(Received for publication, September 6, 1995; and in revised form, November 21, 1995)
From the
Apolipoprotein B (apoB) RNA editing involves site-specific deamination of a cytidine to a uridine. A mooring sequence, a spacer region, and a regulator region are components of the apoB RNA editing motif of which only the mooring sequence is both necessary and sufficient for editosome assembly and editing. The catalytic component of the editosome is APOBEC-1. In rat hepatoma, stable cell lines, overexpression of APOBEC-1 resulted in 3-6-fold stimulation of the editing efficiency on either rat endogenous apoB RNA or transiently expressed human apoB RNA. In these cell lines, cytidines in addition to the one at the wild type site were edited. The occurrence and efficiency of this ``promiscuous'' editing increased with increasing expression of APOBEC-1. Promiscuous editing was restricted to cytidines 5` of the mooring sequence and only occurred on RNAs that had been edited at the wild type site. Moreover, RNAs with mutant editing motifs supported high efficiency but low fidelity editing in the presence of high levels of APOBEC-1. This study demonstrates that overexpression of APOBEC-1 can increase the efficiency of site-specific editing but can also result in promiscuous editing.
Apolipoprotein B (apoB) ()RNA editing (1, 2) is a post-transcriptional(3) ,
site-specific deamination of a cytidine residue to a uridine. This
nucleotide transition (at nt 6666, C
) converts a
glutamine codon (CAA) at amino acid position 2153 to an in-frame STOP
codon (UAA), which results in the translation of a truncated apoB48
protein (reviewed in (4) ). An 11-nucleotide (UGAUCAGUAUA)
mooring sequence (5, 6, 7) is both necessary
and sufficient for site-specific editing in a variety of RNA
backgrounds(6, 8, 9, 10) . A
macromolecular complex or ``editosome'' specifically
assembles upon the mooring sequence (11) and directs editing of
cytidine residues appropriately located in only the 5`
direction(10) . Between the mooring sequence and C
is a region of lax sequence specificity (12, 13, 14) but whose appropriate length is
critical for efficient site-specific editing(12) . Immediately
5` of C
exists a regulator element (UGAUA). Enhancement
of RNA editing efficiency by this element does not require a specific
sequence, although TA immediately adjacent to C
is most
effective(12) .
The deamination reaction involved in apoB RNA editing is a zinc-dependent process mediated by APOBEC-1(15, 16, 17, 18) . This protein has extensive sequence homology to other cytidine deaminases from Escherichia coli and mammals(4, 15, 19) , especially within the zinc coordination domain wherein mutations abolish editing activity(16, 20, 21) . The enzyme stimulates editing activity in vitro when complemented with suitable cell extracts(16) , provides editing activity to human liver cell lines (HepG2) in vitro(22) , and enhances apoB mRNA editing in mice when expressed by adenovirus-mediated gene transfer(23) . The efficiency of apoB RNA editing is an important determinant in the proportion of full-length (apoB100) or truncated (apoB48) protein variants assembled as triglyceride-rich lipoprotein particles(24, 25) .
APOBEC-1 has a weak
and non-sequence-specific binding affinity for RNA (26, 27) . The site-specific RNA editing activity of
APOBEC-1 is absolutely dependent upon its assembly with one or more
auxiliary proteins (15, 16, 18) as an
editosome(11) . Proteins with molecular masses of 66 and 44 kDa
have been identified by ultraviolet light protein-RNA cross-linking as
the presumptive factors for mooring sequence-specific RNA
binding(9, 11, 28) . Extracts from a variety
of tissues, obtained from a number of species can complement APOBEC-1
in RNA editing activity and therefore must contain auxiliary
factors(15, 16, 18, 20) .
Complementation of APOBEC-1 by these factors is not dependent upon
whether the sources of the extract have the ability to either
transcribe or edit apoB RNA. In fact, editing of transfected apoB RNAs
can occur in cell lines that otherwise do not express apoB
RNA(29) . The data suggest that the capacity to edit RNA may be
a more general phenomenon and that there may be RNAs other than apoB
that could support C U RNA editing.
