ARCD-1, an apobec-1-related cytidine deaminase,
exerts a dominant negative effect on C to U RNA
editing
Shrikant
Anant1,
Debnath
Mukhopadhyay1,
Vakadappu
Sankaranand1,
Susan
Kennedy1,
Jeffrey O.
Henderson1, and
Nicholas O.
Davidson1,2
Departments of 1 Internal Medicine and 2 Molecular
Biology and Pharmacology, Washington University School of Medicine,
St. Louis, Missouri 63110
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ABSTRACT |
Mammalian apolipoprotein
B (apoB) C to U RNA editing is catalyzed by a multicomponent holoenzyme
containing a single catalytic subunit, apobec-1. We have characterized
an apobec-1 homologue, ARCD-1, located on chromosome 6p21.1, and
determined its role in apoB mRNA editing. ARCD-1 mRNA is ubiquitously
expressed; phylogenetic analysis reveals it to be a distant member of
the RNA editing family. Recombinant ARCD-1 demonstrates cytidine
deaminase and apoB RNA binding activity but does not catalyze C to U
RNA editing, either in vitro or in vivo. Although not competent itself
to mediate deamination of apoB mRNA, ARCD-1 inhibits apobec-1-mediated
C to U RNA editing. ARCD-1 interacts and heterodimerizes with both apobec-1 and apobec-1 complementation factor (ACF) and localizes to
both the nucleus and cytoplasm of transfected cells. Together, the data
suggest that ARCD-1 is a novel cytidine deaminase that interacts with
apobec-1 and ACF to inhibit apoB mRNA editing, possibly through
interaction with other protein components of the apoB RNA editing holoenzyme.
gene cluster; immunofluorescence; protein-protein interaction; RNA-protein interaction
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INTRODUCTION |
APOLIPOPROTEIN B
(apoB), the major protein component of triglyceride-rich lipoprotein
particles, is present in two forms, apoB100 and apoB48, each the
product of a common structural gene (reviewed in Ref. 41).
ApoB100, generated in the human liver, is present on very low
density and low-density lipoprotein particles. However, in the
mammalian intestine and liver of some species, the genomically
templated apoB mRNA undergoes a single posttranscriptional C to U RNA
editing event, creating a premature stop codon (8, 31).
Translation of the edited mRNA results in the formation of apoB48,
which is a component of chylomicron particles (8, 31).
Accordingly, C to U editing of apoB mRNA plays a major role in the
formation and resulting metabolism of apoB-containing plasma
lipoproteins (reviewed in Ref. 2).
ApoB mRNA editing is catalyzed by a multicomponent enzyme complex,
referred to as an editosome (2, 33). The complex contains a minimal functional core, comprising two proteins, apobec-1 and a
required complementation factor, ACF (21, 26, 36).
Apobec-1 is a 27-kDa RNA-specific cytidine deaminase that binds to the apoB RNA substrate and catalyzes a site-specific C to U deamination reaction in the nuclear transcript (3, 29, 36). In humans, apobec-1 is expressed exclusively in the luminal gastrointestinal tract, but in certain species, including rodents, its expression is
more widespread (10, 13, 20, 28, 38). Apobec-1 is a
zinc-dependent enzyme that exhibits considerable homology to other
members of the cytidine deaminase family, particularly in its catalytic
domain (30). The catalytic domain contains a histidine and
glutamate residue in one alpha helix and a pair of cysteine residues in
a second, closely opposed helix that function to coordinate a molecule
of zinc in a tetrahedral configuration (30). Based on the
crystal structure of Escherichia coli cytidine deaminase, a
model for apobec-1 has been proposed that predicts a head-to-tail orientation of two apobec-1 monomers that are organized as a functional homodimer and thereby enable the glutamate residue to function as a
proton donor in the deamination reaction while simultaneously permitting docking of the protein onto the apoB RNA template
(30). Apobec-1 is an RNA-binding protein that interacts
with the hexamer sequence UUUGAU, which is located immediately
downstream of the edited base in apoB RNA (1). RNase
mapping and computer modeling studies have demonstrated the presence of
a stem-loop structure for apoB RNA with the edited base in the terminal
loop, a structure that is favorable for the binding and editing by
apobec-1 (1, 14, 32).
These characteristics of apobec-1 suggest a series of complex
adaptations that have evolved to permit the exquisite site-specific deamination of a single cytidine in an RNA template of over 14,000 nt.
These suggestions, coupled with the widespread distribution of apobec-1
mRNA in rodent tissues and its relatively recent emergence as an
RNA-specific deaminase (4), imply that other related cytidine deaminases may exist whose structure and function may be
informative with regard to the adaptations alluded to above. Accordingly, we utilized a functional gene database screening approach
to identify apobec-1-like proteins. This led us to identify a family of
cytidine deaminases with significant homology to apobec-1 in the
catalytic domain, suggesting that these proteins are likely to act as
RNA editing proteins.
