an extended RNA duplex within the ADAR2 pre-mRNA formed by base-pairing interactions between regions approximately 1.3-kilobases apart in intron 4 and exon 5. Characterization of ADAR2 pre-mRNA transcripts isolated from adult rat brain identified 16 editing sites within this duplex region, and sites preferentially modified by ADAR1 and ADAR2 have been defined using both tissue culture and in vitro editing systems. Statistical analysis of nucleotide sequences surrounding edited and non-edited adenosine residues have identified a nucleotide sequence bias correlating with ADAR2 site preference and editing efficiency. Among a mixed population of ADAR substrates, ADAR2 preferentially favors its own transcript, yet mutation of a poor substrate to conform to the defined nucleotide bias increases the ability of that substrate to be modified by ADAR2. These data suggest that both sequence and structural elements are required to define adenosine moieties targeted for specific ADAR2-mediated deamination (Dawson, 2004).

The RNA-editing enzyme ADAR1 modifies adenosines by deamination and produces A-to-I mutations in mRNA. ADAR1 has been shown to function in host defense and in embryonic erythropoiesis during fetal liver development. The mechanisms for these phenotypic effects are not yet known. This study reports a novel function of ADAR1 in the regulation of gene expression by interacting with the nuclear factor 90 (NF90) proteins, known regulators that bind the antigen response recognition element (ARRE-2) and have been demonstrated to stimulate transcription and translation. ADAR1 upregulates NF90-mediated gene expression by interacting with the NF90 proteins, including NF110, NF90, and NF45. A knockdown of NF90 with small interfering RNA suppresses this function of ADAR1. Coimmunoprecipitation and double-stranded RNA (dsRNA) digestion demonstrate that ADAR1 is associated with NF110, NF90, and NF45 through the bridge of cellular dsRNA. Studies with ADAR1 deletions demonstrate that the dsRNA binding domain and a region covering the Z-DNA binding domain and the nuclear export signal comprise the complete function of ADAR1 in upregulating NF90-mediated gene expression. These data suggest that ADAR1 has the potential both to change information content through editing of mRNA and to regulate gene expression through interacting with the NF90 family proteins (Mie, 2005).

ADAR2 is a nuclear enzyme essential for GluR2 pre-mRNA editing at Q/R site-607, which gates Ca2+ entry through AMPA receptor channels. Forebrain ischemia in adult rats selectively reduces expression of ADAR2 enzyme and, hence, disrupts RNA Q/R site editing of GluR2 subunit in vulnerable neurons. Recovery of GluR2 Q/R site editing by expression of exogenous ADAR2b gene or a constitutively active CREB, VP16-CREB, which induces expression of endogenous ADAR2, protects vulnerable neurons in the rat hippocampus from forebrain ischemic insult. Generation of a stable ADAR2 gene silencing by delivering small interfering RNA (siRNA) inhibits GluR2 Q/R site editing, leading to degeneration of ischemia-insensitive neurons. Direct introduction of the Q/R site edited GluR2 gene, GluR2(R607), rescues ADAR2 degeneration. Thus, ADAR2-dependent GluR2 Q/R site editing determines vulnerability of neurons in the rat hippocampus to forebrain ischemia (Peng, 2006).

ADAR and translation of mRNA

Translation of mRNA is usually cytoplasmic. The RNA editing enzyme ADAR1, which catalyzes the deamination of adenosine to inosine in double-stranded RNA substrates, induces translation within the nucleus, possibly at the surface of the nucleolus. This activity does not depend on RNA editing. Two regions within ADAR1 are defined that act independently of each other to induce translation: the first includes the double-stranded RNA binding domains (DRBMs) of ADAR1 while the second maps to the C-terminal portion of the catalytic domain. Point mutations within each domain are identified that reduce nuclear translation; those in the DRBM region are also known to diminish RNA binding. This report adds to the growing functionality ascribed to the nucleus (Herbert, 2002).