We have investigated the
effect of APOBEC-1 overexpression on editing site specificity in rat
liver cells. High levels of enzyme expression resulted in increased
efficiency of apoB RNA editing at the wild type site, C.
However, a mooring sequence-dependent phenomenon was observed in which
multiple additional cytidines per RNA substrate were edited. Additional
site editing was a direct consequence of overexpressing APOBEC-1. The
data suggest that site-specific editing cannot be maintained if editing
is up-regulated by only increasing the abundance of APOBEC-1. Moreover,
mutant editing sites, which otherwise edited inefficiently, supported
high efficiency but low fidelity RNA editing under high levels of
APOBEC-1 expression. We propose that regulation of site-specific
editing efficiency in tissues must involve coordinate expression of
APOBEC-1 and auxiliary factors such that the relative abundance of
APOBEC-1 is maintained as rate-limiting.
Editing
was evaluated by the poisoned primer extension analysis described
previously (12) using [-
P] ATP
(6000 mCi/mmol; DuPont NEN) end-labeled DD3 to prime both rat and human
apoB products(31) . Primer extension products were resolved on
a 10% denaturing polyacrylamide gel and quantified by laser
densitometric scanning (PhosphorImager model 425E, Molecular Dynamics).
The editing assay was linear between 50 pg and 10 ng of input 207-base
pair rat apoB PCR substrate. Quantification was performed in triplicate
upon a titration of input PCR products between 0.5 and 10 ng.
McArdle RH7777, a rat hepatoma cell line, was selected for this study of apobec-1 cDNA overexpression as these cells transcribe apoB RNA and edit it with relatively low efficiency(32) . Clonal McArdle stable cell lines were selected under G418 in which apobec-1 mRNA overexpression was directed from the human cytomegalovirus promoter. APOBEC-1 expression in the McArdle stable cell lines was confirmed by Western blotting, and the levels in the cell line with the highest editing efficiency were determined to be at least 8-fold greater than that in the null vector transfected ``control'' McArdle cell line (Fig. 1).
Figure 1:
Overexpression
of APOBEC-1 by apobec-1 cDNA transfection. Approximately 40
µg of total cellular protein from null vector transfected, control
McArdle (1) and ``high'' APOBEC-1-expressing (2) cell lines were Western blotted and reacted with an
anti-APOBEC-1 antibody (see ``Experimental Procedures''), and
APOBEC-1 specific proteins were visualized using I-labeled affinity-purified Protein A. Mr,
migration of molecular weight standard proteins; arrowhead indicates APOBEC-1-specific
immunoreactivity.
The effect of APOBEC-1 overexpression on the editing efficiency of
the endogenous rat apoB RNA was determined in triplicate, in two
independently isolated total cellular RNA populations, from each of 12
independent cell lines (see ``Experimental Procedures'').
Poisoned primer extension analysis of RT-PCR products specific to
endogenous rat apoB RNA demonstrated that the control cell line edited
the wild type site (cytidine at nt position 6666 (C))
with 12% efficiency (n = 8; S.E. = 0.03) (Fig. 2, lane 5). The editing efficiency of C
in three cell lines expressing low, medium, or high levels of
APOBEC-1 was 20% (n = 8; S.E. = 1.0), 55% (n = 8, S.E. = 1.8), and 84% (n = 8,
S.E. = 2.2), respectively (lanes 6-8). The
correlation between increased APOBEC-1 expression and an increase in
RNA editing efficiency corroborates previous data from other
systems(20, 22, 23, 33, 34) .
Figure 2: Additional editing site utilization in cells overexpressing APOBEC-1. Poisoned primer extension analyses were performed upon RT-PCR-amplified apoB DNA templates as described under ``Experimental Procedures.'' Exogenous refers to the transfected human apoB RNA substrate; endogenous refers to the rat apoB RNA substrate. The positions of the primer and the extension products generated from unedited (CAA) and edited (UAA) RNAs are indicated. The length of these products is dependent upon the sequence 5` of the first edited C in the RNA substrate and is therefore different between the rat and human substrates. Longer primer extension products indicative of additional editing site utilization are indicated at 2, 3, and 4.
