©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutagenic Analysis of Double-stranded RNA Adenosine Deaminase, a Candidate Enzyme for RNA Editing of Glutamate-gated Ion Channel Transcripts (*)

(Received for publication, March 10, 1995; and in revised form, May 5, 1995)

Fang Lai , Robert Drakas , Kazuko Nishikura (§)

From the Wistar Institute, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mutagenic analysis of the substrate binding and catalytic domains of double-stranded RNA (dsRNA) adenosine deaminase (DRADA) was carried out. This nuclear enzyme is likely to be involved in the RNA editing of glutamate-gated ion channels that are essential for fast excitatory neurotransmission in mammalian brain. The deletion of the first or the third of the three dsRNA binding motifs within the substrate binding domain dramatically decreases enzyme activity, whereas the second motif seems to be dispensable. The results indicate that the three motifs are not functionally equivalent in the catalytic action of DRADA. Mutation of the putative zinc-coordinating residues, His, Cys, and Cys, abolished the DRADA activity. Similarly, the Glu residue, predicted to be involved in the proton transfer functions of the enzyme, was found to be indispensable. Our results reinforce the previous proposal that the hydrolytic deamination mechanism of DRADA may be more similar to that of the cytidine deaminases than of adenosine deaminases.


INTRODUCTION

Double-stranded RNA (dsRNA)()adenosine deaminase (DRADA) deaminates adenosine residues to inosines in dsRNAs(1, 2) . This nuclear enzyme (3) occurs throughout the animal kingdom(3, 4) . A relatively long double helical structure of either intermolecular or intramolecular duplex RNA is required for efficient modification, but the absolute minimum length of the double-stranded region for substrate recognition may be as short as 15 bp(5) . The mechanism of A to I conversion has been shown to be hydrolytic deamination(6) .

Although the precise biological function(s) for this newly discovered enzyme are currently not known, one possible role DRADA may play is to participate in an RNA editing process(7, 8) . Inosine is treated as G by reverse transcriptase and also by the translation machinery(9) , and, therefore, the A to I base editing introduced by DRADA would be detected as A to G alterations by cDNA sequencing(7, 8) . So far, eight examples of RNA editing that result in alteration of an adenosine residue to guanosine detected by cDNA sequencing have been reported (10, 11, 12) . All eight cases occur in transcripts of certain glutamate-activated ion channel subunits in mammalian brains. These channels respond to L-glutamate, a major neurotransmitter, and mediate fast excitatory synaptic responses(13) . All of the amino acid changes introduced by the RNA editing have a large physiological impact on ion conductance, calcium permeability, and the kinetics of desensitization of the edited glutamate receptor (GluR) channels(10, 11, 14, 15, 16) . In one example, an arginine residue (CGG or CIG), located in the second transmembrane region of both -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) GluR-B subunit and also a subpopulation of kainate receptor GluR-5 and -6 subunits, were generated through an RNA editing process. In this case, an RNA editing event specifically changes the geneencoded glutamine codon (CAG) by modification of adenosine to guanosine(12) . The formation of a short dsRNA structure between the exonic sequence around the editing site and the downstream intron sequence, as well as dsRNA formed between inverted repeat structures in the intron, are essential for RNA editing of these GluR-B transcripts(17, 18) . Furthermore, additional adenosines are converted to guanosines in the intron, and at the third base of another glutamine codon within the dsRNA structure of GluR-B transcripts(17, 18) . These two features, a requirement for dsRNA formation and multiple adenosine modifications are the hallmark of DRADA. Finally, the presence of an inosine residue in the edited GluR-B RNA at the Q/R site was directly demonstrated recently, confirming that the A to G conversion detected by cDNA sequencing is indeed derived from the A to I change of the RNA(19, 20) . The accumulated evidence strongly suggests that DRADA is involved in this particular RNA editing process in mammalian brain (17, 18) .

We have recently cloned and characterized the human DRADA cDNA(21) . The deduced primary structure of the human DRADA revealed a bipartite nuclear localization signal(22) , three repeats of a dsRNA binding motif (DRBM)(23, 24, 25, 26, 27, 28, 29) , and the presence of sequences conserved in the catalytic center of other deaminases(30, 31, 32) . In this study, we have carried out deletion and site-directed mutagenesis of the dsRNA binding domains, and also other amino acid residues that we postulate to be involved in zinc coordination and formation of the catalytic center of DRADA. Our results suggest that the three repeats of the DRBMs are not functionally equivalent to each other, though their amino acid sequences are well conserved. The first and third DRBM seem more important for the DRADA activity, while the second DRBM can be removed without affecting the enzymatic activity. Since the deletion of any one DRBM did not significantly change the K of mutant DRADA proteins for substrate dsRNAs, DRBM1 and DRBM3 may participate in the catalytic mechanism in addition to RNA binding. We identified four amino acid residues essential for the enzyme's catalytic activity. The residues His and Cys, as previously predicted(22) , as well as an additional cysteine residue, Cys, may be involved in zinc chelation. The residue Glu is also found to be indispensable, confirming its possible role in the proton transfer reactions that occur during catalysis by DRADA.


