From The Wistar Institute, Philadelphia, Pennsylvania 19104 and the ¶ Department of Cell and Developmental Biology, University
of Pennsylvania, Philadelphia, Pennsylvania 19104
Received for publication, December 23, 2002, and in revised form, February 24, 2003
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ABSTRACT |
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Adenosine deaminases acting on RNA (ADAR) convert
adenosine residues into inosines in double-stranded RNA. Three
vertebrate ADAR gene family members, ADAR1,
ADAR2, and ADAR3, have been identified. The
catalytic domain of all three ADAR gene family members is very similar
to that of Escherichia coli cytidine deaminase and APOBEC-1. Homodimerization is essential for the enzyme activity of
those cytidine deaminases. In this study, we investigated the formation
of complexes between differentially epitope-tagged ADAR monomers by
sequential affinity chromatography and size exclusion column
chromatography. Both ADAR1 and ADAR2 form a stable enzymatically active
homodimer complex, whereas ADAR3 remains as a monomeric, enzymatically
inactive form. No heterodimer complex formation among different ADAR
gene family members was detected. Analysis of HeLa and mouse brain
nuclear extracts suggested that endogenous ADAR1 and ADAR2 both form a
homodimer complex. Interestingly, endogenous ADAR3 also appears to form
a homodimer complex, indicating the presence of a brain-specific
mechanism for ADAR3 dimerization. Homodimer formation may be necessary
for ADAR to act as active deaminases. Analysis of dimer complexes
consisting of one wild-type and one mutant monomer suggests functional
interactions between the two subunits during site-selective RNA editing.
One type of RNA editing converts adenosine residues into inosine
within the double-stranded RNA
(dsRNA)1 region of substrate
RNAs (1-3). Because inosine is treated as guanosine by the
translational machinery, this A-to-I editing could lead to functional
alterations of the affected genes. For instance, A-to-I RNA editing
results in the expression of editing isoforms of glutamate receptor
(GluR) ion channel subunits (4, 5) and serotonin 2C subtype receptors
(5-HT2CR) (6). Editing of the so-called "Q/R" site of
the Members of the ADAR gene family have been implicated as the enzymes
responsible for A-to-I RNA editing. Three separate mammalian gene
family members (ADAR1 to ADAR3) have been identified (15-22). Data
base search has identified corresponding fish ADARs
revealing the conservation of the complete set of ADAR gene family
members in vertebrates through evolution (23, 24). In invertebrates, a
single Drosophila dADAR, very similar to
mammalian ADAR2 (25), and two less conserved Caenorhabditis
elegans c.e.ADAR1 and c.e.ADAR2 have been
identified (15, 26). Mammalian ADAR1 and ADAR2 are detected
ubiquitously (15, 16, 18-20), whereas the expression of mammalian
ADAR3, Drosophila dADAR, and C. elegans c.e.ADAR1 is restricted mainly to nervous systems (21, 22, 25, 27). Analysis of
ADAR null mutation phenotypes has revealed the importance of A-to-I RNA
editing. Flies with a null mutation of dADAR, although viable, display defective locomotion and behavior accompanied by
various neurological and anatomical changes in the brain. This phenotype is most likely because of the lack of editing in the transcripts of several known target genes such as cac
Ca2+ channel and para Na+ channel
(25). C. elegans strains containing homozygous deletions of
both c.e.ADAR1 and c.e.ADAR2 genes show defects
in chemotaxis, whereas aberrant development of the vulva is
occasionally detected with worms lacking c.e.ADAR1 (27).
Mice with a homozygous ADAR2 null mutation die several weeks
after birth with repeated episodes of epileptic seizures because of
underediting of GluR-B RNA at the Q/R site, a major target of ADAR2
(28). Chimeric mouse embryos derived from
ADAR1+/ Purified recombinant ADAR1 and ADAR2 proteins displayed in
vitro their distinctive editing site selectivity with known RNA substrates (18-20, 30, 31). For instance, ADAR2 edits almost exclusively the D site of 5-HT2CR and the Q/R site of
GluR-B RNA, whereas ADAR1 barely edits these sites. However, ADAR1
selectively edits the A and B sites of 5-HT2CR RNA and the
intronic hot spot (+60 site) of GluR-B RNA. The result of in
vitro editing studies indicate a significant difference among ADAR
gene family members in their interaction with substrate RNA. Specific
structural features of the dsRNA binding domains and their N-terminal
regions may form the molecular basis of this editing site selectivity.
There are only two dsRNA binding motif repeats in the RNA binding
domain of ADAR2 and ADAR3, in contrast to three dsRNA binding motifs in
ADAR1. ADAR2 lacks a part of the N terminus region of ADAR1, just
upstream of its RNA binding domain, where ADAR1 contains two repeats of
a Z-DNA binding motif (32). ADAR3 has a unique N-terminal region
containing the arginine-rich R domain (21, 22). Alternatively, the
deaminase domains and relatively divergent C-terminal regions of ADAR
gene family members may also contribute to the difference observed in
their RNA editing site selectivity as indicated by the studies of
domain exchange between ADAR1 and ADAR2 (21, 33).
A longstanding question with regard to the enzymatic activities of
ADARs is whether they act as monomeric or oligomeric forms and whether
oligomerization plays a role in the site-selective editing mechanism.
The catalytic domain of ADAR is very similar to that of the cytidine
deaminase gene family (1, 2, 15). E. coli cytidine deaminase
forms a homodimer (34), as does APOBEC-1, another cytidine deaminase
involved in C-to-U RNA editing of apolipoprotein B mRNAs (35-37).
In both cases homodimerization is required for enzymatic activity
(34-37). It is possible that the RNA editing site selectivity observed
with ADAR1 and ADAR2 is dependent on their state of oligomerization.
Curiously, the third member of the ADAR gene family, ADAR3, is
incapable of editing all known sites of GluR-B and 5-HT2CR
RNAs. The lack of enzymatic activity may be related to its
oligomerization state. In the present studies, we have investigated
whether ADAR gene family members undergo oligomerization. In addition,
we have examined the possible formation of heteromeric oligomers among
different ADAR gene family members.
