Tuning of RNA editing by ADAR is required in Drosophila
2005; Springer Nature; Volume: 24; Issue: 12 Linguagem: Inglês
10.1038/sj.emboj.7600691
ISSN1460-2075
AutoresLiam P. Keegan, James Brindle, Angela Gallo, Anne Leroy, Robert A. Reenan, Mary A. O’Connell,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle26 May 2005free access Tuning of RNA editing by ADAR is required in Drosophila Liam P Keegan Corresponding Author Liam P Keegan MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK Search for more papers by this author James Brindle James Brindle MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK Search for more papers by this author Angela Gallo Angela Gallo MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK Ospedale Pediatrico 'Bambino Gesù', Piazza S Onofrio, Rome, Italy Search for more papers by this author Anne Leroy Anne Leroy MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK Search for more papers by this author Robert A Reenan Robert A Reenan University of Connecticut Health Center, Genetics, Farmington, CT, USA Search for more papers by this author Mary A O'Connell Mary A O'Connell MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK Search for more papers by this author Liam P Keegan Corresponding Author Liam P Keegan MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK Search for more papers by this author James Brindle James Brindle MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK Search for more papers by this author Angela Gallo Angela Gallo MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK Ospedale Pediatrico 'Bambino Gesù', Piazza S Onofrio, Rome, Italy Search for more papers by this author Anne Leroy Anne Leroy MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK Search for more papers by this author Robert A Reenan Robert A Reenan University of Connecticut Health Center, Genetics, Farmington, CT, USA Search for more papers by this author Mary A O'Connell Mary A O'Connell MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK Search for more papers by this author Author Information Liam P Keegan 1, James Brindle1, Angela Gallo1,2, Anne Leroy1, Robert A Reenan3 and Mary A O'Connell1 1MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK 2Ospedale Pediatrico 'Bambino Gesù', Piazza S Onofrio, Rome, Italy 3University of Connecticut Health Center, Genetics, Farmington, CT, USA *Corresponding author. MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK. Tel.: +44 131 467 8417; Fax: +44 131 467 8456; E-mail: [email protected] The EMBO Journal (2005)24:2183-2193https://doi.org/10.1038/sj.emboj.7600691 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 expressing 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. We show that 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. Introduction The ADAR (adenosine deaminase acting on RNA) enzymes deaminate specific adenosines in transcripts to inosines. Inosine is the base-pairing equivalent of guanosine during translation or cDNA synthesis (Basilio et al, 1962). When the conversion of adenosine to inosine changes codon meaning, it can have dramatic effects on the properties of the encoded protein. Editing of the Q/R site in vertebrate transcripts encoding the glutamate-gated receptor B subunit (GluR-B) affects calcium permeability of AMPA receptors (Sommer et al, 1991), and may slow receptor assembly (Greger et al, 2003). Similarly, RNA editing in transcripts encoding the G-protein-coupled serotonin (5-HT2C) receptor (Burns et al, 1997) produces isoforms with reduced coupling to the target G protein (Price et al, 2001). ADARs recognise short stretches of imperfectly paired RNA duplex formed by pairing of an exon with an editing site complementary sequence (ECS) that is usually located in an adjacent intron (for review, see Keegan et al, 2001). ADAR proteins contain either two or three dsRNA binding domains at the amino terminus and the catalytic deaminase domain is present in the carboxy terminus of the protein (for review, see Keegan et al, 2004). The deaminase domain contains three zinc-chelating motifs and motif I includes an essential glutamate residue. ADAR protein with this glutamate mutated is inactive but is able to compete with active proteins for editing sites in vitro and in vivo (Gallo et al, 2003). The Drosophila ADAR protein forms dimers on RNA substrates through the amino terminus and the first dsRNA binding domain (Gallo et al, 2003). Dimerisation is essential for catalysis, and different isoforms of Drosophila ADAR form heterodimers. The variety of isoforms and their possible interactions suggest complex regulation of RNA editing. In Drosophila melanogaster, there is a single Adar gene, mutations in which produce flies that survive to adulthood but display major defects in walking and mating (Palladino et al, 2000b). Locomotion