From the Department of Internal Medicine and
** Department of Pharmacology and Molecular Biology, Washington
University School of Medicine, St. Louis, Missouri 63110 and the
§ MRC Molecular Medicine Group, Clinical Science Center
and Division of National Heart and Lung Institute, Imperial College
School of Medicine, Hammersmith Hospital,
London W12 0NN, United Kingdom
Received for publication, July 19, 2000, and in revised form, December 12, 2000
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ABSTRACT |
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C to U editing of apolipoprotein B
(apoB) mRNA involves the interaction of a multicomponent editing
enzyme complex with a requisite RNA sequence embedded within an AU-rich
context. This enzyme complex includes apobec-1, an RNA-specific
cytidine deaminase, and apobec-1 complementation factor (ACF), a novel
65-kDa RNA-binding protein, that together represent the minimal core of
the editing enzyme complex. The precise composition of the holo-enzyme,
however, remains unknown. We have previously isolated an enriched
fraction of S100 extracts, prepared from chicken intestinal cells, that displays apoB RNA binding and which, following supplementation with
apobec-1, permits efficient C to U editing. Peptide sequencing of this
most active fraction reveals the presence of ACF as well as GRY-RBP, an
RNA-binding protein with ~50% homology to ACF. GRY-RBP was
independently isolated from a two-hybrid screen of chicken intestinal
cDNA. GRY-RBP binds to ACF, to apobec-1, and also binds apoB RNA.
Experiments using recombinant proteins demonstrate that GRY-RBP binds
to ACF and inhibits both the binding of ACF to apoB RNA and C to U RNA
editing. This competitive inhibition is rescued by addition of ACF,
suggesting that GRY-RBP binds to and sequesters ACF. As further
evidence of the role of GRY-RBP, rat hepatoma cells treated with an
antisense oligonucleotide to GRY-RBP demonstrated an increase in C to U
editing of endogenous apoB RNA. ACF and GRY-RBP colocalize in the
nucleus of transfected cells and, in cotransfection experiments with
apobec-1, each appears to colocalize in a predominantly nuclear
distribution. Taken together, the results indicate that GRY-RBP is a
member of the ACF gene family that may function to modulate C to U RNA
editing through binding either to ACF or to apobec-1 or, alternatively,
to the target RNA itself.
Apolipoprotein B (apoB)1
is an abundant gene product expressed in the mammalian small intestine
and liver and plays a central role in the transport of cholesterol and
triglyceride in plasma (1). Two forms of apoB exist, designated on a
centile scale as apoB100 and apoB48 (2). ApoB48 is generated as a
result of a site-specific, posttranscriptional C to U deamination of the nuclear transcript that in turn results in translational
termination of the (edited) apoB RNA (3, 4). ApoB RNA editing is
particularly active in the mammalian small intestine but also in the
liver of certain species, including the mouse and rat (5). Since apoB
RNA editing eliminates an important functional domain from the C
terminus of the protein (reviewed in Ref. 2), the molecular mechanisms
underlying this organ-specific partitioning have been extensively
investigated to understand the presumed advantage of this specialized
genetic adaptation.
Enzymatic deamination of apoB RNA exhibits important requirements in
both the cis-acting RNA elements and in the trans-acting protein
factors that restrict this reaction largely to a single, canonical site
(6-15). The cis-acting elements have been well characterized and
include an 11-nucleotide (nt) region referred to as a "mooring
sequence," located 4 nt downstream of the edited C, an AU-rich bulk
RNA context, and other "efficiency elements" flanking a ~30-nt
region representing the minimal editing cassette (6-15). This cassette
is located within a region that exhibits important secondary structure,
including a stem-loop conformation that is predicted to position the
edited C in a favorable configuration relative to the active site of
the deaminase (10, 12, 16, 17). C to U editing of apoB RNA is mediated
by an enzyme complex that includes a single catalytic subunit,
apobec-1, as well as additional proteins that together represent the
holoenzyme (18). Apobec-1 is a zinc-dependent cytidine
deaminase that exhibits RNA-binding affinity for AU-rich substrates
(19, 20), such as apoB, as well as other templates including c-Myc,
interleukin-2, granulocyte-macrophage-colony stimulating factor,
and tumor necrosis factor- Over the last several years, many different proteins have been proposed
to function as auxiliary or complementation factors, including
candidates ranging in molecular mass from 40 to 240 kDa (11, 13,
26-30). Despite intensive effort, until very recently none of these
has met the criteria of an authentic enzymatic cofactor in C to U
editing of apoB RNA. The work of Driscoll and colleagues (31) has very
recently resulted in the identification of a candidate protein,
referred to as apobec-1 complementation factor (ACF), that most
plausibly represents, with apobec-1, the minimal editing enzyme
complex. ACF is an ~65-kDa RNA-binding protein, with three distinct
RNA binding domains, that binds both to apobec-1 and to apoB RNA.
Similar findings were also reported by Greeve et al. (32)
who isolated a cDNA identical to
ACF,2 with the exception of
an 8-residue insertion, that exhibits complementation activity that was
enhanced in the presence of additional proteins present in nuclear
extracts. These most recent findings, taken in conjunction with earlier
reports that demonstrated the presence of apoB editing activity in a
higher order complex, suggest that the native editing enzyme complex
may consist of apobec-1 and ACF as well as additional proteins
(32).
We previously reported the presence of apobec-1 complementation
activity in chicken intestinal S100 extracts that fractionated in the
range of 49-65 kDa (17, 33). During the purification of this
complementing activity, using both biochemical and genetic isolation
techniques, we have isolated another RNA-binding protein, glycine-arginine-tyrosine-rich
RNA-binding protein (GRY-RBP), that
exhibits ~50% amino acid sequence similarity to ACF. GRY-RBP binds
ACF both in vitro and in vivo and in addition
binds apoB RNA. GRY-RBP binding to ACF competitively inhibits both its
binding to apoB RNA and C to U RNA editing. ACF and GRY-RBP are both
localized in the nucleus of transfected cells, including cells that are competent to edit apoB RNA as well as cells that lack this activity. Furthermore, in cotransfection experiments, ACF and GRY-RBP each colocalize with apobec-1 in both a cytoplasmic as well as a nuclear distribution. Taken together, the data suggest that GRY-RBP, a member
of the ACF gene family, may function to modulate C to U RNA editing of
mammalian apoB RNA through binding to ACF as well as to apobec-1 and/or
apoB RNA.
