(Received for publication, February 5, 1996; and in revised form, March 19, 1996)
From the
An RNA-binding cytidine deaminase (APOBEC-1) and unidentified
auxiliary protein(s) are required for apolipoprotein (apo) B mRNA
editing. A sequence motif on apoB mRNA (``mooring sequence,''
nucleotides 6671-6681) is obligatory for the editing of cytidine
6666 (C), the only cytidine on apoB mRNA converted to
uridine in normal animals. Transgenic animals with hepatic
overexpression of APOBEC-1 develop liver tumors, and other non-apoB
mRNAs are edited, suggesting a loss of the normally precise
specificity. In this study, we examined apoB mRNA from these transgenic
animals to determine if cytidines aside from C
are
edited. Multiple cytidines downstream from C
in apoB
mRNA were edited extensively by the overexpressed APOBEC-1. This
pathophysiological ``hyperediting'' could be mimicked in
vitro by incubating a synthetic apoB RNA substrate with the
transgenic mouse liver extracts. Multiple cytidines in the synthetic
apoB RNA were edited by recombinant APOBEC-1 but only with
supplementation of the auxiliary protein(s). Mutations in the mooring
sequence markedly decreased the normal editing of C
but,
surprisingly, increased the hyperediting of downstream cytidines.
Furthermore, cytidines in an apoB RNA substrate lacking the mooring
sequence were also edited in vitro. These results indicate
that the hyperediting of apoB mRNA by overexpressed APOBEC-1 depends
upon auxiliary protein(s) but is independent of the mooring sequence
motif. These results suggest that hyperediting may represent the first
step in a two-step recognition model for normal apoB mRNA editing.
Apolipoprotein (apo) ()B48, the amino-terminal 2152
amino acids of apoB100, is synthesized by a novel post-transcriptional
modification of mRNA, designated ``mRNA
editing''(1, 2, 3) . The editing process
deaminates a specific cytidine, nucleotide (nt) 6666, to form
uridine(4, 5) . This deamination changes the codon at
position 2153 from a genomically encoded glutamine (CAA) to an in-frame
stop codon (UAA)(6) . Apolipoprotein B mRNA editing occurs in
the small intestine of all mammals and also in the liver of rats, mice,
dogs, and horses(7) .
A protein that catalyzes the editing reaction has been cloned(8) . This protein, designated ``APOBEC-1'' (apoB mRNA-editing enzyme catalytic polypeptide 1)(9) , is the catalytic subunit of the apoB mRNA-editing complex that deaminates nt 6666 in apoB mRNA(5) . APOBEC-1 is capable of binding to RNA (10, 11) but requires unidentified complementary or auxiliary protein(s), which apparently are part of a multicomponent editing complex, in order to edit apoB RNA (8, 12) . These auxiliary protein(s) have a widespread distribution(12, 13) , have been shown to bind to APOBEC-1, and may bind to apoB mRNA (14) .
Both in vitro and in vivo, cytidine 6666 (C) in apoB
mRNA is deaminated with extraordinary precision. Moreover, apoB mRNA is
the only known substrate for the apoB-editing enzyme in normal animals.
The sequence elements in the apoB mRNA necessary for this high
specificity have been identified by site-directed
mutagenesis(15, 16, 17, 18, 19) .
Mutation of any of 10 of the 11 nucleotides in an 11-nt region (nt
6671-6681) of the apoB mRNA either abolished or greatly reduced in vitro apoB mRNA editing, indicating that mRNA editing is
dependent upon this sequence element (15, 18, 19) (i.e. the
``mooring sequence''). In addition, this mooring sequence
(recognition sequence) is sufficient to promote editing of an
immediately upstream cytidine at a reduced efficiency in vitro or in cultured cells when it is inserted into a heterologous gene (e.g. albumin or luciferase) or heterologous sites in apoB
mRNA(17, 19) .
