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Molecular Cloning of Cdna for Double-Strandedrna Adenosine

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Molecular Cloning of Cdna for Double-Strandedrna Adenosine Proc. Natd. Acad. Sci. USA Vol. 91, pp. 11457-11461, November 1994 Biochemistry Molecular cloning of cDNA for double-stranded RNA adenosine deaminase, a candidate enzyme for nuclear RNA editing UNKYU KIM, YI WANG, TAMARA SANFORD, YONG ZENG, AND KAZUKO NISHIKURA* The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104 Communicated by David Botstein, August 1, 1994 ABSTRACT We have domed human cDNA e Ing dou- Determination of N-Terminal Sequence by Micosequenc- blesranded RNA deue (DRADA). DRADA IS ing. Approximately 100 pmol of purified bovine DRADA a ubtus uear me that conver multiple proteins (10) was fractionated by SDS/PAGE and transferred to inosin In doubeheIdcal RNA bsrat i t apparent onto a poly(vinylidine difluoride) membrane. The 93- and sequencespedfldty. The A-- Icove activity ofthe protein 88-kDa bands were cut out and sequenced by Edman degra- encoded by the coned cDNA was med by b t dation (11). In insect ees. Use ofthe domed DNA as a l r Reverse T s t om ee Chain Reaction (PCR). PlObe de lstate c ation sacros _ and Degenerate sets of oligonucleotides that represented the detected a s e trcri o 7 kb in RNA of ahuantnge codons for N-terminal peptide sequences were synthesized. anald. The deduced ay sr e of human DRADA For the 93-kDa protein, the sense primer was CGG;AAIC revealed a bipartite ner l an dgal, three res of CNGGNAAA/GGTNGA, and the antisense primer was a do t RNA b motf, and the presence of CGGGiATCCNGCT/CTCCTT/CTGGT/CTTNA, which sequences conserved In the ctlyic center ofother d correspond to amino acid residues PGKVE and AEQKL, includig a cytidine da isnvolved in the RNA editing of respectively. For the 88-kDa protein, the sense primer was B. These rucal pr ies are onsisetwith CiGAAIICAAA/GACNGGNTAC/TGTNGA, and the he enymic siture oDRADA, and strengthen the hypoth- antisense primer was CG(KIAICG/ATCG/ATCNGGG/ eds that DRADA carries out the RNA edit of tr pts T/AATG/ATCG/ATC, which correspond to residues KT- e ig gumte-gated ion annels in brain. GYVD and DDPIDD, respectively. Restriction sites (under- lined) for EcoRI for the sense primer and BamHI for the Double-stranded adenosine deaminase (DRADA) is an aden- antisense primer were included at the 5' end. In addition, osine deaminase specific for double-stranded RNA (dsRNA) internalprobes representing the residuesflankedby the sense (1, 2). DRADA deaminates multiple adenosines to inosines by and antisense primers were synthesized. The sequences of a hydrolytic deaminaton reaction (3). Although a relatively the internal probe were for the 93-kfla protein C/TTTG/ long double-helical RNA structure is required for efficient CAGC/CAGC/T/AGGCTCCTG and for the 88-kDa protein modification, the minimum length of the double-stranded CGGGATCCAT/CTGNCCA/GTTC/TTCT/GTT. The region for substrate recognition may be as short as 15 bp (4). first-strand cDNA synthesis was carried out (12) using total Theprecisebiologicalfunction(s) ofDRADA andthefullrange RNA prepared from the cultured bovine endothelial cell line, ofits substrate RNAs are currently not known (5). However, BFA-1C BPT (13). A portion of the PCR-amplified product several examples of in vivo interaction