From the
Apolipoprotein B (apoB) mRNA editing is mediated by an enzyme
complex which includes the catalytic subunit, apobec-1. Recombinant
GST/APOBEC-1 binds with high specificity to a rat apoB RNA template as
demonstrated by UV cross-linking and electrophoretic mobility shift
assay (EMSA). ApoB RNA binding was competed by poly(U), poly(A,U), and
tRNA, but not by poly(A) or other homopolymeric ribonucleotides. UV
cross-linking of GST/APOBEC-1 to an apoB RNA template was uninfluenced
by the binding of proteins of
Apolipoprotein B (apoB),
An important feature of
apobec-1 activity is that preparations of the protein, produced either
from cell-free translation of the cDNA or following cRNA injection into Xenopus oocytes, will not perform apoB RNA editing without a
source of complementation activity (4, 8). Examples of such sources
would be S100 extracts prepared from chicken small intestinal
enterocytes or human liver, neither of which alone has apoB RNA editing
activity(8) . By extension, an important question to emerge from
these studies is whether specific elements of this complementation
activity are involved in directing the catalytic subunit to the
appropriate RNA sequence in the primary apoB transcript for
site-specific deamination(8, 9) . In this regard, much
progress has been made concerning the sequence requirements for apoB
RNA editing (10). The minimal apoB RNA editing cassette consists of
approximately 29 nucleotides flanking the edited base and includes a
critical 11-nucleotide mooring sequence (UGAUCAGUAUA), which is
sufficient to support in vitro RNA editing of both apoB and
heterologous RNA templates(10, 11, 12) .
Virtually all mutations within this 11-nucleotide cassette abolish in vitro apoB RNA editing (10-12). Consistent with these
findings, chicken apoB mRNA, which is not edited in vitro by
mammalian intestinal S100 extracts, contains 3 nucleotide alterations
in this region(13) .
In order to examine the machinery
involved in apoB mRNA editing, several groups have demonstrated the
presence of proteins in either nuclear or S100 extracts which will
cross-link to apoB RNA(14, 15, 16) . Studies
revealed a protein of approximately 40 kDa in nuclear extracts from rat
liver while proteins of
We now present data which demonstrate that a
recombinant glutathione S-transferase/apobec-1 fusion protein
(GST/APOBEC-1) has intrinsic apoB RNA binding activity as inferred from
both UV cross-linking and electrophoretic mobility shift assays.
However, the demonstration that GST/APOBEC-1 binds chicken apoB RNA,
which is not an editable template, suggests that the structural
constraints for RNA binding are distinct from those which determine
editability.
There are no known
RNA binding motifs in the primary amino acid sequence of apobec-1,
suggesting that this is a novel RNA binding
protein(23, 24, 25) . The implications and
biological relevance of this observation in the context of the intact
mammalian apoB mRNA editing enzyme await the further identification of
its component subunits. In the meantime, the functional domains of
apobec-1 involved in this interaction will be the focus of future
investigation.
We thank Susan Skarosi, Trish Glascoff, and Annalise
Hausman for outstanding technical assistance and Federico Giannoni for
discussions and the provision of cell extracts.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
60 and
44 kDa, present in S100
extracts prepared from different sources. The binding of these proteins
was similarly uninfluenced by the simultaneous binding of GST/APOBEC-1.
Moreover, the inclusion of heterologous S100 extracts in the RNA
binding reactions completely abrogated the competitive displacement of
GST/APOBEC-1 by tRNA. EMSA revealed the onset of RNA binding within
1-2 min, and its specificity was confirmed by a supershift with
anti-GST/APOBEC-1 antisera. The structural specificity for apoB RNA
binding, as inferred from EMSA, appears to be distinct from apoB RNA
editing since wild-type chicken apoB RNA, which is not editable, and
several mutant chicken apoB RNAs containing clustered mutations within
the minimal apoB RNA editing cassette, bound with efficiency similar to
the rat apoB RNA template. In conclusion, while the data suggest that
apobec-1 binds AU-rich templates, the importance of this observation in
the context of mammalian apoB mRNA editing remains unknown.