defects in Adar mutant flies are present from eclosion and are succeeded by age-dependent neurodegeneration in the brain (Palladino et al, 2000a). In embryos, Adar is expressed strongly throughout the central nervous system (CNS), but it is also expressed more weakly outside the nervous system in mesoderm and endoderm (Palladino et al, 2000a). Like many Drosophila genes, Adar is expressed from an embryonic promoter and later from an adult promoter activated at metamorphosis. Adar is expressed in the brain of adult flies (Ma et al, 2001), but the complete adult expression pattern has not been described. All of the transcripts that are known to be edited in Drosophila are expressed in neurons but some also show muscle expression. The known editing targets in Drosophila include the cacophony (cac) (Smith et al, 1996) and paralytic (para) (Hanrahan et al, 2000) transcripts encoding the large α1 subunits of the main voltage-gated calcium channel and voltage-gated sodium channel in the CNS, respectively. Editing at all sites in these target transcripts is eliminated in Adar mutant flies (Palladino et al, 2000b). Recently, a further 16 edited transcripts have been described (Hoopengardner et al, 2003), and the list of edited transcripts is likely to be still incomplete (Stapleton et al, 2002). Adar mutants have also been isolated under the name hypnos-2 (hypoxia, anoxia sensitive), in a screen for mutants that recover from anoxic stupor more slowly than wild type (Ma et al, 2001). To investigate how the expression of different ADAR isoforms at different developmental stages and in different tissues contributes to the regulation of RNA editing in Drosophila, we characterised the editing activities of ADAR isoforms in vitro and used the GAL4-UAS system (Brand and Perrimon, 1993) to express cDNA rescue constructs encoding different ADAR isoforms in Adar mutant flies. We find that ADAR isoforms predominant in adult flies are more active than those from embryos and larvae and that editing occurs in muscle as well as within the CNS. We extensively characterise the effect on protein function of one editing event in which ADAR edits its own transcript to produce an ADAR isoform that is less active. At the physiological level, Adar transcript editing appears to be a form of negative autoregulation. An Adar cDNA engineered to resist RNA editing causes lethality when ubiquitously expressed. We present evidence that this lethality is mediated through excessive editing of target transcripts in embryos and larvae. We show that editing levels at sites in the cacophony transcript are lowest in embryos, rise during larval development and are highest in adult flies, but editing is never complete. A similar increase in editing during development occurs at editing sites in the Ca alpha 1D transcript (Zheng et al, 1995; Eberl et al, 1998; Hoopengardner et al, 2003), which encodes an L-type voltage-gated calcium channel expressed in both neurons and muscle. In this transcript, editing at some sites is essentially 100% efficient in adult flies, in muscle as well as in neurons. Results ADAR edits the cacophony transcript and the Adar transcript in vitro RNA editing itself contributes to the generation of ADAR variants in Drosophila. The Adar transcript is edited so that a serine (S) residue close to zinc-chelating motif II in the active site in the deaminase domain is replaced with glycine (G), hence the name Adar S/G site (Palladino et al, 2000a) (Figure 1A). Editing at this site increases during development, being almost completely absent in embryos and rising to 40% in adults. Oddly, RNA editing occurs in the transcript encoding the ADAR 3/4 splice form that predominates after induction of the adult promoter at metamorphosis but not in the 3a splice form that is expressed at all stages (Palladino et al, 2000a). To identify an ECS for the editing site in Adar exon 7, the flanking introns were sequenced but no ECS was found. However, Adar exon 7 itself can fold into a highly duplex structure (Figure 1B) that is conserved even at the third base position in codons in Drosophila pseudoobscura (RA Reenan, unpublished data). Figure 1.Adar protein isoforms expressed, predicted dsRNA structure of Adar exon 7 and in vitro editing of sites in Adar and cacophony transcripts. (A) Structures of ADAR protein isoforms and timings of expression. The Adar 4a promoter is expressed at all stages, whereas the Adar 4b promoter is strongly induced at metamorphosis and is pupal/adult specific. 