Identification of GRY-RBP by Protein Sequence
Analysis--
Chicken enterocyte S100 extracts were prepared as
described previously and fractionated through a 25-ml Blue-Sepharose
column (Amersham Pharmacia Biotech) as detailed in Ref. 17. The
material eluting in fractions 16-19 was pooled and used for UV
cross-linking to a 55-nucleotide 32P-labeled synthetic apoB
RNA (17). Cross-linked proteins were separated by 10% SDS-PAGE, and
the cross-linked bands were identified by autoradiography. The
cross-linked band migrating at ~65 kDa (~p65) was excised and
electroeluted from the gel. This material was subjected to sequence
analysis at the Harvard Microchemistry Facility, using microcapillary
reverse-phase high pressure liquid chromatography and nano-electrospray
tandem mass spectrometry (MS/MS) on a Finnigan LCQ quadrupole ion trap
mass spectrometer. The MS/MS peptide sequences were then analyzed for
consensus to known proteins, and the results were manually confirmed
for fidelity.
Cloning and Expression of Recombinant GRY-RBP--
Full-length
GRY-RBP cDNA was generated by polymerase chain reaction (PCR) from
human liver cDNA and introduced into pPCR-Script (Stratagene). The primers used were GRYsense,
5'-GCGGTCGACATGGCTACAGAACATGTTAATGGAAAT-3', and GRY antisense,
5'-GCGGGGCCCGCGGCCGCCTACTTCCACTGTTGCCCAAAAGTATCCTGATAA-3'. GRY-RBP cDNA was then subcloned into a mammalian expression vector pCMV-Tag3B (Stratagene) using the SalI-ApaI sites
to generate an N-terminal c-Myc-tagged fusion protein. By using
GRY-RBP-specific primers flanked with an NheI restriction
site at the end of the 5' primer, the DNA was PCR-amplified from the
recombinant pPCR-Script vector and introduced into the
NheI-SmaI sites of the expression vector
pTYB2 (Biolabs) to generate the recombinant protein. The primers used were IMP5 sense, 5'-GGTGCGGCTAGCATGGCTACAGAACATGTTAATG-3', and IMP3 antisense, 5'-CTTCCACTGTTGCCCAAAAGTATC-3'. GRY-RBP was fused to a self-cleavable intein tag containing a chitin-binding domain
for affinity purification as a fusion protein. Expression of the fusion
protein was conducted in ER2566 cells, and cultures were grown as
recommended by the manufacturer (New England Biolabs). The cell pellet
obtained from a 1-liter culture was resuspended in 50 ml of lysis
buffer (20 mM HEPES (pH 8.0), 500 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.5 mM
phenylmethylsulfonyl fluoride), and the cells were lysed by sonication,
followed by 30 min of incubation at 4 °C. The lysate was then
centrifuged, and the clarified cell extract was loaded onto a 10-ml
chitin column. The column was washed extensively and GRY-RBP released
in the presence of 50 mM dithiothreitol. Fractions were
analyzed by denaturing 10% SDS-PAGE and Western-blotted using
affinity-purified antipeptide antiserum, generated using residues
105-122 of human GRY-RBP (Research Genetics). Fractions containing
GRY-RBP were pooled and dialyzed against 20 mM HEPES (pH
8.0), 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 20% glycerol, 0.5 mM PMSF.
Cloning and Expression of Recombinant ACF--
ACF cDNA was
amplified by reverse transcriptase-PCR from human (small intestinal and
hepatic) RNA and RACE-ready RNA (CLONTECH) and
cloned into pGem-T easy (Promega), using the following primers: ACF1 sense, 5'-GGATCCCATATGGAATCAAATCACAAATCCG-3'; ACF2 antisense, 5'-CTCGAGTCAGAAGGTGCCATATCCATC-3'. ACF cDNA was then
sequenced and subcloned into pCMV-Tag 2B using the
BamHI-XhoI sites, generating an N-terminal
FLAG-tagged fusion protein. ACF cDNA was also subcloned into
pTYB1 and expressed as an intein fusion protein as described above. Preparations of the recombinant protein were stored at UV Cross-linking Analysis of Protein-RNA Interaction--
A
32P-labeled rat apoB RNA template (50,000-70,000
cpm at 4 × 108 cpm/µg) was incubated with the
indicated amounts of purified recombinant protein 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 unit/µl final concentration) and heparin (2 mg/ml final concentration) prior to UV irradiation on ice in a
Stratalinker (Stratagene) at 250 mJ/cm2. The cross-linked
material was analyzed by 10% SDS-PAGE. Competition experiments were
performed using wild-type or mutants of apoB mRNA (55-mer) or actin
RNA as described previously (13).
Far Western Analysis of Protein-Protein Interaction--
1 µg
of recombinant GRY-RBP was separated by SDS-PAGE and Western-blotted
onto Immobilon P (Millipore). 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 DTT, 0.5 mM PMSF, 0.5 mM benzamidine) at room
temperature for 1 h. Proteins were renatured by diluting the
denaturation buffer 1:1 in buffer A for 12 cycles of 10 min each (29,
34). Filters were blocked overnight at 4 °C with 5% bovine serum
albumin and 5% skim milk in buffer A and then incubated with
35S-labeled apobec-1 (5 × 105 cpm/ml) in
buffer B (buffer A plus 2.5 mM MgCl2, 0.5%
skim milk; 2% bovine serum albumin; 0.1% Tween 20) for 16 h at
30 °C. Filters were washed three times in buffer C (buffer A plus
2.5 mM MgCl2, 0.1% Tween 20), dried at room
temperature, and subjected to autoradiography.
Antisense Oligonucleotide Experiments--
An antisense
morpholino-oligonucleotide to GRY-RBP (5'-GCTCCGTTCATCTTGTTGGCTGTGC-3',
5' located at nt +479) and a scrambled control
morpholino-oligonucleotide were prepared by Gene Tools, LLC, Corvallis,
OR. McArdle rat hepatoma cells were plated on a 35-mm culture dish and
grown to 70% confluence. Oligonucleotides (final concentration, 5 µM) were mixed with delivery reagent (EPEI, Gene Tools)
in serum-free Dulbecco's modified Eagle's medium and layered over the
cells. After incubation for 3 h, the delivery solution was
removed, replaced with complete Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum, and the cells incubated for 48 h. RNA was extracted, and apoB RNA editing was determined by primer
extension assay.
Immunofluorescence Microscopy--
Rat-apobec-1 cDNA was
cloned into pCMV-Tag 2B or 3B and expressed either as an N-terminal
FLAG-tagged or an N-terminal c-Myc-tagged fusion protein, respectively.