In the current paper, we report the characterization of one of the
novel cytidine deaminase, referred to as ARCD-1, encoded on chromosome
6p21.1. ARCD-1 is ubiquitously expressed and has a cytidine deaminase
activity of high specificity. The protein localizes to both nucleus and
cytoplasm in transfected cells. ARCD-1 demonstrates significant
homology to apobec-1 and binds to apoB RNA but does not catalyze apoB
RNA editing. However, ARCD-1 inhibits apoB RNA editing in a
dose-dependent manner and reveals protein-protein interactions with
apobec-1 and with ACF. Together, these data suggest that ARCD-1 is a
ubiquitously expressed novel cytidine deaminase that may act as a
dominant negative inhibitor of apoB RNA editing by interacting with
apobec-1 and/or ACF.
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MATERIALS AND METHODS |
Cloning and tissue distribution of ARCD-1.
ARCD-1 was cloned from adult human small intestinal RNA by RT-PCR using
primers VSARCD-1 (5'-ATG GCC CAG AAG GAA GAG GCT GCT GTG G-3') and
VSARCD-2 (5'-CTA CTT AAG GAT GTC TGC CAA CTT CTC CTC GTA GTA-3'). The
PCR products were cloned into the pPCR-SCRIPT SK(+) amp cloning vector
(Stratagene, La Jolla, CA), and both strands were sequenced.
Full-length antisense ARCD-1 riboprobe was generated in the presence of
[
-32P]UTP (3,000 Ci/mmol) and used to probe a human
multiple-tissue Northern blot (Clontech, Palo Alto, CA) with ExpressHyb
hybridization solution (Clontech) according to the manufacturer's
instructions. A PCR-based approach for assessing gene expression was
performed with a human and a mouse Rapid Scan Gene Expression Panel
(Origene Technologies, Rockville, MD) with primers VSARCD-1 and
VSARCD-2. Control primers for the amplification of
-actin cDNA were
supplied by Origene Technologies. Sequence analysis was conducted using the Clustal W sequence alignment program in the MacVector 6.5 package
(Oxford Molecular Group, Campbell, CA). The presence of the same amino
acid at any given position is defined as "identity," while a
conserved change in the amino acid is represented as
"similarity." The similarity matrix used for determining
amino acid identity and similarity was Blosum 30 (MacVector). The
pairwise alignment parameters were an open gap penalty of 10 and an
extended gap penalty of 0.1.
Recombinant ARCD-1, apobec-1, and
ACF expression.
ARCD-1 was subcloned into the bacterial expression vector pThioHisC
(Invitrogen, Carlsbad, CA), at BamHI and XhoI
sites, after PCR amplification with Pwo polymerase by using
primers SA150 (5'-GAC GAC GAC AGG GGA TCC ATG GCC CAG
AAG GAA GAG GCT GCT GTG G-3'; BamHI site underlined)
and VS21 (5'-GGG CTC GAG CTA CTT AAG GAT GTC TGC CAA CTT
CTC CTC GTA GTA-3'; XhoI site underlined) and sequenced on both strands. Expression and purification of the recombinant protein by nickel-affinity chromatography using a ProBond
resin (Invitrogen) was performed according to the manufacturer's instructions. Apobec-1 was expressed as a
glutathione-S-transferase (GST) fusion protein as previously
described (23). ACF cDNA, isolated from human liver RNA
using primers ACF1 (5'-GGA TCC CCA TAT GGA ATC AAA TCA CAA
ATC CG-3'; BamHI restriction site underlined) and ACF2
(5'-CTC GAG TCA GAA GGT GCC ATA TCC ATC-3'; XhoI
restriction site underlined), was cloned into plasmid PET-28a (Novagen,
Madison, WI) at the BamHI and XhoI sites and
expressed as histidine (His)-tagged protein. Protein expression
was performed according to the manufacturers' recommendations
(Amersham Pharmacia Biotech, Piscataway, NJ; Novagen). The proteins
were size fractionated on 12% SDS-PAGE and silver stained. The purity
of pThioHisC-ARCD-1, GST-apobec-1, and His-ACF was determined to be 99, 70, and 99%, respectively. In vitro apoB RNA editing and primer
extension analyses were performed as previously described
(23). The products of the primer extension analyses were
separated on an 8% urea-PAGE gel and subjected to
phosphorimaging analysis. Stably transfected rat hepatoma cells
(McArdle7777) were generated by selection with 800 µg/ml G418 after
transfection with plasmids pCMV4-apobec-1 and pCMV-neo, as previously
described (23).
Cytidine deaminase activity of recombinant ARCD-1.
Increasing concentrations of recombinant ThioHis-ARCD-1 (1-500 ng)
were incubated with [3H]deoxycytidine in a buffer
containing 45 mM Tris (pH 7.5) and 250 µM unlabeled cytidine for
4 h at 37°C. The reaction mix was subjected to thin-layer
chromatography on polyethyleneimine-cellulose. Bands corresponding to
deoxycytidine and deoxyuridine were excised, and radioactivity was
counted in a beta counter (Beckman Coulter, Fullerton, CA). Where
indicated, inhibitors of cytidine deaminase activity, tetrahydrouridine
(1-40 µM) and 1,10-o-phenanthroline or its inactive
isomer, 1.7-o-phenanthroline (5-25 µM), were added to
the reaction mix.
Generation of recombinant adenovirus expressing
ARCD-1.
