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).
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).
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).
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).
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).
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).
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