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• W2040553608 abstract "In neurons of the mammalian brain primary transcripts of genes encoding subunits of glutamate receptor channels can undergo RNA editing, leading to altered properties of the transmitter-activated channel. Editing of these transcripts is a nuclear process that targets specific adenosines and requires a double-stranded RNA structure configured from complementary exonic and intronic sequences. We show here that the two independent editing sites in α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR-B pre-mRNA are edited with positional accuracy by nuclear extract from HeLa cells. Nucleotide analysis by thin layer chromatography of the edited RNA sequences revealed selective adenosine to inosine conversion, most likely reflecting the participation of double-stranded RNA adenosine deaminase. Our results predict the presence of inosine-containing codons in other mammalian mRNAs. In neurons of the mammalian brain primary transcripts of genes encoding subunits of glutamate receptor channels can undergo RNA editing, leading to altered properties of the transmitter-activated channel. Editing of these transcripts is a nuclear process that targets specific adenosines and requires a double-stranded RNA structure configured from complementary exonic and intronic sequences. We show here that the two independent editing sites in α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR-B pre-mRNA are edited with positional accuracy by nuclear extract from HeLa cells. Nucleotide analysis by thin layer chromatography of the edited RNA sequences revealed selective adenosine to inosine conversion, most likely reflecting the participation of double-stranded RNA adenosine deaminase. Our results predict the presence of inosine-containing codons in other mammalian mRNAs. RNA editing is a posttranscriptional process that changes the sequence of gene transcripts (1Cattaneo R. Annu. Rev. Genet. 1991; 25: 71-88Crossref PubMed Scopus (90) Google Scholar, 2Bass B.L. Gesteland R.F. Atkins J.F. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 383-418Google Scholar). This process is common in mitochondria of plants and protozoa where, as a rule, transcripts undergo multiple editing events to acquire translatable open reading frames. By contrast, RNA editing has been found in only a few mammalian nuclear transcripts where it generates single codon substitutions and, hence, changes in the cognate proteins. Apolipoprotein B (apoB) RNA of the small intestine provided the first example of editing in a nuclear transcript (3Chan L. J. Biol. Chem. 1992; 267: 25621-25624Abstract Full Text PDF PubMed Google Scholar, 4Hodges P. Scott J. Trends Biochem. Sci. 1992; 17: 77-81Abstract Full Text PDF PubMed Scopus (64) Google Scholar). In this RNA a premature stop codon is introduced by cytidine deamination (CAA to UAA), leading to a truncated protein with altered function. A uridine to cytidine substitution changing a leucine to a proline codon occurs in transcripts of the Wilm's tumor susceptibility gene in kidney and testis (5Sharma P.M. Bowman M. Madden S.L. Rauscher III, F.J. Sukumar S. Genes & Dev. 1994; 8: 720-731Crossref PubMed Scopus (152) Google Scholar). Adenosine-specific RNA editing was found in mammalian transcripts of neural origin (6Sommer B. Köhler M. Sprengel R. Seeburg P.H. Cell. 1991; 67: 11-19Abstract Full Text PDF PubMed Scopus (1195) Google Scholar, 7Köhler M. Burnashev N. Sakmann B. Seeburg P.H. Neuron. 1993; 10: 491-500Abstract Full Text PDF PubMed Scopus (367) Google Scholar, 8Lomeli H. Mosbacher J. Melcher T. Höger T. Geiger J. Kuner T. Monyer H. Sprengel R. Higuchi M. Bach A. Seeburg P.H. Science. 1994; 266: 1709-1713Crossref PubMed Scopus (642) Google Scholar). In the central nervous system the principal excitatory neurotransmitter L-glutamate activates postsynaptically located receptor channels (GluRs), 1The abbreviations used are:GluRL-glutamate-activated receptor channeldsRNAdouble-stranded RNAECSediting site complementary sequenceDTTdithiothreitolRTreverse transcriptionPCRpolymerase chain reactionPIPES1,4-piperazinediethanesulfonic acid. the subunits of which form an extended gene family, comprising 16 members to date (9Hollmann M. Heinemann S. Annu. Rev. Neurosci. 