Full-length ARCD-1 with a COOH-terminal FLAG epitope tag was cloned in
pAxCAwt adenoviral vector (PanVera, Madison, WI), at the
SwaI restriction endonuclease site, for expression under the control of the chicken
-actin promoter. This vector was used to
generate recombinant adenovirus in 293 cells according to the manufacturer's instructions. Viral titers were obtained with a multiplicity of infection (MOI) of
109-1010 plaque-forming units/ml.
McArdle7777 cells were infected with increasing titers (2-4 × 103 MOI) of either recombinant adenovirus expressing
ARCD-1 or the bacterial
-galactosidase and were incubated for
48 h. Total RNA and cell lysates were prepared as described
previously (23) and used for primer extension and Western
blot analyses.
Western and Far Western analyses.
Anti-ARCD-1 antibody was generated in rabbits to a synthetic peptide
corresponding to amino acids 206-224 (EDIQENFLYYEEKLADILK; Research Genetics, Huntsville, AL), and IgG was purified by Affi-gel Protein A chromatography (Bio-Rad, Hercules, CA) according to the
manufacturer's instructions. Antibodies generated against apobec-1
have been described previously (23). Western blot analysis was performed for detection of recombinant GST-apobec-1 or ARCD-1, followed by detection with enhanced chemiluminescence (Amersham Pharmacia Biotech). For Far Western analysis, 1 µg of recombinant ThioHis-ARCD-1 was separated on 12% SDS-PAGE and blotted onto Immobilon P membrane (Millipore, Bedford, MA). The immobilized protein
was denatured with 6 M guanidine hydrochloride in buffer A
[20 mM HEPES, pH 7.9, 100 mM KCl, 0.2. mM EDTA, 20% glycerol, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 mM benzamidine] at room temperature for 1 h.
Proteins were renatured by incubation with serial dilutions of the
denaturation buffer (17, 26). The membrane was blocked
overnight at 4°C with 5% BSA and 5% skim milk in buffer
A, followed by incubation with 35S-labeled apobec-1
(5 × 105 cpm/ml) in buffer B (buffer
A plus 2.5 mM MgCl2, 0.5% skim milk, 2% BSA, and
0.1% Tween 20) for 16 h at 30°C. The filters were washed three
times in buffer C (buffer A plus 2.5 mM
MgCl2 and 0.1% Tween 20), dried at room temperature, and
analyzed by autoradiography.
Coimmunoprecipitation assays.
ARCD-1 was cloned into the plasmid pCMV-Tag3B (Stratagene), at
BamHI and XhoI sites, for expression as an
NH2-terminal c-myc-tagged fusion protein.
Cloning of ACF in plasmid pCMV-Tag2B has been published previously
(7). pCMV-Tag3B-ARCD-1 (NH2-terminal
c-myc-tagged) and pCMV-Tag2B-ACF (NH2-terminal
FLAG-tagged) were transcribed and translated in vitro using TNT rabbit
reticulocyte lysate system (Promega, Madison, WI) with T3 RNA
polymerase. The lysates were mixed and incubated at 30°C for
30 min in a binding buffer (20 mM HEPES pH 7.9, 100 mM KCl, 1 mM EDTA,
10% glycerol, 1 mM PMSF, and 0.4% Nonidet P-40). Immunoprecipitations
were subsequently performed for either the FLAG or myc tags by using a
mouse anti-FLAG monoclonal antibody (Stratagene) or a mouse anti-myc
monoclonal antibody 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA),
respectively. The immunoprecipitates were fractionated on 12%
SDS-PAGE and analyzed by autoradiography.
Immunofluorescence studies.
HepG2, McArdle7777, and COS-7 cells, grown on cover glass, were
transiently transfected with pCMV3B-ARCD-1 by using FUGENE (Boehringer-Mannheim) and were incubated for 48 h. The
slides were fixed with 0.5% formaldehyde, permeabilized with 0.5%
Triton X-100, and probed with mouse
-c-myc monoclonal
antibody (Santa Cruz), followed by Cy3-conjugated affinity-purified
donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories, West
Grove, PA). Stained cells were mounted with Vectashield (Vector
Laboratories, Burlingame, CA), and nuclei were imaged with
4,6-diamidino-2-phenylindole (DAPI). Images were obtained with the use
of a Zeiss Axiostop 2 MOT microscope equipped with a ×40 plan-neofluor
objective and a 3CCR camera (DAGE-MTI, Michigan City, IN). A Zeiss
Attoarc variable-intensity lamp was used with filters designed for Cy3
and DAPI. The images were processed using Adobe Photoshop 4.0 software.
Ultraviolet cross-linking and electrophoretic mobility shift
assays.
