(Received for publication, August 26, 1996, and in revised form, October 23, 1996)
From the The double-stranded RNA-specific adenosine
deaminase (ADAR) is an interferon-inducible RNA-editing enzyme
implicated in the site-selective deamination of adenosine to inosine in
viral RNAs and cellular pre-mRNAs. We have isolated and
characterized human genomic clones of the ADAR gene and
cDNA clones encoding splice site variants of the ADAR protein.
Southern blot and sequence analyses revealed that the gene spans about
30 kilobase pairs and consists of 15 exons. The codon phasing of the
splice site junctions of exons 3, 5, and 7 that encode the three copies
of the highly conserved RNA-binding R-motif (RI,
RII, and RIII) was exactly conserved and
identical to those R-motif exons of the interferon-inducible
RNA-dependent protein kinase. Alternative splice site
variants of the 1226-amino acid ADAR-a protein, designated b and c,
were identified that differed in exons 6 and 7. ADAR-b was a 5 Double-stranded RNA-specific adenosine deaminase
(ADAR)1 is an interferon-inducible,
dsRNA-binding protein (1-6). ADAR, also known as dsRAD and DRADA,
catalyzes the covalent modification of double-stranded RNA substrates
by hydrolytic C-6 deamination of adenosine to yield inosine (7, 8).
ADAR is implicated in two types of RNA editing processes. First, A-to-I
modifications are found at multiple sites in viral RNAs, as exemplified
by the biased hypermutations observed in negative-stranded RNA virus
genomes during lytic and persistent infections, as in the case of
measles virus (9, 10). Second, the C-6 adenosine deamination can be
highly site-specific, occurring at one or a few sites in certain viral
and cellular mRNAs as exemplified by hepatitis delta virus (HDV)
RNA (11) and the GluR receptor channel pre-mRNAs (12),
respectively. RNA editing plays an essential role in production of two
HDV proteins from one ORF, proteins which have different functions in
the life cycle of the closed circular single-stranded RNA HDV with the
editing site located in a self-complementary dsRNA structure (11).
Recent re-evaluation of HDV RNA editing demonstrated that the process
occurs on antigenomic RNA and involves a conversion of A to G (13, 14),
raising the possible role of ADAR in the regulation of HDV replication. ADAR is also presumed to be responsible for the specific RNA editing of
pre-mRNA transcripts in the brain encoding the
The molecular cDNA cloning of ADAR from human, rat, and bovine
cells has been described (6, 20, 21). The ADAR cDNA hybridizes to
an interferon (IFN)-inducible 7-kb mRNA and possesses a single long
open reading frame predicted to encode a 1226-amino acid protein (6,
20, 21). We isolated the 6.5-kb human cDNA encoding ADAR during a
screen for cDNA clones of proteins regulated by IFN (5, 6). Genomic
clones corresponding to the ADAR cDNA have likewise been isolated
and the gene mapped by fluorescence in situ hybridization to
human chromosome 1q21.1-21.2 (22).
The presence of two immunologically related forms of the human ADAR
deaminase was demonstrated in a variety of human cell lines using
antisera prepared against three non-overlapping regions of the human
cDNA expressed in Escherichia coli (6); an
interferon-inducible 150-kDa protein present in both the cytoplasm and
nucleus; and a constitutively expressed 110-kDa protein present
predominantly if not exclusively in the nucleus. A second cDNA for
the rat dsRNA adenosine deaminase, designated RED1, likewise hybridizes
to a 7-kb RNA but predicts a 771-amino acid protein (23), substantially different from the 1175-amino acid protein predicted by the originally described rat ADAR cDNA (21). Rat ADAR and RED1 possessed
differential editing activity for the R/G and Q/R sites of GluR (16,
23). Thus, a family of dsRNA adenosine deaminase enzymes with selective substrate specificity may exist that bind and edit different viral or
cellular RNA targets.
The dsRNA-binding properties described for purified ADAR proteins
(1-4) are similar to those elucidated for other known dsRNA-binding proteins, including the interferon-inducible RNA-dependent protein kinase PKR (24) in which the prototype dsRNA-binding R-motif was
discovered (25-27). ADAR possesses in its central region three copies
of the dsRNA-binding R-motif (6, 20, 21), a subdomain now found in
several known double-stranded RNA-binding proteins (28). The core amino
acid residues of the repeated R-motif subdomain of the protein kinase
PKR, established by mutagenesis as crucial in the case of the
dsRNA-binding activity of PKR (26, 29-31), are fully conserved in each
of the three R repeats found in ADAR (6, 32). We have described the
mutational analysis of the three RNA-binding R-motifs of ADAR, in which
the RI, RII, and RIII subdomains
were mutated at a highly conserved and critical lysine residue of the R
core (32). This lysine residue was previously established as one of the
R core residues essential for dsRNA-binding activity of the human PKR
and vaccinia virus E3L proteins (29-31, 33). Examination in
vitro of the various R-motif mutants of ADAR revealed that the
RIII subdomain was essential for both dsRNA deaminase
activity and dsRNA-binding activity, whereas RII was dispensable for both activities (32). Similar conclusions were derived
from the analysis of deletion mutants of ADAR (34). These results
together indicated that the three R-motifs of ADAR are
functionally distinct from each other in the context of binding dsRNA substrates in a functional manner recognized by the enzyme catalytic center for deamination. The catalytic C domain of ADAR was
mapped by mutagenesis to the C-terminal region of the protein (16, 32,
34).
Here we report the identification of three naturally occurring variant
forms of ADAR, including two alternative splice variants that are
differentially expressed in different tissues. In comparison to the
prototype ADAR (6, 20, 21), now designated herein as ADAR-a, one
isoform designated as ADAR-b contains a deletion of 26 amino acids at
the exon 7-intron VII junction which lies between the RIII
motif subdomain and the catalytic C domain. The other variant,
designated as ADAR-c, has an additional deletion of 19 amino acids at
the intron V-exon 6 junction which lies between the RII and
RIII motif subdomains. These splice site variants retain
the R-motif subdomains functionally intact, and all three of the ADAR
variants encode active enzymes that possess comparable deaminase
activity measured with a synthetic dsRNA substrate. Site-directed
mutagenesis of the R-motifs revealed that the presence of one or both
of the deletions in the spacer regions between the R-motifs and the
catalytic domain of the ADAR-b and -c variants altered the functional
importance of each of the individual R-motifs relative to that observed
for ADAR-a.