1994; 17: 31-108Crossref PubMed Scopus (3668) Google Scholar). In this family, the transcripts of five genes are known to undergo RNA editing in a total of eight positions. In each instance, the transmitter-activated channel acquires altered ion permeability or kinetic properties (8Lomeli H. Mosbacher J. Melcher T. Höger T. Geiger J. Kuner T. Monyer H. Sprengel R. Higuchi M. Bach A. Seeburg P.H. Science. 1994; 266: 1709-1713Crossref PubMed Scopus (642) Google Scholar, 9Hollmann M. Heinemann S. Annu. Rev. Neurosci. 1994; 17: 31-108Crossref PubMed Scopus (3668) Google Scholar). GluR transcript editing exclusively targets gene-specified adenosines that appear as guanosines in cloned cDNA. For example, the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR-B pre-mRNA is edited in two independent positions, from a CAG to a CGG codon (Q/R site; Ref. 6Sommer B. Köhler M. Sprengel R. Seeburg P.H. Cell. 1991; 67: 11-19Abstract Full Text PDF PubMed Scopus (1195) Google Scholar) and from an AGA to a GGA codon (R/G site; Ref. 8Lomeli H. Mosbacher J. Melcher T. Höger T. Geiger J. Kuner T. Monyer H. Sprengel R. Higuchi M. Bach A. Seeburg P.H. Science. 1994; 266: 1709-1713Crossref PubMed Scopus (642) Google Scholar). L-glutamate-activated receptor channel double-stranded RNA editing site complementary sequence dithiothreitol reverse transcription polymerase chain reaction 1,4-piperazinediethanesulfonic acid. Our present understanding concerning mechanistic aspects of this enigmatic control over functional channel determinants in central neurons is rudimentary. One recent advance has been the identification of intronic editing site complementary sequences (ECS) that form a short intramolecular dsRNA structure with the to-be-edited exonic sequence (10Higuchi M. Single F.N. Köhler M. Sommer B. Sprengel R. Seeburg P.H. Cell. 1993; 75: 1361-1370Abstract Full Text PDF PubMed Scopus (523) Google Scholar). As documented for both the Q/R and R/G sites in GluR-B pre-mRNA these dsRNA structures serve as substrates for a positionally precise RNA editing (8Lomeli H. Mosbacher J. Melcher T. Höger T. Geiger J. Kuner T. Monyer H. Sprengel R. Higuchi M. Bach A. Seeburg P.H. Science. 1994; 266: 1709-1713Crossref PubMed Scopus (642) Google Scholar, 10Higuchi M. Single F.N. Köhler M. Sommer B. Sprengel R. Seeburg P.H. Cell. 1993; 75: 1361-1370Abstract Full Text PDF PubMed Scopus (523) Google Scholar). It has been proposed that the edited adenosines might constitute inosines generated by adenosine deamination in RNA (2Bass B.L. Gesteland R.F. Atkins J.F. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 383-418Google Scholar, 6Sommer B. Köhler M. Sprengel R. Seeburg P.H. Cell. 1991; 67: 11-19Abstract Full Text PDF PubMed Scopus (1195) Google Scholar). Indeed, the preferred base pairing properties of inosine with cytidine would lead to the incorporation of guanosine in a cDNA position occupied by inosine in the RNA template. The requirement for dsRNA and the fact that only adenosines are targeted in GluR pre-mRNAs have given rise to the hypothesis that dsRNA adenosine deaminase might operate the nucleotide change by hydrolytic deamination of adenosine to inosine (2Bass B.L. Gesteland R.F. Atkins J.F. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 383-418Google Scholar, 10Higuchi M. Single F.N. Köhler M. Sommer B. Sprengel R. Seeburg P.H. Cell. 1993; 75: 1361-1370Abstract Full Text PDF PubMed Scopus (523) Google Scholar, 11Kim U. Nishikura K. Semin. Cell Biol. 1993; 4: 285-293Crossref PubMed Scopus (33) Google Scholar). We have now employed an in vitro system to determine the nature of the edited adenosine in GluR-B pre-mRNA. As shown in this study, editing by nuclear extract displays the positional accuracy and all sequence requirements previously established in cellular assays (8Lomeli H. Mosbacher J. Melcher T. Höger T. Geiger J. Kuner T. Monyer H. Sprengel R. Higuchi M. Bach A. Seeburg P.H. Science. 