A 32P-labeled rat apoB mRNA template, RB105 (50,000 cpm at
4 × 108 cpm/µg) (3), was incubated
with indicated amounts of purified recombinant ThioHis-ARCD-1 and/or
GST-apobec-1 for 15 min at room temperature in a binding buffer
containing 10 mM HEPES (pH 8.0), 100 mM KCl, 1 mM EDTA, 0.25 mM DTT,
and 2.5% glycerol. The RNA was then sequentially treated with RNase T1
(1 U/µl final concentration) and heparin (5 mg/ml final
concentration). For electric mobility shift assay (EMSA) analysis, the
reaction mixtures were fractionated in a 5% native PAGE and
autoradiographed. For competition analysis with either wild-type or
mutant apoB RNAs, a 100-fold excess of competitor was used in the
reaction. For ultraviolet (UV) cross-linking analysis, the reaction
mixture was further subjected to UV cross-linking in a Stratalinker
(Stratagene) at 250 mJ/cm2 and subsequently analyzed by
12% SDS-PAGE under denaturing conditions.
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RESULTS AND DISCUSSION |
Cloning and comparative analysis of ARCD-1.
A search of the National Center for Biotechnology Information's
human expressed sequence tag (EST) database for apobec-1-like cytidine
deaminases containing the conserved signature sequence (H/C)-(A/V)-E-(X)24-30-P-C-(X)2-C revealed
the presence of activation-induced deaminase (AID) (27),
phorbolin (24), and seven novel transcripts, herein
referred to as apobec-1-related cytidine deaminases (ARCD-1 to -7)
(Fig. 1A). On the basis of a
search of human genomic sequences
(http://www.ncbi.nlm.nih.gov), ARCD-1 is located on
chromosome 6p21.1 (BAC clone 18464), whereas ARCD-2 to -7 are clustered
on chromosome 22q12-13.2 (BAC clone bk150C2). Comparative analysis
of residues spanning the putative catalytic domain of the ARCD members
with the known cytidine and deoxycytidine deaminases, using the
distance mapping program PROTDIST (4, 9), suggests that
the ARCDs cluster with AID and phorbolin (24, 27) and
represent a distant evolutionary branch of the RNA editing enzyme
apobec-1 (Fig. 1B). These results suggest that proteins
containing the characteristic cytidine deaminase active site domain
appear similar to the RNA editing enzyme apobec-1 and are encoded in
chromosomes 6 and 22. The functional significance of ARCD-1 in C to U
editing of apoB RNA was further investigated.

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Fig. 1.
Alignment and phylogenetic analysis of the active site of
apobec-1-related proteins with other cytidine deaminases. A:
alignment of the active site. The catalytic domain of cytosine
nucleotide and nucleoside deaminases, blasticidin-S deaminase, the RNA
editing enzymes, and novel apobec-1-related cytidine deaminases (ARCD-1
to -7) were aligned on the basis of the crystal structure of the
Escherichia coli cytidine deaminase (6). The
zinc-coordinating residues are indicated by arrows, and the glutamate
residues, which are the proton donors in the deaminase reaction, are
indicated by arrowheads. B: phylogenetic analysis of ARCDs.
The catalytic domain, shown in A, was used to generate a
distance matrix using the PROTDIST program (9). A tree was
subsequently generated using the KITSCH program and plotted using the
DRAWTREE program.
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Tissue distribution of ARCD-1.
GenBank database searches had earlier revealed ARCD-1 ESTs in skeletal
and cardiac muscle, small intestine, colon, liver, and kidney (data not
shown). To further resolve the distribution of ARCD-1 mRNA, we
probed a human multiple- tissue RNA blot with the full-length
ARCD-1 antisense riboprobe. Similar to the Blast search data, moderate
to abundant expression was observed in kidney, liver, and small
intestine, as well as high levels of expression in cardiac and skeletal
muscle (Fig. 2A). These latter
findings are consistent with the recent report of Liao et al.
(22). The presence of a doublet in cardiac and
skeletal muscle RNA suggests that alternate forms of ARCD-1 mRNA are
transcribed. This may represent differences in the 5' and/or 3'
untranslated regions, since RT-PCR of the full-length coding region
consistently demonstrated a single product (Fig. 2B).
Moreover, with a more sensitive PCR analysis of a multiple-tissue cDNA
panel, ARCD-1 expression was observed to be ubiquitous in both human
and mouse tissues (Fig. 2B). ARCD-1 mRNA also was
detectable in early embryonic mouse tissue, as early as E8.5 (Fig.
2B).

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Fig. 2.
Tissue distribution of ARCD-1. A: Northern blot
analysis. A human multiple-tissue Northern blot was probed sequentially
with riboprobes for human ARCD-1 and -actin and subjected to
autoradiography. Location of the ARCD-1 and -actin mRNAs are shown
(right), and migration of known molecular weight markers are
shown (left). B: PCR analysis for ARCD-1
expression. Multiple-tissue cDNA panels from human and mouse tissue
were subjected to PCR with ARCD-1- and -actin-specific primers. The
products of the PCR were analyzed by 1% agarose gel electrophoresis.
Tissues in both the blot (A) and PCR (B) panels
include brain (B), heart (H), skeletal muscle (SM), stomach (St), colon
(C), testis (T), spleen (Sp), kidney (K), liver (L), small intestine
(SI), pancreas (P), lung (Lu), skin (Sk), placenta (Pl), salivary gland
(SG), thyroid gland (Tg), adrenal gland (Ad), ovary (O), uterus (U),
prostate (Pr), bone marrow (BM), fetal brain (FB), fetal liver (FL),
peripheral blood leukocytes (PB), and mouse breast samples including
virgin (V), pregnant (P), lactating (L), and involuting breast (I).