The poisoned primer extension assay also suggested the presence of
additional C U editing events in the apobec-1 cDNA
transfected cell lines as indicated by additional primer extension
products above the UAA product (indicated as 2 and 3). The length of these products corresponded to editing at
C
and C
. In the low, medium, and high
APOBEC-1 overexpressing cell lines, the combined editing efficiency of
the two additional sites relative to that at C
was 0.9%
(S.E. = 0.1), 4.4% (S.E. = 0.2), and 7.1% (S.E. =
0.3), respectively (n = 7, for each cell line). The
increased editing efficiency at these additional sites correlates with
the increased levels of APOBEC-1 expression, suggesting that their
utilization is dependent on APOBEC-1 abundance (compare lanes
5-8).
Additional site editing was also observed on an
exogenous 448-nt human apoB RNA substrate (wt) transiently transfected
into control McArdle cells and into each of the APOBEC-1 overexpressing
cell lines (Fig. 2, Exogenous substrate). Editing
efficiency at C was 21% (S.E. = 1.1), 30% (S.E.
= 0.8), 55% (S.E. = 1.4), and 85% (S.E. = 2.7) in
the control and low, medium, and high APOBEC-1 overexpressing cell
lines, respectively (n = 6, for each cell line) (Fig. 2, lanes 1-4). Additional primer extension
products (indicated as 2, 3, and 4) were
observed, corresponding to C
U transitions at C
,
C
, and C
, respectively, in the human apoB
RNA. Relative to C
editing in the control and low,
medium, and high APOBEC-1 overexpressing cell lines, the combined
editing efficiencies at these sites were 0.3% (S.E. = 0.1), 6%
(S.E. = 0.5), 17% (S.E. = 0.8), and 42% (S.E. =
2.6), respectively (n = 4, for each cell line). These
data also suggested that the frequency of additional site editing was
dependent on APOBEC-1 abundance.
To evaluate whether the additional
C U transitions were due to RNA editing or inaccuracies in the
assay system, poisoned primer extension analysis was performed on
RT-PCR products amplified from a mixture of in vitro transcribed, unedited and edited apoB RNA substrates. Primer
extension beyond C
(to the additional sites) on RNA
edited only at C
occurred with a frequency of less than
0.2% (n = 6). The proportion of read-through products
was not altered at either extreme low or high concentrations of input
PCR product. This indicates a low but measurable error frequency due to
a nucleotide incorporation error during RT-PCR or primer extension and
may have produced the additional primer extension product (2)
on the control McArdle RT-PCR products (Fig. 2, lanes 1 and 5).
Another limitation of the poisoned primer
extension assay is that it would not be suitable for evaluating
additional site editing on RNAs that where unedited at C due to chain termination at this site. To confirm that the
additional primer extension products were C
U editing events and
not artifacts of the editing assay, the apoB RNA RT-PCR products used
in the primer extension analyses described above were cloned, and
independent isolates were sequenced.
Fig. 3shows the C and T
sequence between nt 6573 and nt 6719 of representative clones of the
transiently transfected exogenous human apoB RNA substrates isolated
from the highest stable APOBEC-1-expressing (A) and control
McArdle (B) cell lines. C U editing at C
was observed in 18 of the 21 exogenous human apoB clones from the
highest APOBEC-1-expressing cell line. This was equivalent to the 85%
C
specific editing efficiency determined by poisoned
primer extension assay (Fig. 2, lane 4). Of the 18
exogenous RNAs that were edited at C
in the highest
expressing APOBEC-1 line, 8 were edited at additional sites 5` of nt
6666 involving a total of 15 C
U transitions. Representative
clones highlighting the nature and extent of additional editing site
utilization are shown (Fig. 3A, lanes
3-8). Lanes 3-6 each demonstrated a single
and different additional editing event at C
,
C
, C
, and C
, respectively. Lanes 7 and 8 are representative of five isolates,
which demonstrated that a single RNA could be additionally edited at
multiple sites involving a wide array of cytidines. Additional
cytidines that were edited included C
, C
,
C
, and C
. Depending on the clone, one or
more cytidines between C
and the additional edited
base(s) appeared to remain unedited, e.g. C
,
C
, C
, and C
. No cytidine
to uridine editing was observed 3` of C
, and no clones
were isolated in which additional site C
U editing had occurred
upon an RNA that was not edited at C
. These data suggest
that additional site editing was mooring sequence-dependent and,
consistent with this model(7, 10) , only cytidines 5`
of the mooring sequence were edited.