(
)an obligate
structural component of lipoproteins secreted by the small intestine
and liver of mammals, circulates in two distinct forms (reviewed in
Ref. 1). ApoB100 contains 4536 residues and is secreted from the liver
while apoB48, which is colinear with the amino-terminal 2152 residues
of apoB100, is secreted from the small intestine(1) . The
molecular mechanism for this organ-specific partitioning is referred to
as apoB mRNA editing(2, 3) . In this process, a
post-transcriptional cytidine deamination of a glutamine (CAA) codon in
the nuclear apoB mRNA produces a UAA stop codon and translational
termination(2, 3) . Numerous experiments conducted over
the last few years have demonstrated that this editing reaction is
mediated by an enzyme complex of which the catalytic subunit, apobec-1,
was recently cloned from rat small intestine(4) . More recently,
homologous gene products have been cloned from human and rabbit small
intestine(5, 6, 7) .
60 and
44 kDa were identified in rat
intestinal and hepatic S100
extracts(14, 15, 16) . Additionally, the
cross-linking pattern demonstrated in rat intestinal S100 extracts was
shown to be most specific for a 4-nucleotide region at the 5` end of
the mooring sequence (UGAU)(15) . Examination of the association
kinetics of these proteins, as inferred by UV cross linking,
demonstrated an interaction prior to the onset of detectable editing of
the target apoB RNA template, findings which have been argued to
support the sequential assembly of a nucleoprotein editing complex
referred to as an editosome(16) . Other workers, however, have
demonstrated that S100 extracts, prepared from either baboon
enterocytes or rat liver, yield proteins of
66 and
44 kDa
following UV cross-linking to a luciferase RNA or an antisense apoB RNA
template(11) . Thus, neither the identity of these proteins nor
their requisite involvement in apoB mRNA editing has been conclusively
demonstrated.
Expression of GST/APOBEC-1
apobec-1 cDNA was
cloned into pGEX-4T3 (Pharmacia Biotech Inc.) and expressed as a
glutathione S-transferase (GST) fusion protein in lon-protease-deficient RB791 Escherichia coli cells.
The fusion protein was extracted from bacterial lysates with 1% Triton
X-100, purified over glutathione-agarose beads (Sigma), and eluted in a
buffer containing 50 mM Tris-HCl, pH 8.0, 10 mM reduced glutathione. Details of the production and functional
analysis of the fusion protein are presented in an accompanying
manuscript(26) . However, the apparent molecular mass of
GST/APOBEC-1, as estimated from 8% SDS-PAGE, is 60 kDa (data not
shown). Antisera to the purified fusion protein was raised in rabbits.
Plasmid Construction
A 105-base pair fragment of
rat apoB cDNA (nt 6639-6743, pRB 105) and a 160-base pair
fragment of wild type and mutant chick apoB cDNA (nt 6608-6768,
pCB 150) were cloned into pGEM3Zf(+) and used as templates for in vitro RNA synthesis (8, 13). Single-point and clustered
mutations were introduced into pCB 150 using a two-step polymerase
chain reaction method and the cloned mutants sequenced on both
strands(17) . A 247-base pair mouse -actin cDNA was used as
previously detailed(18) . In vitro apoB RNA editing
reactions were conducted as previously detailed(8) .
UV Cross-linking and Electrophoretic Mobility Shift
Assays (EMSAs)
Cytoplasmic S100 extracts were prepared from
whole rat liver and from isolated rat or chicken enterocytes as
previously detailed(8) . P-Labeled RNA templates
(50,000 cpm, 2.5-3.0
10
cpm/µg) were
incubated with the indicated amounts of these extracts and/or
GST/APOBEC-1 for 20 min at room temperature in a buffer containing 10
mM Hepes, pH 7.9, 100 mM KCl, 1 mM
dithiothreitol and then treated sequentially with RNase T1 (1
unit/µl final concentration) and heparin (5 mg/ml final
concentration) for 5 min each at room temperature unless otherwise
stated(19) . Competitor RNA templates or homopolymeric RNAs were
added, where indicated, at 1 µg per reaction. In supershift assays,
undiluted anti-GST/APOBEC-1 antisera was added, and the incubation
continued for an additional 20 min before RNase T1 and heparin
addition. For cross-linking studies, the mixture was subjected to UV
irradiation (254 nm) in a Stratalinker cross-linking apparatus
(Stratagene, energy = 250 mJ/cm
) and then separated
by 10% SDS-PAGE under reducing conditions. For RNA gel shift assay, the
mixture was immediately analyzed by 4% native polyacrylamide gel
electrophoresis (37.5:1) using 45 mM Tris borate, 0.1
mM EDTA, pH 8.6. The gels were dried and autoradiographed at
-70 °C. For the zinc-chelation experiments, 500 ng of
GST/APOBEC-1 was preincubated with 1,7- or
1,10-o-phenanthroline (10 mM final concentration) or
ethanol control (0.4% final concentration) for 10 min before being
added to the binding assays for an additional 20-min
incubation(20) .