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Functional analysis of ARCD-1.
Full-length ARCD-1 cDNA is 673 bp long and encodes a protein of 224 amino acids with a predicted molecular weight of 25 kDa (Fig.
3A). Scanning the ARCD-1
protein sequence against the Pfam database (http://pfam.wustl.edu/)
demonstrated the presence of the active site zinc-binding domain
between amino acids 98 and 131 (Fig. 3A) (5).
The human ARCD-1 gene, encoded in BAC clone 18464 spans ~11 kb and
contains three exons, including an untranslated exon 3 (Fig.
3B and Table 1). The catalytic
domain is located within the second exon (Fig. 3B). This
genomic organization is distinct from that previously observed with
apobec-1 (15, 16, 28). The apobec-1 coding region is
encoded in five exons, with the catalytic domain located within exon 3 (15, 16, 28). Alignment of the primary amino acid
sequences of ARCD-1 and apobec-1 demonstrates significant homology
between each of these sequences (Fig. 3B and Table
2), as well as with E. coli
cytidine deaminase (Table 2). Whether apobec-1 and ARCD-1 arose from a
common ancestral gene remains unanswered.

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Fig. 3.
Sequence homology between ARCD-1 and apobec-1. A:
schematic representation of the ARCD-1 coding region. ARCD-1 gene,
located in 6p21.1, is encoded in 3 exons. Translation stop and start
codons are present in exons 1 and 2, respectively. Exon 3 is not
translated. B: alignment of the ARCD-1 and apobec-1 proteins
using the Clustal W program. Identical and similar residues are shown
by a vertical line and a dot, respectively. The active site is boxed,
and the important residues involved in cytidine deaminase activity are
indicated by a star. The RNA binding active site aromatic residues are
indicated by an arrowhead.
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ARCD-1 is a cytidine deaminase.
ARCD-1 was expressed as a fusion protein with a ThioHis tag and was
purified under native conditions by nickel-affinity chromatography. SDS-PAGE analysis of the purified fusion protein revealed a single prominent polypeptide of an apparent size of ~35 kDa, although the
predicted size is ~31 kDa (Fig.
4A). Cytidine deaminase assay revealed that the enzymatic activity of ThioHis-ARCD-1
increased in a dose-dependent manner (Fig. 4B) and was
inhibited upon addition of excess unlabeled cytidine or deoxycytidine
(data not shown). Furthermore, the activity was linear with respect to
protein concentration and time, up to 150 ng (Fig. 4B) and
60 min (data not shown), respectively. The Michaelis-Menten constant
and maximal velocity for ARCD-1 were determined to be 1 × 10
5 M and 4.8 U/mg protein, respectively, which are
comparable to those observed with other cytidine deaminases, including
those isolated from Caenorhabditis elegans and Brugia
pahangi as well as apobec-1 and a related homologue, AID (Table
3). Cytidine deaminase activity was
inhibited in a dose-dependent manner upon the inclusion of either
1,10-o-phenanthroline (Fig. 4C) or
tetrahydrouridine (Fig. 4D). However, addition of
1,7-o-phenanthroline, an inactive isomer that does not
chelate zinc, had no effect on the activity (Fig. 4C). These
data demonstrate that ARCD-1 functions as an authentic, zinc-dependent
cytidine deaminase.

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Fig. 4.
ARCD-1 is an authentic cytidine deaminase. A:
expression and purification of ARCD-1. ARCD-1 cDNA was expressed as a
ThioHis-tagged protein and purified by nickel-affinity chromatography
using imidazole. Proteins in the various fractions were analyzed by
12% SDS-PAGE and silver staining. UI, uninduced; I, isopropyl
-D-thiogalactopyranoside induced; PB, post-binding
column flow-through. B: cytidine deaminase activity with
increasing protein concentrations. Cytidine deaminase activity was
determined by in vitro incubation at 37°C with
[3H]deoxycytidine and increasing concentrations of
recombinant protein (0-500 ng) and assessed by thin-layer
chromatography. C: inhibition of cytidine deaminase activity
by a zinc chelator, 1,10-o-phenanthroline
(1,10), but not by its inactive isomer,
1,7-o-phenanthroline (1,7). Incubation was
performed with 100 ng of ARCD-1 and increasing concentrations of
inhibitor (0-20 mM). D: inhibition of cytidine
deaminase activity with increasing concentrations of tetrahydrouridine
(THU).
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ARCD-1 is an ApoB
RNA-binding protein.
Previous studies have demonstrated that RNA binding activity is
essential for apobec-1 to catalyze C to U editing of apoB RNA
(23, 29). Apobec-1 is an AU-rich RNA-binding protein with affinity for a hexanucleotide sequence, located immediately downstream of the edited base (1). In addition, mutagenesis studies
have identified two aromatic residues within the catalytic domain of apobec-1 that are essential for RNA binding (29, 37). On
the basis of the protein sequence alignment of apobec-1 and ARCD-1, we
observed that these aromatic residues are conserved in ARCD-1, implying
that it also might be an RNA-binding protein (Fig. 3B). Accordingly, EMSAs were performed with purified recombinant ARCD-1 and
32P-labeled wild-type apoB mRNA. As shown in Fig.