Oligonucleotides--
The following oligonucleotides used for
construction of 6His-tagged ADAR baculovirus constructs were
synthesized at the University of Pennsylvania, Cancer Center Nucleic
Acid Facility. All ADAR oligonucleotides correspond to the human
sequence. The nucleotide positions indicated in parentheses are
relative to the initiation codon ATG of ADAR1, ADAR2, and ADAR3
(GenBankTM accession numbers U10439, U76420, and AF034837,
respectively), in which A was assigned as position +1. The 6His epitope
tag sequence is underlined, and all restriction sites within the
oligonucleotides are shown in bold. Not-H-ADAR1UP,
5'-AAGGAAAAAAGCGGCCGCAGAATAAAAATGAATCATCACCATCACCATCACAATCCGCGGCAGGGGTATTCCCTC-3' (+4 to +27); H-ADAR1DW, 5'-GTGGCAGTGACGGTGTCTAG-3 (+196 to +177); Not-H-ADAR2UP,
5'-AAGGAAAAAAGCGGCCGCAGAATAAAAATGAATCATCACCATCACCATCACGATATAGAAGATGAAGAAACATG-3' (+4 to +27); H-ADAR2DW, 5'-GTTGACAGACAGGGTCCTC-3' (+486 to +468); Bam-H-ADAR3UP,
5'-CGCGGATCCAGAATAAAAATGAATCATCACCATCACCATCACGCCTCGGTCCTGGGGAGCGGC-3' (+4 to +24); H-ADAR3DW, 5'-AGACCAGCTGCAGTTTGCACA-3' (+349 to
+329).
ADAR Expression Constructs--
A 6His epitope tag sequence was
introduced into the N termini of the existing FLAG epitope-tagged
expression constructs, pBac-F-ADAR1, pBac-F-ADAR2a, and pBac-F-ADAR3
(20, 22, 38). Different regions of human ADAR1 (amino acids 2-72),
ADAR2a (amino acids 2-35), or ADAR3 (amino acids 2-100) were prepared
by PCR amplification of human ADAR1 (15), ADAR2a (20), and ADAR3
cDNA plasmids (22) using a set of oligonucleotide primers designed
to create NotI and BamHI restriction sites.
Not-H-ADAR1UP and H-ADAR1DW primers were used for PCR amplification of
the ADAR1 sequence, Not-H-ADAR2UP and H-ADAR2DW primers for ADAR2a, and
Bam-H-ADAR3UP and H-ADAR3DW primers for ADAR3. Restriction sites
AflII, StuI, and NotI were utilized
for ligation of the PCR products at their 3' ends into pBac-F-ADAR1,
pBac-F-ADAR2a, and pBac-F-ADAR3, respectively. The resultant
constructs, termed pBac-H-ADAR1, pBac-H-ADAR2a, and pBac-H-ADAR3,
contain a Kozak sequence that is strongly preferred by baculovirus for
protein translation initiation at the N terminus region (39). The
region amplified by PCR was confirmed by sequencing. Baculovirus
expression constructs were then transformed in DH10Bac for
transposition into the bacmid and subjected to blue/white screening for
identification of recombinant baculoviruses.
Expression of the ADAR Recombinant Baculovirus--
Sf9
cells were grown to a density of 2 × 106 cells/ml and
infected with either a single or a combination of two ADAR recombinant viruses (1:1 ratio) at a multiplicity of infection of 10-20. At 48 h post-infection, ~1 × 109 cells were collected.
Extract Preparation--
All procedures were carried out at
4 °C. HeLa cell extract was prepared as described previously (40).
Mouse brain nuclear extract was prepared by the Dignam method (41) with
a minor modification as follows. Fresh mouse brains were minced using a
pair of scissors, and further homogenized by a motor-driven Potter
homogenizer in 3 times the packed cell volume of phosphate-buffered saline. The cell pellet was suspended in a buffer containing 10 mM Hepes (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 1× complete protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN), and
0.5 mM phenylmethylsulfonyl fluoride, and kept on ice for 20 min. Cells were lysed by 10-20 strokes with a glass Dounce homogenizer followed by centrifugation at 10,000 rpm for 15 min in a
Type 65 Ti Beckman rotor. The nuclear pellet was suspended in 3 pellet
volumes of a buffer containing 20 mM Hepes (pH 7.9), 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 1.0 mM DTT, 1×
Complete protease inhibitor mixture, and 0.5 mM
phenylmethylsulfonyl fluoride. After five gentle strokes in a glass
Dounce homogenizer, the protein extract was cleared of debris by
centrifugation at 30,000 rpm for 30 min.
Sequential Epitope Tag Affinity Column Purification of ADAR Dimer
Complexes--
All column chromatography procedures were carried out
at 4 °C. Total cell extract was prepared from Sf9 cells
infected with a single or combination of two recombinant baculoviruses
(38). The cell extract, dialyzed against buffer A (0.05 M
Tris, pH 7.0, 0.15 M NaCl, 5 mM EDTA, 1.0 mM DTT, 20% glycerol, 0.25 mM
phenylmethylsulfonyl fluoride, 0.05% Nonidet P-40) was first passed
through a 1.0-ml (1.0 × 1.3 cm) anti-FLAG M2-monoclonal antibody
(mAb)-agarose gel (Sigma) affinity column equilibrated with buffer A
containing 0.15 M NaCl and 1 mM
In some experiments, the ADAR complex purified on a M2 mAb-agarose
column was treated with RNases prior to its application to the TALON
affinity column. The recombinant ADAR proteins (1× purified) were
treated with single-stranded RNA (ssRNA) specific RNases A (0.5 units/ml) and T1 (10 unit/ml) obtained from Roche Diagnostics or with
dsRNA-specific RNase V1 (1 unit/ml) obtained from Pierce.
RNase-digested ADAR proteins were dialyzed against buffer B, and then
subjected to TALON affinity column chromatography. The RNase digestion
conditions were tested separately with uniformly [ Size Exclusion Column Chromatography Analysis--
Purified ADAR
proteins (1 µg) or crude nuclear extract (2 mg) was applied to a
24-ml (1 × 30 cm) column of Superose 12 HR 10/30 (Amersham
Biosciences) for size exclusion chromatography. The buffer system used
was 0.05 M Tris (pH 7.0), 0.5 M NaCl, 5 mM EDTA, 1 mM DTT, 20% glycerol, and 0.1%
Nonidet P-40. Purified recombinant ADAR proteins were concentrated to
100 µl using Centricon (Amicon) before applying to the column.
Fractions (0.5 ml) were collected at a flow rate of 0.4 ml/min using a
fast protein liquid chromatography system. The molecular weight of ADAR
(monomer or oligomer) was estimated by comparison with molecular weight
standards obtained from Sigma; bovine thyroglobulin (669,000),
horse spleen apoferritin (443,000), sweet potato In Vitro RNA Editing Assay--
Editing of a synthetic
5-HT2C RNA C5 was assayed in vitro as described
previously (22), using 1× or 2× purified recombinant homodimer
complexes as well as 2× purified heterodimer complexes consisting of
one wild-type and another non-functional mutant ADAR monomer. The
standard editing reaction contained 20 fmol of a synthetic C5 RNA
substrate, 10 ng of recombinant ADAR proteins, 0.02 M Hepes
(pH 7.0), 0.1 M NaCl, 10% glycerol, 5 mM EDTA,
1 mM DTT, and 250 units/ml RNasin (Promega). The reactions
were incubated at 30 °C for various times. Quantitation of editing efficiency at five sites of 5-HT2CR RNA was carried out by
dideoxyoligonucleotide/primer extension assay as described previously
(10, 22). The ratio of the edited and unedited RNAs was estimated by
quantifying the radioactivity of the primer-extended products with a
phosphorimaging system (Amersham Biosciences).