EXPERIMENTAL PROCEDURES

Materials

P nuclease was purchased from Pharmacia (Piscataway, NJ). Protein-gold protein quantitation solution was purchased from Integrated Separation Systems (Hyde Park, MA). FLAG epitope-tag peptide and anti-FLAG M2 affinity gel were purchased from Eastman Kodak Co. Proteinase inhibitors phenylmethylsulfonyl fluoride, pepstatin A, leupeptin, and aprotinin were obtained from Sigma.

Oligonucleotides

All oligonucleotides used for deletion and site-directed mutagenesis are as follows: OLStuIm, 5`-AACTACTTCAAAAAGGGCCTGAAGG-3`; OLM1, 5`-AACTGACAGAGTGCCAGCTG-GCCAAGGACAGTGGAAAATC-3`; OLM2, 5`-CAGCCACATCCTTCT-CTGATAACCAGCCTG-3`; OLM3, 5`-TCGTGAGATACCTGA-AGAAGGCAGAACGCA-3`; OLC909-S, 5`-CTGTCAATGACTCCCATGCAGAAATA-3`; OLH910-Y, 5`-GTCAATGACTGCTATGCAGAAATAATC-3`; OLE912-A, 5`-ACTGCCATGCAGCAATAATCTCCCG3-`; OLH958-Y, 5`-GACTGTGTCATTCTATCTGTATATCAG-3`; OLC966-S, 5`-CACTGCTCCGTCTGGAGATGGCG-3`; OLC1036-S, 5`-GTACCATGTCCTCTAGTGACAAAATC-3`; and OLA1051-T, 5`-GGCCTGCAAGGGGCACTGTTGACCC-3`. The mutated bases are underlined. The junction sites for the deletion mutants are indicated by hyphens. For construction of pVL-F-DRADA140, two polymerase chain reaction (PCR) primers were used: FLAGUP, 5`-ATAAGAATGCGGCCGCTAAACATGGCTGACTACAAGGACGACGATGACAAGATGAATCCGCGGCAGGGG-3`, and FLAGDW, 5`-ATCATACGAGGTCACTGTCAGATCTGC-3`. Restriction sites (underlined) for NotI for the sense primer (FLAGUP) and XbaI for the antisense primer (FLAGDW) were included in the 5` end.

Deletion and Site-directed Mutagenesis

A recombinant construct that coded for a full-length DRADA (pVL-DRADA140) was used as a starting plasmid(21) . The total size of the plasmid is about 14 kb with 4.5 kb of cDNA containing the entire translated region of DRADA as an insert in pVL1393 (Pharmingen, San Diego, CA). For preparation of C1, 2, 3, and 4, the wild-type plasmid was subjected to deletion mutagenesis around the putative C-terminal deaminase catalytic domain (amino acids 835-1221) using ExoIII exonuclease (33) after cleavage at the StuI site located near the C-terminal end. The linearized DNA was incubated with ExoIII nuclease (New England Biolabs Inc.) for various times in order to generate nested deletions. The reaction products were then treated with mung bean nuclease (Boehringer Mannheim) to trim the ends of the ExoIII-treated plasmid DNAs. Inserts with various sizes of deletions were released by restriction digestion at the SstI site located between two DRBMs. The digestion products were fractionated on a 1.5% agarose gel, and the purified fragments were subcloned back into the SstI and StuI sites of the wild-type plasmid. The resultant C-terminal deletion clones were sequenced through the junction sites, using the 373A DNA sequencing system (Applied Biosystems, Foster City, CA). The DNA sequences obtained were analyzed by the BESTFIT program of the University of Wisconsin Genetics Computer Group (GCG) sequence analysis software (version 7.0)(34) . Only in-frame deletions were selected for further experiments.

DRBM deletion mutants and also point mutations of the catalytic domain were constructed by using the commercially available Transformer Site-Directed Mutagenesis Kit (Clontech, Palo Alto, CA) with some modifications. The system requires a restriction site unique for the plasmid to be used for designing of a selection oligo. The StuI site located 75 bp upstream of the stop codon was chosen as a selection site. The selection oligo, StuIm, carries a single base substitution that will eliminate the StuI site without changing any amino acid residue. Both selection oligo and mutagenic oligo were phosphorylated using T4 DNA polynucleotide kinase (New England Biolabs Inc.). The plasmid pVL-DRADA140 (0.1 µg) denaturated by 0.2 N NaOH was mixed with 0.1 µg of selection oligo StuIm and 0.1 µg of a mutagenic oligo in a total volume of 20 µl annealing buffer (Clontech), heated at 100 °C for 3 min, and cooled on ice. The mutant strand was synthesized by incubating the reaction mixture with T4 DNA polymerase (Clontech) and T4 DNA ligase (Clontech) at 37 °C for 2 h. The wild-type plasmid DNA was linearized by StuI, while the hybrid of wild-type strand and newly synthesized mutant strand remained uncut. The restriction digestion products were transformed into Escherichia coli BMH 71-18 mutS strain (Clontech). The transformants were suspended in 500 µl of Luria Broth (LB) medium (Bio101), and 20 µl of the cell suspension were inoculated into 1.5 ml of LB containing ampicillin 100 µg/ml, and cultured at 37 °C for 6 h. The plasmid pool was isolated by a quick alkaline method (33) and digested with StuI, once again to linearize the wild-type plasmid. The digestion product was then transformed into E. coli DH5, and 10 clones each of the resultant transformants were screened for the absence of the StuI site. The candidate mutant clones were sequenced using Sequenase version 2.0 (U. S. Biochemical Corp.) to verify the mutation site. All point mutation constructs were designated as C909-S, H910-Y, E912-A, H958-Y, C966-S, C1036-S, and A1051-T in accordance with the names of the mutagenic oligonucleotide.