1994; 266: 1709-1713Crossref PubMed Scopus (642) Google Scholar, 10Higuchi M. Single F.N. Köhler M. Sommer B. Sprengel R. Seeburg P.H. Cell. 1993; 75: 1361-1370Abstract Full Text PDF PubMed Scopus (523) Google Scholar). Importantly, nuclear extract from HeLa cells mediates the site-selective conversion of adenosine to inosine in both the Q/R site and the R/G site. RNAs were transcribed from murine GluR-B minigene constructs. For Q/R site editing DNA constructs were B13 and B13Δ6, which carries a deletion in its ECS sequence and hence is not edited in transfected cells (10Higuchi M. Single F.N. Köhler M. Sommer B. Sprengel R. Seeburg P.H. Cell. 1993; 75: 1361-1370Abstract Full Text PDF PubMed Scopus (523) Google Scholar). DNAs were linearized with XbaI and upon transcription in vitro generated RNAs of an approximate length of 720 nucleotides, including all of exon 11 and 530 nucleotides of intron 11. For R/G site editing, DNA constructs were BglII- BglII (exon 13 into intron 15) carrying the intronic E2 mutation (increased editing relative to wild type sequence) and BglII- BglII carrying the intronic S1 mutation (editing incompetent) (8Lomeli H. Mosbacher J. Melcher T. Höger T. Geiger J. Kuner T. Monyer H. Sprengel R. Higuchi M. Bach A. Seeburg P.H. Science. 1994; 266: 1709-1713Crossref PubMed Scopus (642) Google Scholar). These constructs were linearized with StuI (unique site in exon 14) and yielded RNAs of approximately 1,100 nucleotides containing exon 13, intron 13, and part of exon 14. In vitro transcription was performed for 60 min at 37°C in 25 μl containing 80 m M Hepes-KOH, pH 7.5, 32 m M MgCl2, 2 m M spermine, 40 m M DTT, 5 m M each of ATP, GTP, CTP, and UTP, 10 μCi of α-[32P]GTP (3,000 Ci/mmol), 750 units of SP6 RNA polymerase (Promega), 37.5 units of RNasin (Promega), 475 units of inorganic pyrophosphatase from yeast (Promega), and 1 μg of linearized recombinant vector DNA. Radiolabeled, high specific activity RNAs were synthesized in the presence of 10 μM GTP and 120 μCi of α-[32P]GTP (Amersham Corp., 3,000 Ci/mmol). Unincorporated nucleotides were removed by EtOH precipitation of the phenol/chloroform-extracted reactions. RNAs used for reverse transcription (RT) followed by PCR (12Wahle E. Keller W. Higgins S.J. Hames B.D. RNA Processing. Vol. 2. IRL Oxford University Press, Oxford1994: 1-34Google Scholar) were treated three times for 45 min at 37°C with 10 units of RNase-free DNase (Boehringer Mannheim) in the presence of 20 units of RNasin (Fermentas). Removal of DNA from RNA was demonstrated by PCR amplification (see below). Nuclear extracts were prepared from HeLa cells according to Ref. 13Mullis K.B. Faloona F.A. Methods Enzymol. 1987; 155: 335-340Crossref PubMed Scopus (3876) Google Scholar except that high salt buffer C contained 1.2 M KCl instead of NaCl. Extracts were stored in aliquots at −80°C and had a protein concentration of about 10 μg/μl in 10 m M Tris-HCl, pH 7.9, 12.5% glycerol, 0.75 m M MgCl2, 0.1 m M EDTA, and approximately 270 m M KCl, as measured by conductivity. For standard assays, RNAs (between 10 and 100 fmol) were incubated at 30°C for 3 h in a 25-μl reaction mixture containing 12.5 μl of nuclear extract and having a final buffer composition (“Q buffer”) of 25 m M Tris-HCl, pH 7.9, 135 m M KCl, 2.5 m M EDTA, 6.25% glycerol, 1 m M DTT, 50 μg/ml poly(A). Reactions (25 μl) having 2 μl of nuclear extract yielded in B13 and E2 RNAs approximately half of the editing levels achieved with 12.5 μl of extract. After incubation, reaction mixtures were deproteinized by adding equal volumes of 200 m M Tris-HCl, pH 7.9, 300 m M NaCl, 25 m M EDTA, 2% SDS, 0.8 mg/ml proteinase K for 30 min at 37°C, and RNA was recovered by phenol/chloroform extraction and ethanol precipitation. Preincubation of extract with 