The physiological significance of translation within the nucleus is not addressed in these studies. Nuclear translation would likely represent a very old function of eukaryotic cells. It has been suggested that a coupled transcription-translation mechanism in the nucleus may have developed from prokaryotic origins to become a quality assurance mechanism related to the nonsense-mediated decay of aberrant mRNA. ADAR1 might participate in this mechanism by inducing nuclear translation and using editing to mark flawed mRNAs as defective. Alternatively, the regulated translation of certain proteins in the nucleolus could be important for integrating various signaling pathways. For example, nucleolar pathways are already known to be obligatory for the maturation of numerous small RNAs that are essential for many aspects of RNA processing. Along these lines, a quality control checkpoint for ribosomes in the nucleolus would make sense. Such a pathway might involve the pre-assembly of ribosomes incorporating aminoacylated tRNAs and particular mRNAs. Production of protein from such messages could signal that functional ribosomes have been produced (Herbert, 2002 and references therein).

ADAR and viral infection

The virus-associated VAI RNA of adenovirus is a small highly structured RNA that is required for the efficient translation of cellular and viral mRNAs at late times after infection. VAI RNA antagonizes the activation of the interferon-inducible RNA-dependent protein kinase, PKR, an important regulator of translation. The RNA-specific adenosine deaminase, ADAR, is an interferon-inducible RNA-editing enzyme that catalyzes the site-selective C-6 deamination of adenosine to inosine. ADAR possesses three copies of the highly conserved RNA-binding motif (dsRBM) that are similar to the two copies found in PKR, the enzyme in which the prototype dsRBM motif was discovered. The effect of VAI RNA on ADAR function has been examined. VAI RNA impairs the activity of ADAR deaminase. This inhibition can be observed in extracts prepared from interferon-treated human cells and from monkey COS cells in which wild-type recombinant ADAR was expressed. Analysis of wild-type and mutant forms of VA RNA suggests that the central domain is important in the antagonism of ADAR activity. These results suggest that VAI RNA may modulate viral and cellular gene expression by modulating RNA editing as well as mRNA translation (Lei, 1998).

RNA editing by ADARs in RNAi

Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that deaminate adenosines to create inosines in double-stranded RNA (dsRNA). ADARs are not required for RNA interference (RNAi) in C. elegans and they do not antagonize the pathway to a detectable level when RNAi is initiated by injecting dsRNA. However, transgenes expressed in the somatic tissues of wild-type animals are silenced in strains with deletions in the two genes encoding ADARs, adr-1 and adr-2. Transgene-induced gene silencing in adr-1;adr-2 mutants depends on genes required for RNAi, suggesting that a dsRNA intermediate is involved. In wild-type animals, edited dsRNA corresponding to transgenes are detected, and it is proposed that editing of this dsRNA prevents somatic transgenes from initiating RNAi in wild-type animals (Knight, 2002).

Transgene-induced RNAi in the adr-1;adr-2 mutants appears remarkably similar to cosuppression. One mystery of cosuppression in C. elegans has been that it occurs almost exclusively in the germline. There has been only one previously reported instance of a transgene silencing an endogenous gene in the somatic tissues of a wild-type animal despite the thousands of transgenic strains that have been studied. The rarity of transgene-induced gene silencing in somatic tissues is in some instances attributable to ADAR activity. A distinct difference between cosuppression in the C. elegans germline and transgene-induced RNAi in the adr-1;adr-2 double mutants is their dependence on rde-1 (distantly related to Drosophila argonaute). Cosuppression is rde-1 independent, but transgene-induced RNAi in the somatic tissues of adr-1;adr-2 double mutants requires rde-1, indicating that although these two phenomenon appear related, subtle differences exist. rde-1 belongs to a large gene family in C. elegans, and therefore, it remains a possibility that one of the other gene family members may function in place of rde-1 in cosuppression (Knight, 2002).