4a transcripts are spliced predominantly to produce the 3a splice forms, but 4b transcripts produce only the 3/4 splice form that predominates in adult flies. Exon notations are as described by Palladino et al (2000b). Inclusion of exon 3a arises from splicing to an alternative 5′ splice donor and results in incorporation of 37 extra amino acids between the two double-stranded RNA binding motifs (dsRBMs). Motifs I, II and III in the deaminase domain contain conserved zinc-chelating cysteine and histidine residues. Motif I contains the sequence CHAE and the glutamate residue is essential for catalysis. The S/G RNA editing event in Adar exon 7, indicated by an asterisk, changes a conserved serine codon seven codons after motif II to a glycine codon. (B) RNA structure prediction for all 166 bases of exon 7 of the Adar transcript using the mfold program (Zuker et al, 1999). The edited adenosine 123 is indicated by an arrow labelled S/G site. Editing occurs in transcripts spliced to produce the 3/4 isoform. (C) Poisoned primer extension comparing editing in vitro of Adar exon 7 and cac 15 substrates by the same increasing concentrations of the ADAR 3/4 S/G protein mix. The left lane of each panel has no ADAR protein and 1, 3, 6.25 and 12.5 μl of purified protein was used in the ADAR protein lanes. Arrows indicate radiolabelled primer (P) and the unedited (U) and edited (E) primer extension products. (D) Poisoned primer extension assay on Adar exon 7 comparing purified ADAR 3a S/G or ADAR 3/4 S/G proteins. Lane 1 has no protein, lanes 2 and 3 are duplicate lanes containing ADAR 3a S/G that edits Adar exon 7 transcript to 15% whereas lanes 4 and 5 are duplicate lanes containing ADAR 3/4 S/G with the same concentration of protein as in lanes 2 and 3 that edits the substrate to 33%. Arrows indicate radiolabelled primer (P) and the unedited (U) and edited (E) primer extension products. Download figure Download PowerPoint To determine whether Drosophila ADAR edits the Adar transcript in vitro, ADAR 3/4 protein was expressed in the yeast Pichia pastoris and purified. RNA substrates corresponding to cacophony exon 15+intron 15 (cac15) or Adar exon 7 (Figure 1B) were generated by in vitro transcription, purified and incubated with ADAR proteins. RNA editing was measured using a poisoned primer extension assay (Figure 1C). The RNA editing site in cac 15 was chosen for in vitro editing assays because the edited A is not embedded in a run of A's and the site is efficiently edited. A purified recombinant Drosophila ADAR 3/4 S/G isoform mix (see below) edits the cac 15 site to approximately 20% at saturation, whereas the same protein concentrations edit the Adar exon 7 S/G site with 70% efficiency (Figure 1C). Editing of the Adar exon 7 S/G site to 70% is higher than at any other site that has been measured in vitro and is even higher than the 40% editing that is found in vivo at this position. In addition, the ADAR 3/4 isoform (lanes 4 and 5) edits the Adar exon 7 S/G site more efficiently than the ADAR 3a isoform does in vitro (Figure 1D, lanes 2 and 3). Adar transcript editing generates an ADAR isoform with reduced activity Editing of Adar transcript occurs primarily in adult flies when RNA editing in general is highest. The edited isoform could be contributing to either the increase in overall ADAR activity in adult flies or to an autoregulation acting to restrain editing. Producing a pure preparation of unedited ADAR S protein is complicated by the fact that Adar exon 7 is sufficient as a substrate for RNA editing. Editing of the Adar transcript occurs even in mRNA produced from Adar cDNA constructs expressed in Pichia (Figure 2A, lower chromatogram) that produce an ADAR 3/4 S/G protein mixture. Pichia and other yeasts lack endogenous ADARs or A to I editing activity and an Adar cDNA expressing just the deaminase domain of Drosophila ADAR is not edited (Figure 2A, upper chromatogram). Producing pure unedited ADAR S protein requires an editing-resistant Adar S mRNA. To produce such an ineditable Adar S mRNA, the edited serine codon was changed to another serine codon that does not have an A at the edited first position (Figure 2C). Generating the edited isoform ADAR G using the edited cDNA is trivial. Figure 2.ADAR edits the Adar transcript to generate an edited ADAR isoform with reduced editing activity. (A) Editing of the Adar transcript expressed in Pichia. Sequences of Adar exon 7 RT–PCR product pools from Pichia expressing an active ADAR 3/4 protein (lower chromatogram) or an inactive ADAR protein encoding only the ADAR deaminase domain (upper chromatogram). An arrow marks the edited position. The resulting purified ADAR S/G protein will be a mixture of edited and unedited isoforms as shown in Figure 1C. (B) An SDS polyacrylamide gel of the purified homogeneous unedited S and edited G isoforms of ADAR used in the in vitro assays stained with GelCode Blue Stain Reagent (Pierce). BioRad High Molecular weight markers are on the left; lane 1 is ADAR 3a S, lane 2 is ADAR 3a G, lane 3 is ADAR 3/4 S and lane 4 is ADAR 3/4 G. (C) Strategy for circumventing editing of the Adar