1,000 units/ml micrococcal nuclease (Pharmacia Biotech Inc.) for 30 min at 30°C did not reduce Q/R site editing; incubation was in the presence of 1 m M CaCl2, which was chelated by 2 m M EGTA before adding B13 RNA. Preincubation (30 min, 30°C) with proteinase K (0.8 mg/ml) abolished Q/R site editing. For Q/R site editing, 10 fmol of 5′32P-labeled oligonucleotide primer B-RT (3 × 106dpm/pmol) were hybridized to extract-incubated and purified RNA (approximately 3-5 fmol) in 50 m M Tris-HCl, pH 8.3, 75 m M KCl, 3 m M MgCl2, 10 μM each of dATP, dGTP, and dCTP and 250 μM ddTTP (Pharmacia) by heating for 5 min to 80°C and annealing at 55°C for 4 h in a final volume of 9 μl. After addition of 0.8 units of avian myeloblastosis virus reverse transcriptase (Life Technologies, Inc.) and 1 μl of 0.1 m M DTT the reactions (10.5 μl final volume) were incubated for 1 h at 42°C. For the analysis of RT-PCR products generated from extract-incubated R/G-edited RNAs, approximately 20 ng of agarose gel-purified PCR product was denatured in alkaline, ethanol-precipitated, and resuspended in 50 m M Tris-HCl, pH 8.3, 75 m M KCl, 3 m M MgCl2, 10 μM each of dATP, dGTP, dCTP, 250 μM ddTTP, and 10 fmol of the 32P-labeled oligonucleotide B-RTFF45. After 5 min at 55°C, 0.8 unit of avian myeloblastosis virus reverse transcriptase plus 1 μl of 0.1 M DTT were added, and the reactions (10.5 μl final) were incubated for 45 min at 42°C. Nucleic acid was fractionated on a 15% polyacrylamide, 7 M urea gel, and dried gels were exposed to x-ray film with a DuPont Cronex Lightning intensifying screen. Quantitative analysis was performed on a PhosphorImager (Fuji, BAS1000). Extract-incubated and purified RNAs (3-5 fmol), resuspended in 20 μl of H2O containing 1 μM primer for reverse transcription (KMH3 for Q/R site editing, BFFK3 for R/G editing), were denatured (70°C, 10 min) and added to a 10-μl RT mix (50 m M Tris-HCl, pH 8.5, 75 m M KCl, 3 m M MgCl2, 10 m M DTT, 20 units of RNasin, and 500 μM of each dNTP). A 10-μl aliquot of each reaction was removed for control purposes (“mock RT”). The remainder was incubated with 200 units of reverse transcriptase (Moloney murine leukemia virus, Life Technologies, Inc.) for 1 h at 37°C and 5 min at 95°C. A first PCR amplification using the primers lacZ1/PCRK3 (Q/R site editing) or cis55/PCRK3 (R/G site editing) was in 20 μl of 20 m M Tris-HCl, pH 8.4, 50 m M KCl, 1.5 m M MgCl2, 200 μM dNTPs, 400 n M primers, and 0.5 unit of Taq polymerase (Life Technologies, Inc.). Cycle conditions were: 94°C, 3 min; 20 step cycles (94°C, 20 s; 55°C, 30 s; 72°C, 40 s); and 72°C, 10 min. A second PCR amplification with the nested primer sets MH36/lacZ1 (Q/R site editing) or cis55/intB1 (R/G site editing) was in 50 μl for 35 step cycles. The PCR-generated DNA fragments served as templates for primer extension at the R/G site or were directionally cloned in doubly digested phage M13 RF-DNA (M13 mp19, cleaved with PstI and EcoRI for Q/R site fragments; M13 mp18, cleaved with KpnI and EcoRI for R/G site fragments) (14Yanisch-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11472) Google Scholar). Filter lifts of recombinant phage plaques were hybridized overnight at 22°C in 5 × SSC (1 × SSC: 0.15 M NaCl, 15 m M sodium citrate) with 32P-labeled oligonucleotides B-R (Q/R site) or EDFF-3 (R/G site). Filters were washed in 1 × SSC at 56°C (Q/R site) or 50°C (R/G site) to distinguish edited from unedited sequences. At least 12 recombinant phage DNAs carrying edited and unedited Q/R and R/G forms were sequenced (ABI sequencer 373A). RNAs (150 fmol, ∼109dpm/μg) incubated in nuclear extract were purified (see above) and resuspended in 20 μl of 10 m M PIPES, pH 6.4, 100 m M NaCl, 0.25 m M EDTA containing 50 pmol of the oligonucleotides Bprotect (Q/R site) or FF-Bprotect (R/G site). Oligonucleotide was annealed to RNA (3 min, 90°C; 30 min, 55°C), and 170 μl of 10 m M Tris-HCl, pH 7.5, 300 m M NaCl, 5 m M EDTA containing 0.2 unit of RNase A and 70 units of RNase T1 (Boehringer