Two Efficiency Elements Flanking the Editing Site of Cytidine
6666 in the Apolipoprotein B mRNA Support
Mooring-dependent Editing*
Martin
Hersberger
and
Thomas L.
Innerarity§
From the Gladstone Institute of Cardiovascular Disease,
Cardiovascular Research Institute and the § Department of
Pathology, University of California,
San Francisco, California 94141-9100
 |
ABSTRACT |
Normally, apolipoprotein B (apoB) mRNA
editing deaminates a single cytidine (C6666) in apoB
mRNA. However, when the catalytic subunit of the editing enzyme
complex, APOBEC-1, was overexpressed in transgenic mice and rabbits,
numerous cytidines in the apoB mRNA and in a novel mRNA, NAT1,
were aberrantly hyperedited, and the animals developed liver dysplasia
and hepatocellular carcinomas. To identify the RNA motifs in the apoB
mRNA that support physiological editing and those that support
aberrant hyperediting, we constructed rabbit apoB RNA substrates and
tested them in vitro for physiological editing and
hyperediting. Three previously unrecognized RNA elements that are
critical for efficient physiological editing at C6666 were
identified. In concert with the mooring sequence (6671-6681), the 5'
efficiency element (6609-6628), an A-rich region (6629-6640), and the
3' efficiency element (6717-6747) increased editing at C6666. The 5' efficiency element was the most potent,
elevating physiological editing to wild-type levels in combination with
the mooring sequence. The 3' efficiency element was somewhat less
important but increased physiological editing to levels approaching
wild type. These elements encompass 139 nucleotides on the apoB RNA
transcript and are sufficient for editing with the efficiency of
full-length apoB mRNA. Furthermore, a distal downstream apoB region
(6747-6824) may function as a recognition element in the apoB
mRNA. Hyperediting at C6802 in the rabbit apoB mRNA
is mediated by RNA elements similar to those required for normal
physiological editing at C6666. Similarly sized upstream
and downstream flanking regions of C6802 are necessary for
hyperediting in combination with a degenerate mooring sequence.
 |
INTRODUCTION |
RNA editing is the alteration of the genetic information present
in nascent RNA transcripts. One form of RNA editing, apolipoprotein B
(apoB)1 mRNA editing,
deaminates a specific cytidine (C6666) in the apoB
mRNA, which generates a uridine (1-4) changing codon 2153 from a
genomically encoded CAA (glutamine) to an in-frame stop codon (UAA)
(5, 6).ApoB mRNA editing results in the formation of a truncated
apoB protein (apoB48) that is about half the size of the full-length
genomically encoded apoB (apoB100) (reviewed in Refs. 7 and
8).
Under physiological conditions, apoB mRNA editing is an exquisitely
precise process. No other RNAs are known that undergo this C
U
deamination, and no other cytidines in the apoB mRNA are edited,
except for a minor site in human intestinal apoB mRNA (9). Two
nucleotide sequence elements have been identified that are necessary
for editing: an 11-nucleotide (nt) "mooring sequence" (6671-6681)
(10-12) and a 4-nt spacer sequence between the edited cytidine and the
mooring sequence (10, 11, 13). Integration of 26-63 nt of specific
apoB RNA sequences encompassing the mooring sequence and the spacer
sequence into heterologous mRNAs resulted in editing at levels
substantially less than wild type, suggesting that the RNA environment
is important and that other unidentified elements are necessary for
efficient editing at C6666 (14, 15).
The apoB mRNA editing complex comprises a catalytic subunit
designated APOBEC-1 (16) and other, as yet unidentified, auxiliary proteins (17-19). Overexpression of APOBEC-1 in the livers of mice and
rabbits resulted in liver dysplasia and hepatocellular carcinomas (20).
Overexpression of APOBEC-1 also resulted in the aberrant editing of
several other cytidines in the apoB mRNA (21-23). The highest
degrees of editing occurred at C6738, C6743,
C6762, C6782, and C6802 (21).
Numerous cytidines were also edited in a novel mRNA, NAT1 (22).
This aberrant editing was termed hyperediting to distinguish it from
the physiological editing at C6666. Sequence analysis of
the RNA surrounding the hyperedited cytidines showed that no exact
mooring sequence was present within the correct distance to support
this hyperediting (21). In contrast, other mRNAs containing a
mooring sequence were not hyperedited (20), indicating that elements in
addition to the mooring sequence are necessary for hyperediting.