Human GRY-RBP cDNA was cloned into pCMV-Tag 3B and expressed as an
N-terminal c-Myc-tagged fusion protein. Human ACF was expressed from
pCMV-Tag2B vector as an N-terminal FLAG-tagged fusion protein. COS-7,
HepG2, and McArdle cells (ATCC) were grown on coverslips and
transiently transfected with 1-2 µg of plasmid DNA using FUGENE 6 (Roche Molecular Biochemicals). The cells were fixed 48 h after
transfection with 3.7% formaldehyde, permeabilized with 0.5% Triton
X-100, and probed with mouse monoclonal anti-Myc IgG (SC-40, Santa Cruz
Biotechnology) and rabbit anti-FLAG IgG (71-5400, Zymed
Laboratories Inc.), followed by Cy3- or FITC-conjugated secondary
IgG, respectively (Jackson ImmunoResearch). For confocal microscopy,
nuclei were visualized using TO-PRO-3 iodide (Molecular Probes).
Preparations were imaged with a 63× Zeiss plan apochromatic objective
and a Bio-Rad MRC 1024 confocal adaptor. A krypton-argon laser was used
with epifluorescence filter sets designed for Texas Red (Cy3),
fluorescein (FITC), and cyanine (Cy5). The confocal aperture was set at
1.8. Usually, 15-40 images at planes separated by 0.5 µm were
obtained. This increment allows sectioning of the entire image giving a
range of signals covering every plane of the cells in that image.
Pictures were processed using Adobe Photoshop 4.0 software. For
standard immunofluorescence microscopy, transfection and antibody
staining was carried out as described above using cells grown on
coverslips. Stained cells were mounted with Vectashield and nuclei
imaged with DAPI (Vector). Images were obtained with a Zeiss Axioskop 2 MOT microscope equipped with a 40× plan neofluar objective and a 3CCR
camera (DAGE-MTI, Inc.). A Zeiss Attoarc variable intensity lamp was
used with filter sets designed for Cy3, FITC, and DAPI. Images were
processed using Adobe Photoshop 4.0 software.
Miscellaneous Methods--
Primer extension analysis of apoB RNA
editing was conducted as detailed previously (24).
Coimmunoprecipitation assays were conducted using Myc-GRY-RBP,
FLAG-ACF, apobec-1, and GST-apobec-1, expressed either as recombinant
proteins or, where indicated, from in vitro translation
using a TnT Coupled Reticulocyte Lysate (Promega). Interaction studies
were performed at 30 °C for 30 min in 200 µl of binding buffer (20 mM HEPES (pH 7.9), 100 mM KCl, 1 mM
EDTA, 10% glycerol, 1 mM PMSF, 0.4% Nonidet P-40). ACF and GRY-RBP were coimmunoprecipitated from cell lysates following transient transfection as described above. Following nondenaturing cell
lysis, the target proteins were immunoprecipitated with 5 µl of the
appropriate antisera, including mouse monoclonal anti-FLAG (Sigma) or
rabbit polyclonal anti-Myc (Sigma) antibody at 4 °C for 60 min with
agitation. The complexes were collected on protein A-agarose beads that
were washed extensively and analyzed by 12% SDS-PAGE and
autoradiography. Where indicated, the material coprecipitating on the
protein A-agarose beads was eluted in denaturing buffer, subjected to
SDS-PAGE, and visualized by Western blotting with the appropriate
antisera. Yeast two-hybrid interaction assays were conducted with ACF
and GRY-RBP cDNAs, which were cloned into either pSB202 or pJG
vectors (35) or both, and their interaction with wild-type apobec-1 and
various C- or N-terminal deletion mutants was determined as described
(35). Construction of a yeast two-hybrid library used 5 µg of
poly(A)+ RNA, isolated from chicken enterocytes, packaged
in pAD-GAL4 using the HybriZAP two-hybrid gigapack cloning kit
(Stratagene). Rat apobec-1 cDNA was cloned into pBD-Gal4 as the
bait using standard methods, and 10 µg of each plasmid was used to
cotransform yeast (YRG-2) as detailed by the manufacturer (Stratagene).
Biochemical and Genetic Isolation of Proteins That Bind to ApoB RNA
and to Apobec-1--
We reasoned that proteins that function as
integral components in the apoB RNA editing complex should exhibit
binding activity toward both the substrate (apoB RNA) and also the
catalytic subunit of the holoenzyme (apobec-1). Partially purified
chick intestinal S100 extracts were UV cross-linked to radiolabeled
apoB mRNA (13), and the cross-linked protein(s) of molecular weight
~p65 were identified by autoradiography (Fig.
1). The material contained in these
pooled fractions was previously demonstrated to be enriched in apoB RNA
editing complementation activity, although its composition was unknown
(17). 70 peptides were obtained in this screen of which the sequence of
6, shown in Fig. 1, matched unambiguously to GRY-RBP, whereas 5 others
matched to a sequence recently identified as ACF by Driscoll and
co-workers (31). These results suggest that fractionated chicken
intestinal extracts, a source of enriched complementation activity,
contain at least two proteins that can be identified through
interaction with apoB RNA. The demonstration that ACF copurifies with
other protein(s) is reminiscent of the recent report of Greeve and
colleagues (32) in which fractionated rat liver nuclear extracts
revealed the presence of other proteins in addition to ACF/ASP in the
most highly enriched editing fraction.
Independent examination of interacting proteins was undertaken using a
chicken intestinal library cloned into a yeast two-hybrid expression
system, using rat apobec-1 as bait. This approach also revealed a
strongly interacting clone, which was identified as GRY-RBP (data not
shown). Searches with various regions of GRY-RBP identified several
homologs including the EST image clone (N77737) recently identified as
ACF by Driscoll et al. (31) and ASP by Greeve and colleagues
(32). Clone N77737 was directly sequenced, and five separate nucleotide
differences were identified from the reported sequence of Driscoll and
coworkers (31), (C deletions at 84,191, C insertions at 364, 437, and a
G at 189). After correcting these differences, clone N77737 was found
to align completely with the cDNA of the recently identified
complementation factor, ACF (31). Analysis of the full-length chicken
ACF cDNA demonstrated the predicted amino acid sequence (Fig.
2A) is identical to the human
clone. This apparent conservation suggests an interesting paradox,
since chicken apoB RNA is not edited (33). This functional limitation
reflects at least two components. First, chicken intestine lacks
apobec-1 (33, 36) and thus cannot mediate catalytic deamination of a
target apoB RNA and, second, chicken apoB RNA is itself not an editable
template (33). Thus, the evolutionary and functional significance of
ACF expression in this setting must await further study.
Sequence Alignment and Phylogeny of GRY-RBP and ACF
Isoforms--
Full-length cDNAs encoding GRY-RBP and ACF were
isolated from human liver and intestinal RNA as described, and their
sequence alignments are shown in Fig. 2A. GRY-RBP contains a
distinctive N-terminal extension (Fig. 2A) as well as three
nonidentical RNA recognition motifs (RRMs) that appear conserved with
those found in ACF (Fig. 2A). In addition, GRY-RBP contains
a C-terminal, putative bipartite, nuclear localization sequence
(Lys564-Arg-Lys-X9-Lys-Arg-Arg578)
that differs in sequence and location from that proposed in ACF (31).