Western Immunoblot Analysis--
Proteins were fractionated on
an SDS-8% polyacrylamide gel and transferred to
ImmobilonTM-P nylon membrane (Millipore, Bedford, MA).
Blots were blocked in a buffer containing phosphate-buffered saline and
3% nonfat dry milk. MAbs 15.8.6, 1.3.1, and 3.591 for detection of
native and recombinant ADAR1, ADAR2, and ADAR3 proteins, respectively (22, 42), and mAbs M2 and 6XHN for FLAG- and 6His epitope-tagged recombinant ADAR proteins, respectively, were used. ADAR-specific protein bands were detected by peroxidase-conjugated goat antibodies directed against mouse immunoglobulins (Kirkegaard and Perry Lab., Gaithersburg, MD) and chemiluminescense staining using
RenaissanceTM (PerkinElmer Life Sciences).
Recombinant ADAR1 and ADAR2 but Not ADAR3 Proteins Form Stable
Homodimeric Complexes--
A set of baculovirus constructs for ectopic
expression of ADAR1, ADAR2, and ADAR3 with either a FLAG or a 6His
epitope tag at the N terminus were prepared. Two different sizes of
ADAR1 protein, a full-length 150 kDa and a shorter 110 kDa form (p150 and p110) are synthesized because of differential usage of two Met
initiation codons (17). The full-length ADAR1 (p150), and ADAR2a among
the four known splicing isoforms of ADAR2 (19, 20), were investigated
in the present studies. FLAG- and 6His epitope-tagged ADAR1, ADAR2, or
ADAR3 proteins were coexpressed in Sf9 cells infected with
approximately a 1:1 ratio of two different recombinant baculoviruses,
and purified by sequential affinity chromatography, first on M2
anti-FLAG mAb-agarose gel and then TALON metal resin as schematically
shown in Fig. 1. Each purification step
was monitored by Western analysis using anti-FLAG or anti-6His antibody. Both ADAR1 and ADAR2 were purified as oligomeric complexes containing both FLAG- and 6His-tagged protein (Fig.
2, lanes 3 and 8 for ADAR1 and lanes 4 and 9 for ADAR2). The
binding of FLAG-tagged ADAR1 or ADAR2 protein to the first affinity
column (FLAG mAb column) was nearly complete, whereas a substantial
amount (30 to 50%) of the 6His-tagged ADAR1 or ADAR2 protein was
detected in this first flow-through fraction as expected. The unbound
6His-tagged ADAR protein represents, most likely, the oligomeric
complex consisting of 6His-tagged monomers only (see Fig. 1). In
contrast, only FLAG-tagged ADAR (30 to 50% of the total FLAG-tagged
protein present in the original extracts, again representing complexes
composed entirely of FLAG-tagged monomers) but almost no 6His-tagged
ADAR was detected in the flow-through of the second TALON affinity
column, indicating complete binding of the 6His-tagged ADAR that had
been preselected by FLAG mAb-agarose gel chromatography
(i.e. the oligomeric complex consisting of both FLAG- and
6His-tagged monomers). Overall, the yield of the 2× purified
oligomeric complex was 30 to 50% of the ADAR present in the extracts,
consistent with the amount expected on the basis of monomers sorting
randomly into oligomeric complexes without regard to the FLAG or 6His
tag. This essentially complete recovery establishes that the oligomer
represents the major form of the complex and shows also that our
sequential affinity column purification scheme did not selectively
enrich a rare form of the complex. The copurification of FLAG with 6His
epitope-tagged ADAR1 as well as ADAR2 were confirmed through a similar
sequential affinity column chromatography, but in the reverse order,
i.e. TALON metal resin first and then M2 anti-FLAG
mAb-agarose gel (data not shown). In contrast, recombinant ADAR3
protein was detected in Western analysis only by using the antibody
corresponding to the type of affinity chromatography applied first
(Fig. 2, lane 5) but not by the reciprocal antibody
corresponding to the second affinity chromatography (Fig. 2, lane
10). These results clearly indicate that recombinant ADAR1 and
ADAR2 but not ADAR3 form oligomers.
The apparent molecular mass of ADAR1 (full-length p150 form), ADAR2a,
and ADAR3 have been estimated to be 150, 90, and 80 kDa, respectively,
by SDS-PAGE (18-22, 30, 31). To determine the size(s) of ADAR1 and
ADAR2 recombinant proteins purified through sequential affinity
chromatography, the oligomeric complexes eluted from the second
affinity column were fractionated on a Superose 12 size exclusion
column (Fig. 3, A
and B, top panels). Based on the standard size
markers, the sizes of ADAR1 and ADAR2 oligomeric forms were estimated
to be 300 and 180 kDa, respectively, indicating that they are both
homodimers.
The sequential affinity column chromatography purification procedure as
designed precludes detection of ADAR monomer in the 2× purified
fraction (Fig. 1). Therefore, to check for free monomers we also
examined the apparent sizes of ADAR1 and ADAR2 when expressed as single
epitope-tagged proteins and purified by a single affinity column (1×
purified). We found that the fractionation profiles for both 1×
purified ADAR1 and ADAR2 were identical to those of the 2× purified
ADAR proteins. Distinctive elution peaks anticipated for the monomeric
forms of ADAR1 (150 kDa) and ADAR2 (90 kDa) were not detected (data not
shown). In addition, we carried out the sequential affinity
chromatography of two proteins, each separately tagged with FLAG and
6His epitope, and purified on a single affinity column (1× purified)
following in vitro mixing and incubation. We found that
there was no significant in vitro exchange of two differentially epitope-tagged ADAR1 or ADAR2 proteins, at least during
a 2-h incubation at 30 °C (data not shown). Taken together, our
results suggest that both ADAR1 and ADAR2 recombinant proteins ectopically expressed in Sf9 cells form predominantly a stable homodimer complex.
Superose 12 column chromatography conducted with FLAG epitope-tagged
recombinant ADAR3 proteins purified by a single affinity column
chromatography (1× purified) revealed a complex elution pattern
significantly different from one expected for its monomeric state (Fig.