Oligonucleotides designed from the human
ADAR-a cDNA sequence (6) were used for site-directed mutagenesis of
the three dsRNA-binding R-motif subdomains (RI,
RII, and RIII), for screening cDNA
libraries by PCR, and for the construction of plasmid expression vectors. The following oligonucleotides were synthesized.
NotI(+)31, 5 cDNA libraries were
screened by polymerase chain reaction, using several primer pairs in
different combinations to scan the entire 3.7-kb open reading frame
region of the ADAR cDNA (6). PCR reactions (35) were performed
using native Taq DNA polymerase according to conditions
recommended by the manufacturer (Perkin-Elmer). A random-primed human
kidney cDNA library (Clontech) in The
PCR-based method for site-directed mutagenesis of the prototype human
ADAR cDNA (6), described previously in detail (32), was utilized
for the newly isolated splice site variants. The methods for
construction of the transcription vector plasmids were essentially as
described by Sambrook et al. (37). Chemicals were reagent
grade; enzymes were obtained from New England BioLabs unless otherwise
noted.
Starting with plasmid construction pcDNAI/Neo K88 (amino acids
296-1226) engineered to lack the 5 In
vitro transcription and translation were performed as described
previously (32) to confirm the protein coding capacity of each
construction. Briefly, XhoI-linearized plasmid DNA (5 µg)
of the wild-type or mutant versions of ADAR-a, -b or -c, either M1 or
M296, was transcribed in vitro using phage T7 RNA polymerase (New England BioLabs) according to the manufacturer's instructions; subsequent translation in vitro was carried out using the
nuclease-treated rabbit reticulocyte lysate system (Promega) according
to the manufacturer's recommendations. The reaction mixtures (50 µl)
contained mRNA at a concentration of 20 µg/ml and
[35S]methionine (Amersham Corp., 1000 Ci/mmol) at 15 mCi/ml; incubation was for 60 min at 30 °C. 35S-Labeled
protein products were analyzed by 10% SDS-PAGE and
autoradiography.
Monkey kidney COS-1 cells, maintained in monolayer culture in
Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone), were used for the expression in vivo
of ADAR proteins. Transfection with wild-type or mutant cDNA
expression vectors was carried out by the DEAE-dextran/chloroquine
phosphate method (38), using 5 µg of DNA per ml, when the cell
cultures in 60-mm dishes were about 80% confluent. Transfected cells
were harvested 60 h after transfection, and cell-free extracts
were prepared by Nonidet P-40 lysis with 0.3 ml of buffer A (20 mM HEPES, pH 7.5; 1 mM magnesium chloride; 10 mM potassium chloride; 0.5 mM dithiothreitol;
10% (v/v) glycerol; and 0.25 mM phenylmethylsulfonyl fluoride) containing 0.1% Nonidet P-40. After 10 min on ice and centrifugation at 2,000 rpm for 5 min, the resultant supernatant solution was adjusted to a final concentration of 0.1 M KCl
using a 1 M stock solution of KCl. The nuclear pellet was
washed twice with buffer A, suspended in 0.2 ml of buffer A containing
0.1 M KCl, disrupted by sonication, and then the crude
nuclear extract was recovered by centrifugation at 14,000 rpm for 15 min.
Western immunoblotting was
performed as described previously (6). Typically 20 µl of the
cytoplasmic or nuclear extract from COS-1 cells was fractionated by
7.5% SDS-PAGE, transferred to nitrocellulose filter membranes, and
probed with antiserum (1:500 dilution) generated against recombinant
ADAR protein expressed in E. coli (6). Antibody-antigen
complexes were subsequently detected with 125I-labeled
protein A (0.05 mCi/ml; ICN) and autoradiography; autoradiograms were
quantified by laser densitometry.
Measurement of dsRNA-specific adenosine deaminase activity
was essentially as described previously (3, 6, 32). The 32P-labeled synthetic dsRNA substrate was prepared by
annealing opposing transcripts in 20 mM Tris-HCl, pH 7.9, containing 0.15 M NaCl by first heating at 75 °C for 5 min and then slowly cooling to room temperature. The standard deaminase
reaction mixture contained approximately 10 fmol of
32P-labeled dsRNA and varying amounts of ADAR protein;
following incubation at 30 °C for 2 h, the reaction mixture was
extracted with phenol:chloroform and then chloroform. RNA was recovered by ethanol precipitation in the presence of 0.5 µg of poly(rI) (Sigma) added as carrier, washed with 70% ethanol,
dried, and suspended in 10 µl of nuclease P1 buffer (30 mM potassium acetate, pH 5.3, containing 10 mM
zinc sulfate) before digestion with 1.5 µg of nuclease P1 (Pharmacia
Biotech Inc.) for 2 h at 50 °C (3). IMP and AMP were resolved
from each other by thin layer chromatography (TLC) on cellulose NM 300 glass plates (Macherey and Nagel) in a solvent consisting of saturated
(NH4)2SO4, 100 mM
sodium acetate, pH 6.0, and isopropyl alcohol (79:19:2).
Autoradiography was usually performed for 8 h at A human
genomic library in the Unless otherwise specified, materials and
reagents were as described previously (6, 22, 32).
A human genomic library in
the
Exon-intron sizes and junction sequences of human ADAR gene
Department of Molecular,
-splice
site variant that possessed a 26-amino acid deletion within exon 7;
ADAR-c was a 3
-splice site variant that possessed an additional
19-amino acid deletion within exon 6. The wild-type ADAR-a, -b, and -c
proteins all possessed comparable double-stranded RNA-specific
adenosine deaminase activity. However, mutational analysis of the
R-motifs revealed that the exon 6 and 7 deletions of ADAR-b and -c
variants altered the functional importance of each of the three
R-motifs.
-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate
glutamate-gated ion channel GluR receptor subunits (15, 16). Editing of
the GluR pre-mRNAs results in altered channel properties of the
encoded proteins (12, 17). The highly selective Q to R, and R to G,
amino acid changes in GluR subunits are dependent upon formation of
dsRNA hairpin structures involving sequences derived from exon 11 and intron 11 for the Q/R site (18, 19) and exon 13 and intron 13 for the
R/G site (17). Thus, the A to I deaminations observed both in viral RNA
genomes and cellular pre-mRNA transcripts are dependent upon
double-stranded regions within the substrate RNA.