Oligonucleotides
The following oligonucleotides
were used for mutagenesis of chicken apoB cDNA and for primer extension
assay. Restriction sites are underlined; underlined and bolded
nucleotides represent the mutations introduced. Chicken APOB 5` primer
SH1, 5`-GGGTACCGGAAAAACGCCTGGATAAT-3` (KpnI); chicken APOB 3`
primer SH2, 5`-GAGATCTAGAGCTGTCATTTTTTCA-3` (BglII); chicken
APOB CH150 M 5` mutagenic primer SHM1,
5`-AATTGAT-CAGTATATCAAAG-3`; chicken APOB CH150 M
3` mutagenic primer SHM2,
5`-CTTTGATATACTGATCAATT-3`; chicken APOB CH150 ML
5` mutagenic primer SHM3,
5`-GATACAATTTGATCAGTATATC-3`;
chicken APOB CH150 ML 3` mutagenic primer SHM4,
5`-GATATACTGATCAAATTGTATC-3`;
chicken APOB CH150 MR 5` mutagenic primer SHM5,
5`-ATTGATCAGTATATTAGAGATA-3`;
chicken APOB CH150 MR 3` mutagenic primer SHM6,
5`-TATCTCTAATATACTGATCAAT-3`;
rat primer extension primer [BT7]:
5`-AGTCCTGTGCATCATAATTATCTCTAATATACTGA-3`; chicken WT primer extension
primer [PECH-35], 5`-TGTCAAACTGATCGTAATTCTCTTTGATGTACTGC-3`;
chicken M/ML primer extension primer [PECHM/ML-35],
5`-TGTCAAACTGATCGTAATTCTCTTTGATATACTGA-3`; chicken MR/MLR primer
extension primer [PECHMR/MLR-35],
5`-TGTCAAACTGATCGTAATTATCTCTAATATACTGA-3`.
UV Cross-linking of GST/APOBEC-1 to an ApoB RNA
Template
Incubation of GST/APOBEC-1 with a rat apoB RNA template
followed by UV cross-linking produced a single band, which migrated
with an apparent molecular mass of 72 kDa (Fig. 1A, lanes 3 and 4). The intensity of this band increased
in a dose-dependent manner with increasing quantities of the fusion
protein (Fig. 1B). ApoB RNA binding was competed for by
the addition of excess rat apoB RNA (Fig. 1A, lane
5), demonstrating the specificity of binding. Additionally,
poly(U) and poly(A,U) competed for apoB RNA binding, suggesting that
GST/APOBEC-1 may function similarly to other AU-binding
proteins(19, 21) . This suggestion is also consistent
with the high (69%) AU content of the RB 105 template. Neither poly(A) (Fig. 1A, lane 7), other homopolymeric
ribonucleotides (poly(G), poly(C), and poly(I):poly(C)), nor
single-stranded DNA competed for binding of the apoB RNA template (data
not shown). By contrast, apoB RNA binding was effectively competed for
by tRNA (Fig. 1A, lane 6). This observation was
unexpected since our in vitro apoB RNA editing assays contain
200-250 ng of tRNA(8, 13) . In order to confirm
this observation, a series of incubations was performed in the presence
of increasing quantities of tRNA. The results indicate a dose-dependent
inhibition of apoB RNA binding, beginning with as little as 10 ng of
tRNA (Fig. 1C). The explanation for this observation is
not readily apparent although it should be emphasized that the
orientation of the recombinant GST/APOBEC-1 with respect to the RNA
template is likely to be different from that of the native, endogenous
apobec-1, which exists within the holoenzyme and presumably functions
in the context of apoB mRNA editing complementation factor(s). This
issue is addressed further in experiments detailed below. The
specificity of the apoB RNA binding reaction was further confirmed by
the demonstration that GST/APOBEC-1 binds efficiently to the sense but
not the antisense RB 105 template (Fig. 1D, compare lanes 3 and 6) and with considerably reduced
efficiency to a chicken apoB RNA template or an irrelevant sense RNA
(mouse
-actin, Fig. 1D, lanes 7-12).
Cross-linking of the chicken apoB RNA template to GST/APOBEC-1 was
further explored, as detailed below.