Recently, we have demonstrated that
APOBEC-1 overexpressed in the livers of transgenic mice leads to
hepatocellular dysplasia and carcinoma(20) . The C in apoB mRNA was virtually completely edited in the livers of
these transgenic animals. Other hepatic mRNAs with sequence motifs
similar to that of the mooring sequence were examined in these animals
for cytidine deamination editing. The mRNA of tyrosine kinase was found
to be edited in the transgenic mouse livers but not in those of the
control mice. This mRNA has a sequence motif that differs by 1
nucleotide from the mooring sequence. Surprisingly, two other mRNAs
containing the exact mooring sequence were not edited at cytidines
4-6 nucleotides upstream from this sequence(20) . These
results demonstrated that the mooring sequence is not sufficient for
the APOBEC-1-mediated editing and that other sequence and/or structure
element(s) are also required, even in the transgenic animals
overexpressing APOBEC-1.
In this study, we considered the
possibility that other sites in apoB mRNA might be edited when APOBEC-1
is overexpressed in transgenic animal livers and that the analysis of
the pattern of editing could give us additional insights into the
sequence requirements for apoB mRNA editing. We found that apoB mRNA
was edited at multiple sites downstream from C.
Moreover, the auxiliary protein(s), but not the mooring sequence, were
necessary for this ``hyperediting.'' Based on these results,
we propose a two-step recognition model for normal apoB mRNA editing.
Rabbit APOBEC-1 was overexpressed in transgenic mice and
rabbits, predominantly in the liver(20) . The purpose of the
present study was to determine the effects of APOBEC-1 overexpression
on apoB mRNA isolated from the livers of transgenic mice or rabbits.
RNA from the APOBEC-1 transgenic animal livers and nontransgenic normal
mouse and rabbit livers was amplified using RT-PCR of apoB mRNA in the
vicinity of the normal editing site (C). In addition,
human apoB mRNA was amplified by RT-PCR from transgenic mouse livers
expressing human apoB (21) and double transgenic mouse livers
expressing both human apoB and rabbit APOBEC-1. Previously, it was
shown that, in transgenic mice expressing human apoB in their liver,
human apoB mRNA was edited at the correct site (C
) and
to the same extent as mouse apoB mRNA(25) . The PCR products
were subcloned into pCRII vectors, and multiple clones were sequenced
from each PCR product to look for additional editing sites.
As
expected, apoB mRNA from normal rabbit liver was not edited (data not
shown). The mouse apoB transcript from the normal mice and the human
apoB transcript from the transgenic mice expressing only human apoB
mRNA, but not rabbit APOBEC-1, were edited at the canonical site (nt
6666). In addition, C in the human transcript was
slightly edited. No other cytidines were edited in control animals.
In the transgenic animals expressing rabbit APOBEC-1, the hepatic
apoB mRNAs of all three species were extensively edited at multiple
sites (Fig. 1). We have designated this type of RNA editing
``apoB mRNA hyperediting'' to differentiate it from normal
apoB mRNA editing. As illustrated by four randomly chosen clones, the
pattern of editing appears to be stochastic (Fig. 1A).
There was no apparent 5` to 3` or 3` to 5` preference in the
distribution of the edited sites. There were two major and one minor
clusters of edited cytidines in the apoB mRNAs. One major region of
edited cytidines was C-C
. The other
was C
-C
. The minor cluster of
edited cytidines was in the region of C
-C
(Fig. 1B).