The C-terminal region of GRY-RBP, between residues 446 and 623, contains 20 RG clusters and 8 RGG repeats in an overall context of 36%
arginine or glycine residues. This region, in particular the RGG
repeats and RG clusters, may signify an RNA binding domain (37) that
spans over 170 residues. By contrast, the C-terminal region of ACF
contains 6 RG clusters within a domain spanning residues 311-402 that
is composed of 28% arginine/glycine residues. Further analysis
revealed three isoforms of ACF cDNA in human liver (data not
shown). These include the cDNA clone reported by Driscoll et
al. (31), a second isoform containing an 8-amino acid insertion
(ASP, described by Greeve et al. (32), and a third form with
a 55-amino acid deletion (data not shown). Sequence analysis of 10 independent clones isolated from adult human liver RNA revealed 6 encode the clone identified by Driscoll and co-workers (31) and 2 contain the 8-amino acid insertion and 2 contain the 55-amino acid
deletion. Analysis of ACF cDNAs from human small intestine revealed
that 3 of 9 clones contain the 8-amino acid insertion (data not shown),
recently demonstrated by Greeve and colleagues (32) in their
liver-derived clone. This insertion was present in the single clone
analyzed from chicken small intestine. Phylogenetic analysis suggests
that ACF and GRY-RBP represent two distinct members of a common
ancestral gene family that is conserved from Dictyostelium
to mammals (Fig. 2B). Examination of the UNIGENE data base
reveals that the EST corresponding to ACF is located on human
chromosome 10, whereas GRY-RBP is located on chromosome 20.
Protein-Protein Interaction of GRY-RBP with ACF and with
Apobec-1--
Protein-protein interaction of GRY-RBP with ACF was
examined by several complementary strategies. We first undertook
immunoprecipitation of the products of radiolabeled in vitro
translation mixed with epitope-tagged recombinant, unlabeled protein.
In vitro translated Myc-GRY-RBP yielded a predominant
species of ~80 kDa with smaller products that represent either
internal methionine residues or partial degradation products, whereas
in vitro translation of FLAG-ACF yielded a protein of ~67
kDa (Fig. 3A, lanes 1 and
2, respectively). Mixing radiolabeled ACF with cold,
unlabeled Myc-GRY-RBP followed by immunoprecipitation with anti-Myc IgG
revealed a physical interaction of these two proteins as demonstrated
in lane 2 of Fig. 3B, showing a radiolabeled band
corresponding to the dominant translation product of ACF alone. The
converse experiment, mixing radiolabeled GRY-RBP with cold, unlabeled
FLAG-ACF followed by immunoprecipitation with FLAG IgG, similarly
revealed a single radiolabeled band corresponding to the dominant
translation product of GRY-RBP alone (Fig. 3B, lane 4).
Control immunoprecipitations, performed with the radiolabeled ligand
and anti-epitope IgG, but without the target protein, revealed no
coprecipitation (Fig. 3B, lanes 1 and
3).
To demonstrate the interaction of ACF and GRY-RBP in a physiological
context, cotransfection experiments were conducted in which
epitope-tagged ACF, GRY-RBP, and apobec-1 were introduced either alone
or in combinations into COS cells. Cell lysates were prepared and
examined by Western blotting to demonstrate expression of the relevant
protein. Cells transfected with ACF, apobec-1, or GRY-RBP cDNA
alone demonstrated comparable expression of the cognate protein (Fig.
3C, lanes 2-4). Cotransfection of ACF and apobec-1 (Fig.
3C, lane 5) or of ACF and GRY-RBP (Fig. 3C,
lane 6) also revealed expression of the relevant proteins in
cell lysates. Immunoprecipitation of cell lysates was then conducted to
demonstrate protein-protein interaction in vivo. Apobec-1
and ACF was coimmunoprecipitated from COS cells transfected with both
cDNAs (Fig. 3D, lane 5), confirming the recent findings
of Driscoll and colleagues (31). Extending these findings, COS cells
transfected with ACF, and GRY-RBP demonstrated coprecipitation of
GRY-RBP following immunoprecipitation of ACF (Fig. 3D, lane
6). These cumulative findings provide further evidence for a
physical interaction of ACF and GRY-RBP in vivo.
The interaction of GRY-RBP, and of ACF, with apobec-1 was further
examined using a yeast two-hybrid binding assay. As indicated in Fig.
3E, the data reveal strong (++) interaction between ACF and
apobec-1 and comparable interaction between apobec-1 and GRY-RBP. Apobec-1 has been demonstrated previously to exist as a homodimer (35,
38), and its self-interaction (+++ in this assay) is shown by way of a
positive control. Both C- and N-terminal deletions of apobec-1 failed
to support an interaction with GRY-RBP, suggesting that these domains
may be of importance in stabilizing and maintaining the interaction
with additional proteins in the holoenzyme. This speculation is
supported by the earlier demonstration that these very N- and
C-terminal deletions in apobec-1 eliminate homodimerization, RNA
binding, and apoB RNA editing activity (35). Nevertheless, it bears
emphasis that other interpretations, such as an effect on apobec-1
folding, cannot be excluded.
Finally, the interaction of GRY-RBP and apobec-1 was examined by far
Western blotting using immobilized GRY-RBP and 35S-labeled
apobec-1, generated from in vitro translation (Fig. 3F). The ability of apobec-1 to bind to GRY-RBP in this
renaturation assay further suggests that these two proteins have the
capacity to interact biochemically.
Taken together, the results from several independent lines of evidence
indicate that GRY-RBP interacts with ACF and with apobec-1.
RNA Binding Activity of ACF and GRY-RBP--
Recombinant ACF and
GRY-RBP (Fig. 4A) were used to
establish the RNA binding activity of the respective proteins with an
apoB template. GRY-RBP binds to apoB RNA as evidenced by the dominant UV cross-linked band (Fig. 4B). The binding of ACF to apoB
RNA, previously demonstrated by Driscoll and colleagues (31), was confirmed in our hands (Fig. 4C). Additionally, incremental
supplementation with ACF inhibited GRY-RBP binding to apoB (Fig.
4C). GRY-RBP additions in turn appeared to interfere with
ACF binding to apoB RNA, the data suggesting that binding of GRY-RBP to
apoB RNA was of lower affinity than that of ACF (compare 1000 ng of
GRY-RBP in D to 500 ng of ACF in C of Fig. 4).