All single DRBM (amino acids 502-798) deletions, M1, M2, and M3, were achieved as described above using the same kit and selection oligo. The mutagenic oligos were designed in such a way that each has 15-20 bases of homology to both flanking sequences of the deletion. The precise deletions were confirmed by sequencing. Double DRBM deletion mutants M1M3 and M2M3 were reconstructed from single deletion mutants using a unique restriction site AflII located near the NH-terminal and SstI located between DRBM2 and DRBM3. The AflII-SstI fragments of M1 and M2 were subcloned into M3 at AflII and SstI sites, respectively, resulting in double DRBM deletions of M1M3 and M2M3.

pVL-F-DRADA140 carrying an additional FLAG epitope-tag (35) at the NH-terminal of the full-length DRADA wild-type protein was constructed as follows: the entire coding region of DRADA was amplified using a plasmid pVL-DRADA140 (21) and a set of PCR primers FLAGUP and FLAGDW. This PCR procedure created a methionine residue followed by the polypeptide sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG epitope-tag), at the 5` of the NH-terminal of the authentic DRADA140 protein. In addition, it also removed the GC-rich 5`-untranslated region and long 3`-untranslated region, and created a new Kozak (36) sequence that is strongly preferred by baculovirus for protein translation initiation (37) at the new NH-terminal region. The PCR products were digested with NotI and XbaI, and the 3.9 kb NotI/XbaI DNA fragment was gel purified, and then ligated into NotI and XbaI sites of pVL1392 vector (InVitrogen, San Diego, CA). DRBM deletions with FLAG epitope, pVL-F-M1, pVL-F-M2, and pVL-F-M3 plasmids, were prepared by excising the AflII-XbaI fragments (3.7 kb) containing deletions from the original mutant constructs and ligating them into the AflII and XbaI sites of pVL-F-DRADA140. A point mutation with FLAG epitope, pVL-F-E912-A, was created in the same way as other point mutations except that pVL-F-DRADA140 was used as a starting plasmid. The DRADA proteins containing the FLAG epitope-tag prepared from these constructs were purified by a monoclonal antibody M2 affinity column chromatography (Kodak).

Cell Culture

Spodoptera frugiperdera (Sf9) cells were obtained from The Wistar Institute Expression Core Facility. The insect medium TMN-FH with 10% fetal bovine serum was purchased from Pharmingen, and supplemented with 0.1% Pluronic-F68 (Life Technologies, Inc.), and 1% antibiotic-antimycotic (Life Technologies, Inc.).

Recombinant Baculovirus

To obtain recombinant baculovirus expressing human DRADA proteins, 2 10 Sf9 cells were co-transfected with a mixture of 3 µg of recombinant plasmid and 2.5 µg of BaculoGold linearized baculovirus DNA following the manufacturer's instructions (Pharmingen). The transfected cells were incubated at 27 °C for 5 days. Primary recombinant viruses collected as supernatant of the culture were then reamplified by infecting 1 10 Sf9 cells with 500 µl of primary recombinant virus stocks. Amplified viruses were collected after 3 days of incubation at 27 °C.

Virus Infection and Cell Extract Preparation

Approximately 1 10 Sf9 cells were infected with 500 µl of amplified recombinant virus, incubated at 27 °C for 48 h. The infected cells were collected and pelleted by centrifugation. The infected cells were washed once with 5 ml of ice-cold phosphate-buffered saline without CaCl or MgCl and 1 ml of TEN (0.15 M NaCl, 40 mM Tris-Cl, pH 7.5, 1 mM dithiothreitol (DTT), and 1 proteinase inhibitor mix), then resuspended in 150 µl of 0.25 M Tris-Cl, pH 7.8, containing 1 mM DTT and 1 proteinase inhibitor mix. Following sonication, the cell debris was pelleted at 4 °C by centrifugation. The supernatant was mixed with an equal volume of buffer containing 0.1 M Tris-Cl, pH 7.5, 0.2 M NaCl, 10 mM EDTA, 40% glycerol, 2 mM DTT, and 2 proteinase inhibitor mix. The proteinase inhibitor mix (500) was made with phenylmethylsulfonyl fluoride (0.2 M), pepstatin (1.5 mM), leupeptin (4.2 mM), and aprotinin (0.3 mM). For each construct, three to six independent infections and cell extract preparations were carried out in order to obtain a reliable quantitative estimate for the mutated DRADA enzyme activity.

In Vivo Labeling of Recombinant DRADA Protein

The production of recombinant DRADA protein was assessed by [S]methionine labeling of infected cells(37) . Sf9 cells (2 10) were infected with 500 µl of each amplified recombinant virus stock at multiplicity of 10-20 for 46 h. The infected cells were collected and resuspended in 1 ml of methionine-free Grace's medium (Life Technologies, Inc.) with 10% heat-inactivated fetal bovine serum. Following incubation at 27 °C for 1 h, the medium was replaced with 1 ml of fresh methionine-free medium containing 40 µCi of TranS-label (ICN Biomedicals, Costa Mesa, CA) and incubated for another 1 h. The cells were washed with 1 ml of phosphate-buffered saline three times. The labeled protein was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (8%) and fluorography.