3C, upper panel). Because the silver staining of
the recombinant ADAR3 proteins indicated that they were more than 90%
homogeneous, the results were surprising. Although the presence of a
minor peak at the position expected for the monomer form was clearly
detected (80 kDa, indicated by an arrow), the majority of
recombinant ADAR3 proteins eluted as a broad smear covering a range
from ~400 kDa to much smaller than the monomeric form, suggesting
possible nonspecific interaction with the Superose 12 matrix. Using
high salt (2 M NaCl) and different pH buffers did not
change the elution pattern.
We previously have reported the presence of a ssRNA binding domain
located within the arginine-rich R domain (22). Thus, we reasoned that
binding of RNA molecules from the insect cells might be responsible for
the unusual migration of ADAR3. However, size fractionation of the
ADAR3 after treating extensively with RNases specific for both ssRNA
and dsRNA (see below) was identical to that of untreated protein (data
not shown). In conclusion, the results of sequential affinity column
purification strongly suggest a monomeric state for recombinant ADAR3,
but we currently do not understand the reason for its unusual migration
on Superose 12.
Interestingly, FLAG epitope-tagged ADAR1 and ADAR2 expressed in
Sf9 cells and purified on M2 anti-FLAG mAb-agarose gels
have been demonstrated to be enzymatically active in deamination of adenosines on a long synthetic dsRNA substrate or in the site-selective in vitro RNA editing of GluR or 5-HT2CR
substrate RNAs (20, 30), whereas recombinant ADAR3 proteins are
inactive in these assays (22). We now realize that the ADAR1 and ADAR2
recombinant proteins used for our previous studies were predominantly
homodimeric forms, and thus it may be that the enzymatic activity of
these two recombinant ADARs are related to a dimer formation.
RNA Independent Homodimerization of ADAR1 and
ADAR2--
The size exclusion chromatography with doubly
purified fractions revealed no large oligomeric complexes that would
form via binding of multiple ADAR monomers to a long dsRNA. However,
homodimerization of ADAR1 and ADAR2 recombinant protein could be
dependent on the presence of a short dsRNA substrate of Sf9 cell
origin serving to bridge two monomers of ADAR1 or ADAR2. Both
RNA-dependent and -independent homodimerization of the
dsRNA-activated protein kinase PKR has been reported (43-45). The
overall arrangement of functional domains in PKR, two dsRNA binding
domains at the N terminus, and a separate catalytic domain, is
somewhat similar to ADAR. We therefore tested the homodimer complex
copurified by sequential affinity chromatography for their sensitivity
to single-strand and double-strand specific ribonuclease treatments
(Fig. 4). FLAG and 6His epitope-tagged ADAR1 or ADAR2 recombinant proteins remained together as a dimer regardless of RNase A and T1 (ssRNA specific) or RNase V1 (dsRNA specific) treatment. Thus, the association of two different
epitope-tagged monomers is unlikely to be mediated through an RNA
molecule(s) (Fig. 4B). On the other hand, it is still
possible that an RNA molecule directly involved in formation of the
ADAR homodimer may be resistant to the ribonuclease digestion because
of its close contact with ADAR proteins. To eliminate the possibility, we looked for RNA molecules bound to the ADAR homodimer complex by
32P labeling and PAGE analysis of the labeled RNAs.
Approximately 2 µg of 2× purified ADAR1 or ADAR2 was subjected to
proteinase K digestion and subsequent RNA extraction. The total RNA
extracted was then labeled using 32P-labeled pCp and T4 RNA
ligase and analyzed by 7 M urea-PAGE. By including known
amounts of a synthetic 21-nucleotide RNA molecule as an internal
control, we concluded that no significant level of RNA is present (less
than 0.1 ng of RNA for 2 µg of the doubly purified ADAR homodimer).
This is less than one RNA base per dimer, clearly insufficient to act
as a bridge between monomeric proteins (data not shown).
Homodimerization of Native ADARs--
To confirm
homodimerization of native ADAR1 and ADAR2, HeLa cell and mouse
brain nuclear extracts were subjected to Superose 12 size exclusion
column chromatography. In HeLa cells, both p150 and p110 forms of ADAR1
were detected (Fig. 3A, middle panel). The peak
of native ADAR1 p150 coincided with that of the recombinant p150
homodimer complex (300 kDa), and ADAR1 p110 proteins also eluted in the
fractions expected for its homodimer complex (220 kDa). In mouse brain,
only the p110 form of ADAR1 was detected in nuclear extracts, and it
appeared in the fractions expected for the homodimer (Fig.
3A, lower panel). Native ADAR2 proteins appear
also to exist mainly as a complex of homodimer size (Fig. 3B, middle and lower panels). Both the
ADAR2a and ADAR2b splicing isoforms of native human ADAR2 differing in
size by 40 amino acid residues (19, 20) were detected in the HeLa
nuclear extract. In mouse, the size difference, only 10 amino acid
residues, was not sufficient for detection of these two isoforms
separately (18). We noted a slightly broader elution peak of native
ADAR1 and ADAR2 especially with a tailing toward smaller molecular
weight regions in comparison with recombinant homodimer complexes,
possibly indicating the presence of some monomer form (Fig. 3,
A and B, middle and lower
panels). As with the recombinant protein, fractionation of the
native mouse brain ADAR3 resulted in a complex elution pattern, but
with a minor peak that coincides with the size for the anticipated
ADAR3 homodimer (160 kDa), suggesting the possibility that native ADAR3
may also form a homodimeric complex in brain (Fig. 3C,
lower panel). Unlike the recombinant protein, smearing into
fractions corresponding to small molecules was not observed with native
ADAR3. More importantly, no obvious peak of monomer was detected for
native ADAR3 (Fig. 3C, lower panel), in contrast to recombinant ADAR3 (Fig. 3C, upper panel). It
should be pointed that a fraction of native ADAR1 (detected for certain
extract preparations but not seen clearly in Fig. 3A,
middle and lower panels) and ADAR3 proteins (Fig.
3C, lower panel) migrated with an apparent
molecular weight of >600,000, whereas no larger complex of native
ADAR2 was detected other than the homodimer size complex in the
extracts of HeLa cells and mouse brain. Interestingly, digestion of the
extracts with RNase A and T1 prior to Superose 12 size exclusion column
chromatography resulted in shifting of at least a part of the larger
ADAR1 or ADAR3 complexes to complexes of homodimer size. Most
importantly, however, RNase digestion (ssRNA and dsRNA specific) did
not affect the size of the homodimer-like complex of ADAR1, ADAR2, or
ADAR3, indicating RNA independent homodimerization of native ADARs
(data not shown). We have recently reported the association of both
ADAR1 and ADAR2 with large nuclear ribonucleoprotein particles,
consisting of four splicesomal subunits that assemble together with the
pre-mRNA. However, the size of the complexes observed in the
present studies are far smaller than the large nuclear RNP particles
(200 S), which can be detected with the HeLa nuclear extract prepared
through a specific gentle extraction procedure in the presence of 2 mM vanadyl ribonucleoside RNase inhibitor (42). Thus, the
nature of the larger complexes of native ADAR1 and ADAR3 detected in
the present studies (both RNase digestion sensitive and resistant) is
not clear at this time.