Oligonucleotides
-AATGCCTCG
CAATGAAT-3
(nt
31-53); BamHI(+)44,
5
-CTC
GCAATG(
/G)ATCCGCGGCAGG-3
(nt
36-63); (
)968, 5
-GAGATAGTCGCAGATTTTCTCC-3
(nt 968-947); NdeI(+), 5
-CAACCCCTCC
GC-3
(nt
1090-1107); RI*(+), 5
-GCTGGAAGC
AGAAAGTGGC-3
(nt 1698-1717); RI*(
),
5
-GCCACTTTCT
GCTTCCAGC-3
(nt 1717-1698); (+)1900,
5
-CACTGCTTGAGTGTATGC-3
(nt 1900-1917); RII*(+),
5
-GTGTGAGTGCTCCCAGC
AG-3
(nt 2023-2042);
RII*(
), 5
-CCACTTTCT
GCTGGGAGCAC-3
(nt
2049-2029); SalI(+), 5
-CAAGTTG
CAGTCCG-3
(nt 2279- 2298); SalI(
), 5
-GGTCCGGACTG
CAAC-3
(nt 2302-2282);
RIII*(+), 5
-CTGCGCACACAGC
AGAAGC-3
(nt
2363-2382); RIII*(
),
5
-GCCTTGCTTCT
GCTGTGTGC-3
(nt 2387-2367); (
)2532,
5
-GTGCTTCTGGGGACCTTGAGAG-3
(nt 2532-2511);
(
)2558, 5
-GCCAGTGAGAGGGA(G/C)TCTGTG-3
(nt 2558-2544/2465-2459);
(
)2577, 5
-TCTGGTCATGGAAGGTGC-3
(nt 2577-2560); HE*(+),
5
-CAATGACTGCCA
GCAG
AATAATCTC-3
(nt
2765-2791); HE*(
),
5
-GGGAGATTATT
CTGC
TGGCAGTC-3
(nt
2793-2769); DK*(+),
5
-CATGTCCTGTAGTG
C
AAATCCTAC-3
(nt
3146-3171); DK*(
), 5
-CAGCGTAGGATTT
G
CACTACAGG-3
(nt
3175-3151); KpnI(
),
5
-CAGTGCCTCT
GTCC-3
(nt 3483-3464); (
)3644,
5
-GAAGTAGTTCTTGGCCGTCTCG-3
(nt 3644-3623); (
)4090,
5
-GGTCAGTGTAGCAAACAC-3
(nt 4090-4073). The symbol (+) indicates a sense primer, and (
) an antisense primer. The numbers in
the parentheses correspond to the nt position in the ADAR-a cDNA
sequence (6, GenBank accession number U18121[GenBank]). The sequence in
lowercase type represents substitution of the ADAR cDNA nucleotides
to generate the indicated restriction site; the (A/G) nucleotides in
parenthesis for BamHI(+)44 show the alternative bases within
the mixed oligomer at the +4 site flanking the ATG. Underlined
sequences identify the specified restriction sites used for subcloning;
an underlined single nucleotide indicates an engineered mutation.
gt10 was initially screened
with the following pairs: NotI(+)31/(
)968 and
NotI(+)31/RI*(
) were used respectively for the
N-terminal portion of the ADAR ORF;
NdeI(+)/SalI(
) and NdeI(+)/(
)2577
for the central region containing the three R-motifs;
NdeI(+)/KpnI(
), SalI(+)/KpnI(
), HE*(+)/KpnI(
),
DK*(+)/(
)3644, and DK*(+)/(
)4090 for scanning from the central to
the C-terminal portion. PCR products initially were characterized by
agarose gel electrophoresis and staining with ethidium bromide, and
analyzed by restriction endonuclease digestion; fragments of interest
were subsequently subcloned into pBluescript SK
vector
(Stratagene) for sequence analysis by the dideoxy chain termination
method (36) using modified T7 DNA polymerase Sequenase version 2.0 according to the manufacturer's instructions (U.S. Biochemical Corp.).
For those regions of ADAR where differences were identified from the
originally described ADAR cDNA (6, 20), a
ZAP human placenta
library (Stratagene) and a mouse pY2 cDNA library derived from
J774.1 mouse macrophage-like cell line (kindly provided by Drs. F. Perier and C. Vandenberg, University of California, Santa Barbara) were
also analyzed by PCR with multiple primer pairs.
-GC-rich portion of the ORF and to
initiate at Met-296 (6) as the wild-type parent, constructions
possessing mutations in one or more of the three R-motifs had
previously been described in the form of M296 ADAR (32). These included
constructs with the RI(K554E), RII(K665E), and
RIII(K776E) substitutions, the three single mutants each
with one of the R-motifs altered, and with
RI(K554E)RII(K665E)RIII(K776E), the
triple mutant in which all three of the R-motifs were altered. Combined
PCR and sequence analyses, as described under the "Results," revealed the presence of two naturally occurring splice site variants of the prototype deaminase cDNA (6, 20), designated herein as
ADAR-a. One of the variants (termed ADAR-b) possessed a 78-nt in-frame
deletion between the RIII motif and the catalytic C domain; the other (termed ADAR-c) possessed an additional 57-nt in-frame deletion between the RII and RIII motifs. The
K88 3.7-kb starting parent previously constructed from overlapping
cDNA clones (6), utilized because of the presence of convenient
restriction sites that facilitated preparation of expression vector
constructs, was found to correspond to ADAR-b. Using the previously
reported mutagenesis strategy (32), PCR fragments corresponding to
ADAR-a and -c generated with primer pairs
SalI(+)/KpnI(
) and (+)1900/SalI(
) from the human kidney
cDNA library, respectively, were used to
produce the wild-type, the three single R mutants, and the triple R
mutant for M296 ADAR-a and -c. The wild-type construct encoding ADAR-d
which contained only the 57-nt deletion between the RI and
RIII motifs was also engineered likewise. To obtain constructs encoding the full-length M1 ADAR protein, a PCR product corresponding to the N-terminal region of ADAR was amplified from the
human kidney cDNA library using primers BamHI(+)44 and
(
)968. The BamHI-BamHI fragment which contained
the Met-1 codon engineered to be in optimal flanking nt context was
subcloned back into the BamHI site of the parental M296
constructs to generate the three M1 full-length versions of the
deaminase, ADAR-a, ADAR-b, and ADAR-c.