Purification of Recombinant DRADA Proteins

The recombinant DRADA proteins, F-DRADA140, F-DRADAM1, F-DRADAM2, F-DRADAM3, and F-DRADAE912-A containing a FLAG epitope-tag were purified on an anti-FLAG M2 immunoaffinity gel column. Approximately 20 mg of crude extract protein made from Sf9 cells infected with various recombinant baculoviruses were applied to 1 ml of anti-FLAG M2 affinity gel (Eastman) equilibrated with buffer A (0.05 M Tris, pH 7.0, 0.05% Nonidet P-40, 20% (v/v) glycerol, and 1 mM of -mercaptoethonol plus 0.15 M NaCl). The flow-through fraction was applied two more times to the same column to increase the binding efficiency of the recombinant protein. The column was washed with 30 ml of buffer A containing 0.15 M NaCl, then with 30 ml of buffer A containing 0.75 M NaCl and once again with 30 ml of buffer A containing 0.15 M NaCl. The recombinant DRADA protein was extracted with 6 ml of buffer A containing 75 µg/ml of FLAG peptide. The eluted proteins were pooled and concentrated by Centricon 30 (Amicon, Beverly, MA) and stored at -70 °C.

Protein Assay

Protein concentration was determined using the Bio-Rad protein assay reagent or the integrated separation systems protein-gold assay system with bovine serum albumin as a standard. The purified proteins were analyzed by one-dimensional SDS-PAGE (4% acrylamide stacking gel, 8% acrylamide resolving gel)(38) . Protein bands were visualized by silver staining(39) .

DRADA Assay

The DRADA enzymatic activity was assayed in vitro(4, 40) . Unless specified otherwise, the reaction was carried out in 100 µl, which contained 10 fmol of [-P]ATP-labeled c-myc dsRNA, 0.05 mM Tris (pH 7.0), 0.2 M NaCl, 5 mM EDTA, 1 mM DTT, 20% glycerol, and various amounts of cell extract or purified proteins. After incubation at 37 °C, the reaction products were deproteinized and then precipitated with ethanol as described previously. The DRADA reaction products were digested with nuclease P into 5`-mononucleotides and analyzed by one-dimensional TLC. The solvent system used was 0.1 M sodium phosphate, pH 6.8, ammonium sulfate, 1-propanol, 100:60:2 (v/w/v), as described previously(41) . The radioactivity of the adenosine and inosine spots on TLC plates was quantified by a PhosphorImaging system (Molecular Dynamics, Sunnyvale, CA).

RNA-binding Assay

A filter-binding assay (42) was carried out by using recombinant DRADA proteins containing the FLAG epitope-tag at their NH-terminal and [-P]ATP-labeled c-myc dsRNA (575 bp), or c-myc antisense single-stranded RNA (ssRNA) (605 nucleotides)(4) . Each reaction mixture, kept on ice, contained 10 ng of DRADA and varying quantities of dsRNA or ssRNA (0.05-5 nM) in 100 µl of the DRADA assay buffer containing 0.05 M Tris (pH 7.0), 0.1 M NaCl, 5 mM EDTA, 1 mM DTT, 20% glycerol, 5 µg/ml E. coli tRNA, and 5 µg/ml bovine serum albumin. The reactions were incubated for 10 min at 37 °C and immediately filtered through a nitrocellulose membrane (25 mm and 0.45 µm; Millipore, Bedford, MA) on a manifold filtration apparatus (Schleicher & Schuell) at a slow flow rate. The membranes were subsequently washed five times with 0.5 ml each of the ice-cold DRADA assay buffer, dried, and subjected to liquid scintillation counting.


RESULTS

Identification of Four Critical Residues Likely to Be Involved in Zinc Chelation and Proton Transfer of DRADA

We aligned the human DRADA sequence with other known deaminases and with a recently identified nematode gene, T20H4.4(43) , that has a high degree of homology to DRADA and is postulated to be a prototype of the non-vertebrate DRADA gene(21) . We found that several short stretches of contiguous amino acid residues, including two tripeptide sequences HAE and PCG located in the C-terminal region, are highly conserved (Fig. 1). We believe these conserved amino acid residues are directly involved in the catalytic mechanism of DRADA(21) . Among these residues, of special interest are the amino acids histidine and cysteine, which can participate in coordination of a zinc atom and formation of a catalytic center, as was predicted by examination of the three-dimensional structure of adenosine deaminase (44) and cytidine deaminase(45) . We first prepared four serial deletion mutations in the C-terminal region as recombinant baculovirus DRADA C1, C2, C3, and C4 (Fig. 1). The mutated DRADA proteins were expressed as recombinant baculovirus in insect cells (Fig. 2A), and the A to I conversion activity of altered enzymes were tested in vitro using crude extract proteins made from Sf9 cells infected with four different DRADA mutant constructs (Fig. 2B). Although host Sf9 cells also contain a trace amount of the DRADA activity (0.2% A to I conversion in one experiment), its very low level did not interfere with our quantitation of the enzymatic activity for each mutant protein. We found that all of the C-terminal deletion mutants lost enzyme activity (Fig. 2B), probably due to a drastic change in the folding of the enzyme structure. Therefore, for mutagenesis of this putative catalytic domain, site-directed point mutations at some of the conserved amino acid residues were introduced (Fig. 1). Based on alignment between human DRADA and T20H4.4(43) , all histidines (His and His) and cysteines (Cys, Cys, and Cys) found in the regions conserved between these two genes were mutated to conservative replacements, tyrosine and serine, respectively. Among these residues, His and Cys are within the two tripeptide sequences highly conserved among different cytidine and deoxycytidylate deaminases(30, 31, 32, 46) . These residues are known to be involved in zinc-chelation at least in the case of cytidine deaminase(45) , whereas Cys, His, and Cys were targeted as the potential third and fourth zinc-coordinating amino acid residues.