These findings are of interest in light of the recent demonstration by
Greeve and colleagues (32) that binding of ACF to apoB RNA was
competitively displaced by KSRP, suggesting that other RNA-binding
proteins, in addition to ACF and apobec-1, may regulate the assembly
and composition of the holoediting enzyme. The findings do not allow us
to distinguish whether this apparent displacement is mediated directly,
through competitive binding for a shared site on the RNA, or
alternatively through the binding of ACF and GRY-RBP to one another
indirectly modulating RNA binding affinity. However, we will return to
the functional consequences of ACF and GRY-RBP binding below.
RNA Binding Specificity of GRY-RBP and ACF--
The RNA binding
activity of ACF and GRY-RBP was further examined using homopolymeric
RNAs to compete their binding to apoB RNA. Poly(U), poly(AU), poly(I),
and poly(G) competed both proteins but poly(A) competed only ACF (Fig.
5A). Poly(C) and poly(IC) failed to compete RNA binding with either ACF or GRY-RBP (Fig. 5A). These data suggest that there are differences in the
specificity of RNA binding between GRY-RBP and ACF, particularly with
respect to A-rich templates. Despite these differences, however, the
results suggest that both proteins bind to U- and AU-rich targets. This feature would be anticipated in light of their binding to the apoB RNA
template, which is ~70% AU-rich in the region flanking the edited
base (14).
To refine the binding specificity, the recombinant proteins were used
in a binding assay with apoB RNAs, into which various scrambling
mutations have been introduced. These apoB mutants, each of identical
length and spanning the minimal editing cassette, were created by
changing 6-nucleotide sections to the complementary sequence, as
previously described (Fig. 5B) (13, 17). The results of
these experiments suggest that GRY-RBP binds with low specificity
throughout the apoB template, as evidenced by the comparable reduction
in binding with all the scrambled mutations, compared with that
observed with a nonspecific, actin RNA (Fig. 5B). On the
other hand, ACF appears to bind preferentially to two regions within
the minimal apoB RNA, as evidenced by the abrogation of competition
with mutants C and D (Fig. 5B). One site is upstream of the
edited base (nt 6660-6665) and another is downstream (nt 6667-6673),
the latter spanning the proximal end of the mooring sequence and
partially overlapping the binding site recently identified for apobec-1
(16, 17, 20). These results are similar to those obtained using crude
rat enterocyte S100 extracts where binding activity of p60 was
localized to a region spanning nts 6671-6674 (25). The current data
also extend the results of Driscoll and colleagues (29, 31) who
demonstrated that ACF failed to bind to a 280-nt baboon apoB RNA
containing three mutations within the mooring sequence (6671, 6675, and
6678). The cumulative interpretation of these earlier findings, along
with the current data, suggests that regions immediately flanking the
edited base may represent binding sites for ACF. Nevertheless, fine
mapping of the binding site of ACF and its role in the cooperative
assembly of the apoB RNA editing enzyme will require further study.
Regulation of C to U RNA Editing Activity through ACF-GRY-RBP
Interaction--
ACF and apobec-1 were used in an in vitro
editing assay to confirm the demonstration that these two proteins
represent the minimal editing enzyme complex for C to U deamination of
apoB RNA. The data reveal a linear increase in C to U editing activity in an assay using 250 ng of GST-apobec-1 and 1-40 ng of ACF (Fig. 6A). Optimal editing activity
(>80% C to U conversion) was noted in assays containing ~40 ng of
ACF and ~250 ng of GST-apobec-1 (Fig. 6A). This
corresponds to a predicted molar ratio of apobec-1:ACF of 3:1, assuming
that all the available protein exists in a functionally active complex.
As noted previously in assays utilizing chicken intestinal S100
extracts, further addition of complementation activity (in the present
studies, authentic recombinant ACF) led to a progressive decline in
editing activity (Fig. 6A). These findings emphasize the
crucial stoichiometry of apobec-1 and ACF in this assay and lend
indirect support to the hypothesis that other proteins may participate
in a regulatory capacity in vivo. Although antisera against
GRY-RBP, ACF, and apobec-1 are available, the abundance of ACF and
GRY-RBP is below detection levels by Western blotting of fractionated
cell or tissue extracts (data not shown, but in agreement with Driscoll
et al. (29)), precluding estimates of their relative
proportions within a functional editing enzyme. Accordingly, the
precise molar concentrations of apobec-1, ACF, and potentially GRY-RBP
within the holoenzyme remain to be ascertained directly.
The addition of increasing amounts of recombinant GRY-RBP to assays
containing ACF and apobec-1 demonstrated progressive inhibition of RNA
editing, with complete abrogation of C to U deamination of apoB RNA
noted at the highest amounts tested (Fig. 6B). By contrast,
assays containing apobec-1 and up to 100 ng of GRY-RBP failed to
demonstrate evidence of C to U editing activity, indicating that
GRY-RBP itself lacks the ability to complement apobec-1 (Fig. 6B). Assays were then conducted using a range of input RNA
in the presence of apobec-1 and ACF and with increasing amounts of GRY-RBP. C to U conversion demonstrated saturable kinetics with an
apparent Km of 7 ± 1.1 nM (Fig.
6C). The presence of increasing amounts of GRY-RBP (10 and
100 nM) altered the Km for this reaction
to 24 and 40 nM, respectively. Lineweaver-Burk plots of the
data suggest that this is the result of competitive inhibition (Fig.
6C, inset). To examine further the mechanism of this
inhibition, experiments were conducted in which assays, containing
amounts of ACF and apobec-1 sufficient to yield ~25-50% editing,
were modified through addition of GRY-RBP and then rescued with the
addition of ACF, apobec-1, or both. The results of a representative
series of such experiments indicate that the addition of ACF (Fig.
6D) but not apobec-1 (data not shown) rescues the inhibition
produced by GRY-RBP. Furthermore, supplemental apobec-1 failed to
produce incremental effects in assays rescued with ACF (data not
shown), suggesting that GRY-RBP exerts its inhibitory effects through
binding to and sequestering ACF. This suggestion may also account for
the effects noted on apoB RNA binding, alluded to above (Fig. 4).
Effects of Other ApoB RNA-binding Proteins on C to U Editing
Activity--
The demonstration that GRY-RBP binds to ACF and to apoB
RNA and also inhibits C to U RNA editing raised the possibility that other apoB RNA-binding proteins may also modulate this process, as
previously demonstrated with hnRNP C and D (26, 39). However, as
demonstrated in Fig. 7, pyrimidine
tract-binding protein (PTB), hnRNP-A, and hnRNP-F, all bind to
apoB RNA (Fig. 7, upper panel), yet none produce significant
inhibition of apoB RNA editing (Fig. 7, lower panel). These
results imply that the interaction of GRY-RBP with apoB RNA and the
consequent inhibition of C to U editing is not a general phenomenon
associated with AU-rich RNA-binding proteins.