Figure 1:
Hyperediting of apoB mRNA in apoB
mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1) transgenic
animal livers as detected by sequence analysis. An approximately
350-base pair fragment of apoB mRNA was amplified by RT-PCR, subcloned
into pCRII vectors, and sequenced. By comparing these cDNA sequences
with the genomic sequence, edited cytidines were determined. A, edited cytidines detected in four random cDNA clones of
apoB from transgenic mouse livers are indicated by solid circles (). B, the percentage of edited clones for each
cytidine from nucleotides (nt) 6543 to 6851 is shown by shaded
bars. Mouse apoB represents the mouse sequence from an APOBEC-1
transgenic mouse (n = 25); rabbit apoB, the rabbit
sequence from an APOBEC-1 transgenic rabbit (n = 18);
human apoB, the human sequence from a double transgenic mouse
expressing both human apoB and rabbit APOBEC-1 (n =
10). No editing was detected in the upstream region from nt 6504 to
6642. Cytidines whose editing in the nt 6643-6851 region was not
detected by sequence analyses are indicated by asterisks.
Many of the sites shown by
sequencing to be edited also were examined by primer extension analysis
with three different primers (Fig. 2). One primer (M52)
demonstrated that C and C
were edited in
APOBEC-1 transgenic mice (Fig. 2) and rabbit (data not shown)
but not in control animals. The band corresponding to the editing of
C
is much stronger than that corresponding to the
editing of C
. This suggests that these two cytidines are
edited simultaneously in most transcripts, which is consistent with the
results of the sequence analyses. The second primer (M53) verified that
C
, C
, C
, and
C
were edited in the transgenic mice but not in the
nontransgenic mice. Another primer extension analysis with the third
primer (M54) confirmed that C
was deaminated in the apoB
mRNA from the APOBEC-1 transgenic mice but not in the control mice (Fig. 2).
Figure 2:
Primer extension analysis showing the
editing of multiple cytidines of apoB mRNA in APOBEC-1 transgenic mouse
livers. A fragment of mouse apoB mRNA was amplified by RT-PCR from
control and APOBEC-1 transgenic mouse livers and analyzed by primer
extension analyses with three different primers. The first primer (M54) detected the editing of C in transgenic
mouse livers. The second one (M53) detected the editing of
C
, C
, C
, and
C
. The third one (M52) revealed the editing of
C
and C
.
Three findings are noteworthy and differentiate
apoB mRNA hyperediting from normal apoB mRNA editing. First,
C, which is in the center of the mooring sequence, was
edited in transcripts from all three species in APOBEC-1 transgenic
animals. Previously, Shah et al.(18) demonstrated
that cytidine at this site was crucial for normal apoB mRNA editing in vitro since the mutation of this residue to guanidine
abolished editing at C
. However, our results suggest
that this nucleotide is not critical for the hyperediting of apoB RNA.
The second surprising finding was that most of the other alternate
editing sites were downstream from the mooring sequence. Only a few
cytidines were found by sequence analyses to be edited at low frequency
(less than 10%) in the upstream region we examined (nt
6504-6665). Based on numerous in vitro studies, the
accepted working model for apoB mRNA editing predicts that a protein or
proteins (auxiliary protein(s)) bind to the mooring sequence and direct
editing immediately upstream from the mooring sequence
element(26, 27) . The third finding is that apoB RNA
hyperediting does not deaminate random cytidines but prefers cytidines
surrounded by thymidine or adenosine. In the three apoB mRNAs examined,
a total of 40 different cytidines were edited. The nucleotide
immediately upstream from the editing site was either thymidine (70%)
or adenosine (30%). The nucleotide immediately downstream usually was
adenosine (58%) or thymidine (27%) and, less frequently, cytidine (10%)
or guanosine (5%). This same pattern of base preference also was found
in another mRNA that we found to be edited at multiple sites. (
)Thus, when APOBEC-1 is overexpressed in vivo, there is a striking ``nearest neighbor'' A or T
preference, at least for apoB mRNA hyperediting. In contrast, Chen et al.(16) concluded that the bases in the immediate
vicinity of the editing site are unimportant for normal apoB mRNA
editing. In their study, 22 different mutants were created in which the
bases immediately flanking C
in apoB mRNA were altered.