80 °C with a
screen; radioactivity associated with the excised TLC spots was
quantified using a Beckman LS1801 liquid scintillation system.
phage vector EMBL-3 SP6/T7 prepared from
human placenta DNA (Clontech) was screened initially by filter
hybridization using random primed 32P-labeled cDNA
fragments of the human ADAR-a cDNA as the probes (6, 37).
Subsequently, probes included random-primed 32P-labeled
genomic fragments. Lambda phage DNA was prepared from twice-rescreened
plaques, and genomic inserts were characterized by restriction mapping
and Southern blot analysis (6, 39). A human genomic P1 library in the
pAD10SacBII vector (40) was screened by PCR using synthetic
oligonucleotide primers based on sequences determined from previously
isolated
genomic clones. The library screening by PCR followed the
protocol described previously (22). Restriction fragments of positive
genomic clones were subcloned into the pBluescript plasmid (Stratagene)
for detailed restriction mapping and DNA sequencing by the
dideoxynucleotide procedure (36).
Isolation of Genomic Clones and Determination of the Structural
Organization of the Human ADAR Gene
phage vector EMBL3 was screened using as probes restriction
fragments of the human ADAR cDNA, previously designated as the K88
or DSRAD cDNA (6). Several overlapping
phage clones were
isolated (Fig. 1C). These genomic clones were
characterized by restriction mapping and Southern blot analysis.
However, because the typical insert size of the
phage genomic
clones was about 15 kb and the overlap between
clones was limited,
a P1 phage genomic library was subsequently screened. Three overlapping
P1 clones with inserts of about 85 kb, designated as clones 249, 652, and 959, were isolated that covered the entire ADAR gene. A
composite map of the human ADAR gene was determined (Fig.
1B). The precise exon-intron organization was established by
sequencing plasmid subclones and by comparison of the genomic sequences
to the previously determined cDNA sequences (6, 20). The 5
-region
of the published ADAR cDNA (6, 20) was verified and extended by the
5
-rapid amplification of cDNA ends procedure. The human
ADAR gene contains 15 exons and spans about 30 kb (Fig. 1).
The AUG translation initiation site for the 1226-amino acid ADAR
protein is located in exon 1, the A of which corresponds to +1 of the
cDNA sequence. The 3
-terminal exon number 15, the largest exon,
includes the UAG translation termination site and the 3
-untranslated
region. The complete exon-intron boundaries of the human
ADAR gene are summarized in Table I. Exons
range in size from 94 to 2984 base pairs. Introns range from 127 base
pairs to about 6.5 kb pairs. All splice site junctions conform to the
GT
AG rule (41).
Fig. 1.
Physical map of the human ADAR
gene. A, the structure of the gene is represented with
regard to the organization of the exons and introns. Exons are
indicated to scale by filled boxes, numbered
1-15; introns and the 5- and 3
-flanking regions are indicated by the solid lines. The entire gene spans
approximately 30 kb in length and contains 15 exons. Translation
initiates (ATG) in exon 1 and terminates in exon 15 as indicated.
R refers to the dsRNA-binding subdomain motif, the three
copies of which are found in exons 3, 5, and 7. B, the
restriction map shows cleavage sites for endonucleases BamHI
(B), EcoRI (E), HindIII
(H), and XhoI (X). C, five
of the overlapping
phage genomic clones (6, 7, 143, 151, and 176)
are shown to scale, along with the three P1 genomic clones (249, 652, and 959) that span the entire length of the ADAR gene and
continue into flanking sequences.
[View Larger Version of this Image (16K GIF file)]
Exon
Junction (cDNA
Position)
Intron
Junction (cDNA
position)
Exon no.
No.
Size
No.
Size
bp
kb
1
202
CGGCAGgtaagccgggct
(15)
I
5.4
cttattctgcagGGGTAT (16)
2
2
1586
ACCTCGgtaagagaccac
(1601)
II
2.5
ctttccgtcaagATTTAA (1602)
3
3
184
GAGAAGgtaggtgtcctc
(1785)
III
0.4
cattttctctagACTGCA (1786)
4
4
149
ACCCAAgtatgtcctacg
(1934)
IV
0.6
atctcctgtcagGTTCCA (1935)
5
5
145
AACCAGgtagggcgtttt
(2079)
V
0.1
attctcctttagCCTGAA (2080)
6
atgcccaacaagGTCAGG (2137)
6
6
191
GCCCAAgtgagtgtccta
(2270)
VI
6.5
ctcatcccaaagGTTCGT (2271)
7
6
134
GCCCAAgtgagtgtccta
(2270)
ctcatcccaaagGTTCGT (2271)
7
7
226
AAGACAgttaagacgtct
(2496)
VII
0.3
ttttccccacagCTCCCT (2497)
8
7
148
ACAGAGgtaaccccagtg (2418)
8
172
GAACAGgtgagtgaggct
(2668)
VIII
0.3
acaccttcctagGGAATC (2669)
9
9
94
CATCAGgtgagcgaggtc
(2762)
IX
0.7
ttctttttgtagGTTTCT (2763)
10
10
123
TATCAGgtctgtacagtt
(2885)
X
0.3
cctgtttttcagCACTGC (2886)
11
11
134
AGAACGgtgagtgataca
(3019)
XI
1.8
tctctcacacagGAGAAG (3020)
12
12
183
CATTGGgtaaggggcctg
(3202)
XII
0.3
ttttgtactcagGTTACC (3203)
13
13
113
CCCAAGgtgctataaccc
(3315)
XIII
0.4
tgggattcctagGTTGGC (3316)
14
14
128
GGATGGgtaaggagacag
(3443)
XIV
0.15
ttgtttctctagGCCACG (3444)
15
15
2984
AAAAAACAAGAATCTG
(6427)
Analysis of the position of introns within the codons of the human
ADAR gene revealed a non-random distribution. Intron phase zero before the first base of the codon occurred in 4 out of 14 introns
(29%) in the protein coding region of the ADAR gene; 3 out
of 14 (21%) were intron phase 1 after the first base, and 7 out of 14 (50%) were intron phase 2 after the second base of the junction codon.