Different Members of the ADAR Gene Family Do Not Associate as
Heterodimers--
We next investigated the possibility of heterodimer
complex formation between two different members of the ADAR gene family (Fig. 5). Two differentially
epitope-tagged ADAR gene family members, i.e. ADAR1 and
ADAR2 (lanes 1 and 7), ADAR1 and ADAR3
(lanes 2 and 8), or ADAR2 and ADAR3 (lanes
3 and 9), were coexpressed in the same cell
(Sf9) and purified by sequential affinity chromatography. The
presence of both ADAR proteins tagged with FLAG and 6His was confirmed
in the extracts, but no formation of heterodimer complexes between any
combination of two different ADAR gene family members was detected
(lanes 10-12). Although these experiments with
the recombinant ADAR proteins suggest no formation of heterodimer complexes, it is possible that a heterodimer complex may form in
vivo, i.e. between ADAR2 and ADAR3 in brain. We
therefore conducted coimmunoprecipitation experiments using nuclear
extracts of mouse brain in an attempt to detect heterodimer complex
formation between two different ADAR native proteins. Mouse nuclear
extract immunoprecipitated with a mAb specific to an ADAR gene family
member was examined by Western blotting analysis with mAbs specific to
the remaining two ADAR gene family members. All the ADAR gene family
member-specific mAbs successfully immunoprecipitated the anticipated
cognate ADAR protein but did not coimmunoprecipitate any of the
remaining ADAR gene family members (data not shown). These results
suggest that formation of heteromeric complexes among different ADAR
gene family members is unlikely.
Functional Interaction between the Two Monomers of the ADAR
Homodimer--
The two monomers within an ADAR1 or ADAR2 homodimer may
bind to two separate substrate RNAs and deaminate adenosine residues at
each catalytic center independently. Alternatively, the two monomers
may act cooperatively for conversion of an adenosine residue of a
single substrate RNA. To obtain some insights into possible functional
interaction between the two monomers, we examined the enzymatic
activities of a heterodimer complex made between wild-type and a
non-functional ADAR mutant in comparison to those of mutant or
wild-type homodimers (Fig. 6). ADAR1
E912A with a Glu912 Homodimerization of ADAR1 and ADAR2--
In this study, we have
demonstrated that recombinant ADAR1 and ADAR2 both exist predominantly
as stable homodimers. Purification of the complexes via sequential
affinity chromatography with two different epitope tags revealed for
the first time the presence of oligomeric complexes, whereas size
exclusion column chromatography identified the complexes as homodimers.
The homodimer formation is mediated by protein-protein interaction
between two monomers and is independent of binding to RNA. Although we
originally assumed that both oligomeric as well as monomeric forms of
ADAR proteins might be present in equilibrium, our results suggest that
recombinant ADAR1 or ADAR2 ectopically expressed in Sf9 cells
appears to form predominantly a homodimer, possibly immediately after
translation or even during translation as occurs for some dimeric
proteins (e.g. tubulin). Furthermore, homodimers once formed
appear to be very stable without detectable exchange of their monomer
components under physiological conditions.
Native ADAR1 biochemically purified from various sources (bovine liver,
calf thymus, and Xenopus oocytes) has been reported to have
various sizes (80 to 120 kDa), smaller than the full-length p150 form,
probably because of translation initiation at the internal methionine
codon (equivalent to our p110 form) or truncation of the N terminus as
a result of nonspecific proteolysis (47-49). In contrast to our
findings in this study with recombinant ADAR1, biochemically purified
native ADAR1 was found to be a monomer by size exclusion column
chromatography and by glycerol gradient sedimentation analysis
(47-49). In addition to the differences in protein size and possible
species differences, another possible explanation for the discrepancy
is that native ADAR1, predominantly in the homodimer state, might
become dissociated into monomers because of the rather vigorous
biochemical purification procedures applied. Interestingly, it has been
reported that the monomeric form of Xenopus ADAR1, once
biochemically purified, does not re-dimerize even under low ionic
strength conditions (49). In all previous studies, the purification of
native ADAR1 was assayed by enzymatic activity, suggesting that the
biochemically purified monomeric ADAR1 was capable of deaminating the
dsRNA substrate used in their assay (46-48). An alternative
interpretation consistent with that data is that ADAR1 protein
dissociated into monomers because of the non-physiological conditions
applied during purification, but was reconstituted into homodimers upon
binding to dsRNA during the A-to-I base modification assay, which
restored its enzymatic activity. Recently, homodimerization of ADAR2 on
a dsRNA substrate has been proposed based on the results of kinetic
analysis (50). A-to-I RNA editing was observed only under conditions
allowing ternary complex formation between the ADAR2 homodimer and
GluR-B RNA substrate, indicating a requirement for ADAR2 dimer
formation for its site-selective editing activity (50). Binding of a
substrate RNA and acceleration of homodimerization of PKR has been
reported also (43-45).
Our results on recombinant ADAR1 and ADAR2 are supported by size
exclusion column chromatography analysis of HeLa and mouse brain
nuclear extracts, which detected the anticipated homodimer size complex
of native ADAR1 and ADAR2. The native complexes were detected by
Western blotting analysis of the crude extracts using ADAR1- or
ADAR2-specific antibodies, and thus we cannot exclude the possibility
that they represent the monomer associated with some currently unknown
molecule (protein or RNA). However, the identical sizes of the native
complexes and recombinant homodimers suggest that a large fraction
of native ADAR1 or ADAR2 most likely also exist as homodimers.
Possible Dimerization of Native ADAR3 in Brain--
In contrast to
the results with ADAR1 and ADAR2, we could not detect the homodimer of
recombinant ADAR3. Recombinant ADAR3 ectopically expressed in
Sf9 cells apparently remains as a monomer, which may explain its
lack of enzymatic activity (22). The behavior of the recombinant ADAR3
during size exclusion column chromatography was also aberrant, eluting
from a Superose 12 column as a broad smear, including fractions
corresponding to physically impossible sizes. It may be that a majority
of recombinant ADAR3, incapable of homodimerizing, does not have a
uniform structure and migrates through the column without forming a
distinctive fractionation peak. Alternatively, monomeric recombinant
ADAR3 may interact non-specifically with the matrix material of the
sizing column used, possibly because of lack of a required
post-translational modification that takes place in brain but not in
Sf9 insect cells. This hypothesis then predicts that native
ADAR3 protein may form a homodimer and behave differently from the
recombinant proteins during size exclusion column chromatography.
