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• W2109282318 abstract "ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine. We have demonstrated previously that ADAR2 can modify its own pre-mRNA, leading to the creation of a proximal 3′-splice junction containing a non-canonical adenosine-inosine (A-I) dinucleotide. Alternative splicing to this proximal acceptor shifts the reading frame of the mature mRNA transcript, resulting in the loss of functional ADAR2 expression. Both evolutionary sequence conservation and mutational analysis support the existence of an extended RNA duplex within the ADAR2 pre-mRNA formed by base-pairing interactions between regions ∼1.3-kilobases apart in intron 4 and exon 5. Characterization of ADAR2 pre-mRNA transcripts isolated from adult rat brain identified 16 editing sites within this duplex region, and sites preferentially modified by ADAR1 and ADAR2 have been defined using both tissue culture and in vitro editing systems. Statistical analysis of nucleotide sequences surrounding edited and non-edited adenosine residues have identified a nucleotide sequence bias correlating with ADAR2 site preference and editing efficiency. Among a mixed population of ADAR substrates, ADAR2 preferentially favors its own transcript, yet mutation of a poor substrate to conform to the defined nucleotide bias increases the ability of that substrate to be modified by ADAR2. These data suggest that both sequence and structural elements are required to define adenosine moieties targeted for specific ADAR2-mediated deamination. ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine. We have demonstrated previously that ADAR2 can modify its own pre-mRNA, leading to the creation of a proximal 3′-splice junction containing a non-canonical adenosine-inosine (A-I) dinucleotide. Alternative splicing to this proximal acceptor shifts the reading frame of the mature mRNA transcript, resulting in the loss of functional ADAR2 expression. Both evolutionary sequence conservation and mutational analysis support the existence of an extended RNA duplex within the ADAR2 pre-mRNA formed by base-pairing interactions between regions ∼1.3-kilobases apart in intron 4 and exon 5. Characterization of ADAR2 pre-mRNA transcripts isolated from adult rat brain identified 16 editing sites within this duplex region, and sites preferentially modified by ADAR1 and ADAR2 have been defined using both tissue culture and in vitro editing systems. Statistical analysis of nucleotide sequences surrounding edited and non-edited adenosine residues have identified a nucleotide sequence bias correlating with ADAR2 site preference and editing efficiency. Among a mixed population of ADAR substrates, ADAR2 preferentially favors its own transcript, yet mutation of a poor substrate to conform to the defined nucleotide bias increases the ability of that substrate to be modified by ADAR2. These data suggest that both sequence and structural elements are required to define adenosine moieties targeted for specific ADAR2-mediated deamination. The conversion of adenosine to inosine (A-to-I) by RNA editing is catalyzed by hydrolytic deamination (1Polson A.G. Crain P.F. Pomerantz S.C. McCloskey J.A. Bass B.L. Biochemistry. 1991; 30: 11507-11514Crossref PubMed Scopus (107) Google Scholar) via the enzymatic activity of a family of adenosine deaminases that act on RNA (ADARs) 1The abbreviations used are: ADARadenosine deaminases that act on RNAdsdouble-strandedntnucleotideBSAbovine serum albuminDRBMdsRNA-binding motif.1The abbreviations used are: ADARadenosine deaminases that act on RNAdsdouble-strandedntnucleotideBSAbovine serum albuminDRBMdsRNA-binding motif. (2Bass B.L. Annu. Rev. Biochem. 2002; 71: 817-846Crossref PubMed Scopus (942) Google Scholar, 3Bass B.L. Nishikura K. Keller W. Seeburg P.H. Emeson R.B. O'Connell M.A. Samuel C.E. Herbert A. RNA. 1997; 3: 947-949PubMed Google Scholar). These proteins are double-stranded RNA (dsRNA)-specific enzymes that contain variable N termini, multiple copies of a dsRNA-binding motif (DRBM) and conserved C-terminal sequences encoding a catalytic adenosine deaminase domain (2Bass B.L. Annu. Rev. Biochem. 