Figure 3:
Confirmation of additional editing site
utilization on human apoB RNA by RT-PCR product sequencing. A,
APOBEC-1-overexpressing McArdle cell line; B, null
vector-transfected, control McArdle cell line. The human apoB sequence
is given (as C and T only), and site-specific (C) and
additional editing sites are indicated with an arrowhead. Only
the C and T lanes are shown for clarity. Nucleotide coordinates are
from the published human sequence (39) .
Only 4 of 23 exogenous clones
isolated from the control McArdle cell line demonstrated C U
editing at C
by direct DNA sequencing (Fig. 3B) This was equivalent to an editing efficiency
of 17%, comparable with that determined by the poisoned primer
extension assay (Fig. 2, lane 1). None of the 23 clones
showed additional site editing, supporting the possibility that the
additional primer extension product seen on apoB RNA from control
McArdle cells was due to an assay system read-through error.
Importantly, however, these control data underscore the importance of
APOBEC-1 overexpression in additional site editing.
Fig. 4shows the C and T sequence between nt 6572 and nt 6683
of representative clones of endogenous rat apoB RNA substrates isolated
from the highest APOBEC-1-expressing (lanes 1-3) and
control McArdle (lanes 4 and 5) cell lines. C
U editing at C
was observed in 18 of 22 endogenous rat
apoB clones isolated from the highest APOBEC-1-expressing cell line.
This was equivalent to the 82% C
specific editing
efficiency determined by poisoned primer extension assay (Fig. 2, lane 8). Two of the 18 clones edited at
C
were additionally edited at C
,
C
and C
for a total of four C
U
transitions (Fig. 4, lanes 2 and 3). Lane
1 shows the sequence of an isolate only edited at
C
. Similar to the exogenous substrates isolated from
this cell line, additional editing site utilization was only observed
upon RNAs that were edited at C
, and no C
U
transitions were observed 3` of the mooring sequence. Fig. 4, lanes 4 and 5, show representative endogenous
sequences from 24 clones isolated from the control McArdle cell line. Lane 4 is an unedited isolate, and lane 5 is
representative of the four C
edited clones. None of
these four clones showed editing at additional sites, consistent with
the role of APOBEC-1 overexpression in this process.
Figure 4:
Confirmation of additional editing site
utilization on rat apoB RNA by RT-PCR product sequencing. Lanes
1-3, APOBEC-1-overexpressing McArdle cell line; lanes 4 and 5, control McArdle cell line. The rat apoB sequence
is given (as C and T only), and site-specific (C) and
additional editing sites are indicated with an arrowhead. Only
the C and T lanes are shown for clarity. Nucleotide coordinates are
from the published rat sequence using the wild type editing site
(C
) as an alignment point (40) .
The disparate
spacing of additional editing events relative to the mooring sequence
suggests that the constraints on the spacer (and perhaps regulator)
elements of the tripartite editing motif had become lax in cells
expressing high levels of APOBEC-1. Previous studies have shown that a
mutant human apoB RNA substrate (TL), which contains a perfect mooring
sequence but a five-base pair spacer element (compared with four in the
wt) and no 5` regulator element(6, 12) , supported 3%
editing in transfected McArdle cells ()and less than 7% of
wild type levels in vitro(6, 12) . We
therefore chose to examine the potential of this mutant RNA substrate
to support additional site editing in the highest APOBEC-1-expressing
cell line. ApoB RNAs from duplicate transient transfections of the wt
(positive control) and TL expression constructs were analyzed by
poisoned primer extension assay as described above. Remarkably, greater
than 60% editing efficiency (n = 6, S.E. = 2.2)
was observed at the primary editing site
(C
(6, 12) ) compared with 67% (n = 3, S.E. = 1.4) at C
observed on the
wt substrate (Fig. 5, lanes 2 and 1,
respectively). Additional primer extension products were also observed
on the wt and TL RNA substrates at a combined efficiency of 23% (n = 4, S.E. = 4.0) and 26% (n = 4,
S.E. = 1.2) relative to that at C
and
C
, respectively. These products corresponded to editing
of additional 5`-located cytidines. These results corroborate those in Fig. 2and extend them to further highlight that additional
editing site utilization is not constrained by the length of the spacer
element and that the five-nt (UGAUA) regulator element is unnecessary
for high levels of editing of RNA substrates in high level
APOBEC-1-expressing cell lines.