Indeed, size fractionation analysis of mouse brain extracts on a
Superose 12 column revealed an elution peak corresponding to a complex
of ~160 kDa, the anticipated size of the ADAR3 homodimer, in addition
to a separate peak corresponding to a much larger molecular mass
complex (>600 kDa). The size of the 160-kDa complex also corresponds
to the size of a potential ADAR2/ADAR3 heterodimer (Fig.
3C, lower panel).
Heterodimerization among proteins related to the human ADAR family has
been observed in other organisms. The ADAT gene family (tRNA-specific
A-to-I editing enzymes) has been identified in yeast because of their
deaminase domain sequence homology to ADAR (1). ADAT1 edits
A37 of tRNAAla as a monomer (51), whereas
A34 at the first anticodon position of tRNAAla
is edited specifically by a heterodimer formed by ADAT2 and ADAT3 (52).
ADAT2 is the catalytically active subunit, whereas ADAT3 is the
regulatory subunit not directly involved in the A-to-I deamination
mechanism (52). More recently, the possibility of heterodimer formation
between c.e.ADAR1 and c.e.ADAR2 has also been indicated (27). We were
intrigued by the possibility that enzymatically inactive ADAR3 might
form a heterodimer complex with an enzymatically active ADAR member
(e.g. ADAR2), and therefore investigated possible
heterodimer formation among three different ADARs. We found no
indication of any heterodimer formation between two different ADAR gene
family members, including the heterodimer consisting of ADAR2 and
ADAR3, at least among recombinant proteins co-expressed in the same
Sf9 insect cell. Taken together with our preliminary results
from coimmunoprecipitation experiments of mouse brain extracts, we
conclude that the complex eluting around 160 kDa is likely to represent
a ADAR3 homodimer that forms only in mouse brain.
Interaction of Two Monomers--
The structural basis of the
interactions between two monomer subunits of ADAR1 or ADAR2 is
currently unknown. The deaminase domain structure of ADAR is predicted
to have a similarity to E. coli cytidine deaminase (1, 15).
In the x-ray crystal structure of E. coli cytidine deaminase
the homodimer has a 2-fold symmetry axis, indicating that the two
monomers are structurally indistinguishable and predicted to be
functionally equivalent (34). Molecular modeling of another cytidine
deaminase APOBEC-1, ApoB RNA editing enzyme, predicts a structural
configuration of the homodimer very similar to that of E. coli cytidine deaminase (37). Extensive interactions widely spread
over many regions are involved in the formation of the monomer-monomer
interface of E. coli cytidine deaminase and APOBEC-1
homodimers (34, 37). The active site in each monomer is completed only
with contributions from the other partner subunit (34, 37). We have
recently conducted studies to map the regions required for formation of the homodimer using the ADAR1 mutant baculovirus constructs (38) coexpressed with the wild-type construct and sequential affinity chromatography purification of the dimer
complex.2 Although the
conclusions of those studies remain preliminary because of the unstable
nature of certain deletion constructs, it appears that the interface
interactions of the two monomers occurs over a widespread region
including the deaminase domain as well as the dsRNA binding domains. In
contrast, the N-terminal region containing the Z-DNA binding domain
(amino acids 1 to 295) is not required, because formation of the
heterodimer between p150 and p110 of ADAR1 can be detected. The regions
critical for nuclear import or export of ADAR1 have been mapped
recently (53, 54). It would be interesting to know if formation of the
homodimer is involved also in the nuclear-cytoplasmic shuttling
mechanism. The stable nature of the recombinant ADAR1 and ADAR2
homodimers predict its de novo formation during translation
and transport as a homodimer unit. However, it is also possible that
the monomer is used as a transport form (nuclear import or export),
whereas homodimer formation may be a part of a mechanism to concentrate the active complex in one compartment of the cell.
Dimerization is known to affect the enzymatic activity as well as
substrate specificity (45, 52). Each dsRNA binding domain of ADAR is
independently capable of binding to a dsRNA region as short as 15 to 20 bp. A number of ADAR proteins bind to multiple sites of a long
completely complementary dsRNA (55), but also to a discrete site of a
specific hairpin RNA (56). Thus, each monomer of ADAR1 or ADAR2 may
bind to a separate dsRNA molecule through its own dsRNA binding
domains. However, it is also possible that one homodimer binds to a
single substrate RNA whereas the dsRNA binding domains of the two
monomers make cooperative interactions. In an attempt to understand the
functional interactions of the two monomers, enzymatic activity of a
heterodimer consisting of one wild-type and one inactive mutant monomer
was compared with the homodimers consisting of only wild-type
functional monomers. The point mutation was in a position that is
equivalent to the residue in APOBEC-1 (E63) predicted to play a
critical role in both the catalysis of the deamination reaction and in
monomer interface interactions (37). Interestingly, the site-selective editing activity of ADAR1 or ADAR2 heterodimer complexes revealed substantial cooperation of the two monomers. Our results hint that the
glutamate residue, Glu912 of ADAR1 or Glu396 of
ADAR2, may contribute to the formation of the active site for the
partner monomer in addition to the catalysis mechanism. Surprisingly,
the enzymatic activity of the heterodimers suggested that each monomer
can independently catalyze non-selective deamination of a long dsRNA
substrate. We do not presently have enough information to distinguish
several possible models for the interactions of the two monomers or
each monomer with a substrate RNA. A model for the interaction of two
monomer subunits of APOBEC-1 with a single substrate RNA (ApoB RNA) has
been proposed (37). In this model, the targeted cytidine residue
is deaminated at the catalytic center of one monomer, whereas a
specific downstream uridine residue of the same RNA is bound by the
partner subunit. In contrast, each monomer of the E. coli
cytidine deaminase homodimer catalyzes deamination of one cytidine
independently (34). On the analogy of the model for APOBEC-1 and ApoB
RNA interaction, one possible interpretation of our results is that the
number of substrate RNAs interacting with each ADAR homodimer may vary
depending on the tertiary structure of the RNA. A long uninterrupted
and completely complementary dsRNA (A-form double helix, and most
likely rod shaped) may be independently bound and deaminated by each
monomer. In contrast, a substrate RNA with relatively short dsRNA
regions, loops, and bulges such as 5-HT2CR may be bound by
the dsRNA binding domains of both monomers, and deaminated after
formation of an active complex in which interactions between the two
monomers brings about alignment of a select adenosine residue with one of the two catalytic centers. Our future studies will define
interactions of the two subunits of ADAR1 and ADAR2 homodimers and
address their significance for enzymatic activity as well as
intracellular localization.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
GluR-B subunit dramatically decreases the Ca2+ permeability
of the channel (7). Substantial reduction in G-protein coupling
efficiency is noted with A-to-I editing of 5-HT2CR RNA at
five positions (A to E sites) located in the intracellular II loop
region (6, 8-10). A-to-I RNA editing also occurs in non-coding
sequences. Editing of its own intron sequence by adenosine deaminase
acting on RNA (ADAR) 2 creates an alternative splice acceptor site
leading to synthesis of a truncated translation product, which may be a
negative feedback mechanism to regulate the activity of ADAR2 (11). In
all these examples, a dsRNA structure formed between the exonic
sequences containing an editing site(s) and downstream or upstream
intronic sequences has been proven to be essential for editing (4-6,
12). Systematic search with a recently devised method for cloning of
inosine-containing RNAs has led to identification of more than two
dozen editing sites occurring in the intron and 3'-untranslated regions
of new target genes. A-to-I RNA editing of these non-coding regions may
affect the splicing rate, the translation efficacy, or stability of the edited mRNAs (13). Furthermore, the intronic and untranslated region sequences subjected to A-to-I RNA editing often contain common
repetitive elements such as Alu and LINE1 repeats forming a long dsRNA
structure, raising the possibility that A-to-I RNA editing may be
involved in a mechanism regulating the very abundant repetitive
sequences in mammalian genomes (2, 3, 13). Finally, A-to-I RNA editing
of dsRNAs derived from transgenes appears to prevent silencing of the
transgenes regulated by RNA interference, revealing the potential
intersection of the two mechanisms, RNA editing and RNA interference
both evolved to deal with dsRNA (14).