Twenty of these mutants were edited at C
in
vitro. The conclusion from this study was that, in the immediate
vicinity of the editing site at C
, there is a relatively
lax sequence specificity for apoB mRNA editing. One of the two mutants
not edited had thymidine immediately before and after the edited
cytidine(16) . However, in apoB mRNA hyperediting, several
cytidines that were highly edited in apoB mRNA were flanked by
thymidines.
We previously have examined whether the apoB
mRNA-editing enzyme can edit other mRNAs with the same mooring sequence (i.e. fatty acid synthase and P1 protein) and mRNAs with
motifs that differ from the apoB mooring sequence by a single
nucleotide (i.e. prostaglandin synthase homologue and tyrosine
kinase)(20) . These mRNAs have cytidines 4-6 nt upstream
from the mooring-like sequence motifs, which were not edited in the
normal mouse liver. Only the cytidine upstream from the mooring-like
sequence of tyrosine kinase was slightly edited (1%) in transgenic
mice overexpressing APOBEC-1(20) . However, the hyperediting of
apoB mRNA by overexpressed APOBEC-1 raised the possibility that other
cytidines in these mRNAs, such as those downstream from the
mooring-like sequences, might be edited in APOBEC-1 transgenic animals.
To examine this possibility, 250-300 base pair fragments around
the mooring-like sequences of fatty acid synthase, P1 protein, and
tyrosine kinase were amplified by RT-PCR from APOBEC-1 transgenic mouse
livers. The PCR products were subcloned, and 10 subclones of each mRNA
(cDNA) were sequenced. In contrast to apoB mRNA, no cytidine at any
position was edited in these mRNAs (data not shown), suggesting that
sequence and/or structure element(s) other than the mooring sequence
are required for both normal and hyperediting.
Normal apoB mRNA
editing requires APOBEC-1, auxiliary protein(s), and the mooring
sequence (wild-type: TGATCAGTATA) of apoB. Previously, we demonstrated
that an apoB RNA with a double mutation in the mooring sequence (mutant
118: TGgTCACTtTA) was edited at only 11% of the level of that of the
wild-type apoB RNA(19) . The mutant 124 (gGATgAGaATA), with a
triple mutation in the mooring sequence, was not edited in vitro by a rat enterocyte extract(19) . By adjusting the
conditions described under ``Materials and Methods,'' we were
able to achieve apoB RNA hyperediting in vitro and were
therefore able to determine if hyperediting is dependent upon auxiliary
protein(s) and the mooring sequence of apoB. As shown in Fig. 3A, liver extract from a normal mouse edited 26
± 6% (n = 5) of the C of
wild-type RNA substrate but failed to edit the C
of apoB
RNA with mutations in the mooring sequence (Fig. 3A,
mutants 118 and 124). Liver extract from a transgenic mouse
overexpressing rabbit APOBEC-1 edited 70 ± 18% (n = 5) of C
from wild-type apoB RNA, which was
reduced to 38 ± 18% (n = 5) and 22 ± 15% (n = 5) for RNA mutants 118 and 124, respectively. As
expected, the maltose-binding protein (MBP)-APOBEC-1 fusion protein
expressed in Escherichia coli was unable to edit any of the
RNAs (Fig. 3A). With the addition of rabbit liver
extract that contains auxiliary protein(s), the C
on the
wild-type apoB RNA was edited 71 ± 13% (n = 5),
with reduced editing (33 ± 7%; n = 5) for the
mutant 118 with a double mutation and further reduction (16 ±
6%; n = 5) for the mutant 124 with a triple mutation in
the mooring sequence. The apoB RNA hyperediting of C
and
C
demonstrated a different pattern (Fig. 3B). The control mouse liver extract edited
little, if any, C
and C
in the RNA
substrates. However, the liver extract from the APOBEC-1 transgenic
mice modestly edited C
and C
in the
wild-type apoB RNA substrates (4 ± 2%; n = 5).