of the enzyme with was analyzed by Southern blot hybridization using the inter- cellular and viral gene transcripts have been reported in recent nal probe. The 75-bp cDNA that hybridized to the internal years (5). Forinstance, DRADA seems responsible forgenesis probe was purified from the agarose gel, digested with EcoRI of defective measles virus with biased hypermutation, which and BamHI, and ligated with pBluescript KS+ plasmid. results in the lethal central nervous system disease measles Selected subclones, BUCi and BUC2, were sequenced to inclusion body encephalitis (6). Furthermore, the codingofthe confirm the identity. DRADA-treated mRNAs is expected to be s ficantly al- Screening of the Recombinant Library. A human natural tered, and therefore, the involvement ofDRADA in the RNA killer cell cDNA library was screened using BUC2 as a editing process has been suggested (5, 7, 8). Indeed, DRADA specific probe. The positive cDNA plasmid, HUC1, con- now seems to be responsible at least for the RNA editing of tained -4 kb ofinsert DNA, which hybridized to both BUCi glutamate-gated ion channel subunits (glutamate receptor, and BUC2 by Southern blot analysis. The insert of HUC1 GluR) in mammalian brains (9). was then used to rescreen the original cDNA library, from Toward our ultimate goal of better understanding the which additional overlapping cDNA clones, HUC2, HUC3, physiological functions of DRADA, we have isolated and and HUC4, were obtained. characterized cloned cDNA encoding human DRADA.J The Southern Blot Analyis. Southern blot hybridization was deduced primary structure ofDRADA revealed the presence carried out in buffer containing 2x SSC, lx Denhardt's of sequence motifs that underlie the functional and catalytic solution, 40% (vol/vol) formamide, 10% (wt/vol) dextran properties of this intriguing enzyme. sulfate, 1% SDS, denatured salmon sperm DNA (0.05 mg/ ml), and heat-denatured 32P-labeled probe (106 cpm/ml) at 3TC for 18 hr. The membrane was washed with 2x SSC/1% MATERIALS AND METHODS SDS at 370C for 30 mm. DRADA Assay. The DRADA enzymatic activity was as- of the Recom t Bacuviru DRADA Pro- sayed in vitro as described (10). The radioactivity of the tein. For afull-length construct, pVLDRADA140, Xba I-Kpn adenosine and inosine spots on TLC plates was quantified by a phosphor imaging system (Molecular Dynamics). Abbreviations: dsRNA, double-stranded RNA; DRADA, dsRNA adenosine deaminase; DRBM, dsRNA binding motif; GluR, gluta- mate receptor; RNP, ribonucleoprotein. The publication costs ofthis article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U10439). 11457 Downloaded by guest on September 27, 2021 11458 Biochemistry: Kim et al. Proc. Natl. Acad. Sci. USA 91 (1994) A B 1 MNPRQQYSL8 GYYYHPFQ ZHQLRYQQP GPG88P8SFL LKQII1LKGQ 51 LPK&PVIGKQ TPSLPPBLPG LRPRIPVLL& 8SYTRGRVDI RGVPRGVHA 101 SQGLQRGFQH PGPKGRSLPQ RGVDCL88HF QILSIYQDQZ QRILKILZZL kDa 151 CKQLAT!AED LBGKLGTYI ZINRVLYBLL TPPLWKIAVS 93 201 TOMQOiQSQOV VRPDQS8QGA PUSDPSLZPZ DRX8!TVBKD LZPIFIAV8A 88 251 QA&UIQBSGVV RPD8HSQO8P NSDPGLZPZD SNTSALZDP LBFLDNABIX 4,41-0=_ 83 301 NXICDYLFNV 8DSSALNLAK NIGLTKARDI XAVLIDIRQ Q RITP 351 PIWNLTDKR IURMQIKRMN 8VPZTAPA&X PITRA18CNIP? r-OwNkD r6 kD 0 401 NN TT?'NUKVZ GQIQPVIKLZ NMEARUPZPA RLKPPVNYNG PS8G 451 NGQWADDIP DDLN8IRAAP QIIRIIMIRIP SFY8HGL&P&&JYKILTZCQ FIG. 1. Cloning of cDNA en- 501 LINPIBOLL YAQFAQUC _ IZQBGPP PRFQVV INGEZPPPAN coding