ES cells die at the midgestation stage
with a phenotype indicative of dyserythropoietic defects (29). It has
not yet been ruled out that antisense effects generated by transcripts
derived from the ADAR1-targeted allele may contribute to the
observed embryonic lethal phenotype (29).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol instead of 1 mM DTT. After washing the
column with 10 ml each of buffer A containing 0.15 M NaCl,
0.75 M NaCl, and again 0.15 M NaCl, the complex
was eluted with 5 ml of buffer A containing 0.15 M NaCl and
200 µg/ml FLAG peptide. The pooled peak fractions were dialyzed
against buffer B (10 mM Tris, pH 7.5, 0.3 M
NaCl, 20% glycerol, 0.05% Nonidet P-40, 1 mM
-mercaptoethanol) and then applied to a TALON metal resin (BD
Biosciences, Palo Alto, CA) affinity column. Following extensive
washing with buffer B containing 10 mM imidazole, proteins were eluted with 150 mM imidazole. The yield of recombinant
proteins during the sequential affinity chromatography was followed by Western blotting analysis using an anti-FLAG M2 mAb (Sigma) or anti-6His 6XHN mAb (BD Biosciences). The purity of recombinant proteins
purified by the first ("1× purified") and second ("2× purified") affinity column chromatography were determined by
electrophoresis on a 10% SDS-PAGE gel followed by silver staining.
-32P]ATP-labeled c-myc antisense ssRNA or
dsRNA (40), confirming their complete digestion with the relevant RNase(s).
-amylase (200,000),
yeast alcohol dehydrogenase (150,000), bovine serum albumin (66,000), and bovine carbonic anhydrase (29,000). The peak for the ADAR complex
was confirmed by Western blotting analysis, and the peak position of
the marker proteins was determined by measuring the optical
absorption at 280 nm.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (23K):
[in a new window]
Fig. 1.
Purification of differentially epitope-tagged
ADAR proteins by sequential affinity chromatography.
Recombinant ADAR proteins purified through single or double affinity
column chromatography are denoted as 1× Purified or
2× Purified, respectively.
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Fig. 2.
Oligomerization of recombinant ADAR1 and
ADAR2 but not ADAR3. Two ADAR recombinant proteins, epitope-tagged
at their N termini with either FLAG or 6His, were expressed together in
Sf9 cells (transfection I for ADAR1, II
for ADAR2, and III for ADAR3). Oligomeric forms of ADAR1
(lanes 3 and 8), ADAR2 (lanes 4 and
9), or ADAR3 (lanes 5 and 10),
purified by sequential column chromatography based on both epitope
tags, were identified by Western analysis using mAbs specific to the
epitope tag. Anti-FLAG mAb was used for Western analysis following the
first M2 anti-FLAG mAb affinity column chromatography purification
(lanes 3-5), whereas anti-6His mAb was used after the
second TALON affinity column chromatography purification (lanes
8-10). Recombinant ADAR2 tagged with one epitope only, FLAG or
6His (F-ADAR2 and H-ADAR2), were included as controls to show the
specificity of the two mAbs used for Western analysis (lanes
1, 2, 6, and 7). ADAR1 or ADAR2
oligomeric complexes containing both FLAG and 6His epitope tags
(F/H-ADAR1 and F/H-ADAR2)
were identified with anti-6His mAb (lanes 8-10) and also
with anti-FLAG mAb (not shown). In contrast, ADAR3 was detected only
with mAb matching the epitope used for the first affinity
chromatography, i.e. FLAG in this set of experiments
(lane 5), but not with mAb matching the epitope used for the
second affinity chromatography, i.e. 6His (lane
10). When the order of the sequential affinity chromatography was
reversed, i.e. TALON first and M2-FLAG mAb-agarose second,
ADAR3 (6His-tagged) was detected again only after the first affinity
chromatography with anti-6His mAb (not shown). Proteins purified by
single (or first) affinity chromatography are indicated as
1X, whereas those purified by two sequential affinity column
are designated as 2X. Approximately 10 ng each of 1×
purified and 20 ng each of 2× purified proteins were loaded onto 8%
SDS-polyacrylamide gels.
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Fig. 3.
Analysis of ADAR oligomeric complexes by
Superose 12 gel filtration column chromatography. Oligomeric forms
of ADAR proteins fractionated by Superose 12 gel filtration column
chromatography were analyzed by Western blotting analysis using mAb
specific to ADAR1 (panel A), ADAR2 (panel B), or ADAR3
(panel C). Recombinant ADAR1 (A, upper
panel) and ADAR2 (B, upper panel) proteins,
differentially epitope-tagged and purified by sequential affinity
column chromatography (2X), and recombinant ADAR3 proteins
(C, upper panel) purified by a single M2 FLAG mAb
affinity column (1X), were analyzed. Extracts made from HeLa
cells or mouse brain were also investigated. The positions of size
marker proteins are indicated by open arrowheads. Expected
positions of the ADAR1 homodimer (300 kDa for p150 and 220 kDa for
p110) and the ADAR2 homodimer (180 kDa) are indicated by
arrows (panels A and B). Positions of
ADAR3 monomer (80 kDa) and homodimer (160 kDa) are also indicated by
arrows (panel C). Recombinant ADAR3 proteins were
detected also in all fractions collected beyond the 30 fractions shown,
up to fraction 50.