2002; 71: 817-846Crossref PubMed Scopus (942) Google Scholar, 4Rueter S. Emeson R. Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998: 343-361Google Scholar, 5Schaub M. Keller W. Biochimie (Paris). 2002; 84: 791-803Crossref PubMed Scopus (83) Google Scholar). Two enzymes in this family, ADAR1 and ADAR2, have been shown to be involved in the site-selective deamination of adenosines within multiple RNA transcripts (2Bass B.L. Annu. Rev. Biochem. 2002; 71: 817-846Crossref PubMed Scopus (942) Google Scholar, 4Rueter S. Emeson R. Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998: 343-361Google Scholar), yet the basis for their distinct, but overlapping, specificities at particular adenosine moieties is not well understood (6Melcher T. Maas S. Herb A. Sprengel R. Seeburg P.H. Higuchi M. Nature. 1996; 379: 460-464Crossref PubMed Scopus (428) Google Scholar, 7Lehmann K.A. Bass B.L. Biochemistry. 2000; 39: 12875-12884Crossref PubMed Scopus (206) Google Scholar). The DRBMs of ADAR1 and ADAR2 are similar to the domains that mediate dsRNA interactions in a large variety of proteins, including dsRNA-dependent protein kinase, Drosophila staufen and Escherichia coli RNase III (8St Johnston D. Brown N.H. Gall J.G. Jantsch M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10979-10983Crossref PubMed Scopus (482) Google Scholar), yet binding of these DRBMs appears to be independent of RNA sequence (8St Johnston D. Brown N.H. Gall J.G. Jantsch M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10979-10983Crossref PubMed Scopus (482) Google Scholar, 9Ryter J.M. Schultz S.C. EMBO J. 1998; 17: 7505-7513Crossref PubMed Scopus (393) Google Scholar, 10Eckmann C.R. Jantsch M.F. J. Cell Biol. 1997; 138: 239-253Crossref PubMed Scopus (44) Google Scholar, 11Manche L. Green S.R. Schmedt C. Mathews M.B. Mol. Cell Biol. 1992; 12: 5238-5248Crossref PubMed Scopus (414) Google Scholar). Previous studies of A-to-I editing have revealed that an RNA duplex interrupted by single-strand bulges and loops is critical for site-specific A-to-I conversion (2Bass B.L. Annu. Rev. Biochem. 2002; 71: 817-846Crossref PubMed Scopus (942) Google Scholar, 5Schaub M. Keller W. Biochimie (Paris). 2002; 84: 791-803Crossref PubMed Scopus (83) Google Scholar, 12Gott J.M. Emeson R.B. Annu. Rev. Genet. 2000; 34: 499-531Crossref PubMed Scopus (341) Google Scholar, 13Emeson R. Singh M. Bass B. RNA Editing. Oxford University Press, Oxford2000: 109-138Google Scholar). This imperfect duplex structure is generally formed by intramolecular base-pairing interactions between exon and intron sequences in pre-mRNA transcripts that can be in close proximity to one another or as many as 1700 nucleotides (nt) apart (14Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (853) Google Scholar, 15Higuchi M. Single F.N. Kohler M. Sommer B. Sprengel R. Seeburg P.H. Cell. 1993; 75: 1361-1370Abstract Full Text PDF PubMed Scopus (509) Google Scholar, 16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar, 17Lomeli H. Mosbacher J. Melcher T. Hoger T. Geiger J.R. Kuner T. Monyer H. Higuchi M. Bach A. Seeburg P.H. Science. 1994; 266: 1709-1713Crossref PubMed Scopus (636) Google Scholar, 18Herb A. Higuchi M. Sprengel R. Seeburg P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1875-1880Crossref PubMed Scopus (135) Google Scholar). It has been proposed that selective binding of the RNA by the DRBM, as well as complementarity between the RNA structure surrounding the targeted adenosine and the catalytic domain, may determine the adenosine(s) that are selectively modified in vivo (19Yi-Brunozzi H.Y. Stephens O.M. Beal P.A. J. Biol. Chem. 2001; 276: 37827-37833Abstract Full Text Full Text PDF PubMed Google Scholar). While there is no discrete sequence motif common among substrates to direct the deamination of specific adenosine residues, distinct 5′-nearest neighbor sequence preferences have been identified for both ADAR1 (U = A > C > G) and ADAR2 (U = A > C = G) using in vitro editing assays with artificial RNA duplexes and a 3′-nearest neighbor preference for ADAR2 has also been suggested (U = G > C = A) (7Lehmann K.A. Bass B.L. Biochemistry. 