Mannheim) were added. RNase digestion was for 30 min at 37°C (Bprotect) or 32°C (FF-Bprotect), followed by phenol/chloroform extraction and ethanol precipitation with 10 μg of glycogen as carrier. Precipitated nucleic acid was resuspended in 10 μl of 0.1% bromphenol blue, 0.1% xylene cyanol and electrophoresed on a native 15% polyacrylamide gel in 1 × TBE (90 m M Tris borate, 2 m M EDTA, pH 8.0) at 200 V for 8-9 h. Protected bands were gel-eluted overnight at 22°C in 0.5 M NH4Ac, 1 m M MgCl2, 0.1 m M EDTA, 0.1% SDS. Nucleic acid was collected by ethanol precipitation after sterile filtration (Millipore, 0.22 μM) of the eluate, denatured (5 min, 70°C), and digested to 3′-nucleoside monophosphates in 20 μl of 10 m M NH4Ac, pH 4.5, containing 0.5 unit of ribonuclease T2 (Life Technologies, Inc.) for 4 h at 37°C. The digest was dried in a SpeedVac concentrator, resuspended in 3 μl of water containing 3′-XMPs (where X is A, I, G, C, or U; 1 μg/μl each; Sigma), spotted on a cellulose plate (Sigma), and chromatographed in saturated (NH4)2SO4, 0.1 M NaAc, pH 6, 2-propyl alcohol (79:19:2, by volume). Internal labeling of RNA by α-[32P]GMP combined with ribonuclease T2-mediated label transfer permitted an evaluation of label distribution in 3′-XMPs derived from the oligonucleotide-protected RNA moieties. In the protected B13 RNA moiety (Fig. 2 a), the 5′-32P groups of nine GMPs are transferred by T2 to three 3′-GMPs, three 3′-UMPs, two 3′-AMPs (unedited), or one 3′-IMP (fully edited). Within the E2 RNA moiety (Fig. 2 b) these numbers are five 3′-GMPs, two 3′-UMPs, one 3′-AMP (unedited), or one 3′-IMP (edited). Radioactive spots on one-dimensional TLCs (3′-UMP and 3′-CMP co-migrate) were quantified with a PhosphorImager (Fuji, BAS 1000), and the relative abundance of each 3′-XMP was determined. In four experiments the averaged ratios ± S.D. for the labeled 3′-XMPs from protected B13 RNA were G/C + U/A/I, 2.6 ± 0.1/3.2 ± 0.1/2.1 ± 0.1/0.11 ± 0.06 (theoretical values: 3/3/1.7/0.3, assuming 30% Q/R site editing) and for E2, 4.1 ± 0.3/2.1 ± 0.2/1.6 ± 0.2/0.2 ± 0.1 (theoretical: 5/2/0.9/0.1, assuming 10% R/G site editing). Deviations from predicted values result from contamination by heterodisperse RNA fragments of the gel-isolated heteroduplexes, as evidenced by radioactive material in gel lanes resolving the unprotected ribonuclease A and T1-treated RNAs (Fig. 2) and the 3′-[32P]CMP spot on two-dimensional TLC (Fig. 3).FIG. 3Two-dimensional thin-layer chromatography of ribonuclease T2 digestion products of the oligodeoxynucleotide-protected in vitro edited B13 (a) and E2 (b) RNAs. Isobutyric acid/NH4OH/water (66:1:33, by volume) was used as solvent for the first dimension (migration, bottom to top) and saturated (NH4)2SO4, 0.1 M NaAc pH 6, 2-propanol (79:19:2, by volume) for the second dimension (migration, left to right). The individual 3′-XMPs (Gp, Ap, Ip, Up, Cp) are indicated on the chromatograms. The presence of 3′-[32P]CMP is likely to reflect a contamination by heterodisperse RNA fragments of the protected, gel-isolated RNAs (Fig. 2).View Large Image Figure ViewerDownload Hi-res image Download (PPT) B-RT, 5′-GGCGAAATATCGCATCCTTG-3′, antisense, GluR-B, exon 11; B-RTFF45, 5′-ATTGTTATACTATTCCACCC-3′, antisense, GluR-B, intron 13; Bprotect, 5′-CATCCTTGCCGCATAAAGGCACCC-3′, antisense, GluR-B, exon 11, complementary to edited Q/R site; FF-Bprotect, 5′-CCACCCACCCTAATGAGGATCC-3′, antisense, GluR-B, intron 13/exon 13 border, complementary to edited R/G site; KMH3, 5′-GACACGGTACCACACAACGGCATTTCCATGAATTGATGTTAGAG-3′, revT primer, anti-sense, GluR-B, intron 11; BFFK3,5′-GACACGGTACCACACAACGGATTGTGAGTTACCTCATATCCG-3′, revT primer, antisense, GluR-B, intron 13; lacZ1, 5′-GCCTGCAGCCATGGTGAATCAACTAACGAATTTGG-3′, sense, GluR-B, exon 11; PCRK3, 5′-GACACGGTACCACACAACGG-3′, PCR primer for cDNA primed with KMH3 or BFFK3; cis55, 5′-CTCTGCGAGCTCAGGTCCAACTGCACCTCGG-3′, vector-specific 5′ primer; MH36, 5′-TCACCAGGGAAACACATGATCAAC-3′, antisense, GluR-B, intron 11; intB1, 