transcript during protein expression in P. pastoris. To produce a pure preparation of the genome-encoded, unedited ADAR S isoform, the edited serine codon is mutated to a different serine codon that does not have A at the edited position. The mRNA expressed from this construct is ineditable and the encoded protein is the S isoform. Specific editing activities of purified unedited S or edited G isoforms of ADAR on cac 15 and Adar exon 7 transcripts are shown. The specific activity is the amount of inosine generated per minute per microgram ADAR protein on the specific substrates, measured using poisoned primer extension assays. Download figure Download PowerPoint Proteins were purified following overexpression in P. pastoris (Figure 2B), and their specific activities measured by poisoned-primer extension assays on cac 15 and Adar exon 7 transcripts (Figure 2C). The edited isoform is less active in RNA editing on both substrates. An eight-fold decrease in editing the Adar transcript was observed, whereas the decrease in specific activity in editing the cac transcript was approximately three-fold. The effect of editing is seen in the context of either the ADAR 3a or ADAR 3/4 splice forms; the difference between splice forms is less than the effect of editing but the ADAR 3/4 splice form is more active. These assays were performed with two different purified preparations of each ADAR protein, and the specific activities measured over a range of protein concentrations were the same in both preparations. Therefore, the ADAR exon 7 site is a high-affinity site for the ADAR enzyme and by editing it, subsequent translation produces an enzyme that is less active in editing. One effect of own-transcript editing is to limit RNA editing activity. The embryonic isoform ADAR 3a can also edit its own transcript in vitro (Figures 1D and 2C); however, this editing is not observed in wild-type flies. Editing at the Adar (S/G) site is also observed in the UAS-Adar 3a transgenic fly lines (data not shown). Developmental regulation of site-specific RNA editing activity Given these differences in editing efficiency in vitro between different ADAR isoforms, is there a correlation between ADAR isoform expression and editing of other transcripts in vivo? We chose to study editing of the cacophony (cac) transcript. This was the first transcript reported to be edited in Drosophila and 10 different sites are edited to give codon changes (Smith et al, 1996). We wished to determine if there is developmental regulation of cacophony transcript editing in vivo that reflects the expression of the more active ADAR isoforms in adults. The cac gene encodes the pore-forming α1 subunit of a voltage-gated calcium channel expressed in the CNS (Smith et al, 1996; Peixoto et al, 1997). The protein is 1851, amino acids long containing four internal repeats (I–IV) each with six proposed membrane-spanning segments (S1–S6). Figure 3A shows the locations within the predicted protein of amino-acid residues altered by RNA editing. Each editing site is named by the exon in which it is located. Figure 3.Developmental regulation of site-specific RNA editing in the cacophony transcript. (A) Percentage of individual sequenced cDNA clones edited at 10 sites in the cacophony transcript in embryos (E), larvae (L) and adult flies (A). The number of individual clones sequenced in each case is indicated above the error bars, which show the standard error of the percentage. Each editing site is named by the exon in which it is located. The locations within the protein of amino-acid residues changed by RNA editing are indicated on a conventional structure for this class of channel (Catterall, 2000). The cac 15 site is within the paddle structure involved in voltage gating. Black filled circles indicate three residue changes resulting from five RNA editing events in the Ca-alpha 1D transcript encoding a muscle voltage-gated calcium channel (see Figure 6). (B) Average editing level at all tested sites in the cacophony transcript at each developmental stage. The number of individual clones sequenced is indicated above the bars. Download figure Download PowerPoint The developmental profile of RNA editing in the cac transcript is shown as the percentage of edited clones among individual sequenced cDNA clones from total RNA of embryos (E), larvae (L) and adult flies (A) (Figure 3A and B). All sites show an increase in RNA editing through development, consistent with increasing levels of ADAR expression through development and with the Adar mutant phenotype, which is most evident in adult flies. One editing site in the amino terminus was not studied as it is translationally silent and a second site in this region has been recently identified, raising the number of editing sites to 12 (Smith et al, 1996; Kawasaki et al, 2002). A