Figure 1: Structure of DRADA and mutants. The structures of the wild-type (WT) human DRADA and all deletion and point mutants used in this study are schematically shown. The locations of the bipartite nuclear localization signal, Nuc (filled box), three repeats of DRBM1, 2, and 3 (hatched boxes), and the deaminase catalytic domain (striped box) are indicated. The location of a unique StuI restriction site used for designing the selection oligo as well as AflII and SstI sites also utilized for preparation of mutant constructs are indicated. The lines depict the DRBM (M1, M2, M3, M1M3, and M2M3) and the deaminase catalytic domain (C1-C4) deletion mutants. The numbers below each line indicate the amino acid residues present in the deletion mutant proteins. In the lower panel, the residues within the deaminase catalytic domain changed by site-directed mutagenesis are indicated by arrows below the mutated wild-type residues. The sequence of 15 short stretches of contiguous amino acid residues, found to be conserved between the human DRADA and a nematode gene T20H4.4 and proposed to form the deaminase catalytic domain (amino acids 889-1160), is shown. The two tripeptide sequences, HAE and PCG, which are highly conserved among deaminases and predicted to participate in the formation of the catalytic center, are highlighted. A summary of the DRADA enzymatic activity (A to I conversion) of each mutant observed is indicated at right or bottom. The DRADA activity is coded in the range +++ to +, where +++ is wild-type activity and + indicates a low but significantly higher than background level of activity.




Figure 2: Characterization of the C-terminal deletion mutants. A, fluorography of the SDS-PAGE analysis for Sf9 cells infected with the C-terminal deletion mutants is shown. Forty-six h after infection with recombinant baculoviruses, Sf9 cells were labeled with [S]methionine, and proteins were fractionated by SDS-8% polyacrylamide gel electrophoresis. In addition to the expected size band, the C1 construct produced discrete lower molecular weight protein bands which may be the products of proteolysis of the full-length mutant protein. B, DRADA activity for the C-terminal deletion mutants was determined by a base modification assay. The 100-µl reaction contained 50 µg of extract proteins and 10 fmol of [-P]ATP-labeled c-myc dsRNA in the standard assay buffer (see ``Experimental Procedures''). Following incubation for 1 h at 37 °C, the reaction product was deproteinized and digested with P nuclease. The digests were analyzed by one-dimensional TLC. The relative positions for inosine 5`-monophosphate (pI) and adenosine 5`-monophosphate (pA) are indicated. Inosines converted from adenosines were estimated by quantitating the ratio of pI and pA spots on TLC plates with the PhosphorImaging system (Molecular Dynamics).



We found that substitutions at His and Cys abolished the enzyme activity completely, whereas DRADA mutants with alterations at Cys and His still exhibited some enzyme activity (32 and 7% of the wild-type, respectively), suggesting the critical importance of His and Cys in zinc coordination (Fig. 3). Substitutions of Cys also abolished the enzyme activity, and, therefore, Cys residue may act as the third protein ligand of the zinc, together with His and Cys. Cys and His may also play important roles for the DRADA catalytic mechanism, but probably not as zinc-chelating ligands.


Figure 3: Characterization of the deaminase domain mutants. A, fluorography of the SDS-PAGE analysis; B, analysis of the DRADA activity. The percentage of adenosine 5`-monophosphate (pA) to 5`-monophosphate (pI) conversion by the wild-type DRADA was treated as 100; the relative activity of the mutants is shown at the bottom. The assay conditions were as described in Fig. 2.



Glu, which is expected from the three-dimensional structure of E. coli cytidine deaminase (45) to subserve the critical proton transfer function of the enzyme, was changed to alanine. The DRADA mutant E912-A with a Glu Ala substitution exhibited no enzyme activity (Fig. 3). Mutant A1051-T retained activity almost equal to that of wild-type (Fig. 3).