2000; 39: 12875-12884Crossref PubMed Scopus (206) Google Scholar). While up to 50% of the adenosine residues within a perfect RNA duplex may be modified by ADAR1 and ADAR2 in vitro, RNA substrates whose duplex structures are interrupted by mismatches, bulges and loops are edited more selectively (20Nishikura K. Yoo C. Kim U. Murray J.M. Estes P.A. Cash F.E. Liebhaber S.A. EMBO J. 1991; 10: 3523-3532Crossref PubMed Scopus (133) Google Scholar, 21Polson A.G. Bass B.L. EMBO J. 1994; 13: 5701-5711Crossref PubMed Scopus (234) Google Scholar). Recent in vitro studies with synthetic RNA substrates have indicated that internal loops within ADAR substrates may serve to uncouple adjacent helices to convert long, promiscuously deaminated substrates into a series of short, selectively modified RNA targets (22Lehmann K.A. Bass B.L. J. Mol. Biol. 1999; 291: 1-13Crossref PubMed Scopus (130) Google Scholar).Previous studies have demonstrated that ADAR2 edits a specific adenosine moiety within intron 4 (also referred to as intron 1, Ref. 23Slavov D. Gardiner K. Gene (Amst.). 2002; 299: 83-94Crossref PubMed Scopus (40) Google Scholar) of its own pre-mRNA to generate a non-canonical 3′-splice acceptor containing an adenosine-inosine dinucleotide that effectively mimics the highly conserved AG sequence normally found at 3′-splice junctions (16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar). Splicing to this proximal acceptor site (site -1) alters the reading frame of the ADAR2 mRNA and results in loss of expression for the catalytically active 78 kDa protein, suggesting that autoediting may represent a negative feedback loop by which ADAR2 can modulate its own level of protein expression (16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar). Further support for this model was recently reported by Maas et al. (24Maas S. Patt S. Schrey M. Rich A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14687-14692Crossref PubMed Scopus (308) Google Scholar) in which a direct and specific correlation between ADAR2 activity and the extent of autoediting at the -1 position was observed. A-to-I editing has also been identified in mRNAs encoding Drosophila ADAR (dADAR) at a conserved residue of the catalytic domain, suggesting a common paradigm for ADAR regulation by autoediting (25Palladino M.J. Keegan L.P. O'Connell M.A. Reenan R.A. RNA. 2000; 6: 1004-1018Crossref PubMed Scopus (139) Google Scholar). Like all characterized ADAR substrates, ADAR2 pre-mRNA is predicted to form an RNA duplex as a result of intramolecular base pairing between two halves of an imperfect inverted repeat (-1512 to -1416 and -61 to +34, relative to the proximal 3′-splice site) (16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar, 23Slavov D. Gardiner K. Gene (Amst.). 2002; 299: 83-94Crossref PubMed Scopus (40) Google Scholar).In this study, we report that nucleotide sequence conservation between five vertebrate ADAR2 pre-mRNAs supports the base-pairing features underlying the predicted RNA secondary structure and we identify a total of 16 editing sites within ADAR2 pre-mRNA transcripts isolated from adult rat brain. The effects of mutations designed to disrupt base-pairing interactions were consistent with local structures surrounding editing sites and further suggested a model of two independent ADAR2 editing domains separated by a large internal loop. Competition analyses with mutant RNAs revealed that ADAR2 preferentially modifies its own pre-mRNA (site -1) based upon sequence/structure information contained immediately surrounding this region of the duplex. These findings correlate with increased evolutionary sequence conservation surrounding site -1 and suggest that ADAR2 is specifically targeted to the region of the alternate 3′-splice acceptor. Analysis of sequences surrounding fourteen ADAR2 sites within the ADAR2 pre-mRNA revealed a significant nucleotide bias at eight positions and three of these positions coincided with increased levels of