5A, ARCD-1 bound apoB RNA,
which was supershifted when apobec-1 was also added to the reaction
(Fig. 5A). To further determine the binding specificity, we
performed EMSA analysis with radiolabeled human apoB RNA in the
presence of cold apoB transcripts of the same size into which two
different scrambled 6-nt mutations had been introduced (Fig. 5B, mutants C and D) (32). Mutant D was
especially chosen for the experiment because it contains part of the
apobec-1 binding site (1). In addition, tRNA was added as
competitor because previous studies from our laboratory have
demonstrated that tRNA inhibits apobec-1 binding to apoB RNA
(3). Both of the apoB RNA scrambling mutants inhibited
ARCD-1 binding, suggesting that ARCD-1 and apobec-1 may bind to
different sites in apoB RNA. In UV cross-linking assays, both ARCD-1
and apobec-1 demonstrated cross-linking to apoB RNA (Fig.
5C). It should be noted that the difference in size of the
cross-linked bands in Fig. 5C reflects the size of the
respective fusion proteins (MATERIALS AND METHODS). No
competition for binding was observed between the two proteins when they
were added simultaneously to the radiolabeled transcript (Fig.
5C), again suggesting, among other possibilities, that
ARCD-1 and apobec-1 may bind to different sites in apoB RNA. On the
basis of the intensity of the ARCD-1 band in both the UV cross-linking and EMSA analyses, we speculate that the binding affinity of ARCD-1 for
apoB RNA is lower than that of apobec-1. The finding that apoB RNA
binding is also abrogated in the presence of 100-200 ng of tRNA
(Fig. 5B) further suggests that this interaction is of low
specificity. Further study of this issue, however, will require fine
mapping of the binding site coupled with kinetic measurements of
RNA-binding affinity before such speculation can be formally tested.

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Fig. 5.
ARCD-1 is an apoB RNA-binding protein. A:
electrophoretic mobility shift assays (EMSA) were performed by using a
rat apoB RNA template in the presence of 250 ng of GST-apobec-1 and/or
500 ng of ThioHis-tagged ARCD-1. The products were analyzed by native
PAGE. Location of ARCD-1, glutathione-S-transferase
(GST)-apobec-1, and the combined ARCD-1/GST-apobec-1 bands are
indicated by an open arrow, arrowhead, and closed arrow, respectively.
B: EMSA was carried out in the absence ( ) or presence (+)
of competitor RNA. Competitors include the tRNA, wild-type (WT), and
scanning mutants of apoB RNA representing 6 nucleotide changes either
upstream (C) or downstream (D) of the edited base (bottom).
Location of ARCD-1 is indicated by an arrow. C: ultraviolet
cross-linking was performed with a radiolabeled apoB RNA template in
the presence of the indicated protein and was analyzed by 12%
SDS-PAGE. Location of the cross-linked bands corresponding to apobec-1
and ARCD-1 is indicated by an arrow and arrowhead, respectively.
Migration of protein standards (kDa) are shown (left).
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ARCD-1 inhibits apoB mRNA editing
both in vitro and in vivo.
The observation that ARCD-1 is a cytidine deaminase and an apoB
RNA-binding protein raised the possibility that it might either substitute for apobec-1 in editing apoB RNA or modulate the editing process. Accordingly, recombinant ARCD-1 was analyzed for its ability
to catalyze in vitro apoB mRNA editing, as previously described
(23). Increasing amounts (0-500 ng) of ARCD-1, added to bovine liver S-100 extracts, failed to catalyze C to U editing of
apoB RNA, suggesting that ARCD-1 cannot substitute for apobec-1 in the
editing reaction (Fig. 6A,
lane 3, and data not shown). However, the addition of
increasing quantities of recombinant ARCD-1 to assays containing 250 ng
of GST-apobec-1 and bovine liver S-100 extracts demonstrated
competitive inhibition of apoB RNA editing, with complete inhibition
observed upon addition of 300 ng of ARCD-1 (Fig. 6A). We
previously demonstrated that the addition of up to 2 µg of other apoB
RNA-binding proteins, including hnRNP-A1, hnRNP-F, and hnRNP-I/PTB,
fails to modulate C to U editing of apoB RNA in this assay
(7). We conclude that the inhibition observed with ARCD-1
is therefore not a general phenomenon observed with the addition of
AU-rich RNA-binding proteins.

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Fig. 6.