The First and Third DRBM Repeats Are Critical for DRADA Catalytic Activity

The three repeats of DRBM present in the central region of DRADA are predicted to serve as a substrate binding domain for this enzyme(21) , which has a strict specificity for dsRNAs(1, 2, 5) . We confirmed this region as the dsRNA binding domain by expressing this region containing only the three DRBMs as a glutathione S-transferase fusion protein in E. coli and testing it by the filter binding assay. The glutathione S-transferase-3DRBM truncated DRADA protein exhibited a strong affinity for dsRNA (data not shown). In order to examine the roles played by these motifs in the DRADA enzyme activity, we then generated five deletion mutants in which each of the three DRBMs, or a combination of two DRBMs was deleted from the intact wild-type DRADA ( Fig. 1and Fig. 4A). We assayed these DRBM mutants for their enzyme activity, and found that two deletion mutants M1M3 and M2M3, which have only a single DRBM, lost activity completely (Fig. 4B), suggesting that the DRADA enzymatic activity requires more than one DRBM. In contrast to the results obtained with single DRBM mutants, unexpected results were obtained with the DRBM mutants in which only one of the three DRBMs had been eliminated. While the mutants M1 and M3 exhibited, if any, very little activity, the mutant M2 retained enzymatic activity comparable to that of wild type (Fig. 4B). These results suggest not only that DRADA requires at least two DRBMs, but also that the first and third repeats may provide indispensable functions for its enzymatic action. This result, indicating the nonequivalent nature of the three DRBMs, is surprising in light of the highly conserved amino acid sequences of the three DRBMs(21) , and also the notion that the DRBMs found in other dsRNA-binding proteins may be equivalent to each other(28, 29) .


Figure 4: Characterization of the DRBM deletion mutants. A, fluorography of the SDS-PAGE analysis; B, analysis of the DRADA activity. The assay conditions were as described in Fig. 2.



The Expression Levels of Mutant Proteins

In vivo labeling and analysis of the recombinant DRADA proteins by SDS-PAGE confirmed that, in general, the mutant proteins and the wild-type protein were present at similar levels in the infected Sf9 cells (Figs. 2A, 3A, and 4A). All deletion mutants showed a decreased size as expected. For certain mutants, especially C909-S, the decreased protein level could be a factor in the reduced activity of crude extracts of Sf9 cells (32% of wild-type level). However, the complete loss of the enzymatic activity observed with H910-Y, E912-A, C966-S, and some deletion mutants cannot be due to a reduced protein level. These mutants did not show any A to I conversion activity even after increase of the extract proteins or the incubation time for the enzyme assay (data not shown). In addition, Sf9 cells were infected three to six times separately with individual recombinant viruses at various multiplicity. The base modification assays were carried out with cell extracts made from these separate infections. We found that the relative DRADA activities among wild-type and mutants, M2, C909-S, H958-Y, and A1051-T, were very similar (within ± 20%) among different extracts (data not shown). The complete loss or dramatic decrease of the DRADA activities for H910-Y, E912-A, C966-S, M1, M3, M1M3, M2M3, and all of C-terminal region deletion mutants was also confirmed by separate infection experiments. Thus, the total loss of the enzyme activity observed with certain mutants cannot be attributed to variation of virus infection efficiency nor a decrease in the levels of their protein expression in the Sf9 host cells.

Binding of DRBM Deletion Mutants to dsRNA

Because our results with DRBM mutants raised the possibility that each of the three DRBMs may have significantly different functions in the DRADA reaction, we next examined binding of these DRBM deletion mutants to dsRNA using purified recombinant DRADA proteins, a synthetic c-myc dsRNA (575 bp), and a nitrocellulose filter binding assay traditionally used for quantitation of interactions of nucleic acid binding proteins with K stronger than 1 nM(42) . This assay was used previously to determine the substrate dissociation constant of wild-type DRADA purified from bovine liver, which turned out to have a much higher affinity for substrate than other deaminases (40) .

Purified recombinant DRADA proteins were used for the binding assay instead of crude cell extracts (Fig. 5). New baculovirus expression plasmids carrying an epitope-tag peptide FLAG (35) at the NH-terminal end were prepared from original constructs DRADA-140 (WT), M1, M2, and M3 as well as the catalytic domain mutant E912-A. The recombinant FLAG-DRADA fusion proteins expressed in infected Sf9 cells were purified using the FLAG epitope by anti-FLAG M2 antibody affinity column chromatography (Fig. 5B). The binding of all DRADA mutants maintained specific binding to dsRNA, and did not bind to ssRNA (Fig. 5A). The affinity constant was obtained by Scatchard-type analysis (Fig. 5A). The reverse Scatchard plots for wild-type and all mutant DRADA proteins tested were almost superimposable and, therefore, plots for only wild-type DRADA, M1, and M2 are shown in Fig. 5. The K, derived from the intercept on the abscissa of the Scatchard plot, was identical (0.11 nM) for wild-type and all four of the mutant DRADA proteins. It is of special interest that DRBM deletion mutants F-M1, F-M2, and F-M3 all have K values identical to that of the wild-type protein. This shows that the dramatically decreased DRADA enzymatic activity observed with M1 and M3 is not due to their altered dsRNA binding capacity.