editing in vitro. Furthermore, the ability of natural ADAR2 substrates to compete for ADAR2 editing (site -1) correlated with their conformity to this nucleotide bias, suggesting that both a consensus sequence and structural elements are required to define the preference and efficiency of ADAR2-mediated deamination.EXPERIMENTAL PROCEDURESAnalysis of ADAR2 Secondary Structure and Nucleotide Distribution—The secondary structure of rat ADAR2 pre-mRNA, in a region encompassing previously identified A-to-I editing events (-1668 to +200 relative to the proximal 3′-splice site) (16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar), was predicted using two RNA-folding algorithms, RNAFOLD (26Zuker M. Methods Mol Biol. 1994; 25: 267-294PubMed Google Scholar) and mfold (27Mathews D.H. Sabina J. Zuker M. Turner D.H. J. Mol. Biol. 1999; 288: 911-940Crossref PubMed Scopus (3198) Google Scholar, 28Zuker M. Mathews D.H. Turner D.H. Barciszewski J. Clark B.F.C. RNA Biochemistry and Biotechnology. Kluwer Academic, 1999: 11-43Google Scholar). Sequences from ADAR2 genes extending from nucleotides -1512 to -1416 and -61 to +34 (relative to the proximal 3′-splice site for rat ADAR2) from human (accession no. AL133499), rat (16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar), mouse (accession no. AF411054), and pufferfish (Takifugu rubripes) (accession nos. AF124050 and AF124049) were aligned using the Megalign program of the DNASTAR software suite (DNASTAR, Inc.). To identify potential nucleotide preferences in sequences surrounding ADAR2 editing sites, the distribution of sequence information was analyzed surrounding 14 adenosine moieties that were edited by ADAR2 in vitro (Table I). Forty nucleotides flanking each edited adenosine were manually aligned and the nucleotide distribution, purine/pyrimidine ratio and GC content at each position were determined. This information was then compared by a χ2-test of independence to the nucleotide distribution observed at equivalent positions for all 50 non-edited adenosines in the predicted region of the ADAR2 duplex. Positions at which the χ2-analysis indicated a nucleotide preference (p < 0.05) were considered statistically significant.Table IEditing frequency in rADAR2 pre-mRNA* Brackets indicate sites in which editing events were linked according to a χ2-test of independence. nd, not determined. Open table in a new tab Tissue Culture and Transfection—Human embryonic kidney (HEK293) cells were transiently co-transfected by calcium phosphate precipitation (29Ausubel F. Brent R. Kingston R. Moore D. Seidman J. Smith J. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1998Google Scholar) with rat ADAR1a (rADAR1a) or rat ADAR2b (rADAR2b) cDNAs containing an N-terminal epitope (FLAG) tag in the presence of a 3606-nt rADAR2 minigene, a 3360-nt rADAR2 minigene, or a control eukaryotic expression vector (pRC-CMV; Invitrogen) (Fig. 3). Crude nuclear extracts and total RNA were prepared ∼60 h post-transfection, as previously described (16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar).Western Blotting—Expression levels of ADAR proteins in crude nuclear extracts from transfected cells were monitored by Western blotting analysis (29Ausubel F. Brent R. Kingston R. Moore D. Seidman J. Smith J. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1998Google Scholar) using an anti-FLAG M2 monoclonal antibody (Sigma) followed by secondary antisera conjugated to horseradish peroxidase (Jackson Immunoresearch). The secondary antibody was detected using the SuperSignal West Dura Extended chemiluminescence reaction kit (Pierce, Inc.) in accordance with the manufacturer's instructions. Chemiluminescence was monitored using the Bio-Rad image detection system and quantitation was performed using Quantity One® software (Bio-Rad) on serially diluted samples that fell within the linear range of detection.Quantification of RNA Editing—To quantify the editing of ADAR2 transcripts in RNAs isolated from rat brain or transiently transfected HEK293 cells, first-strand cDNA was synthesized from 3-5 μg of total RNA, amplified