Comparison of cDNA and genomic sequences revealed that the three
RNA-binding subdomain motifs RI, RII, and
RIII of the ADAR deaminase (6, 32) are located in exons 3, 5, and 7, respectively (Fig. 1A). The
RNA-dependent protein kinase PKR possesses two copies of
the dsRNA-binding subdomain R-motif (24, 26). Interestingly, the codon
phasing was exactly conserved between the exons that specify the R
subdomains in the ADAR and PKR genes in the
human, and the pkr gene in the mouse (Fig.
2). This conserved phasing would readily accommodate
exon skipping involving one of the R-motif exons, while still retaining
the open reading frame of the mRNA encoding the downstream enzyme catalytic region.
Multiple Forms of ADAR Due to Alternative Splicing
Previous studies revealed that two forms of the human dsRNA-specific adenosine deaminase are likely to exist, as demonstrated by Western immunoblot analysis of nuclear extracts from cultured cells (6). This earlier finding, together with the present observation that the intron phases prior to the R-motif exons of ADAR were all phase two (Fig. 2), raised the possibility that alternative splice site selection could result in an exon-skipping event that would give rise to distinct ADAR proteins lacking one or more of the R-motif-containing exons. To explore this possibility, cDNA libraries were screened by PCR using a series of primer pairs that covered the entire ADAR cDNA. No evidence was obtained for ADAR variants lacking one or more of the R-motifs (data not shown). Surprisingly, however, two ADAR variants were discovered which appeared to be products of an entirely different type of alternative splicing than that anticipated for skipping an R-motif-containing exon.
Initially primers SalI(+) and ()2577 produced two PCR
products, both from human and mouse cDNA libraries (data not
shown). Sequence analysis of the subcloned PCR products revealed that the smaller product contained a 78-nt deletion, positioned between the
RIII motif and the C domain. This is a region of high
similarity between the human and mouse ADAR cDNA sequences, and the
78-nt deletion was found in both human kidney and mouse macrophage
cDNA libraries as illustrated with primer pair PP1 (Fig.
3A). Comparison of cDNA sequence with the
human ADAR gene structure revealed that the 78-nt deletion
terminated precisely at the splicing junction, between exon 7 and
intron VII (Fig. 4). This variant was designated as
ADAR-b. The 26-amino acid region deleted from exon 7 in ADAR-b corresponds to amino acid residues 807-832 of the prototype ADAR cDNA (6, 20). The 5
end of the nucleotide sequence deleted from
exon 7 satisfies the requirement to serve as a cryptic 5
-splicing site, suggesting that the deletion to generate exon 7
arises as a
result of alternative splicing (Fig. 4, Table I). Similarly, primer
pair PP2, NdeI(+)/SalI(
), generated two
products from the human kidney library (Fig. 3B, lane e).
Sequence analysis established that the smaller PCR product contained a
57-nt deletion between the RII and RIII motifs,
precisely at the splicing junction between intron V and exon 6 (Fig.
4). This deleted 19 amino acids, residues 695-712, generating exon
6
. The 3
-terminal sequence displayed the characteristics of a
3
-splicing site, conforming to the GT-AG rule (41) as shown in Table
I.
Four ADAR variants are theoretically possible if two exons, exons 6 and
7, are present in two (,
) forms (Table I). Therefore, a number
of PCR primer pairs that could distinguish the potential combinations
(Fig. 4, lower) were utilized to analyze cDNA libraries prepared from human kidney, human placenta, and mouse macrophages. As
shown in Fig. 3A, the human kidney library yielded three
products (lane b), whereas the mouse library gave only two
(lane c) upon PCR analysis with the primer pair PP1 that
flanked both deletions. As compared with the human kidney library, the
human placenta library did not include a detectable ADAR message
possessing the 57-nt deleted exon 6
(Fig. 3B, lane f). As
shown in Fig. 3B, PCR products obtained with two primer
pairs, PP3 (lanes g and i) and PP4 (lanes
h and j), indicated that human kidney (lanes g and h) expressed three ADAR species, one with
full-length exons 6
and 7
, designated as ADAR-a, one with
full-length exon 6
and the 78-nt deleted exon 7
, designated as
ADAR-b, and one with both the 78- and 57-nt deleted exons 6
and
7
, respectively, designated as ADAR-c. By contrast, the human
placenta library only yielded the ADAR-a and -b forms (lanes
i and j). The fourth theoretical variant combination,
containing only the 57-nt deletion exon 6
and the full-length exon
7
(denoted ADAR-d), was not detected in the cDNA libraries
examined (lanes h and j). These results
demonstrated the utilization of two alternative splice sites for exon 6 and exon 7, yielding three variant forms of ADAR as summarized by the
schematic diagram of Fig. 4. The variant forms of ADAR are
differentially expressed in placenta and kidney tissues and macrophage
cells, suggesting the possibility of a tissue-specific function for
variant ADAR isoforms.
Four
versions of the dsRNA-specific adenosine deaminase protein, the three
naturally occurring variants ADAR-a, -b and -c, and an engineered
variant not yet detected naturally (ADAR-d), were expressed both
in vitro and in vivo. The rabbit reticulocyte lysate cell-free protein synthesizing system, programmed with ADAR
mRNA transcribed in vitro from the ADAR cDNA
templates, was utilized initially to confirm the protein coding
capacity of the constructs. The ADAR protein products synthesized
in vitro showed the predicted sizes as analyzed by SDS-PAGE
(data not shown). Because the full-length ADAR constructs possessing
the natural 5-untranslated region from the cDNA were expressed
poorly (6, 32), a truncated 5
-terminal untranslated region was
generated by PCR using the BamHI(+)44 primer; this
engineered construct also included the AUG1 translation start in
optimal context. ADAR-a mRNA transcribed from this construct was
efficiently translated in vitro (data not shown). This ADAR
construction, in the pcDNAI/neo vector, was used for expression of
the M1 full-length 1226-amino acid ADAR proteins in COS cells.
Expression of ADAR proteins in vivo was detected by Western
immunoblot analysis, using antibodies 2 and 3 generated against
recombinant ADAR proteins produced in E. coli (6). Antibody
3 generated against the N-terminal region of the cDNA ORF was
previously shown to specifically recognize an interferon-inducible p150
ADAR protein found in the cytoplasm and nucleus but not the p110
nuclear protein (6). This is illustrated in Fig.