Figure 5: Binding of DRADA mutants to dsRNA. Binding of purified recombinant DRADA mutants as well as wild-type protein to [-P]ATP labeled c-myc dsRNA (575 bp) or antisense ssRNA (nt 605) was analyzed by a nitrocellulose binding assay. Ten ng of purified FLAG-DRADA fusion protein for each mutant were incubated in duplicate with various concentrations of dsRNA or ssRNA, as described under ``Experimental Procedures.'' The reverse Scatchard plots of c-myc dsRNA binding to wild-type and various DRADA mutants were obtained. Since the plots for all mutants analyzed were almost identical to that of wild-type DRADA, plots for only wild type, M1, and M2 are shown. The intercept at the abscissa defines the K, which was 0.11 for wild type and all four mutants (M1, M2, M3, and E912-A). B, the purified recombinant DRADA proteins used in this study were examined by SDS-PAGE and silver staining.




DISCUSSION

The Catalytic Mechanism of the DRADA Reaction

Site-directed mutagenesis of His and Cys completely abolished the enzyme activity of the mutated DRADA proteins, despite the fact that both amino residues were substituted by conservative replacements. This result reinforced our hypothesis that these two amino acid residues, both located within the tripeptide sequence HAE and PCG conserved among different cytidine and deoxycytidylate deaminase family members(31, 32, 45, 46) , play a critical role for the DRADA catalytic mechanism. Although the presence of a zinc atom within DRADA has not yet been demonstrated directly, the presence of short stretches of ``deaminase'' consensus tripeptide sequences strongly suggests that these two residues participate in zinc chelation. The recent x-ray crystallography of E. coli cytidine deaminase confirmed the postulated zinc-chelation site for that enzyme(45) .

Three other amino acid residues, potentially able to participate in zinc chelation, were also mutated in this study. Of these, the mutation of Cys also resulted in the loss of enzyme activity. Since the coordination of the zinc atom requires at least three protein ligands for both adenosine deaminase (44) and cytidine deaminase(45) , it is reasonable to postulate that this Cys residue is also involved in zinc chelation. Our site-directed mutagenesis of Glu confirmed the importance of this residue for the enzymatic action. It most likely plays a critical role in proton transfer functions required for the deamination reaction, predicted once again by analogy from the three-dimensional structure of E. coli cytidine deaminase(45) . Interestingly, a similar mutation for the cytidine deaminase subunit (REPR) of apolipoprotein B100 RNA editing enzyme has also been reported to abolish the enzymatic activity (47) . A1051-T represents an example of a residue which can tolerate a conservative substitution and maintain almost full activity of the DRADA enzyme (Fig. 3), even though Ala is one of the amino acid residues conserved between the catalytic domain of DRADA and T20H4.4(21) . The result suggests that some of the conserved residues such as His and Glu are absolutely necessary for the enzyme function, while others contribute only to the maintenance of the overall protein folding and structure, and accommodate conservative sequence alterations. Obviously, the final functional assignment of amino acid residues shown as indispensable in this study must wait the structural studies of DRADA complexed with dsRNA.

Distinctive Functions of the Three DRBMs in the DRADA Enzyme Action

The DRBM is approximately 70 amino acid residues. It may be divided into a less conserved NH-terminal two-thirds and a more conserved C-terminal region. The C-terminal region enriched for positively charged amino acids, especially arginine, and predicted to form an -helix, may interact directly with the dsRNA helix structure(26, 28) . This new type of RNA binding motif is found in a growing family of proteins(24, 25, 26, 27, 28, 29, 47) . The DRBM elements of these proteins have already been tested for their dsRNA binding capacity with recombinantly expressed DRBMs(23, 24, 26, 27, 28, 29) . It appears that each DRBM is usually capable of binding to the dsRNA independently(28, 29) , except in certain cases where at least two repeats were needed for efficient binding(25, 26) . There are two different types of DRBM, a full-length long repeat and a short repeat(29) . Some previously sequenced DRBMs have only the C-terminal -helix region, which led to the hypothesis that the N-terminal two-thirds of the motif are less important for dsRNA binding(29) . DRADA contains three full-length DRBM repeats(21) .

Although an increasing number of proteins that interact with RNA have been discovered in recent years(48) , the exact nature of interaction between these proteins and RNA is poorly understood. However, sequence-specific interaction of DNA or RNA with protein often accompanies conformational change of the nucleic acid so that specific bases in the nucleic acids are in close contact with the amino acid residues responsible for the interaction(49, 50) . Such conformational changes upon protein binding include melting of base pairings in the double-stranded region(51) . DRBMs not only act as the substrate binding domain but also may generate critical conformation distortion in the dsRNA, including disruption of the limited base pairings around the modification site, to allow the adenosines to be exposed and accessible to the catalytic domain of DRADA as we postulated previously (21) .

Because of the very similar amino acid sequence of three DRBMs (Fig. 6), we originally speculated that these three motifs together contribute evenly to the unusually high affinity of the enzyme binding to its substrate(21) . However, our results presented in this study suggest that the three motifs are not equivalent and that DRBM1 and DRBM3 may play more important roles for the enzyme action than DRBM2. In fact, the DRBM2 is dispensable at least for the A to I conversion activity of DRADA. One possible explanation for this unexpected result is that DRBM2 may provide a spacer or hinge in the wild-type enzyme to give structural flexibility for the other two DRBMs located on either sides of this motif, DRBM1 and DRBM3. The space between DRBM1 and DRBM2 or DRBM2 and DRBM3 are almost identical, 40 and 41 amino acids, respectively, which may not be long enough for two neighboring motifs to interact with each other upon binding to a substrate dsRNA. Alternatively, the spacer may be necessary to position the -helix forming region of the two DRBMs on the same side of the RNA duplex. Such flexible interaction and orientation of the two DRBMs may be necessary for strong binding affinity of DRADA to dsRNA(5, 40) . Because of the way the deletion was made, the DRBM deletion mutant M2 contains a longer spacer region (71 amino acids) between the remaining DRBM1 and DRBM3, which may give a similar spacer effect of the DRBM2 played in the wild-type enzyme. However, if this is the case, one may expect significant differences in the dsRNA binding affinity between wild-type or M2 and M1 or M3. In contrast, K values for all three DRBM deletion mutants were nearly the same as that of wild-type DRADA protein, ruling out a simple spacer effect.