Antisense Inhibition of GRY-RBP Expression, Effects on ApoB
mRNA Editing--
To demonstrate a potential physiological role
for GRY-RBP in regulating apoB RNA editing, we undertook antisense
oligonucleotide treatment of rat hepatoma cells to decrease expression
of GRY-RBP. McA cells were selected since they express the requisite
trans-acting proteins to edit endogenous apoB mRNA. Our prediction,
based upon the findings reported above, was that decreased
expression of GRY-RBP might result in increased editing
activity. Control, untransfected cells demonstrated ~15% C to U
editing of endogenous apoB mRNA (Fig.
8, lane 1), a range frequently
encountered with this cell line (24). Cells transfected with an
antisense oligonucleotide to GRY-RBP, by contrast, demonstrated a
2-fold increase in endogenous apoB RNA editing (>30%, Fig. 8A,
lanes 2-4), whereas a scrambled oligonucleotide was without
effect. The results are summarized in Fig. 8B and support
our prediction that GRY-RBP may play a physiological role in the
regulation of apoB RNA editing in vivo. Further study will
be required to address the regulatory mechanisms involved.
Colocalization of ACF, Apobec-1, and GRY-RBP in Transfected
Cells--
To characterize further the interaction of ACF, GRY-RBP,
and apobec-1 within the cell, epitope-tagged proteins were introduced into HepG2 and McA cells and localized using immunofluorescence microscopy. Transfection of epitope-tagged proteins permitted the
detection of exogenous protein in cells that express endogenous ACF,
with (McA) or without (HepG2) coexpression of endogenous apobec-1.
Cotransfection of ACF and GRY-RBP suggests a nuclear localization of
both proteins in HepG2 and McA cells (Fig.
9, upper left panel).
Cotransfection of GRY-RBP and apobec-1 suggests that GRY-RBP is again
found in a nuclear distribution, whereas apobec-1 is distributed in a
predominantly nuclear localization pattern also but with evidence of
cytoplasmic staining (Fig. 9, upper right panel).
Cotransfection of ACF and apobec-1 indicates a predominantly nuclear
staining pattern of ACF, again with evidence of cytoplasmic staining of
apobec-1 along with the intense nuclear staining pattern (Fig. 9,
lower panel). It must be emphasized, however, that the
interpretation of these results is based upon the distribution of
epitope-tagged proteins within cells that express a small but
presumably functional pool of endogenous ACF, GRY-RBP, and for McA
cells, apobec-1. In all cases, the size of this pool is unknown, since
these proteins defy quantitation using conventional immunochemical
approaches (data not shown). Accordingly, resolution of the crucial
question of whether these findings reflect the pattern of endogenous
proteins must await the development of more sensitive methodology and
reagents.
To refine the colocalization data hinted at above, confocal microscopy
was undertaken in COS-7 cells, which express very low levels of ACF,
GRY-RBP, and undetectable levels of apobec-1 (data not shown). Apobec-1
was found predominantly in the nucleus, although some staining
was noted within in the cytoplasm (Fig.
10A). In both locations,
apobec-1 staining colocalized with ACF (Fig. 10, A-C).
Similarly, cotransfection of apobec-1 and GRY-RBP demonstrated a
predominantly nuclear colocalization of both proteins, with diffuse
cytoplasmic staining also evident (Fig. 10, E-G). By
contrast, cotransfection of ACF and GRY-RBP demonstrated almost
exclusively nuclear staining, with both proteins again colocalizing in
the confocal, merged image (Fig. 10, I-K). Taken together,
the imaging results support the proposal that ACF and GRY-RBP are
colocalized nuclear proteins and demonstrate that apobec-1 associates
in vivo with both GRY-RBP and with ACF.
The pattern of apobec-1 distribution (predominantly nuclear with some
cytoplasmic staining) is consistent with the working model of C to U
editing of apoB RNA as a nuclear event and raises the additional
possibility that apobec-1 may shuttle from a cytoplasmic to nuclear
pool. Such speculation requires formal proof but is consistent with
recent data demonstrating a role for apobec-1 in regulating the
stability of AU-rich transcripts through its binding activity, a
function presumed to imply a cytoplasmic location (15). In addition,
recent results from Smith and colleagues (40) imply the
possibility that apobec-1 may edit apoB RNA in the cytoplasm of rat
hepatoma cells, again consistent with its localization in this
compartment rather than the nucleus, as had been earlier concluded
(37). However, the current findings are somewhat at variance with those
of Smith and colleagues (40) in regard to the localization of
ACF. Their findings suggest both a cytoplasmic and nuclear
distribution as evidenced by standard immunofluorescence microscopy,
whereas our data, using confocal microscopy, suggest that ACF
exhibits a predominantly nuclear localization (Figs. 8 and 9). Further
examination of this apparent compartmentalization is clearly warranted.
In summary, the findings of this report indicate that the S100 fraction
of chicken enterocyte extracts, a source established to be enriched for
apoB editing complementation activity, contains ACF as well as a
related protein, GRY-RBP. Both proteins bind to apoB RNA and also bind
to one another. GRY-RBP and ACF both exhibit binding activity for apoB
RNA, the binding site for ACF localizing to a region flanking the
edited base. In addition, these proteins colocalize with one another in
the nucleus of transfected cells, and each appears to colocalize with
apobec-1. These data, taken together with the finding that addition of
GRY-RBP to the minimal editing reaction components (apobec-1 plus ACF)
produces a competitive inhibition of C to U editing and the
demonstration that antisense inhibition of GRY-RBP expression increases
apoB RNA editing in rat hepatoma cells, suggest that GRY-RBP may play a
role in the regulation of apoB RNA editing. One possible interpretation is that this regulation may be exerted through a complex interaction that reflects the role of additional components that compose the holoediting enzyme. The mechanism of interaction of these and other
candidate genes will be the focus of future reports.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
(16). Targeted deletion of apobec-1
eliminated C to U editing of apoB mRNA, demonstrating that no
functional redundancy exists in the catalytic deamination of this RNA
(21-23). Nevertheless, although absolutely required for C to U editing
of apoB RNA, apobec-1 alone is not sufficient (11, 18). Specifically,
recombinant apobec-1 will deaminate a monomeric cytidine substrate but
alone exhibits no deaminase activity on an RNA template (24, 25). This
observation coupled with earlier studies in which C to U editing
activity was found to exist in a higher order complex provide support
for the proposal that apoB RNA editing is mediated by a heteromeric
enzyme complex whose composition and functional organization
facilitates catalytic deamination of the targeted base (11, 26,
27).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
20 °C in small aliquots and used at the indicated
concentrations. ACF cDNA was also cloned into the expression vector
pET28a using the BamHI-XhoI sites to generate a
His-tagged protein. The protein was overexpressed in Novablue (DE3)
cells according to the manufacturer's instructions (Novagen). The
bacterial supernatant was incubated with 2 ml of
Ni2+-nitrilotriacetic acid metal affinity resin
(Qiagen) and His6-tagged ACF eluted with sequential
imidazole steps up to 400 mM. Fractions containing
His6-ACF were pooled and dialyzed. A full-length chicken intestinal ACF cDNA was also amplified by reverse transcriptase-PCR using the human primers, above. Antisera against human ACF (N- and
C-terminal antipeptide antisera) were generously provided by D. Driscoll as detailed previously (31).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Peptide sequencing from fractionated chicken
S100 extracts. Chicken enterocyte S100 extracts were subjected to
10-70% ammonium sulfate precipitation and fractionated through
Blue-Sepharose (Amersham Pharmacia Biotech) to enrich for RNA binding
and C to U editing complementation activity (13). The most enriched
fractions (fractions 16-19) (13) were used for UV cross-linking
to a 55-mer apoB RNA and then autoradiographed. The cross-linking
material was silver-stained and eluted for peptide sequence. The
peptide sequences obtained from the mass spectroscopy analysis are
indicated. The assignments of leucine and isoleucine were not
distinguished and are indicated as alternatives (L/I).