Figure 1:
Identification of
GST/APOBEC-1 following UV cross-linking to a rat apoB cRNA. 500 ng of
either GST- or GST/APOBEC-1 was incubated for 20 min with a P-labeled rat apoB cRNA template (RB 105), the resulting
complex was cross-linked under UV for 1.5 min and analyzed by 10%
SDS-PAGE. A, binding was performed either in the absence of
competitor RNA or in the presence of 1 µg of unlabeled RB 105 cRNA,
125 ng of tRNA, or 1 µg of poly(A), poly(U), or poly(AU). B, binding of increasing concentrations of GST/APOBEC-1 to rat
apoB cRNA (RB 105). C, competition for binding of GST/APOBEC-1
with increasing concentrations of tRNA. D, specificity of
binding of GST/APOBEC-1 to a rat apoB cRNA. Incubations were conducted
as above using either sense (RB 105 S) or antisense (RB
105 AS) rat apoB RNA, chicken apoB cRNA (CB 150 S), or a
mouse
-actin cRNA (ACT).
In vitro apoB RNA
editing reactions were conducted with GST/APOBEC-1 and chicken
enterocyte S100 extracts, in the presence or absence of 250 ng of tRNA.
These reactions produced similar in vitro conversion
efficiency of the RB 105 template (data not shown), suggesting that the
inclusion of tRNA is not detrimental to the apoB RNA editing reaction.
By contrast, no apoB RNA editing activity was demonstrated when
incubations of GST/APOBEC-1 were conducted without a source of
complementation activity, even in the absence of tRNA (data not shown).
This suggests that, despite its ability to bind the RNA template,
GST/APOBEC-1 cannot perform in vitro editing alone. Since apoB
RNA editing has been demonstrated to proceed at high efficiency in
vitro in the presence of tRNA, the possibility that apoB RNA
binding by GST/APOBEC-1 is stabilized by the presence of
complementation factors was addressed.
GST/APOBEC-1 Binds to an ApoB RNA Template in the
Presence of Complementation Factors and Independent of the Binding of
p60 and p44
Previous studies have demonstrated UV cross-linked
proteins of 60 and
44 kDa in S100 extracts from mammalian
enterocytes and rat
liver(11, 14, 15, 16) . UV cross-linking
experiments performed in the absence of GST/APOBEC-1 yielded proteins
of
44 kDa in enterocyte extracts from chicken (Fig. 2, lane 1, closed arrowhead) and rat small intestine (Fig. 2, lane 8, open arrowhead) while both
60- (open arrow, Fig. 2) and
44-kDa proteins
were identified in rat liver S100 extracts (Fig. 2, lane
5). The identity of the strongly reactive cross-linked protein of
28 kDa in rat intestinal S100 extracts (Fig. 2, lanes
8-10) is unknown, but Western blotting with anti-apobec-1
antisera was unreactive (data not shown). UV cross-linking of these
S100 extracts performed in the presence of GST/APOBEC-1 resulted in an
indistinguishable pattern of protein binding to that found with S100
extracts alone (Fig. 2, lanes 2-4, 6, 7, and 9, 10). Conversely, the pattern of UV
cross-linking noted above for GST/APOBEC-1 alone (Fig. 1) was
uninfluenced by the binding of the
60- and
44-kDa proteins (Fig. 2, closed arrow) present in S100 extracts. Taken
together, this suggests that apoB RNA binding by GST/APOBEC-1 is not
competed for by either of the two previously identified cross-linking
proteins present in S100 extracts. Furthermore, these findings imply
the presence of distinct RNA binding sites on the template and also
that the binding of GST/APOBEC-1 and either the
60- or
44-kDa
proteins is not cooperative. In addition, apoB RNA binding by
GST/APOBEC-1 is not competed for by endogenous apobec-1 in rat
intestinal or hepatic S100 extracts suggesting, among other
possibilities, that its concentration in these sources may be extremely
low or that its presence in association with other components of the
apoB mRNA editing enzyme precludes competitive interaction with the
recombinant GST-fusion protein.
Figure 2:
S100 extracts prepared from rat liver or
intestine or from chicken intestine do not compete out cross-linking of
GST/APOBEC-1. Chicken (CHICK), rat enterocyte (SI),
and rat liver (LIV) S100 extracts (20 µg) were incubated
with the RB 105 probe, either alone (-) or in the presence of 500
ng of GST/APOBEC-1 (+). The position of GST/APOBEC-1 is shown by
the solid arrow. The position of p60 is shown by the open
arrow. The position of bands corresponding to p44 is shown by arrowheads (closed = chicken, open = rat).