human DRADA. (A) The 551 AWQ8KVAKD AUNANTILL NIAKASX B8BHYS= K lC final purified fraction was ana- 601 TTP'P8AT8FF 8QBPVYLL HG RLLBKUGP ANZPKIQYCV lyzed by SDS/PAGE (7% gel) and 651 AVGAQTFP8V 8APSVQ MANZAKAL GAN8A DQPI 8 three protein bands were visual- 4W'1 701 BLDULiN VUKIQZLV RYLWEPVGG LLZYAPR.BF AABFUNDQB ized by silver staining. Lanes: 1 VLI 751 GPPNNPKIVY QAKVGGRNPP AVCABSKKQQKO DR and 2, molecular mass standards; 801 RlNfGPKVYPV TQABLRRTU LL8RBPZAQP KLPLTG8TF HDQE=IAiE 3, bovine DRADA. (B) The de- 851 CFIUL!81Q PsL8RKIL& AZImI8ZD UGVVVSLQG NUCVKGD8LB duced amino acid sequence of the I 901 IKGZITVIDCN ABIISRRGFI RPLYBL Y NSAEDBIF ZPAOGQ 136-kDa form of human DRADA 951 IXKTVSFNLY IZTAPCGDGA L1DKC8DRA KI8Y8OBYP VPUPIQQK is shown. The putative bipartite 1001 RUEVINOZOT IPVZ8BDIVP TDGIRLGZR LRTKSCBDKI LRUNVWLQG nuclear localization signal is 1051 ALLNIFLQPI YLKSVTLGYL FSQGHLTRAI CCRVTRDG8A FPDGLRRPFI boxed. The N-terminal sequences 1101 VIIHIPJKVQVS IYDKRQ8QK ?UT8VNWIL ADGYDLUILD TRNGVDGIPR of 93- and 88-kDa proteins are 1151 UKL8RVSIKN ILIZKKLC8 IPRYRRDTLLRL BGZAR DYNITARFIz indicated by arrows; three DRBM 1 2 3 1201 IGLIDUGYO WZSKPQINKN FYLCPV* repeats are indicated by lines. I (the 5'-end 3.7 kb of HUCI) and Kpn I-Xba I (the 3'-end 1 by PILEUP, BESTFIT, and GAP programs, and identification of kb of HUC2) fragments were ligated with pVL1393 (Invitro- various protein sequence motifs was by the MOTIFS program gen). For pVLDRADAA, a mutant lacking the C-terminal 346 (15). A homology search of the National Center for Biotech- amino acids, a new termination codon was created at residue nology Information peptide sequence database (Release 82.0) 880 by filling in the overhang ofXma III located downstream was performed by Internet electronic mail. ofthe dsRNA binding motifs (DRBMs). Infected Spodoptera frugiperdera (Sf9) cells were labeled with 50 pCi of [35S]me- RESULTS thionine (1 Ci = 37 GBq) for 1 hr (14), and the labeled protein was analyzed by SDS/PAGE and fluorography. Purification of DRADA Proteins and Isolation of DRADA Determination of the cDNA Sequence. DNA sequencing of cDNAs. By following the A I modification activity, we sets of nested deletion mutants of the cDNA clones was have purified (10) DRADA to homogeneity from bovine liver carried out with the model 373A DNA sequencing system nuclear extracts. The final fraction contained three DRADA (Applied Biosystems). polypeptides of93, 88, and 83 kDa (Fig. 1A). The N-terminal Sequence Analysis. The overlapping sequences were amino acid sequences of the 93-kDa and 88-kDa proteins aligned and combined by the FRAGMENT ASSEMBLY program were determined by microsequencing, and two short bovine (15). Alignments ofDRBM and deaminases were determined cDNA clones encoding the N termini were obtained. Using B - DRADA1 40 - DRADA A A Nuc DRBM pVLDRADA1 40 pVLDRADAA FIG. 2. Characterization of the recombinant DRADA pro- C teins. (A) Full-length DRADA140 and C-terminally truncated DRADAA constructs are shown. * - - pi (B) Forty-eight hours after infec- tion with recombinant or wild- type baculoviruses, SO9 cells were labeled with P5S]methionine and proteins were separated by SDS/ * * * * * -pA PAGE. (C) Crude extracts made after infection at various time Polyhedrin points were analyzed by a modi- fied-base assay using 20 ,g of extract as described (10).
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