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Fig. 4.
RNase-resistant homodimerization of
recombinant ADAR1 and ADAR2. A, digestion of ssRNA with
RNases A and T1 and dsRNA with RNase V1. Digestion of
32P-labeled c-myc RNAs, antisense ssRNA by A and
T1 (lanes 1-5), and dsRNA by V1 (lanes 6-10),
were tested at 20 °C. Both ssRNA and dsRNA were digested completely
within 30 min. B, F/H-ADAR1 and F/H-ADAR2 dimer complexes
purified by affinity chromatography on anti-FLAG M2 mAb-agarose beads
(1× purified) were subjected to RNase digestion using either A and T1
or V1, at 20 °C for 30 min, further purified by TALON affinity
chromatography, and then tested with Western analysis using anti-FLAG
mAb. Controls (C) were done exactly the same but without
RNase treatments.
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Fig. 5.
Absence of oligomerization among different
ADAR gene family members. Three separate extracts made from
Sf9 cells infected with a combination of two recombinant
baculovirus: transfection I, F-ADAR2 and H-ADAR1
(lanes 1 and 7), transfection II,
F-ADAR3 and H-ADAR1 (lanes 2 and 8), and
transfection III, F-ADAR3 and H-ADAR2 (lanes 6 and 12), were purified first by M2 anti-FLAG mAb
(lanes 4-6) and then by TALON affinity column
chromatography (lanes 10-12). Two recombinant proteins are
detected in the extracts with anti-FLAG (lanes 1-3) and
anti-6His mAb (lanes 7-9). FLAG-tagged proteins were
detected with anti-FLAG mAb (lanes 4-6) following the first
affinity chromatography (1X), but no 6His-tagged proteins
(i.e. no heterodimer) were detected with anti-6His mAb
(lanes 10-12) after the second affinity chromatography
(2X).
Ala substitution and ADAR2a E396A
with a Glu396
Ala substitution were used as the mutant
monomers. The glutamate residues Glu912 of ADAR1 and
Glu396 of ADAR2 are located within the tripeptide sequences
HAE and PCG, which are highly conserved among
ADAR gene family members as well as cytidine and deoxycytidylate
deaminase gene family members. These residues are believed to play a
critical role in proton transfer functions required for the hydrolytic
deamination reaction (15, 38). We have previously shown that
site-directed mutagenesis of Glu912 of ADAR1 (E912A)
results in complete abolishment of the deaminase activity without
affecting substrate RNA binding capability (38). Formation of the
heterodimer between one wild-type and one mutant monomer was first
confirmed by their copurification through sequential affinity column
chromatography as above (data not shown). Non-selective ADAR activity,
which converts multiple adenosines to inosines in a
sequence-independent manner, was determined on a long 575-bp synthetic
c-myc dsRNA (Fig. 6A). Site selective A-to-I RNA
editing activity was monitored by determining the editing of
5-HT2CR RNA at the A site by ADAR1 and the D site by ADAR2.
Preferential editing of the A and D sites by ADAR1 and ADAR2,
respectively, has been demonstrated previously in vitro
using recombinant proteins (6, 22, 46). Preliminary time course
experiments were conducted separately to choose conditions under which
the enzymatic reaction remains first-order in enzyme concentration, so
that the results can be compared quantitatively. The enzymatic
activities of heterodimers consisting of one wild-type and one
non-functional mutant monomer were found to be approximately half (55%
for ADAR1 and 52% for ADAR2) of the wild-type homodimer activity when
tested with the long c-myc dsRNA substrate (Fig.
6A). In contrast, site-selective RNA editing activity by the
heterodimer, determined on 5-HT2CR RNA, decreased to
~30% of the wild-type homodimer activity (Fig. 6B). These
results may indicate that natural substrate RNAs induce cooperative
interactions between the two monomers in the wild-type homodimer
complex. Presumably, the three-dimensional structure of natural
substrates such as 5-HT2CR RNA, which includes short dsRNA
regions, loops, and bulges, facilitate simultaneous interactions with
both monomers in the dimer complex.
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Fig. 6.
Functional interaction between two subunits
of the dimer complex in A-to-I RNA editing. Enzymatic activities
of wild-type (both 1× and 2× purified), non-functional mutants (ADAR1
E912A and ADAR2 E396A), and heterodimer complexes consisting of
6His-tagged wild-type and FLAG-tagged mutant subunits were tested.
By conducting a preliminary time course experiment, an appropriate
incubation time within the linear reaction range was selected. ADAR1,
open bars. ADAR2, filled bars. Three independent
experiments were conducted (n = 3), and standard errors
are indicated. A, A-to-I base modification activities were
determined with a synthetic long c-myc dsRNA (575 bp) by
using 10 ng each of recombinant enzyme for 15 min at 37 °C. The
results were normalized relative to the values obtained with wild-type
1× purified enzymes. A-to-I base modification by ADAR1 and ADAR2
wild-type enzymes (1× purified) was 11.5 and 21.9%, respectively.
B, site-specific editing of 5-HT2CR RNA was
monitored. In vitro editing of 5-HT2CR RNA at
the A site by ADAR1 and at the D site by ADAR2 was carried out for 30 and 10 min, respectively, at 30 °C. A site editing by ADAR1
wild-type (1× purified) was 25.2%, whereas D site editing by ADAR2
wild-type (1× purified) was 51.6%.
DISCUSSION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank C-X. Chen for preparation of epitope-tagged ADAR expression constructs and the Wistar Genomics/Microarray, Expression Vector-Recombinant Protein Production, and Hybridoma facilities for excellent technical assistance. We also thank the Wistar editorial services department for preparing the manuscript.
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FOOTNOTES |
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* This work was supported in part by grants from the National Institutes of Health, the Doris Duke Charitable Foundation, the Israel-US Binational Science Foundation, and the March of Dimes (to K. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to the results of this work.
§ Supported by a training grant from the NCI, National Institutes of Health.
** To whom correspondence should be addressed: The Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104. Tel.: 215-898-3828; Fax: 215-898-3911; E-mail: kazuko@wistar.upenn.edu.
Published, JBC Papers in Press, March 4, 2003, DOI 10.1074/jbc.M213127200
2 D-S. C. Cho and K. Nishikura, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: dsRNA, double-stranded RNA; ADAR, adenosine deaminase acting on RNA; GluR, glutamate receptor; 5-HT, 5-hydroxytryptamine or serotonin; 5-HT2CR, serotonin receptor subtype 2C; ssRNA, single-stranded RNA; mAb, monoclonal antibody; DTT, dithiothreitol; c.e.ADAR, Caenorhabditis elegans adenosine deaminase acting on RNA.
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