Further studies conducted in the
presence of high concentrations (250 ng) of tRNA and GST/APOBEC-1,
together with 20 µg of rat liver or chicken intestinal S100
extract, produced an indistinguishable UV cross-linking pattern to that
shown in Fig. 2(data not shown). This suggests either that the
complementation factors stabilize or position GST/APOBEC-1 in a more
favorable orientation with respect to the apoB RNA template, despite
the presence of tRNA, or in some way sequester the tRNA and abrogate
its inhibitory effects. It is equally possible that the complementation
factors function as adaptors to bring the RNA template and apobec-1
into proximity. Further studies will be necessary to resolve these
possibilities. It also bears emphasis that, under the conditions of in vitro editing (high concentrations of tRNA and the presence
of complementation factors), apobec-1 would be anticipated to bind the
RNA template.
EMSA of GST/APOBEC-1 following Incubation with an ApoB
RNA Template
The finding that GST/APOBEC-1 binds an apoB RNA
template was further pursued by means of EMSA. A single-shifted band
was found with increasing amounts of GST/APOBEC-1 (Fig. 3A). A supershifted band was demonstrated
following the addition of anti-GST/APOBEC-1 (Fig. 3B, open arrow). The supershifted complex contains virtually 100%
of the GST/APOBEC-1, confirming the identity of the EMSA gel shift.
Timed incubations demonstrated an RNA gel shift within 3 min of the
addition of GST/APOBEC-1 (Fig. 3C). Further analysis of
the time course of apoB RNA binding demonstrated a detectable gel shift
as early as 1-2 min of incubation (Fig. 3D).
Although the time frame for apoB RNA editing was not specifically
addressed in these studies, previous work from our own and other
laboratories suggest that at least a 5-min incubation is required for
the detection of C to U conversion(13, 16, 22) .
The current findings are thus consistent with previous suggestions that
the binding of proteins to the apoB RNA template precedes
editing(16) . The addition of 10 mM 1,10-o-phenanthroline, but not the inactive isomer or
ethanol vehicle, decreased, but did not abolish, apoB RNA binding, as
inferred by EMSA (Fig. 3E). These findings are broadly
consistent with the recent demonstration that apobec-1 is a
zinc-dependent cytidine deaminase(20) . However, the observation
that 10 mM 1,10-o-phenanthroline abolishes in
vitro apoB RNA editing (20) but does not completely inhibit
apoB RNA binding suggests that these properties are distinct.
Figure 3:
Formation of an apoB RNAGST/APOBEC-1
complex demonstrated by EMSA. Either GST- or GST/APOBEC-1 was incubated
with the
P-labeled apoB cRNA template, and the complex was
analyzed by nondenaturing polyacrylamide gel electrophoresis. A, GST- (500 ng, lane 2) and GST/APOBEC-1 (250 ng, lane 3; 500 ng, lane 4). B, supershift of
the GST/APOBEC-1
apoB RNA complex with increasing concentrations
of anti-apobec-1 antisera (
APOBEC-1) (open
arrow, lanes 3-6). The shifted
GST/APOBEC-1
apoB RNA complex is shown by the closed
arrow. A nonspecific band (arrowhead) is also seen due to
interaction with a protein in serum (lane 2, NRS,
normal rabbit serum). C, time course of GST/APOBEC-1 binding
from 0-30 min. RNase T1 and heparin incubations were performed
simultaneously for 5 min following incubation. D, time course
of binding from 0-3 min. RNase T1 and heparin incubations were
performed simultaneously for 1 min following incubation. Bold
arrows indicate shifted complex; two smaller arrows indicate nonspecific artifact. E, effect of zinc
chelation with o-phenanthroline on apoB RNA binding by
GST/APOBEC-1. The protein was incubated with 10 mM 1,7- (lane 2) or 1,10-o-phenanthroline (lane 3)
or with ethanol control (lane 1) prior to addition of the RNA
template. Migration of the free transcript (F) and position of
the complex formed with GST/APOBEC-1 (closed arrow) are
indicated in all panels.