5′-GCGGTACCGTGAGTTACCTCATATCCGTAT-3′, antisense, GluR-B, intron 13; B-R, 5′-GCATCCTTGCCGCATAAAGGC-3′, antisense, GluR-B, exon 11, complementary to edited Q/R site; EDFF-3, 5′-CCACCCTAATGAGGATCCTT-3′, antisense, GluR-B, intron 13/exon 13 border, complementary to edited R/G site. Nuclear extract prepared from HeLa cells (12Wahle E. Keller W. Higgins S.J. Hames B.D. RNA Processing. Vol. 2. IRL Oxford University Press, Oxford1994: 1-34Google Scholar) was analyzed for RNA editing activity. RNAs transcribed in vitro from editing competent as well as incompetent GluR-B minigenes for both the Q/R (10Higuchi M. Single F.N. Köhler M. Sommer B. Sprengel R. Seeburg P.H. Cell. 1993; 75: 1361-1370Abstract Full Text PDF PubMed Scopus (523) Google Scholar) and the R/G (8Lomeli H. Mosbacher J. Melcher T. Höger T. Geiger J. Kuner T. Monyer H. Sprengel R. Higuchi M. Bach A. Seeburg P.H. Science. 1994; 266: 1709-1713Crossref PubMed Scopus (642) Google Scholar) site were incubated with nuclear extract. Site-selective editing was determined by primer extension (Fig. 1) performed on the recovered RNAs (Q/R site) or on their RT-PCR products (R/G site). RNAs from minigenes (B13, Q/R site; E2, R/G site) edited in transfected cells (8Lomeli H. Mosbacher J. Melcher T. Höger T. Geiger J. Kuner T. Monyer H. Sprengel R. Higuchi M. Bach A. Seeburg P.H. Science. 1994; 266: 1709-1713Crossref PubMed Scopus (642) Google Scholar, 10Higuchi M. Single F.N. Köhler M. Sommer B. Sprengel R. Seeburg P.H. Cell. 1993; 75: 1361-1370Abstract Full Text PDF PubMed Scopus (523) Google Scholar) were also edited by the nuclear extract, whereas RNAs from minigenes rendered editing incompetent by nucleotide deletions (B13Δ6, Q/R site) or substitutions (S1, R/G site) in the intronic ECS site were not (Fig. 1,Table I). The extent of site-specific editing was time-dependent and, after 3 h, reached (mean ± S.D.) 32 ± 6% (n = 10) for the Q/R site and 10 ± 2% (n = 8) for the R/G site (Fig. 1, Table I). These editing efficiencies were confirmed by an assay (10Higuchi M. Single F.N. Köhler M. Sommer B. Sprengel R. Seeburg P.H. Cell. 1993; 75: 1361-1370Abstract Full Text PDF PubMed Scopus (523) Google Scholar) based on PCR-mediated sequence amplification (13Mullis K.B. Faloona F.A. Methods Enzymol. 1987; 155: 335-340Crossref PubMed Scopus (3876) Google Scholar), directional cloning of amplified DNAs, and probing the recombinant phage plaques with oligonucleotides for edited and unedited sequence forms. Furthermore and importantly, DNA sequencing of sets of recombinant phage DNAs derived from RNAs incubated in nuclear extract (Fig. 1, legend) demonstrated that the only edited positions concerned the adenosines of the Q/R and R/G sites (not shown). Collectively these data indicate that the sequence requirements for the in vitro editing of two independent positions in GluR-B pre-mRNAs are those previously established by cell transfection. Similarly, the positional selectivity of editing observed for GluR-B minigenes in transfected cells is strictly maintained by the nuclear extract. Analysis of 60 cloned DNA sequences derived from in vitro edited B13 RNA showed no evidence for the low level editing of additional adenosines observed around the Q/R site in native GluR-B pre-mRNA (10Higuchi M. Single F.N. Köhler M. Sommer B. Sprengel R. Seeburg P.H. Cell. 1993; 75: 1361-1370Abstract Full Text PDF PubMed Scopus (523) Google Scholar).Table I:RNA editing of GluR-B RNAs by nuclear extract from HeLa cellsTable I:RNA editing of GluR-B RNAs by nuclear extract from HeLa cellsAll reactions (25 μl) contained 12.5 μl of nuclear extract and were incubated for 3 h at 30°C. Numbers in parentheses indicate independent determinations; standard deviations of values listed were ≤20%. ND, not determined. All reactions (25 μl) contained 12.5 μl of nuclear extract and were incubated for 3 h at 30°C. Numbers in parentheses indicate independent determinations; standard deviations of values listed were ≤20%. ND, not determined. Editing was