5A by analysis of extracts prepared from
human SY5Y cells, which were included as a positive control and
reference marker; p150 was greatly enhanced in both nuclear
(lanes a and b) and cytoplasmic (lanes
c and d) extracts prepared from IFN-treated (Fig.
5A, lanes b and d) as compared with untreated
(lanes a and c) cells. Only the p150 protein was
detected with antibody 3 (lanes c and d), whereas
both p150 and p110 were detected by antibody 2 (lanes a and
b) as previously reported (6).
The three cDNA constructs encoding the wild-type M1 full-length deaminase variants, ADAR-a, -b and -c, were all efficiently expressed in COS-1 cells (Fig. 5A, lanes f-h). The ADAR-a, -b, and -c proteins were present in both the cytoplasmic (Fig. 5A, lanes f-h) and nuclear fractions (data not shown) of transfected COS cells. Although antibodies prepared against recombinant human ADAR react poorly with monkey COS ADAR (6), a COS cell p150-like protein was detectable in both the cytoplasmic and nuclear fractions in cells transfected with vector alone (lanes e and i). The IFN-inducible p150 protein of human SY5Y cells (lanes c and d) possessed an electrophoretic mobility comparable to the full-length p150 ADAR-a variant expressed in COS cells (lane f). Curiously, both vector-coded and endogenous chromosome-coded ADAR proteins were detected as two bands on Western immunoblots. The lower signal is believed to represent a proteolytic cleavage product (2, 6).
All four variants of the deaminase cDNA, ADAR-a, -b, -c, and -d, were also efficiently expressed in transfected COS cells as the M296 N-terminally truncated form of ADAR (Fig. 5B, lanes k-n). This is illustrated by the Western blot of the cytoplasmic fractions (Fig. 5B, lanes k-n), using antibody 2 which was previously shown to recognize both the p150 and the p110 human proteins (6) as shown also in Fig. 5A (lanes a and b). Similar to previously reported findings for human cells (6), COS cells also expressed high level of the nuclear p110-like protein (compare lane o with j), which was comparably sized to the M296 ADAR-a variant (lane k) as shown in Fig. 5B.
Because the nuclear fractions prepared from COS cells exhibited high
deaminase activity due to the presence of the endogenous M296-like p110
protein (3, 6, 42), the cytoplasmic fractions prepared from transfected
cells were employed to examine the enzymic activity of the
cDNA-encoded ADAR splice variants. All three of the full-length
wild-type versions of the recombinant ADAR variants, ADAR-a, -b and -c,
and all four N-terminally truncated M-296 wild-type versions of the
ADAR variants, ADAR-a, -b, -c and -d, possessed dsRNA adenosine
deaminase activity (Fig. 6, b-h). By
contrast, the cytoplasmic fraction prepared from control COS cells
transfected with the expression vector alone without an ADAR cDNA
insert showed much lower endogenous deaminase activity (Fig. 6,
lane a). Relative specific enzyme activities were calculated
for the ADAR variants by quantifying the extent of A-to-I conversion
relative to the amount of expressed ADAR protein (Fig.
7). The wild-type ADAR-a, -b, and -c variants of the
full-length p150 form (Fig. 7A), as well as the wild-type
ADAR-a, -b, -c, and -d variants of the M296 form (Fig. 7B),
displayed comparable specific deaminase activity measured with a
synthetic dsRNA substrate. These results indicate that the deletions in
exons 6 and 7 resulting from alternative splicing caused no discernible
effect on the enzyme activity of the wild-type ADAR.
Functionally Distinct R-motif Subdomains Associated with Exon 6 and 7 Variants Resulting from Alternative Splicing
Previously we introduced an equivalent mutation into each of the three repeated R-motif subdomains of ADAR in the M296 form by substituting a highly conserved R-core lysine residue that had been shown by mutagenesis studies of PKR and E3L to be essential for RNA binding activity (29-33). Analysis of R-motif mutants of ADAR revealed that the three R-motif subdomains are functionally distinct (32, 34); RIII was the most important for enzyme function, whereas the single substitution mutants at either the RI or RII subdomain retained significant enzyme activity.
In order to investigate the biochemical properties of the two newly
identified alternative splice variants ADAR-b and -c relative to the
originally identified 1226 ADAR-a protein (6, 20, 21), the same Lys to
Glu substitution (see Fig. 2 schematic) was introduced into
each of the R-motifs of the three versions of ADAR, utilizing the
PCR-based mutagenesis strategy we previously described (32). All three
of the single R mutants, and the triple R mutant, were constructed and
analyzed for ADAR-a, -b and -c in the M296 form, and for ADAR-a in the
M1 full-length form. The constructs were expressed in transfected COS
cells, and cytoplasmic fractions were prepared and analyzed for ADAR
protein and dsRNA-specific adenosine deaminase activity. For ADAR-a,
either in its full-length p150 form (Fig. 7A) or the
truncated M296 form (Fig. 8), mutation of any of the
three R-motif subdomains reduced but did not abolish enzyme activity.
By contrast, the ADAR-a RIRIIRIII
triple mutant showed no detectable deaminase activity either as the
full-length p150 protein (Fig. 7A) or the M296 protein (Fig.
8), relative to the respective wild-type ADAR-a parent. However,
comparison of R-motif subdomain mutants of the ADAR-a, -b, and -c
variants in the M296 form revealed distinctly different profiles of
enzyme activity. No substantial difference was observed for ADAR-a in the M296 form among the three single R-motif mutants (Fig. 8), comparable to the finding for ADAR-a in the full-length p150 form (Fig.
7A). In striking contrast to these results for ADAR-a, in the case of the ADAR-c variant the RIII subdomain was
absolutely essential for the deaminase activity whereas RII
appeared to be dispensable (Fig. 8). In fact, mutation of the
RII core lysine even appeared to enhance enzymic activity
relative to the wild-type parent expressed in vivo (Fig. 8).
For the ADAR-b variants, the RI mutant retained partial
activity, approximately 50% relative to wild-type, whereas the triple
RIRIIRIII mutant showed little activity (Fig. 8). These results obtained with in vivo
expressed ADAR-b are similar to results obtained using in
vitro synthesized ADAR proteins (32).