ARCD-1 inhibits apoB mRNA editing. A: in vitro RNA
editing. Increasing amounts of ARCD-1 (0-500 ng) were added to
assays containing 20 fmol of apoB RNA, 250 ng of GST-apobec-1, and 10 µg of 30% ammonium sulfate precipitate of bovine liver S-100 (Bov
Liv S100) extracts. The RNA was extracted and C to U editing
quantitated by primer extension analysis. As control, 250 ng of ARCD-1
were added to an assay containing GST-apobec-1 and apoB RNA but no
S-100 extracts. Edited RNA was quantitated by phosphorimaging. Data are
representative of 3 such experiments. B: endogenous apoB
mRNA editing. McArdle7777 cells, a rat hepatoma cell line, were stably
transfected with apobec-1, and individual clones were selected that
demonstrated 85-90% endogenous apoB mRNA editing. These cells
were infected with increasing multiplicity of infection (2-4 × 103 MOI) of a replication-defective recombinant
adenovirus encoding either -galactosidase (LacZ) or FLAG-tagged
ARCD-1. Lysates were subjected to Western blot analysis with -FLAG
antibody (WB: -FLAG) to detect expression of exogenous ARCD-1
(top). In addition, total RNA from the cells was isolated
and used as a template for RT-PCR to amplify apoB RNA sequences around
the edited site. The RT-PCR products were subsequently subjected to
primer extension analysis to quantitate C to U editing. ApoB mRNA
editing was quantitated by phosphorimaging analysis and represented as
a percentage of control (bottom). Data are representative of
3 such experiments.
|
|
To pursue this inhibition of C to U RNA editing and to determine
whether ARCD-1 exerts inhibitory activity in vivo, we generated a
replication-defective adenovirus expressing either the bacterial LacZ
protein or FLAG-tagged ARCD-1. Preliminary studies were undertaken with
McArdle7777 cells, a rat hepatoma cell line that edits ~10-15% endogenous apoB RNA (7). Transduction of McArdle7777 cells with the adenovirus encoding ARCD-1 reduced C to U editing to undetectable levels (data not shown). Nevertheless, because viral toxicity and the low levels of endogenous apoB RNA editing could contribute to this effect, a more stringent approach was sought to
address the question of whether ARCD-1 could inhibit apobec-1-mediated C to U editing. The recombinant virus was therefore transduced into McArdle7777 cells stably transfected with apobec-1, which demonstrate ~85-90% apoB mRNA editing (Fig. 6B), the
result of overexpression of apobec-1 (23). As demonstrated
in Fig. 6B, endogenous apoB mRNA editing was inhibited by
ARCD-1 in a dose-dependent manner, whereas no effect on RNA editing was
observed when the cells were infected a recombinant virus expressing
-galactosidase. These results confirm the findings from the in vitro
studies suggesting that ARCD-1 inhibits apoB mRNA editing catalyzed by
apobec-1.
Protein-protein interaction of ARCD-1 with apobec-1
and with ACF.
Apobec-1 has been shown to bind to a number of candidate editosomal
proteins, including ACF, GRY-RBP, ABBP-1, and hnRNP-C (7, 12, 19,
21, 26). Some of these, including GRY-RBP, ABBP-1, and hnRNP-C,
have been demonstrated to inhibit apoB RNA editing (7, 12,
19). Because ARCD-1 inhibited C to U RNA editing activity, we
reasoned that it might interact with key components of the editing
holoenzyme, including apobec-1 and ACF. To address this question, we
evaluated protein-protein interaction between ARCD-1 and apobec-1 by
Far Western analysis. Recombinant ThioHis-ARCD-1 and GST-apobec-1 were
resolved by SDS-PAGE, transferred to polyvinylidene difluoride
membranes, and incubated with 35S-labeled apobec-1. The
results, shown in Fig. 7A,
demonstrate heterodimerization between apobec-1 and ARCD-1, suggesting
that apobec-1 and ARCD-1 can associate in the absence of apoB RNA. By
way of control, homodimerization of apobec-1 was confirmed in this
assay as previously reported (7, 18, 26). The migration of
apobec-1 and ARCD-1 was confirmed by Western blotting with
-apobec-1
and
-ARCD-1 antibodies, respectively (Fig. 7A).

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Fig. 7.
ARCD-1
interacts with apobec-1 and apobec-1 complementation factor (ACF).
A: recombinant GST-apobec-1 and ThioHis-ARCD-1 were
separated on 12% SDS-PAGE and subjected to Western blot (WB) analysis
with -apobec-1 and -ARCD-1 IgG. Subsequently, the membrane was
subjected to Far Western analysis (FW) with 35S-labeled
apobec-1 and then autoradiographed. Locations of the bands
corresponding to apobec-1 and ARCD-1 are indicated (right), and
molecular mass markers (kDa) are shown (left). B:
myc-ARCD-1 and FLAG-ACF were generated in vitro by using a coupled
2,4,6-trinitrotoluene lysate in the presence of
[35S]methionine, resulting in the production of a single
major product for the two proteins (lanes 1 and
2). The products were mixed and subjected to
immunoprecipitation (IP) with either -FLAG ( -F; lane
3) or -myc ( -m; lane 4) antibodies. Mobility of
the 2 species is indicated (right), with molecular mass
markers (kDa) indicated (left).
|
|
To further demonstrate interaction between ARCD-1 and ACF, we undertook
immunoprecipitation analysis of radiolabeled in vitro translation
products. In vitro-translated myc-tagged ACF yielded a predominant
species of ~67 kDa, as described previously (7), whereas that of FLAG-tagged ARCD-1 yielded a protein of ~26 kDa (Fig. 7B). Mixing the proteins and immunoprecipitating with
either
-FLAG or
-myc antibodies revealed the presence of both
proteins, suggesting that ARCD-1 and ACF are present as
heteromers in solution. Control immunoprecipitations, performed
with radiolabeled ligand and anti-epitope antibody in the absence
of its cognate protein, revealed no product (Fig. 7B).