Surprisingly, the editing of C
and C
by
the transgenic mouse liver extracts was enhanced in the mutant 118 (11
± 8%; n = 5) and in the mutant 124 (11 ±
6%; n = 5). Likewise, compared to the wild-type
substrate (15 ± 5%; n = 5), C
and
C
were more efficiently edited in mutant 118 (19
± 5%; n = 5) and mutant 124 (17 ± 4%; n = 5) by the MBP-APOBEC-1, but only with the addition
of the auxiliary protein(s) from rabbit liver extracts. Thus, apoB RNA
hyperediting is dependent upon auxiliary protein(s) but independent of
the canonical mooring sequence.
Figure 3:
Effects of mutations in the mooring
sequence on normal editing (C) and hyperediting
(C
and C
) of apoB mRNA in vitro. A and B, synthetic baboon apoB RNA (280 base pairs)
of either a wild-type (wt), mutant 118, or mutant 124 was
incubated with either 100 µg of normal mouse liver extract (control liver), 100 µg of APOBEC-1 transgenic mouse liver
extract (APOBEC-1 transgenic liver), 5 µg of a fusion
protein consisting of maltose-binding protein and APOBEC-1 (APOBEC-1), or 5 µg of MBP-APOBEC-1 + 100 µg of
normal rabbit liver extract containing the auxiliary protein(s) (APOBEC-1 + auxiliary protein(s)) at 30 °C for 16 h.
The editing of C
(A) or C
and
C
(B) was determined by primer extension
analyses.
To further confirm that hyperediting
is independent of the mooring sequence, we synthesized a shorter apoB
RNA substrate (nt 6687-6824 of rabbit apoB mRNA) lacking the
mooring sequence. When incubated with the recombinant MBP-APOBEC-1 and
the auxiliary protein(s) in vitro, 12.5% of C of this substrate RNA was edited (data not shown), demonstrating
that hyperediting does not require the mooring sequence, at least for
C
.
That the hyperediting is independent of the
mooring sequence was further supported by experiments using mutant apoB
RNA into which a second mooring sequence was introduced upstream at nt
6597 (mutant SR2). Synthetic RNA of wild-type baboon apoB and the
mutant SR2 were incubated with MBP-APOBEC-1 and rabbit liver extract
containing the auxiliary protein(s) in vitro, and editing was
determined by primer extension analysis. As reported
previously(19) , the introduction of the second mooring
sequence led to the editing of C, which was immediately
upstream from the second mooring sequence (data not shown). In
contrast, C
, C
, and C
,
which were downstream from the second mooring sequence, were not edited
in either wild-type or SR2 RNA, showing that the introduction of the
second mooring sequence did not lead to the hyperediting. These results
are consistent with the concept that hyperediting is independent of the
mooring sequence.
Another finding from the in vitro study
is that the editing of C is much more efficient than the
hyperediting of C
. We studied the time-course of
the editing and hyperediting by performing in vitro assays for
2-16 h (Fig. 4). The hyperediting of C
was detected after 2 h (4%) and gradually increased up to 16 h
(23%). In contrast, C
was edited 65% at 2 h and 81% at 4
h. There was no further increase in the editing of C
after 4 h. Thus, the editing of C
is more
efficient and rapid than the hyperediting.
Figure 4:
Time-course curve of the editing of
C and C
in vitro. The
baboon synthetic apoB RNA was incubated with 5 µg of MBP-APOBEC-1
and 100 µg of rabbit liver extract containing the auxiliary
protein(s) for 2-16 h, and editing of C
(
)
and C
(
) was determined by primer extension
analyses.
A model for the mechanism of apoB RNA hyperediting can be derived based on our knowledge of normal apoB mRNA editing and the results of this study. The apoB mRNA-editing complex consists of the catalytic cytidine deaminase subunit (APOBEC-1) and auxiliary protein(s). The auxiliary protein(s) bind to APOBEC-1 and, most likely, bind to the apoB mRNA. In normal animal livers, apoB mRNA editing requires both the mooring sequence and other mooring-independent element(s). When APOBEC-1 is overexpressed in transgenic animal livers, the hyperediting does not require the canonical mooring sequence, and the editing occurs at multiple sites.