Figure 5: Overexpression of APOBEC-1 overcomes the constraints of the spacer and regulator elements. The control wild type human apoB (wt) and the mutant substrate with imperfect spacer and regulator elements (TL) were independently, transiently transfected into the high APOBEC-1-expressing cell line. Poisoned primer extension analyses were performed as described in Fig. 2. The positions of the primer and the extension products generated from unedited (CAA) and edited (UAA) RNAs are indicated. Longer primer extension products indicative of additional editing site utilization are indicated (2, 3, and 4), their length being dependent upon RNA transcript sequence.
This study has revealed C U editing at sites other
than C
whose efficiency of utilization was dependent
upon APOBEC-1 abundance. These sites were not utilized in normal
tissues or cells but were unique to cells that overexpress APOBEC-1
following transfection with apobec-1 cDNA. We therefore
consider this activity as ``promiscuous editing.'' The data
also demonstrated that promiscuous editing was mooring
sequence-dependent, as only RNAs that were edited at C
were further edited at the promiscuous sites. Furthermore,
consistent with the orientation specificity of the mooring
sequence(10) , only cytidines 5` of the mooring sequence were
promiscuously edited.
The occurrence of the promiscuous editing
events has been documented by primer extension and dideoxy sequencing
analyses. Both assays were dependent on RT-PCR and could therefore have
errors due to nucleotide misincorporation. We have determined that the
cumulative error frequency in the primer extension assay was 0.2%.
Only the additional primer extension product seen on apoB RNA from
control McArdle cells fell within this range. The efficiency of
promiscuous site utilization by APOBEC-1-overexpressing cells was much
higher and ranged from 0.9 to 42%. More importantly, the efficiency of
both C
editing and promiscuous site utilization
increased with the degree to which APOBEC-1 was overexpressed in stable
transfected cell lines.
The C U transitions proposed as
promiscuous editing sites also occurred with significantly greater
efficiency than the assay error rate involved in the DNA sequencing
analyses. 9.1% of the total cytidine residues 5` of C
in
the exogenous substrates and 1.4% of the total cytidine residues 5` of
C
in the endogenous substrates were converted to uridine
in the clones isolated from the highest APOBEC-1-expressing cell line.
A total of 24 base errors (not including editing site conversions) were
observed following the sequencing of all four nucleotides of 90
independent exogenous and endogenous RNA isolates for a total of over
11,500 bases read. This was equivalent to 0.21%, or 1 base error in
every 476 bases, resulting from reverse transcriptase, PCR, or dideoxy
sequencing nucleotide misincorporation. These errors were assorted, e.g. A
G, T
C, G
A, were randomly located
throughout the different substrates, and were not specific to edited
substrates. Notably, there were no C
T errors observed either 3`
of or beyond 100 bases 5` of C
. No cytidines were
observed that had been converted to nucleotides other than uridine.
DNA sequence analyses clearly demonstrated the extent and variety of
sites promiscuously edited. These data confirmed those from primer
extension analyses and extended the information by demonstrating that
promiscuous site utilization only occurred on RNAs that had been edited
at C and that all of these sites resided 5` of the
mooring sequence. The analysis also revealed that cytidines were not
edited in a processive manner. cDNAs from RNA substrates were cloned in
which a variety of cytidines 5` of C
were selected for
editing. For example, editing of C
and C
occurred in some but not all clones, whereas no clones were
sequenced in which C
, C
,
C
, C
, and C
were edited.