using the polymerase chain reaction (PCR), and assessed by either direct DNA sequencing of individual cDNA isolates subcloned into pBKSII- (Stratagene) or by a modified primer-extension analysis (16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar). Primer extension analysis of site -1 was performed as previously described (16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar). For site -1428, primer-extension was performed using a sense primer (5′-CTTTGTCAGCTGGGAG-3′) in the presence of 1.2 mm dATP, 1.2 mm dCTP, 1.2 mm dTTP, and 5 mm ddGTP. Site -1476 editing was measured by extension of an antisense primer (5′-TCTTTTGCTGGAGGATG-3′) in the presence of 1.2 mm dATP, 1.2 mm dCTP, 1.2 mm dGTP and 5 mm ddTTP. Analysis of editing site concurrence was determined by direct sequence analysis of 100 independent rat brain ADAR2 cDNA clones, extending the full-length of the predicted duplex, and assessed by a χ2-test of independence.Site-directed Mutagenesis—ADAR2 intron 4 mutations were introduced by oligonucleotide-directed site mutagenesis in pBSKII- (Stratagene) as described (30Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4886) Google Scholar) using synthetic oligonucleotides to introduce restriction site-based mutations.In Vitro Transcription of RNA Substrates—All RNA substrates were synthesized in vitro using the Megascript T7 RNA transcription kit (Ambion). To determine RNA concentrations, RNA synthesis was performed in the presence of [α-32P] adenosine 5′-triphosphate as a trace label (final specific activity = 6 × 105 cpm/mmole UTP); RNA was precipitated with 2-propyl alcohol and quantified by scintillation spectrometry. The ADAR2 substrate was synthesized from an 873 bp template linearized at an FspI restriction site, while ADAR2 competitor RNA was transcribed from the 257-bp template linearized at ApaI (Fig. 2A). A PCR fragment containing the R/G duplex sequence was amplified from mouse genomic DNA using the synthetic oligonucleotide primers (5′-ACACCTAAAGGATCCTCATTAAGG-3′) and (5′-TAAGAGTCTTAAAGACACATCAGGG-3′), and cloned into pBSKII- (Stratagene). This construct was linearized at an EcoRI restriction site and used as template for R/G competitor RNA synthesis. 5-HT2CR competitor RNA was transcribed from a 288-bp genomic DNA fragment as previously described (14Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (853) Google Scholar). The sense and antisense strands of the nonspecific dsRNA substrate were synthesized separately from a fragment of the rat α2-adrenergic receptor and the single-stranded RNAs were annealed as described (31Rueter S.M. Burns C.M. Coode S.A. Mookherjee P. Emeson R.B. Science. 1995; 267: 1491-1494Crossref PubMed Scopus (115) Google Scholar) to generate the nonspecific duplex substrate.Fig. 2In vitro time course analysis of ADAR2 editing.A, schematic diagram is presented indicating the structure of ADAR2 genomic fragments (3606- and 3360-nt minigenes) used for transfection studies and corresponding 704 and 232 nt transcripts used for in vitro editing analysis; the position of exon and intron sequences are indicated by closed boxes and solid lines, respectively, while dashed lines indicate sequence information deleted from the in vitro transcripts. The position of the imperfect inverted repeat, predicted to form an extended RNA duplex (see Fig. 1), is designated with arrows; the specific coordinates of the inverted repeat and sequences included in the in vitro substrates are indicated relative to the proximal 3′-splice site. B, time course of in vitro editing (site -1) for ADAR2 substrates using recombinant FLAG-rADAR2b under single turnover conditions (ADAR2 RNA = 0.3 nm, FLAG-rADAR2b protein = 7.5 nm). The inset represents linear regression analysis of initial rates determined between 0 and 10 min.