We have formulated a plausible two-step recognition model to explain normal editing and hyperediting of apoB mRNA. The first step is the relatively loose recognition of the apoB mRNA sequence by either the auxiliary protein(s), APOBEC-1, or a complex of APOBEC-1 and the auxiliary protein(s). This step does not require the specific mooring sequence and is probably dependent upon unidentified sequence and/or structure element(s). Our hypothesis is that this first recognition step permits the apoB mRNA-editing complex to recognize a relatively short segment of apoB mRNA from tens of thousands of different transcripts. Transcripts that lack the unidentified element, such as P1 protein and fatty acid synthase, are not edited at canonical or multiple sites even though these mRNAs contain the exact mooring sequence.
The second step of normal apoB mRNA editing requires the
specific mooring sequence that ``anchors'' the editing
complex at the correct site on apoB for the deamination of
C. In normal animals, where APOBEC-1 exists at
relatively low levels, the first step alone is not sufficient to
support editing; without the canonical mooring sequence, normally there
is insufficient editing complex concentrated at any one site to support
the deamination reaction. However, when APOBEC-1 is overexpressed,
sufficient editing complex is concentrated by the first recognition
step on a short fragment of apoB mRNA to permit the hyperediting of
multiple cytidines. The hyperediting seems to be independent of the
second step. At the least, it does not require the specific mooring
sequence, which is essential for normal editing of C
.
That the mutants in the mooring sequence enhanced the hyperediting is
consistent with our two-step model. When the mooring sequence is
mutated, the binding equilibrium of the editing complex naturally
favoring the C
is disrupted, allowing the less favorable
interaction with downstream sites to occur more often.
If both the first (mooring-independent) step and the second (mooring-dependent) step are required for normal editing, why was the mooring sequence sufficient to elicit editing when inserted into heterologous RNAs such as luciferase (19) and albumin(17) ? These RNAs probably contain the sequence and/or structure element(s) necessary in the first step. When the mooring sequence was inserted into apoE RNA, it failed to support editing(28) . Apparently, apoE RNA lacks the element(s) necessary in the first step.
What are the element(s) required for the first step? One may be an AU-rich sequence element, as suggested by Backus et al.(17) , who showed that a 3` AU-rich sequence flanking the mooring sequence was important for efficient editing. The fragment of mouse apoB mRNA we amplified by RT-PCR contains 69.7% AU. In contrast, the mRNAs of P1 protein and fatty acid synthase contain only 47.7 and 41.9% AU, respectively. Furthermore, APOBEC-1 has been shown to bind to AU-rich mRNA(10, 11) . However, the first-step recognition sequence element is more complex than simply the AU content of the mRNA, since the 3`-untranslated region of N-myc RNA is not edited in the APOBEC-1 transgenic animal livers (data not shown) despite the fact that this region contains 68.5% AU and a sequence motif only two nucleotides different from that of the mooring sequence.
In conclusion, we have reported that overexpressed APOBEC-1 edits non-random multiple cytidines of apoB mRNA in transgenic animals. The hyperediting is dependent upon auxiliary protein(s) but independent of the specific mooring sequence. We also have proposed a two-step recognition model for normal apoB mRNA editing that consists of an initial mooring sequence-independent step and a second, mooring sequence-dependent step. When APOBEC-1 is overexpressed in the liver, the first step becomes sufficient to support the editing of multiple cytidines on certain RNA transcripts. This may play an important role in the development of hepatocellular carcinoma in these animals. If this hypothesis is correct, the elucidation of the sequence and/or structure element(s) that support hyperediting should provide insight into the first critical step in the mechanism of apoB mRNA editing.