It is somewhat surprising that cosuppression functions in the C. elegans germline since there is evidence that adr-2 is expressed in this tissue. Perhaps the level of ADAR activity in the germline is insufficient to prevent cosuppression, or cosuppression may not be mediated by dsRNA. If cosuppression involves a dsRNA intermediate, it may be protected from editing by localization to the cytoplasm, where ADARs are rarely found, or rapidly cleaved to siRNAs that may be too short to serve as ADAR substrates. In plants the correlation between the effectiveness of cosuppression and the absence of ADARs is strong. Plants are highly susceptible to cosuppression and also lack genes encoding ADARs (Knight, 2002).

Perhaps the simplest interpretation of these data is that the activities of ADARs and Dicer are physically separated. ADARs are primarily localized to the nucleus, but Dicer is found in the cytoplasm of embryonal carcinoma and HeLa cells. If the two activities are separated, dsRNA delivered by injection may trigger RNAi before ever being exposed to ADARs in the nucleus. When dsRNA is produced in the nucleus at low concentrations from transgenes, however, ADARs may first edit the dsRNA before the duplex is exposed to Dicer. In other organisms, highly edited RNAs are retained in the nucleus. If a similar retention mechanism exists in C. elegans, it could further prevent edited dsRNAs from entering the cytoplasm and being cleaved by Dicer. At high concentrations of dsRNA, however, ADARs are subject to substrate inhibition, and this may explain why RNAi that is triggered in wild-type animals by inducing synthesis of dsRNA is not inhibited by ADARs (Knight, 2002).

Alternatively, it is possible that both ADARs and Dicer are present in the nucleus. In C. elegans the intracellular localization of Dicer remains unknown. dcr-1 is required for RNAi in the C. elegans germline, and reducing dcr-1 levels by RNAi results in a decreased sensitivity to RNAi in the somatic tissues. C. elegans dcr-1 homozygous strains, however, only have defects in somatic RNAi when the triggering dsRNA is provided from a transgene. Although the reason dcr-1 homozygotes only have defects in somatic RNAi triggered from transgenes remains unclear, it is intriguing that deletions in both dcr-1 and the two genes encoding ADARs only effect somatic RNAi initiated in the nucleus. If ADARs and Dicer are nuclear, then at low concentrations of dsRNA, ADARs may simply have a higher affinity for dsRNA than Dicer (Knight, 2002).

In C. elegans, several ADAR substrates have been identified, but it is unclear exactly how editing of these substrates leads to the phenotypes of animals lacking ADARs. Many ADAR substrates in C. elegans and humans are edited in noncoding regions that are predicted to form long double-stranded regions, suggesting that, in addition to changing the coding potential of mRNAs, editing may also play other roles. It is intriguing to speculate that editing of naturally occurring dsRNAs may prevent these dsRNAs from triggering gene silencing as observes with dsRNAs originating from transgenes (Knight, 2002).

Adar and heterochromatin

The fate of double-stranded RNA (dsRNA) in the cell depends on both its length and location. The expression of dsRNA in the nucleus leads to several distinct consequences: (1) the promiscuous deamination of adenosines to inosines by dsRNA-specific adenosine deaminase (ADAR) can lead to the nuclear retention of edited transcripts; (2) dsRNAs might induce heterochromatic gene silencing through an RNAi-related mechanism. Is RNA editing also connected to heterochromatin? Members of the conserved Vigilin class of proteins have a high affinity for inosine-containing RNAs. In agreement with other work, these proteins were found to localize to heterochromatin and mutation or depletion of the Drosophila Vigilin, DDP1, leads to altered nuclear morphology and defects in heterochromatin and chromosome segregation. Furthermore, nuclear Vigilin is found in complexes containing not only the editing enzyme ADAR1 but also RNA helicase A and Ku86/70. In the presence of RNA, the Vigilin complex recruits the DNA-PKcs enzyme, which appears to phosphorylate a discrete set of targets, some or all of which are known to participate in chromatin silencing. These results are consistent with a mechanistic link between components of the DNA-repair machinery and RNA-mediated gene silencing (Wang, 2005).


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