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Rat ADAR2 Expression and Purification—The isolation and expression of recombinant FLAG-rADAR2b was achieved using the Multi-Copy Pichia expression kit (Invitrogen). The FLAG-rADAR2b cDNA (16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar) was subcloned into the Pichia pastoris pPIC3.5K vector, introduced into the GS115 yeast strain by electroporation and selected in 3 mg/ml neomycin; yeast cell lysates were prepared at 4 °C per manufacturer's guidelines. Briefly, cell pellets from 1-liter cultures were resuspended in 100 ml breaking buffer (50 mm NaPO4, pH 7.4, 1 mm PMSF, 1 mm EDTA, 5% glycerol), homogenized with glass beads by 20 cycles of vortexing for 30 s and clarified by high speed centrifugation. The supernatant was then exchanged into standard purification buffer (20 mm HEPES, 100 mm NaCl, 2 mm EDTA, 1 mm DTT + 10% glycerol). Recombinant FLAG-rADAR2b protein was purified from 100 ml of supernatant using a protocol modified from O'Connell et al. (32O'Connell M.A. Gerber A. Keegan L.P. Methods. 1998; 15: 51-62Crossref PubMed Scopus (19) Google Scholar) for the purification of human ADAR2 from HeLa cell nuclear extracts. Recombinant proteins were first resolved by SP-Sepharose cation exchange followed by Source Q anion exchange fast-performance liquid chromatography (Amersham Biosciences). For the 25-ml SP-Sepharose column, proteins were eluted in 240 ml of purification buffer over a gradient of 100-500 mm NaCl. Peak fractions were then resolved on a 1-ml Source Q column over a gradient of 50-500 mm NaCl in a volume of 35 ml of buffer. FLAG-rADAR2b peak fractions, as assessed by Western blotting analysis, eluted between 280 and 350 mm NaCl. Finally, the recombinant protein was purified by Poly(I)*-Poly(C)* dsRNA affinity chromatography as described (32O'Connell M.A. Gerber A. Keegan L.P. Methods. 1998; 15: 51-62Crossref PubMed Scopus (19) Google Scholar) and dialyzed into standard purification buffer containing 0.05% Igepal and 50% glycerol.The peak fraction of FLAG-rADAR2b was purified to near homogeneity according to silver staining and Western blotting analysis using the M2 monoclonal anti-FLAG antibody (Sigma). The peak fraction of FLAG-rADAR2b was serially diluted in the presence of 100 μg/ml bovine serum albumin (BSA) and resolved by SDS-PAGE alongside BSA standards of known concentration. The protein concentration was then quantified on a Molecular Dynamics PhosphorImager by comparing the density of the ADAR2 band to the BSA standard curve.In Vitro Editing/Deamination assays—Editing assays were performed under single turnover conditions at 37 °C with 7.5 nm FLAG-rADAR2b protein and 0.3 nmin vitro transcribed RNA as substrate in a buffer containing 25 mm Tris, pH 7.8, 100 mm NaCl, 1 mm dithiothreitol, 6% glycerol, 100 μg/ml RNase-free BSA, 0.003% Igepal, and 0.8 units of RNasin/μl. Reactions were stopped by addition of Proteinase K, SDS and EDTA as described previously (33Hough R.F. Bass B.L. J. Biol. Chem. 1994; 269: 9933-9939Abstract Full Text PDF PubMed Google Scholar). To analyze the editing of ADAR2 at site -1, first-strand cDNA was synthesized with an oligonucleotide primer complementary to the unique 3′-extension of the ADAR2 RNA substrate that was not present in competitor RNAs. First-strand cDNA was amplified by PCR and the extent of editing was quantified by primer-extension analysis as described (16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar). Apparent Km values were measured for ADAR2, 5-HT2CR and R/G duplex substrates by assessing the A-to-I conversion at 5 min over a concentration range of 0-316 nm RNA by primer-extension analysis. Primer extension of 5-HT2CR transcripts was performed as previously reported (14Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (853) Google Scholar). For the R/G site, extension proceeded using an antisense primer (5′-GTTATAC TATTCCACCC-3′) in the presence of 1.2 mm dATP, 1.2 mm dCTP, 1.2 mm dGTP, and 5 mm ddTTP. For competition studies, the editing of 0.3 nm ADAR2 (site -1) was assessed in the presence of competitor at concentrations ranging from 10-13m to 10-5m RNA.RESULTSPredicted Secondary Structure for ADAR2 pre-mRNA—We demonstrated previously that intronic sequences critical for ADAR2 pre-mRNA editing (site -1) were contained between -1668 and -1154, relative to the proximal 3′-splice