Sequence Requirements for ApoB RNA Binding Are Distinct
from the Requirements for ApoB RNA Editing
Previous studies have
demonstrated that a conserved 29-nucleotide cassette flanking the
edited base contains sufficient structural information to confer
editing capacity upon heterologous RNA
templates(10, 11) . Chicken apoB RNA, which is not
edited, contains several differences from the conserved mammalian
sequence within the minimal apoB RNA editing cassette, including three
nucleotide alterations within the 11-nucleotide mooring sequence
previously shown to be essential for apoB RNA
editing(10, 11, 12) . The nucleotide sequence of
rat and chicken apoB RNA are illustrated in Fig. 4A, in
order to demonstrate these differences and also to illustrate the
mutations that were introduced into the chicken apoB cDNA fragment in
order to revert groups of nucleotides back to the rat sequence. Four
groups of mutations were tested for RNA editing and RNA binding, each
centering on the mooring sequence, for reasons detailed above. These
were (i) mooring sequence alone (M); (ii) mooring sequence and 3
nucleotides upstream (i.e. to the left (ML)); (iii) mooring
sequence and 3 nucleotides downstream (i.e. to the right
(MR)); (iv) the entire 29-nucleotide cassette, containing both upstream
and downstream alterations (MLR). Each of these mutants, along with the
wild-type chicken apoB RNA and the control rat apoB RNA, was incubated
in an in vitro RNA editing assay. As shown in Fig. 4B, the rat apoB RNA template is effectively edited in vitro (35% UAA), but of the chicken apoB RNA
templates, only the MLR mutant supports editing activity (
4% UAA).
These findings demonstrate that the entire 29-nucleotide mammalian apoB
RNA cassette is necessary for editing of a chicken apoB RNA template, a
conclusion broadly consistent with the results of other
studies(10, 11, 12) . Further studies of the
chimeric chicken/rat apoB RNA templates will be necessary to determine
the structural requirements in the regions flanking the minimal editing
cassette which restore wild type levels of RNA editing.
Figure 4:
Binding of GST/APOBEC-1 to an apoB RNA
template is distinct from editing. A, nucleotide sequence of
rat and chicken apoB RNA in the homologous region flanking the edited
base. The 11-nucleotide mooring sequence is bracketed.
Mutations were performed as detailed under ``Materials and
Methods'' to introduce clustered revertants in the chicken
template back to the rat sequence. B, in vitro RNA
editing reactions were performed on the various templates using
GST/APOBEC-1 and chicken intestinal S100 extracts (see ``Materials
and Methods''). The reaction products were resolved on an 8%
polyacrylamide gel, and the mobility of unedited (CAA) and edited
templates (UAA and UAA
) are shown by the arrows. C, EMSA was performed using the various RNA
templates and GST/APOBEC-1. The arrow shows the mobility of
the shifted band. In the right panel, various competitor RNAs
were examined for their ability to displace the rat apoB cRNA template
(RB 105). These included excess sense (S) or antisense (AS) rat apoB cRNA and excess wild-type chicken apoB cRNA (CB
150). Antisense rat apoB cRNA forms a duplex with the sense template (open arrowhead). F = free probe. D,
supershift of the GST/APOBEC-1 complexes demonstrated following the
addition of antisera as detailed in Fig. 3. The open arrow indicates the supershifted band while the closed arrow shows the remaining unshifted probe. The open arrowhead is a nonspecific band. F = free
probe.
In order to
examine the relationship of the structural constraints on apoB RNA
editing to apoB RNA binding, these templates were used in gel-shift and
super-shift assays. GST/APOBEC-1 produced an indistinguishable gel
shift with all the apoB RNA templates examined (Fig. 4C), suggesting that there is a loose stringency
for apoB RNA binding. Additionally, the gel shift with the rat apoB RNA
template was competed with either excess unlabeled rat or chicken apoB
RNA (Fig. 4C, right panel). The absence of a
shift in the presence of excess antisense rat apoB RNA suggests that
the double-stranded RNA is not a target for apobec-1 binding. Finally,
a comparable supershift was obtained using antisera to GST/APOBEC-1 in
the presence of the different RNA templates (Fig. 4D),
demonstrating that the recombinant protein binds to all the templates
examined, regardless of their intrinsic ability to undergo editing. The
features of these chicken apoB RNA templates which allow binding of
GST/APOBEC-1 have not been elucidated, but the implication arising from
the data in this study is that the AU content of the template is of
fundamental importance. In this regard, the AU content of the chicken
apoB RNA templates used in this study range from 63-67%. A
further conclusion is that the stringency required for apoB RNA binding
by apobec-1 is looser than that for RNA editing.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.