abolished by heat treatment (10 min, 65°C) or proteinase K digestion of nuclear extract but not by micrococcal nuclease. GluR-B pre-mRNAs when incubated in the absence of extract under conditions used to reveal RNA self-modification (15Shub D.A. Peebles C.L. Hampel A. Higgins S.J. Hames B.D. RNA Processing. Vol. 2. IRL Oxford University Press, Oxford1994: 211-239Google Scholar) showed no site-selective editing. Thus, editing appears not to be mediated by the RNA itself nor does it require other RNAs but is mediated by protein components provided by the nuclear extract. Indeed, the extent of editing depended on the amount of extract (see “Materials and Methods”). Failure of the editing reaction to go to completion did not appear to result from limiting amounts of editing factor(s) in extract since editing efficiencies remained constant over a 10-fold concentration range of RNA (Table I). The addition of ATP to 100 μM did not alter the extent of editing (not shown), indicating that editing is not dependent on ATP as an energy source. Editing was not affected by poly(I), poly(C), and poly(dI)˙poly(dC) but was efficiently inhibited by poly(I)˙poly(C) (inhibitory constant, IC50∼0.3 n M), revealing the participation of a factor with high affinity for dsRNA. This factor is likely to be dsRNA adenosine deaminase (2Bass B.L. Gesteland R.F. Atkins J.F. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 383-418Google Scholar, 11Kim U. Nishikura K. Semin. Cell Biol. 1993; 4: 285-293Crossref PubMed Scopus (33) Google Scholar), which converts adenosines to inosines in extended dsRNA with limited positional specificity (16Polson A.G. Bass B.L. EMBO J. 1994; 13: 5701-5711Crossref PubMed Scopus (237) Google Scholar). dsRNA adenosine deaminase displays such high affinity binding to dsRNA (equilibrium binding constant, Kd∼0.2 n M; Ref. 17Kim U. Garner T.L. Sanford T. Speicher D. Murray J.M. Nishikura K. J. Biol. Chem. 1994; 269: 13480-13489Abstract Full Text PDF PubMed Google Scholar), and the optimal KCl concentration (approximately 100 m M, Table I) for in vitro editing of the Q/R site in GluR-B pre-mRNA was that required by purified dsRNA adenosine deaminase (18O'Connell M.A. Keller W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10596-10601Crossref PubMed Scopus (78) Google Scholar) for optimal adenosine to inosine conversion in dsRNA. However, B13 RNA is not edited by the purified enzyme, 2M. A. O'Connell and W. Keler, unpublished data. suggesting that site-selective editing may involve additional factor(s). To determine the chemical nature of the edited adenosine, 3′32P-labeled mononucleosides (XMPs) generated by ribonuclease digestion of a short sequence within internally labeled, extract-incubated RNAs were analyzed by TLC (19Kuchino Y. Hanyu N. Nishimura S. Methods Enzymol. 1987; 155: 379-396Crossref PubMed Scopus (74) Google Scholar). Different GluR-B-specific RNAs were enzymatically synthesized in the presence of α-[32P]GTP and were incubated with nuclear extract. The radiolabeled, edited RNAs, recovered intact as judged by gel migration (not shown), were hybridized with a molar excess of an oligodeoxynucleotide (24-mer) complementary to the RNA sequence around the edited nucleotide position (Fig. 2). After digestion with the single-stranded specific ribonucleases A and T1 the resultant RNA-DNA heteroduplexes were resolved by native polyacrylamide gel electrophoresis. The heteroduplex containing the protected B13 RNA moiety appeared as three closely spaced bands of different intensities. The same triplet of bands was generated by ribonuclease digestion of a synthetic oligomeric RNA-DNA hybrid (Fig. 2 a) and probably reflects the removal by ribonuclease of terminal bases from the oligodeoxynucleotide-protected RNA. Gel-recovered heteroduplexes were subjected to digestion by ribonuclease T2, which generates 3′-XMPs from RNA (20Rushizky G.W. Sober H.A. J. Biol. Chem. 1963; 238: 371-376Abstract Full Text PDF PubMed Google Scholar), leading to the transfer