site (16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar). Further analysis of ADAR2 pre-mRNA sequences using RNA-folding algorithms (26Zuker M. Methods Mol Biol. 1994; 25: 267-294PubMed Google Scholar, 27Mathews D.H. Sabina J. Zuker M. Turner D.H. J. Mol. Biol. 1999; 288: 911-940Crossref PubMed Scopus (3198) Google Scholar, 28Zuker M. Mathews D.H. Turner D.H. Barciszewski J. Clark B.F.C. RNA Biochemistry and Biotechnology. Kluwer Academic, 1999: 11-43Google Scholar) identified an imperfect inverted repeat that formed a putative RNA duplex as a result of base-pairing interactions between a portion of this critical upstream region and the 3′-end of the intron (Fig. 1). Comparisons of ADAR2 genomic sequences between rat, mouse, human, and pufferfish revealed >90% intronic sequence conservation in the predicted region of the inverted repeats (Fig. 1), with a majority of the nucleotide differences clustered in predicted bulge regions within the duplex. Furthermore, 100% sequence identity was maintained in both halves of the inverted repeat for a region extending 18-nt upstream and 16-nt downstream from the edited splice junction (site -1) (Fig. 1), suggesting that the structure of this region is conserved to maintain ADAR2 autoediting among multiple vertebrate species (23Slavov D. Gardiner K. Gene (Amst.). 2002; 299: 83-94Crossref PubMed Scopus (40) Google Scholar).Fig. 1Predicted secondary structure and evolutionary conservation of ADAR2 pre-mRNA sequence in the region of major A-to-I editing modifications. The nucleotide sequence and predicted RNA secondary structure for the rat ADAR2 pre-mRNA is presented showing the positions of 16 editing sites identified in ADAR2 pre-mRNA transcripts isolated from adult rat brain; coordinates of the editing sites are relative to the proximal 3′-splice junction in intron 4 (16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar) and the number of nucleotides omitted from the figure is indicated in the loop. Comparisons of evolutionary sequence conservation between human, rat, mouse and two pufferfish ADAR2 genes are presented in which the percentage of species demonstrating 75-100% (black), 25-50% (green), and 0% (red) sequence identity to the rat ADAR2 gene are indicated by colored lettering. The shaded box denotes a region of the predicted RNA duplex where 100% sequence identity has been maintained in all five vertebrate ADAR2 genes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Editing Sites within ADAR2 pre-mRNA Transcripts—In addition to the ADAR2 editing event responsible for generating a proximal 3′-splice site within intron 4 (site -1), four additional A-to-I modifications were observed previously at positions -2, +10, +23 and +24 (relative to the proximal 3′-splice site) (16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar). To further examine editing events within the predicted rADAR2 duplex, sequence comparisons of rat ADAR2 genomic DNA and adult rat brain cDNA clones, generated from RT-PCR amplification of ADAR2 pre-mRNA, were performed to identify additional A-to-G discrepancies. In total, 16 RNA editing sites were identified (Fig. 1) and the frequency of editing at each position was quantified (Table I); nucleotide discrepancies that were observed in less than 5% of the isolated cDNA clones were not considered for subsequent analysis. In addition to the five editing sites previously identified within the ADAR2 duplex (16Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar), three additional sites were observed in the 3′-portion of the inverted repeat (positions -4, -27, and -28) and eight sites in the 5′-half of the duplex. Th" @default.
• W2109282318 created "2016-06-24" @default.
• W2109282318 creator A5000961399 @default.
• W2109282318 creator A5023046863 @default.
• W2109282318 creator A5051937522 @default.
• W2109282318 date "2004-02-01" @default.
• W2109282318 modified "2023-10-12" @default.
• W2109282318 title "Structure and Sequence Determinants Required for the RNA Editing of ADAR2 Substrates" @default.
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