Poisoned primer extension data suggested, however, that C
must have been edited in some proportion of the total cellular
RNA population (Fig. 2, Exogenous substrate band 3).
Potential explanations for this discrepancy between assay systems are
that a nonrandom transition (T
C) occurred at position 6651 due
to an incorporation error or that the clones edited at C
were not among those that had been isolated. Taken together,
however, the data support a ``moor and touch down'' mechanism (7, 12, 24) for promiscuous editing rather
than a processive, 5`-directional scanning mechanism.
Despite
multiple additional editing events, none of the cell lines edited
C in either the endogenous or exogenous total cellular
apoB RNA populations with 100% efficiency. In other studies, the
abundance of APOBEC-1 in tissues and transfected human and rodent
hepatoma cell lines correlates with the proportion of endogenous apoB
RNA that is
edited(16, 20, 21, 22, 23, 24, 25, 33) .
In these systems editing efficiencies were determined to range from 10
to 90%, and in only one example was a low level utilization of a
secondary editing site reported(25) . A system must therefore
exist to ensure site-specific editing at C
. We speculate
that such a system may result from coordinate regulation of apobec-1 and auxiliary factor gene expression. Promiscuous
editing, such as that reported here, may be the consequence of
overexpression of APOBEC-1 without proportionate expression of other
editosomal proteins. In this regard, site-specific editing may only be
possible when APOBEC-1 is rate-limiting relative to the auxiliary
factors. At high APOBEC-1 abundance, editosome assembly may be
aberrant, resulting in a stoichiometry of subunits or conformation that
permits a range of interactions with the RNA substrate. In this
scenario, editing of the promiscuous sites could be directed from a
single editosome assembled at the wild type mooring sequence (nt
6670-6681).
The unique occurrence of promiscuous editing sites
5` of the mooring sequence suggests that cis-acting elements must also
be mechanistically important. The predominance of a four-nucleotide
UGAU motif has been noted as a curious feature of RNA flanking the apoB
RNA editing site (5, 10, 28) . This motif,
which comprises the 5` head of the 11-nt mooring sequence would be
expected to only occur randomly every 256 nucleotides (once in 4 nt). In human apoB RNA, the motif occurs 12 times in a
400-nucleotide region (nt 6440-6839), an average of once every 33
nucleotides. More surprisingly, this motif occurs eight times in the
193-nucleotide region analyzed in these sequencing studies. It has been
recently demonstrated that APOBEC-1 fusion proteins can interact with a
similar RNA motif with low affinity in the absence of auxiliary
factors(26, 27) . Overexpression of APOBEC-1 in our
study may have resulted in C
U editing due to inappropriate
binding of APOBEC-1 homodimers (35) at one or more of these
motifs.
In support of this possibility, the promiscuous editing
sites on the human exogenous RNA at C and C
are 6 and 13 nt (respectively) 5` of UGAU motifs. Edited
cytidines C
, C
, C
,
C
, C
, and C
are located
between 5 and 21 nt 5` of a third UGAU motif. Also consistent with this
possibility, from the point of view of distance constraints, was the
finding that C
, C
, C
, or
C
, which lie more than 30 nt 5` of a UGAU motif, were
not edited. Only three UGAU motifs exist within the 207-base pair
region of the endogenous rat apoB RNA that was analyzed. One of these
motifs is located within the mooring sequence, and it could have been
responsible for the promiscuous editing at C
and
C
. Consistent with the potential quantitative
relationship between UGAU motifs and sites of promiscuous editing, less
extensive promiscuous editing occurred on rat apoB RNA compared with
human apoB RNA.
Contrary to the data supporting multiple foci for
editing complex assembly was the finding that C on the
exogenous human transcript was not edited, although it only resides six
nt 5` of a UGAU motif. Moreover, five UGAU motifs exist within 140 nt
3` of the mooring sequence, yet no editing occurred within this region.