of the 5′-32P group in GMPs to the 3′ position of the preceding nucleoside. Digested material was resolved by one-dimensional TLC. As a result, radiolabel co-migrating with 3′-IMP was readily detected in the appropriate in vitro edited RNA species (Fig. 2). The nature of the inosine in the edited B13 and E2 RNAs was further confirmed by two-dimensional TLC (Fig. 3). In four experiments editing levels as assessed by 3′-[32P]IMP/3′-[32P]AMP ratios on one-dimensional TLC were 11 ± 0.6% for B13 RNA and 13.2 ± 0.9% for E2 RNA. The low level editing of B13 RNA in these experiments is apparently due to sequence contamination of the gel-isolated B13 heteroduplex, as evidenced by the 3′-[32P]CMP spot on the two-dimensional chromatogram (Fig. 3) (see also “Materials and Methods”). The sequence specificity of in vitro adenosine to inosine conversion was clearly demonstrated by the absence of inosine in B13Δ6 RNA (Fig. 2 a) in a B13 RNA mutant in which a guanosine residue had been substituted for the Q/R site adenosine (not shown) and in S1 RNA (Fig. 2 b). Thus, nuclear extracts can be employed to characterize the biochemical machinery responsible for specific adenosine editing. As a first milestone, this in vitro system permitted the identification of edited adenosines in GluR-B pre-mRNAs as inosine residues. Formation of inosine is most likely due to enzymatic adenosine deamination since upon dialysis of nuclear extract editing efficiencies remained unchanged, indicating that nucleotides required for transglycosylation or nucleotide replacement are not necessary for GluR editing. Our results are compatible with the notion (2Bass B.L. Gesteland R.F. Atkins J.F. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 383-418Google Scholar, 10Higuchi M. Single F.N. Köhler M. Sommer B. Sprengel R. Seeburg P.H. Cell. 1993; 75: 1361-1370Abstract Full Text PDF PubMed Scopus (523) Google Scholar, 21Kim U. Wang Y. Sanford T. Zeng Y. Nishikura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11457-11461Crossref PubMed Scopus (372) Google Scholar) that dsRNA adenosine deaminase is a candidate enzyme for the selective adenosine editing in GluR transcripts of central neurons. In view of the ubiquitous expression of this recently cloned (21Kim U. Wang Y. Sanford T. Zeng Y. Nishikura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11457-11461Crossref PubMed Scopus (372) Google Scholar, 22O'Connell M.A. Krause S. Higuchi M. Hsuan J.J. Totty N.F. Jenny A. Keller W. Mol. Cell. Biol. 1995; 15: 1389-1397Crossref PubMed Google Scholar) enzyme, adenosine deamination should be more widespread in nuclear pre-mRNAs than anticipated on the basis of GluR transcript editing. This conclusion is further strengthened by the observation that many cell lines, including HeLa cells, contain the machinery for the correct editing of GluR-B pre-mRNA even in the absence of endogenous GluR gene expression (10Higuchi M. Single F.N. Köhler M. Sommer B. Sprengel R. Seeburg P.H. Cell. 1993; 75: 1361-1370Abstract Full Text PDF PubMed Scopus (523) Google Scholar). We now anticipate the unprecedented occurrence of inosine in select codons of other mammalian mRNAs. As established for GluRs, these codons (23Basillo C. Wahba A.J. Lengyel P. Speyer J.F. Ochoa S. Proc. Natl. Acad. Sci. U. S. A. 1962; 48: 613-616Crossref PubMed Scopus (140) Google Scholar) are likely to specify functionally critical residues in the cognate protein products. Hence, we propose that site-selective adenosine deamination may be a general mechanism for increasing the functional diversity of mammalian gene products. We thank Annette Herold for DNA sequencing, Andrea Bürer (Basel) for cell culture and preparation of nuclear extracts, and Jutta Rami for assistance with the manuscript. The help of Drs. Mario Mörl and Rainer Frank and the critical input by our colleagues Drs. Rolf Sprengel, Mary O'Connell, Hartmut Lüddens, and Thomas Kuner are gratefully acknowledged." @default.
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