Comparing the RI mutants of the ADAR-a, -b, and -c variants, the 26-amino acid deletion of exon 7 between the RIII motif and the C domain in ADAR-b, as well as the additional 19-amino acid deletion of exon 6 between the RII and RIII motifs in ADAR-c, appeared to gradually reduce the functional significance of the RI motif. This was suggested by the progressive increase in deaminase activity of ADAR-a as compared with ADAR-b to ADAR-c, when RI was mutated. By contrast, the deletions in exons 6 and 7 seemed to gradually enhance the functional importance of the RIII subdomain, as shown by the relative decrease in enzyme activity for the ADAR-b and -c variant RIII mutants (Fig. 8). The most dramatic alteration was observed for the RII mutants; the presence of the exon 7 deletion rendered the RII motif dispensable, as demonstrated by the higher enzyme activities for ADAR-b and -c variants relative to the corresponding wild-type parents (Fig. 8).
We have isolated and characterized genomic clones of human ADAR, the interferon-inducible dsRNA-specific adenosine deaminase also known as DRADA and dsRAD (6). Three important points emerge from the results reported herein. First, and most important, is that previously unknown splice site variants of the ADAR editing enzyme were identified that are expressed in human and mouse tissues and cell lines; the two ADAR variants result from alternative splicing of exons 6 and 7. Second, that the variants encode active editing enzymes in which the three RNA-binding R-motif subdomains are functionally distinct. Third, that the ADAR gene structure involving 15 coding exons possesses a precisely maintained codon phasing for the three R-motif exons of human ADAR that is exactly conserved in the human and mouse genes of the dsRNA-dependent protein kinase PKR that possesses two R-motif exons.
We earlier reported the isolation of genomic clones for the ADAR
deaminase, 6 and
151, which were mapped
to a single locus on human chromosome 1q21.1-21.2 by fluorescence
in situ hybridization (22). We now have isolated additional
overlapping
phage clones as well as P1 phage genomic clones for
human ADAR. The exon-intron organization of the 30-kb human
ADAR gene was defined by Southern blot and direct sequence
analyses. The exon structure for the ADAR gene that we
determined is in general agreement with that described by Nishikura and
co-workers (43), although small apparent differences exist between some
intron sizes. Comparison of the genomic sequence with the cDNA
sequence revealed that the coding region of the human ADAR
gene is specified by 15 exons. The intron positions within the codons
of the human ADAR gene were not evenly distributed; 50%
were intron phase 2 after the second base of the junction codon (Table
I). By contrast, results obtained for a large set of animal genes
revealed that phase two was represented by only 22% of the total (44).
Curiously, the intron phases prior to the three R-motif-containing
exons of the human ADAR gene, exons 3, 5, and 7, were all
phase two. Even more striking is the finding that this codon phasing
observed for the human ADAR gene R-motif exons was conserved
exactly for the two R-motif exons of the mouse pkr (39) and
human (45) PKR genes (Fig. 2). This conserved phasing is
consistent with a common ancestor for the R-motif coding exons of the
ADAR and PKR genes and the occurrence of early
exon shuffling events. The conserved phasing would readily accommodate
exon skipping involving R-motif exons, while still retaining the ORF of
the mRNA encoding the downstream catalytic domain of the deaminase
in the case of ADAR, or the kinase in the case of PKR. However, PCR
analysis did not detect the existence of PKR splice variants lacking
one or both R-motif exons (45). Although our PCR analyses of human
placenta and kidney and mouse macrophage cDNA libraries likewise
did not reveal the presence of ADAR splice variants lacking one or more
of the R-motif exons as predicted for such an exon-skipping event, we did identify two other different kinds of ADAR splice variants.
One of the splice variants that we identified, ADAR-b, possessed a
26-amino acid deletion in exon 7; the other splice variant that we
identified, ADAR-c, contained an additional 19-amino acid deletion in
exon 6. The 26-amino acid deletion of ADAR-b was located between the
RIII motif and the C-terminal catalytic domain; the 19-amino acid deletion of ADAR-c was located between the
RII and RIII motifs. In both the ADAR-b and -c
variants, the integrity of all three R-motifs was retained. What was
altered was the spacing between the R-motifs themselves or between the
R-motifs and the catalytic domain of the variant editing enzyme forms.
The prototype ADAR, now designated as ADAR-a, corresponds to the form
of the editing enzyme for which cDNA clones were isolated (6, 20, 21). The human ADAR-a cDNA predicts a 1226-amino acid protein that
is inducible by IFN (5, 6). Three lines of evidence are consistent with
the conclusion that the newly discovered ADAR variants are indeed
splicing variants and not products of separate genes. First, the
positions of the deletions in exons 6 and 7 identified for the ADAR-b
and ADAR-c variants coincide exactly with the previously determined
exon-intron junctions at one terminus and conform at the other terminus
to the GTAG rule (41) for splice sites. Second, the deduced amino
acid sequence of the ADAR-b and -c variant cDNA clones agrees
exactly to the amino acid sequence previously determined for the
prototype ADAR-a cDNA clones (6, 20). Third, PCR analyses of
genomic DNA, either of the
library, the P1 genomic clones, or high
molecular weight chromosomal DNA isolated from human U cells, yields
only a single PCR fragment of predicted size and does not yield smaller
products that would be expected if the deletions found in cDNA
clones also existed in the genome (data not shown).
Recent studies indicate that the dsRNA-specific adenosine deaminase ADAR plays a central role in the A-to-I RNA editing process (10, 46). However, how the site selectivity is achieved for the precise editing required in the cases of some of the natural cellular and viral RNA substrates is an important issue that remains unresolved. The highly conserved RNA-binding subdomain R present in triplicate in ADAR is found in duplicate in the interferon-inducible RNA-dependent protein kinase PKR (24-28, 47). The fact that no RNA-binding sequence specificity has been identified for such R-motif subdomains in the case of the PKR kinase (24, 48) raises the question how ADAR catalyzes the site-selective deamination of adenosine in natural substrates, for example in GluR pre-mRNA (12, 15, 16, 49) and HDV antigenomic RNA (11, 14). The existence of multiple forms of the dsRNA adenosine deaminase would provide one possible mechanism by which the specificity problem could be resolved (6, 23). Purified ADAR (dsRAD) from Xenopus oocyte nuclei will catalyze the deamination of GluR-B RNA in vitro, although the deamination of the biologically relevant Q/R editing site occurs much less frequently in vitro by the Xenopus enzyme than observed in vivo (15). One possibility is the absence of accessory factors that may play indirect roles in vivo; another possibility is the presence of multiple closely related variants of the editing enzyme in the purified enzyme preparation, only one of which is relevant for in vivo editing. Although dsRNA-specific adenosine deaminase enzymes have been purified extensively and characterized from bovine liver (2), calf thymus (3), and chicken lung (50) in addition to Xenopus oocytes (1), the apparent size of the enzymes varies considerably among the four sources, and the purified preparations often contain multiple bands as determined by gel electrophoresis.