similar developmental increase in editing has been reported for the para transcript (Hanrahan et al, 2000). In addition, we have also analysed approximately 10 other transcripts and this developmental increase in editing is observed in most cases (data not shown). In transgenic flies, the ADAR 3/4 isoform efficiently rescues both Adar locomotion defects and editing of cac transcripts Are the large differences in editing activity between the different ADAR isoforms in vitro reflected in their ability to rescue the Adar mutant phenotype? To test rescue of the Adar mutant phenotype by ADAR isoforms in vivo, cDNAs encoding ADAR 3a or ADAR 3/4 were fused to a truncated hsp70 promoter with five GAL4 binding sites upstream, in the vector pUAST (Phelps and Brand, 1998), and transgenic Drosophila lines were generated. The ADAR isoforms were expressed by crossing three independent transgenic lines for each UAS-Adar construct to another line that is heterozygous for the Adar 1F4 deletion and also has an actin-5C-GAL4 driver construct (Ito et al, 1997) that expresses GAL4 in all cells. We chose this GAL4 driver that directs ubiquitous expression of ADAR because an initial screen of drivers showed that this driver gave the most efficient rescue. Rescue of the Adar mutant phenotype in male flies was quantitated using open field locomotion across lines in a gridded plate to measure restoration of walking ability. This is the simplest test that can be applied to the Adar mutant flies (three 2 min measurements on each of 10 or more flies for each transgenic UAS-Adar line). The data are presented as the average number of lines crossed in the assay period. More specific behavioural tests are unsuitable because they require normal locomotion. Figure 4A shows rescue of the X-chromosome Adar 1F4 locomotion defect in male progeny by either the UAS-Adar 3a or UAS-Adar 3/4 constructs with the actin-5C-GAL4 driver. Adar mutant flies are very defective in the locomotion test (Figure 4A) (Palladino et al, 2000b). Clearly, the ADAR 3/4 isoform gives a more effective rescue than the ADAR 3a isoform and this correlates with their in vitro editing activities (Figure 1D). Locomotion rescue correlates very well with in vitro editing by ADAR splice forms because a very large number of locomotion measurements were carried out (90 or more measurements of locomotion for each UAS-Adar construct). The Adar wild-type strain used for comparison in these experiments contains the actin-5C-GAL4 driver construct, which reduces locomotion compared to Canton S without affecting editing of cac 15 or cac 17c sites significantly. Figure 4.Phenotypic rescue and lethality associated with expression of ADAR isoforms in Drosophila. (A) The ADAR 3/4 isoform that is enriched in adult flies rescues Adar IF4 locomotion defects and RNA editing more efficiently than the ADAR 3a isoform. Open field locomotion measurements on male Adar wild-type; actin-5C-GAL4 flies (1) or mutant male Adar 1F4 flies or rescued male flies of the general genotype y1, Adar 1F4, w; act-5C-GAL4; UAS-Adar isoform. Average 2 min locomotion measurements are three measurements on each of 10 flies from three independent transgenic lines for each UAS-Adar construct. Standard errors are indicated. Percentage editing at the cac 15 site and at the cac 17c site, plotted on the right Y-axis, is presented beside the locomotion rescue data for each isoform. The percentage editing at the cac editing sites was determined by sequencing individual cDNAs from wild-type, Adar 1F4 or rescued flies and the number of individual cDNA clones sequenced is above the error bar. Standard error of the percentage is indicated. The inset shows a Western blot with an anti-FLAG antibody of normalised fly extracts of wild-type, Adar 1F4 and rescue genotypes. The lanes in the inset are numbered to correspond to the rescue lines in the main figure. The arrow indicates ADAR and the band underneath is nonspecific. (B) Expression of a Drosophila Adar transcript resistant to editing is lethal. Free range locomotion assays on Adar IF4 mutant flies expressing UAS-Adar constructs under the control of an actin-5C-GAL4 driver and rescue of editing at the cac15 and cac 17 sites measured as in panel A. The inset shows a Western blot with an anti-FLAG antibody of normalised fly extracts of wild-type, Adar 1F4 and the viable rescue genotype. The lanes in the inset are numbered to correspond to the rescue lines in the main figure; however, there is no lane 3 as it is lethal and extract from male y1, Adar 1F4, w; Cy; UAS-Adar 3/4 flies is included as it shows that very little protein is expressed in the absence of the GAL4 driver. Download figure Download PowerPoint Rescue of RNA editing at the cac 15 site and the cac 17c site was measured by sequencing individual cDNA clones from two or