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Fig. 2.
Sequence alignment of GRY-RBP and ACF.
A, the deduced amino acid sequence of GRY-RBP and
human/chicken (h/Ch) ACF (see text) are aligned. The three
RNA recognition motifs (RRMs) are indicated in shaded text.
Peptides identified through mass spectroscopy are indicated by the
bold line above the sequence. The putative
nuclear localization motif in GRY-RBP is indicated by a broken
line. B, phylogenetic analysis of GRY-RBP and ACF. The
RRM domains were aligned, and a distance matrix was calculated using
the PROTDIST and Phylip programs.
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Fig. 3.
GRY-RBP interacts with ACF and with
apobec-1. A, Myc-GRY-RBP and FLAG-ACF were synthesized
in vitro using a coupled TnT lysate (Promega) in the
presence of [35S]methionine. Lanes 1 and
2 are representative examples of the products revealed by
autoradiography of 10% denaturing SDS-PAGE. The mobility of the
respective species is indicated by the arrowhead or the
bold arrow with molecular weight markers indicated on the
left. B, co-Immunoprecipitation of ACF and
GRY-RBP. The products of 35S-radiolabeled in
vitro translation of ACF (lanes 1 and 2) or
GRY-RBP (lanes 3 and 4) were mixed with unlabeled
Myc-tagged GRY-RBP (lane 2) or FLAG-tagged ACF (lane
4), respectively. After mixing for 30 min at 30 °C, the
indicated IgG ( -Myc or
-FLAG, respectively) was added to all
incubations and then was gently rotated at 4 °C for 90 min and
collected on protein A-Sepharose beads. Lanes 2 and
4 indicate the coimmunoprecipitation of each species.
C, COS cells were transfected with vector DNA (lane
1) or with DNA encoding ACF, apobec-1, or GRY-RBP (lanes
2-4). Other transfections were conducted in which apobec-1 and
ACF or GRY-RBP and ACF were simultaneously introduced (lanes
5 and 6). After 48 h, cell lysates were prepared
and extracts analyzed by Western blotting with anti-FLAG or anti-Myc
IgG. D, ACF coimmunoprecipitates with GRY-RBP and with
apobec-1 in transfected cells. The extracts from COS cells prepared as
in C were immunoprecipitated with anti-FLAG IgG, and the
immunoprecipitates were resolved by SDS-PAGE and Western-blotted with
anti-Myc IgG. The data indicate coimmunoprecipitation of apobec-1 with
ACF (lane 5) and of GRY-RBP with ACF (lane 6),
with specificity demonstrated by the absence of corresponding bands in
the other lanes. E, protein-protein interaction of GRY-RBP
with apobec-1 using yeast two-hybrid assay. Apobec-1 or mutants
thereof, GRY-RBP and ACF cDNAs, were cloned into the indicated
yeast vectors (see under "Materials and Methods") and interactions
were determined using
O-nitrophenyl-
-D-galactopyranoside staining
as previously validated (33). Descriptions of the different apobec-1
mutations are detailed (33). F, far Western blotting reveals
interaction between GRY-RBP and apobec-1. Left panel,
GRY-RBP (1 µg) was resolved on a 10% SDS-PAGE and stained with
Coomassie Blue. Middle panel, GRY-RBP was resolved by
SDS-PAGE and transferred to a polyvinylidene difluoride membrane.
Immobilized GRY-RBP was submitted to 12 cycles of
denaturation-renaturation and probed with 35S-labeled
apobec-1. Right panel, the identity of GRY-RBP was confirmed
by Western blot analysis using affinity-purified GRY-RBP IgG. In each
case, a representative result is shown from replicate
experiments.
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Fig. 4.
RNA binding activity of GRY-RBP and ACF.
A, 1 µg of protein was analyzed by denaturing SDS-PAGE and
stained with Coomassie Blue. B, UV cross-linking was
performed by incubating increasing amounts of GRY-RBP (50 ng-2 µg)
with a 32P-labeled 105-nt rat apoB cRNA (RB105) flanking
the edited base. C, 100 ng of GRY-RBP was cross-linked to
radiolabeled RB105 RNA in the presence of increasing amounts of His-ACF
(50-500 ng), representing up to a 10-fold molar excess of recombinant
ACF. D, 100 ng of recombinant His-ACF was incubated with
increasing amounts of GRY-RBP (50-1000 ng), representing up to 20-fold
molar excess and cross-linked to RB 105 RNA. This is a representative
of three such experiments.
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Fig. 5.
ApoB RNA binding activity of GRY-RBP and ACF,
competition with homopolymeric RNA and apoB RNA mutants.
A, 250 ng of unlabeled homopolymeric RNA was added to
binding reactions containing radiolabeled RB105 and 200 ng of either
GRY-RBP (left panel) or ACF (right panel). After
treatment with RNase T1 and UV irradiation, the cross-linked products
were analyzed on a 10% SDS-PAGE. Molecular weight markers are
indicated at the left of each gel. B, competition
with mutant apoB RNAs. Upper panel, cross-linking was
carried out with a 55-nt apoB RNA flanking the edited base, in the
absence ( ) or presence of competitor RNA; 55-nt wild-type RNA
(WT), scanning mutants B-I, representing 6 nucleotide sections immediately upstream (B and
C) or downstream (D-I), of the edited base (shown
in lower panel). As nonspecific control, an actin cRNA was
used at equivalent concentrations. Lower panel, the
cross-linked material was stained with Coomassie Blue to demonstrate
equivalent amounts of protein in each lane. These results are
representative of triplicate experiments.