Immunolocalization of ARCD-1 in transfected cells.
ApoB mRNA editing is a nuclear event that occurs either during or after
mRNA splicing (18, 35). Recent studies have demonstrated that apobec-1 is present in both the nucleus and cytoplasm, suggesting that its function may reflect shuttling between these compartments. (7, 40). Mutagenesis studies identified a bipartite
nuclear localization and leucine-rich nuclear export signal sequences in the NH-2 and COOH terminus of apobec-1,
respectively (40). Similarly, human cytidine deaminase was
found to contain a bipartite nuclear localization signal, and cytidine
deaminase was observed in both compartments of the cell
(34). A search of the predicted ARCD-1 sequence did not
reveal the presence of any known nuclear import or export sequences
(data not shown), and the conserved nuclear localization sequence in
apobec-1 was not present in ARCD-1. To determine the intracellular
localization of ARCD-1, we introduced FLAG-tagged ARCD-1 into COS-7,
HepG2, and McArdle7777 cells. These three cell lines were chosen to
reflect intrinsic differences in the expression of apoB RNA and the
transacting proteins required for C to U RNA editing. Specifically,
COS-7 cells do not express apoB RNA or the editing factors apobec-1 and
ACF (7, 26, 40). HepG2 cells express endogenous apoB RNA
and ACF but not apobec-1 (7, 11). McArdle7777 cells, on
the other hand, express endogenous apoB RNA, apobec-1, and ACF and
support C to U editing activity (7, 25, 26, 39). Indirect
immunofluorescence staining of the transfected cells for the FLAG
epitope demonstrated diffuse nuclear and cytoplasmic ARCD-1 staining in
all three cell lines (Fig. 8), a pattern
of distribution similar to that observed with apobec-1
(40). These data suggest that the cellular distribution of
ARCD-1 is neither dependent on nor apparently influenced by the
expression of either of ACF, apobec-1, or the target substrate RNA.
Attempts to localize the endogenous protein by using either anti-peptide antisera or affinity-purified IgG were unsuccessful, suggesting that the expression of endogenous ARCD-1 is below the detection limit of these reagents (data not shown).

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Fig. 8.
Subcellular localization of ARCD-1. HepG2 (A and
D), COS-7 (B and E), and McArdle7777
(C and F) cells were transfected with
FLAG-tagged ARCD-1. The slides were stained with -FLAG monoclonal
antibody and 4,6-diamidino-2-phenylindole to identify ARCD-1 and
nuclei, respectively. Location of the nuclei is indicated by an arrow.
Images are representative of 3 independent transfections.
|
|
In conclusion, the current results demonstrate that ARCD-1 is an
authentic cytidine deaminase whose in vitro enzymatic kinetics are
similar to those of apobec-1. ARCD-1 binds to but does not catalyze C
to U editing of apoB RNA; rather, it inhibits apobec-1 in a
dose-dependent manner. Together, these data suggest that ARCD-1 may
function in a dominant negative fashion to constrain C to U editing,
although its role in the regulation of apoB RNA editing remains to be
determined. Further studies are necessary to determine whether ARCD-1
is an authentic component of the apoB RNA editing holoenzyme. In
addition, the observation that ARCD-1 expression is ubiquitous raises
the question of its role in other tissues. In this regard, ARCD-1 is
only the second example of a eukaryotic protein that encodes both
enzymatic cytidine deaminase and RNA-binding activities, albeit of low
specificity in regard to this latter function (2).
However, it should be emphasized that neither phorbolin nor AID (other
recently described homologues of apobec-1) expresses RNA binding
activity (24, 27). We suspect that the target transcript
(if any) of ARCD-1 would most likely be expressed in one of the tissues
where ARCD-1 is highly expressed, namely, cardiac and skeletal muscle.
These and other questions concerning the function of this gene product
may be the focus of future reports.
 |
ACKNOWLEDGEMENTS |
We thank Karen Hutton of the morphology core of the Digestive
Disease Research Core Center (DDRCC) for help with the
immunofluorescence studies.
 |
FOOTNOTES |
This work was supported by National Institutes of Health (NIH) Grants
HL-38180 and DK-56260 (to N. O. Davidson), NIH DDRCC Grant
DK-52574 (N. O. Davidson, and a Pilot and Feasibility Award to S. Anant), and an American Gastroenterology Association Research Scholars
Award (to S. Anant).
Address for reprint requests and other correspondence: S. Anant
or N. Davidson, Washington Univ. School of Medicine, Dept. of Internal
Medicine, Division of Gastroenterology, Campus Box 8124, 660 South
Euclid Ave, St. Louis, MO 63110 (E-mail: sanant{at}im.wustl.edu or
nod{at}im.wustl.edu).
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.
Received 14 May 2001; accepted in final form 7 August 2001.
 |
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