Figure 6: Difference among the three repeats of the DRBM in DRADA. The amino acid sequences of DRADA DRBMs are shown together with the consensus sequence obtained from alignment of 23 different repeats found in 11 DRBM-containing proteins(21, 29) . The C-terminal region of the DRBM, predicted to form an -helix(26, 28) , is indicated. Certain amino acid residues, part of the consensus sequence, and present only in DRBM1 and DRBM3 but missing from DRBM2, are indicated by arrowheads.



By comparing the amino acid sequences for the three DRBM, we noticed that there are several amino acid residues of the DRBM consensus sequence present in DRBM1 and DRBM3 but missing in DRBM2 (Fig. 6). In addition, we recently compared the amino acid sequence of human and mouse DRADA deduced from cDNA sequences.()Although all three DRBM sequences are highly conserved between human and mouse DRADA, DRBM1 and DRBM3 contain 10 and 9 amino acid residue extensions of evolutionarily conserved sequence upstream of the N-terminal boundary of the 72-amino acid motif, whereas DRBM2 does not contain this 5` extension. The evolutionarily conserved sequence present upstream of DRBM1 and DRBM3 may add to these two repeats additional functions other than dsRNA binding, which distinguishes them from DRBM2.

One enigma concerning the DRADA reaction is the fact that the amino group of adenosine to be removed by DRADA through hydrolytic deamination is engaged in Watson-Crick base pairing with uracil, forming the RNA duplex structure of the substrate RNA. The A-U base pairing must be disrupted prior to initiation of the deamination reaction(21) . It may be that the specific amino acid sequence unique to DRBM1 and DRBM3 allows them, possibly acting in concert, to destabilize or unwind A-U base pairs, creating a local ssRNA region and non-base-paired adenosine residues and providing easy access for the deaminase catalytic domain of DRADA. The hypotheses discussed will be tested by creating more mutant constructs in our future studies.

Separate Functional Domains of DRADA

As we predicted previously(21) , results from the present studies suggest that DRADA indeed contains at least two separate functional domains: a substrate binding domain consisting of three repeats of DRBM and the C-terminal region consisting of the catalytic or deaminase domain. These two separate functional domains likely interact with each other, especially if adenosine residues to be deaminated are located within the dsRNA region to which the binding domain binds. As already mentioned in the previous section, some DRBM repeats may play active roles in the deamination process. DRADA seems to be self-contained, with all of the necessary functional domains within it, and recombinantly expressed and purified enzyme is indeed capable of carrying out deamination of multiple adenosines to inosines in vitro by itself. This structural feature of DRADA is in contrast to that of the recently cloned REPR, the cytidine deaminase component of the multisubunit complex (editosome) for editing of apoliprotein B100 RNA(46) . In this editing system, the substrate binding subunit, which recognizes a specific cis-RNA element termed the ``mooring sequence'' essential for precise editing, seems to be required in addition to REPR (52) . Moreover, an additional auxiliary factor may also be required for REPR to carry out precise editing of apoliprotein B100 RNAs(46, 52) . In view of the high degree of precision necessary for selecting specific adenosine residues at the GluR RNA editing sites(10, 11, 12, 18) , the lack of sequence specificity of DRADA has been a puzzle(7, 8, 18) . It remains possible that the apparently self-contained enzyme, DRADA, may yet require additional factor(s) for site-selective editing of natural substrate RNAs such as GluR transcripts in vivo. DRADA mutants generated in this study are likely to be useful for our future studies of RNA editing by DRADA.


FOOTNOTES

*
This work was supported by National Institutes of Health grants GM40536, CA09171, and CA10815, and grant PRG-94-156 from the Alzheimer's Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: The Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104. Tel.: 215-898-3828; Fax: 215-898-3911.

The abbreviations used are: dsRNA, double-stranded RNA; DRADA, dsRNA adenosine deaminase; DRBM, dsRNA binding motif; Sf9, Spodoptera frugiperdera; ssRNA, single-stranded RNA; PAGE, polyacrylamide gel electrophoresis; GluR, glutamate receptor; DTT, dithiothreitol; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s).

R. Drakas and K. Nishikura, unpublished data.


ACKNOWLEDGEMENTS

We thank Dawn H. Marchadier in the Wistar Protein Expression Core Facility for her excellent technical assistance with recombinant baculovirus expression, and Drs. Roger M. Burnett and John M. Murray for helpful discussion and critical reading of this manuscript. We also thank the Wistar Editorial Services Department for preparing the manuscript.


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