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Fig. 6.
In vitro RNA editing with ACF and
apobec-1, competitive inhibition by GRY-RBP. A,
apobec-1 and ACF alone edit an apoB RNA template. Increasing amounts of
recombinant ACF were added to 250 ng of GST-apobec-1 together with 20 fmol of a 470-nt rat apoB cRNA and in vitro C to U editing
assayed by primer extension (see under "Materials and Methods").
B, increasing amounts of purified GRY-RBP were added to
assays containing 20 fmol of apoB RNA and 250 ng of recombinant
GST-apobec-1 together with 2 ng of purified ACF (lanes
1-5). The RNA was extracted and C to U editing quantitated by
primer extension assay. The relative mobility of the edited (UAA) and
unedited (CAA) products are indicated. Editing is expressed as % U. This is a representative of 2-4 experiments at each concentration of
GRY-RBP. As control, 10 or 100 ng GRY-RBP was added to an assay
containing 250 ng of GST-apobec-1 and apoB RNA but without ACF
(lanes 6 and 7). C, substrate
dependence of C to U editing activity. Editing assays were conducted
with increasing concentrations of RNA substrate under optimal
conditions, using a 3-h time point. Edited RNA was quantitated by
phosphorimaging. Regression analysis using Lineweaver-Burk kinetics was
performed at increasing concentrations of GRY-RBP (inset) to
demonstrate competitive inhibition. Each point is the average of three
independent determinations. D, rescue of GRY-RBP inhibition
with ACF. C to U editing assays were conducted using 250 ng of
GST-apobec-1 and 2 ng of ACF together with 20 fmol of apoB RNA. 4 ng of
GRY-RBP produced ~50% inhibition, which was overcome by rescue with
8 or 16 ng of ACF. A representative experiment from three independent
determinations is shown.
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Fig. 7.
Other apoB RNA-binding proteins fail to
modulate C to U editing. Upper panel, UV cross-linking
assays were conducted with recombinant hnRNP A1, F, and PTB together
with a radiolabeled apoB RNA (see under "Materials and Methods").
The bound transcript was resolved by denaturing SDS-PAGE. Lower
panel, C to U editing assays were conducted with recombinant
apobec-1 and ACF (lane 3) to which was added 2 µg
recombinant hnRNP A1 (lanes 4 and 5), F
(lanes 6 and 7), or PTB (lanes 8 and
9). There was no detectable difference in the extent of C to
U editing in any of the incubations containing ACF and apobec-1. A
representative assay is shown.
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Fig. 8.
Antisense oligonucleotide inhibition of
GRY-RBP expression increases apoB RNA editing in McA rat hepatoma
cells. A, McA hepatoma cells were incubated with 5 µM antisense GRY-RBP ( -GRY-RBP, lanes 2-4)
or a scrambled oligonucleotide (lanes 5-7) and RNA analyzed
by primer extension. The products were resolved by PAGE and
fluorography; p, primer; C, unedited apoB RNA;
U, edited apoB RNA. B, apoB RNA editing was
quantitated by phosphorimaging of the gel and expressed as % U. Antisense GRY-RBP treated cells showed a significant increase in apoB
RNA editing.
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Fig. 9.
Immunofluorescence microscopy of transfected
GRY-RBP, ACF, and apobec-1 in HepG2 and McA cells. The indicated
cells were grown on coverslips and transfected with epitope-tagged
protein as indicated under "Materials and Methods." Left
upper panel cotransfection of GRY-RBP (A) and ACF
(B). Right upper panel, cotransfection of GRY-RBP
(A) and apobec-1 (B). Lower panel,
cotransfection of apobec-1 (A) and ACF (B). In
all cases, these are representative images derived from three
independent experiments. Nuclear counterstaining was performed with
DAPI (C), the arrows indicating cells expressing
the relevant protein products for each panel.
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Fig. 10.
Immunofluorescence confocal microscopy of
transfected apobec-1, ACF, and GRY-RBP. COS-7 cells were
transfected with Myc-apobec-1 (A) and FLAG-ACF
(B) or with FLAG-apobec-1 (E) and Myc-GRY-RBP
(F). In both cases, the merged images (C and
G) indicate colocalization of the signals. In addition,
FLAG-ACF (I) was coexpressed with Myc-GRY-RBP (J)
and the confocal images merged (K) revealing colocalization
in a nuclear distribution (compare To-Pro 3 iodide nuclear staining in
D, H and L). These images are representative of
three independent transfections.
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ACKNOWLEDGEMENTS |
---|
We acknowledge the initial contributions of Jing Min in the two-hybrid screen. We thank Karen Hutton (Digestive Disease Research Core Center) for technical assistance in the confocal microscopy studies, W. Lane (Harvard Microchemistry Facility, Harvard University) for protein sequencing, and D. Driscoll for providing antiserum to ACF. We also thank Clare Gooding and Chris Smith for supplying purified PTB and Shern Chew and Ian Eperon for hnRNP A1.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants HL-38180 and DK-56260 (to N. O. D.) and the Morphology Core of the Digestive Disease Research Core Center Grant DK-52574.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.
¶ Supported by the Medical Research Council (UK).
Both laboratories contributed equally to this work.
To whom correspondence should be addressed: Gastroenterology
Division, Washington University Medical School, Box 8124, 660 S. Euclid
Ave., St. Louis, MO 63110. Tel.: 314-362-2027; Fax: 314-362-2023;
E-mail: NOD@IM.WUSTL.EDU.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M006435200
2 We use the term ACF to denote the complementation factor identified by Driscoll and colleagues (31). ACF is of almost identical sequence to the cDNA recently cloned by Greeve and colleagues (32), referred to as ASP. For the purposes of this report, we refer to ACF to describe this protein and its functions.
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ABBREVIATIONS |
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The abbreviations used are: apoB, apolipoprotein B; nt, nucleotide; PAGE, polyacrylamide gel electrophoresis; ACF, apobec-1 complementation factor; MS, mass spectrometry; PMSF, phenylmethylsulfonyl fluoride; PCR, polymerase chain reaction; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; DAPI, 4,6-diamidino-2-phenylindole; PTB, pyrimidine tract-binding protein; hnRNP, heterogeneous nuclear ribonucleoprotein.
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