more independent RT–PCR reactions on RNA from rescue flies of one transgenic line for each construct (Figure 4A). The number of individual clones sequenced is presented above the error bars. We chose to measure in vivo editing in the cac transcript as it was the first transcript that was found to be site-specifically edited in Drosophila; however, we do not believe that the unedited cac transcript is the cause of the Adar mutant phenotype. Rescue of editing is substantial but it does not reach or exceed wild-type levels. In addition, sites in a number of other edited transcripts are also edited at levels approaching but not exceeding wild-type editing levels in the UAS-Adar 3/4 rescue line (data not shown). The expressed ADAR proteins bear FLAG and 6xHis epitope tags and Western blots with anti-FLAG antibody have been performed in the rescue lines to confirm that expression levels of constructs are comparable (Figure 4A, inset), given the variations between lines that arise due to position effects on transgene insertions. The variation between lines will not affect locomotion rescue data, which are based on multiple lines, but needs to be considered for in vivo editing, which is based on single lines. Nevertheless, the differences in locomotion rescue and the level of editing observed in vivo between Adar 3a S/G and Adar 3/4 S/G are similar to that observed in vitro (Figure 1D). The difference in locomotion rescue between the ADAR splice forms is greater when a less effective rescue driver is substituted (data not shown). ADAR must act prior to splicing at many sites. Editing sites are frequently found close to splice sites within predicted RNA duplexes that include a splice junction. Loss of vertebrate ADAR2 causes accumulation of an incompletely spliced GluR-B (Higuchi et al, 2000). It is possible that RNA editing could assist splicing by simply binding the duplex at splice sites and recruiting helicases to disrupt the duplex. Therefore, we wished to confirm that the Adar mutant phenotype in Drosophila is due to loss of adenosine deaminase activity. A UAS-ADAR 3/4 EA construct encoding a protein with a mutation in the active site glutamate (Lai et al, 1995) gave minimal rescue of locomotion, and very low levels of editing were found at the cac 15 site and the cac 17c site in RNA from these flies even though this protein is efficiently expressed (Figure 4A). Therefore, deamination is required for rescue of the Adar mutant phenotype; the deaminated base itself could also facilitate splicing in cases where it destabilises a dsRNA editing structure that occludes a splice junction. Ubiquitous expression of the genome-encoded isoform of ADAR is lethal in Drosophila If the function of self-editing in Adar is to limit overall RNA editing activity, then expressing pure unedited ADAR 3/4 S isoform in Drosophila would bypass this regulation and could be toxic. The ineditable Adar 3/4 S and the edited Adar 3/4 G cDNA constructs used for expression in Pichia were introduced into the Drosophila pUAST vector. Transgenic fly lines bearing UAS-Adar 3/4 S and UAS-Adar 3/4 G constructs were generated, and crossed to the actin 5C-GAL4 driver to compare their ability to rescue the Adar 1F4 locomotion defect (Figure 4B). Crossing flies bearing the ineditable UAS-Adar 3/4 S construct to flies bearing an actin-5C-GAL4 driver construct produced no progeny having both constructs. The complete lethality obtained with the ineditable UAS-Adar 3/4 S construct is an exacerbation of a partial loss of viability seen with the UAS-Adar 3/4 S/G construct. Among male progeny of the rescue crosses that carry both the Adar 1F4 mutation and the relevant UAS-Adar construct, the number of actin 5C-GAL4 driver flies obtained, expressed as a percentage of the number of Cy balancer flies obtained that lack the GAL4 driver, was UAS-Adar 3/4 EA 72%, UAS-Adar 3/4 G 69%, UAS-Adar 3/4 S/G 20% and UAS-Adar 3/4 S 0%. Decreases in viability correlate with levels of ADAR 3/4 S isoform expressed. When editing of Adar exon 7 was examined in UAS-Adar 3a S/G and UAS-Adar 3/4 S/G rescue flies (Figure 4A), we found that the transcript expressed from the transgene was 70% edited, that is, only 30% of the transcripts still expressed the ADAR 3/4 S protein. The ineditable UAS-Adar 3/4 S construct is lethal because it produces more of the genome-encoded form. Rescue of the Adar 1F4 locomotion defect was less efficient with the UAS-Adar 3/4 G construct than with the unedited UAS-Adar S/G construct that generates a mixture of edited and unedited isoforms. The weaker rescue is not due to weaker expression of ADAR from the UAS-Adar 3/4 G construct (Figure 4B, inset). Editing of the cac15 and cac 17c sites was efficient in these rescue flies. The UAS-Adar 3/4 S constru
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