ADAR has been shown to be present ubiquitously in primary tissues and cell lines by Northern analysis or direct enzyme assay (6, 20, 21, 42). However, our results establish that at least one alternative splice variant, ADAR-c, was expressed differentially in different tissues (Fig. 3). Both ADAR-a and -b were expressed in human kidney and placenta, as well as in a mouse macrophage-like cell line. By contrast, ADAR-c was detected only in the human kidney library from the three cDNA libraries examined. All of the ADAR variants were efficiently expressed in COS cells (Fig. 5). The various splice variants of ADAR were active enzymes, with comparable specific activity when examined as the wild-type version of the three R-motif subdomains (Figs. 6 and 7).
As an approach to examining the functional importance of each of the three copies of the R-motif subdomains, we constructed a family of ADAR point mutants in which each of the R-motifs was mutated in each of the splice variants. A highly conserved lysine residue (Fig. 2) established as essential for the R-motif mediated RNA-binding activity of the cellular PKR kinase and the viral E3L protein (29, 30, 33) was substituted with glutamic acid in either any one, or all three, of the R copies in the ADAR splice variants. For ADAR-a, either in its full-length p150 form or the truncated M296 form, mutation of any of the three R-motif subdomains reduced but did not abolish enzyme activity (Figs. 7A and 8). By contrast, the ADAR-a triple RIRIIRIII mutant showed no detectable deaminase activity either as the full-length p150 protein or the M296 protein relative to the respective wild-type ADAR-a prototype parent. This suggests that each of the three R-motifs is comparably important for enzyme function in the case of ADAR-a, and the N-terminal portion of ADAR-a upstream of M296 and the R-motifs has little effect on the function of the dsRNA-binding domain.
Results obtained for the RI mutant of variants ADAR-a, -b,
and -c suggest that functionally distinct R-motif subdomains result from the alternative splicing of exon 6 and exon 7 (Fig. 8). When RI was mutated, the 26-amino acid deletion (exon 7 form)
between the RIII motif and the C domain in ADAR-b, as well
as the additional 19-amino acid deletion (exon 6
form) between the
RII and RIII motifs in ADAR-c, appeared to
gradually reduce the functional significance of RI. By
contrast, exons 6
and exon 7
seemed to gradually enhance the
functional importance of the RIII subdomain, as revealed by
the relative decrease in enzyme activity for the RIII
mutants of the ADAR-b and -c variants relative to ADAR-a containing
exons 6
and 7
. The most dramatic alteration was observed for the
RII mutants; the presence of the exon 7
rendered the RII motif dispensable, as demonstrated by the higher enzyme
activities for ADAR-b and -c variant RII mutants relative
to the corresponding wild-type parents, and also to ADAR-a. These
results suggest that the presence of deletions flanking the
RIII motif due to alternative splicing, especially the one
located between the RIII motif and the C domain, leads to
distinct functionality of each of the three R-motifs. This implies that
shortening the distance between the dsRNA substrate binding domain and
the catalytic domain, as well as the distance within the three R
subdomains, can consequently change the manner by which the three
repeated R-motifs and the C domain interact, thus altering the way by
which dsRNA substrates are bound and correctly positioned at the
catalytic center for deamination. These results are consistent with the
notion that the splice variants of ADAR likely contribute to mediating
the required site selectivity in editing of natural substrates.
Although preferential deamination of certain adenosine residues has been demonstrated in vitro using synthetic RNA substrates (4, 51), the basis of the site selectivity presumed to occur in vivo as illustrated by the postulated modification of hepatitis delta virus and human immunodeficiency virus RNAs (13, 14, 52) and also the cellular mRNAs encoding the glutamate-activated cation channel proteins where one or two adenosine residues are modified (12, 19) has not been resolved. In support of the speculation that the repeated nature of the R-motif subdomain found within ADAR may be important in determining the relative substrate selectivity of the enzyme, there exist multiple forms of ADAR with distinct positioning of the three R-motifs relative to the C domain that showed distinct involvement of each individual R-motif in the process of deamination. They are most likely to represent ADAR enzymes differing in substrate specificity, which are involved in recognizing distinct natural dsRNA substrates via interacting with dsRNA region in distinct structural environment. A similar situation has been described for the Wilms tumor susceptibility gene WT1, where the alternative splicing between the third and fourth zinc fingers results in an insertion of KTS that is of great physiological importance (53, 54). This insertion of three amino acids altered the binding properties of the WT1 isoforms for their DNA targets (55) as well as their subnuclear localization (56).
The demonstration of multiple splice variants of ADAR with functionally distinct R-motif subdomains, as shown herein, when taken together with the reported rat RED1 dsRNA adenosine deaminase presumably encoded by a different gene than the prototype rat ADAR deaminase (23), clearly establishes that there is indeed a family of dsRNA adenosine deaminase enzymes. These ADAR enzymes undoubtedly act to recognize a variety of natural viral and cellular RNA substrates with distinct structures. What is not yet clear is whether a single deaminase protein is sufficient to confer the necessary site selectivity required in the editing reaction, or whether the process is more complex at the level of structural organization. For example, whether an accessory protein interacts with either the substrate RNA or the deaminase to help confer the required editing specificity, or whether an editing complex formed by interactions among a family of ADAR deaminases and accessory proteins contributes to the specificity (49), remains to be established.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U75489[GenBank], U75490[GenBank], U75491[GenBank], U75492[GenBank], U75493[GenBank], U75494[GenBank], U75495[GenBank], U75496[GenBank], U75497[GenBank], U75498[GenBank], U75499[GenBank], U75500[GenBank], U75501[GenBank], U75502[GenBank], U75503[GenBank], U75504[GenBank], U75505[GenBank].