In the endogenous rat apoB RNA, C
and C
were not edited, although they were proximal to a UGAU motif at
nt 6590. Interestingly, there was no UGAU motif suitably located to
support the observed editing at C
and C
on the rat endogenous apoB RNA transcript. Moreover, none of the
promiscuous editing sites on the human or rat apoB RNAs are flanked by
optimal spacer or regulatory elements. The data suggest therefore that
although cis-acting elements must play an important role, promiscuous
editing cannot be solely accounted for by the occurrence of multiple
UGAU motifs, individually capable of binding to editing complexes. We
cannot currently distinguish between the two alternate hypotheses for
promiscuous editing, although it is clear that APOBEC-1 overexpression
is of central importance.
Promiscuous editing upon the endogenous rat apoB RNA can be predicted to induce a number of silent amino acid changes, some conservative amino acid changes, several alterations anticipated to effect protein structure, and even the introduction of a translation STOP codon seven amino acids N-terminal to that which would be created by utilization of the normal editing site. A similar array of amino acid alterations were predicted as a result of promiscuous editing upon the human RNA including the introduction of translation STOP codons at amino acid residues 2145 and 2148. A number of different apoB mutations associated with hypobetalipoproteinemia have been mapped that include the introduction of numerous STOP codons throughout the apoB gene (reviewed in (36) ). Under normal circumstances, it is unlikely that promiscuous editing would occur in human liver, as it does not express sufficient APOBEC-1 to edit its own apoB RNA(22) . The emphasis that is currently being placed on regulating serum low density lipoprotein levels as a means of reducing the risk of atherogenic diseases raises the possibility of using apobec-1 cDNA in gene therapy (23, 34) . The current studies suggest that such transfections in humans would be inappropriate due to the potential of inducing promiscuous editing.
To this extent, we believe that promiscuous editing may not be
limited to apoB RNA. Induction of site-specific editing within
heterologous RNA contexts both in vitro and in cells has been
described through the insertion of the 11-nucleotide mooring
sequence(6, 8, 9, 37) . The high
efficiency with which C in the TL mutant RNA was edited
and the occurrence of promiscuous editing suggest that RNAs containing
regions with low homology to the apoB RNA editing motif might become
editing substrates if APOBEC-1 was overexpressed without the concordant
increase in auxiliary factor expression. The potential that RNAs other
than apoB are edited has also been suggested by the finding that cell
lines of diverse origin that did not express apoB RNA had the ability
to edit a transfected apoB cDNA(29) .
A search of
GenBank using the Pearson and Lipman type FASTA algorithm (38) for human and rodent RNA transcripts that contain homology
to the 11-nt mooring sequence demonstrated that only apoB contains a
perfect match for a complete 21-nt tripartite editing motif. Six human
and rodent RNAs were identified that contained a perfect 11-nt mooring
sequence with a 5`-located cytidine residue (mouse P1 protein, mouse
fatty acid synthase, human CD44, human serine/threonine kinase
receptor, human coagulation factor XI, and human serum albumin
(GenBank
accession numbers X62154, X13135, M59040, L31848,
M18304, and M12523, respectively). Over 20 RNAs were also identified
with perfect homology to the 5` head of the mooring sequence but only
partial homology to the 3` end. Based on the results from apoB editing
site mutagenesis studies (5, 6, 12, 13) a consensus editing
motif (UGAUY(A/T)NN(A/T)YN) was compared with GenBank
and
resulted in the identification of over 3000 human and rodent RNAs as
potential editing substrates. When considered together with our
analysis of wild type and mutant editing motif utilization, the results
of the data base search suggest that the potential for promiscuous
editing following overexpression of APOBEC-1 is extremely high. The
uncertain phenotypic outcome from promiscuous editing indicates that apobec-1 cDNA expression alone may be unsuitable for gene
therapy.
Addendum-While this manuscript was under
review Yamanaka et al.(41) reported liver-specific
overexpression of APOBEC-1 in transgenic mice and rabbits that resulted
in mooring sequence-dependent C U editing of alternative RNA
substrates and genotoxicity resulting in hepatic dysplasia.