Here we report results from in vitro studies of
physiological editing at C6666 and hyperediting at
C6802 in the apoB RNA. Our results show that the essential
elements for physiological editing at C6666 of the rabbit
apoB mRNA encompass 139 nt, consisting of defined upstream
efficiency elements, the mooring sequence, and the 3' efficiency
element. Similar RNA features are necessary for the hyperediting of
cytidines when APOBEC-1 is overexpressed.
 |
EXPERIMENTAL PROCEDURES |
Generation of ApoB RNAs--
ApoB RNAs were produced by in
vitro transcription from polymerase chain reaction (PCR)
constructs, that included a T7 promoter. The apoB cDNA constructs
were generated by PCR amplification from the plasmid pRabSK, a
derivative of pRab-1 (24), encoding a 354-base pair rabbit apoB
cDNA segment. The PCR products were purified by agarose gel
electrophoresis and eluted by Qiaex II gel extraction (Qiagen, Santa
Clarita, CA). The primers designed on the computer program OLIGO 4.0 were 18-25-nt-long and were purchased from Life Technologies, Inc. The
T7 promoter sequence with GGA (5'-GGATCCTAATACGACTCACTATAGGGA-3') added
to achieve efficient expression was incorporated into the PCR
constructs by a second PCR amplification. RNA was produced by the
T7-MEGA shortscript in vitro transcription kit (Ambion,
Austin, TX). The RNA was analyzed by agarose gels, treated with DNase
1, and purified by phenol-chloroform extraction and ethanol
precipitation. The names of the RNAs refer to the first nucleotide of
the RNA according to the apoB cDNA position (e.g. B6629
starts at nt 6629), and the length of the RNAs is given in
nucleotides.
The DNA constructs for the P1 (GenBankTM accession number
X62154) and the N-myc RNA (GenBankTM accession number
M12731) were produced by reverse transcription-PCR from normal mouse
liver RNA. The primers for the N-myc amplification were
MU6879:5'-ACGGCCTGTATACTTTTGTATG-3' and
ML7053:5'-AACAAATACAGTAAACAAGGAA-3'. For the P1 amplification, the
primers were P1U1279:5'-GCCCGCTGCAGTGTTTTGG-3' and
P1L1454:5'-CCCTGTATTGGTGCATCCTAA-3'. The N-myc and the
P1-PCR fragments were TA subcloned (Invitrogen, San Diego, CA) and
sequenced. Chimeric RNAs were produced from PCR-derived DNA constructs
by recombinant PCR (25). The PCR products were purified, and the RNAs
were produced as described for the apoB RNAs. Substitution mutagenesis
was accomplished by incorporating the alterations in the primers used
to produce the PCR constructs.
The chimeric RNA MS-P1 (196 nt) consists of 58 nt of apoB RNA
(6629-6686) and 138 nt of P1 RNA (1337-1474). The N-myc
chimeric RNA MS-M (197 nt) has 58 nt of apoB RNA (6629-6686) and 139 nt of N-myc RNA (6936-7074). The second set of
N-myc chimeric RNAs used the 138-nt N-myc RNA MY
(6859-6996) as a control. M-MOOR-M (138 nt) has 51 nt of
N-myc RNA (6859-6909), 26 nt of apoB RNA (6662-6687), and
61 nt of N-myc RNA (6936-6996). EFF-MOOR-EFF (138 nt) has
20 nt of apoB RNA (6609-6628), 31 nt of N-myc RNA (6879-6909), 26 nt of apoB RNA (6662-6687), 30 nt of N-myc
(6936-6965), and 31 nt of apoB RNA (6717-6747). The third set
consists of the F-M-F RNA (139 nt), which has 53 nt of apoB RNA
(6609-6661), 26 nt of N-myc RNA (6910-6935), and 60 nt of
apoB RNA (6688-6747). M-F RNA (137 nt) has 77 nt of N-myc
RNA (6859-6935) and 60 nt of apoB RNA (6688-6747). F-M RNA (140 nt)
has 53 nt of apoB RNA (6609-6661) and 87 nt of N-myc RNA
(6910-6996). EFF-M-EFF RNA (137 nt) has 20 nt of apoB RNA
(6609-6628), 86 nt of N-myc RNA (6879-6964), and 31 nt of
apoB RNA (6717-6747). The fourth set of apoB/N-myc chimeric
RNAs used the 196-nt N-myc RNA M (6879-7074) as a control.
M-B6747 has 118 nt of N-myc RNA (6879-6996) and 78 nt of
apoB RNA (6747-6824). The PCR templates for all the
apoB/N-myc chimeric RNAs were sequenced.
In Vitro ApoB mRNA Editing Assay--
For the in
vitro apoB mRNA editing assay (26), 100 pg of synthetic RNA
prepared as described above, 100 µg of rabbit liver S100 extract, 5 µg of recombinant APOBEC-1 (MBP-APOBEC-1) (21), 1 µg of
Escherichia coli tRNA, and 40 units of RNasin (Promega, Madison, WI) in buffer D containing 1 mM dithiothreitol in
a reaction volume of 100 µl were incubated at 30 °C for the
indicated times and then extracted as described (26). In control
experiments, the RNAs were incubated in this assay mixture lacking
rabbit liver S100 extract. All RNAs were amplified by reverse
transcription-PCR, and the resulting single-band PCR products were
purified over a microspin S-300 HR column (Amersham Pharmacia
Biotech).
Primer Extension Analysis--
Primer extension was performed
essentially as described (1). Except in the experiments with primers
PE6762, PE6782/3, and PE6802, the coding strand instead of the
noncoding strand was analyzed for hyperediting. For these experiments,
1 mM each dATP, dGTP, and dTTP and 5 mM ddCTP
were used for the poisonous primer extension. The extent of editing was
determined with a radioanalytic imaging system (AMBIS, San Diego, CA).
The primers used for primer extension in apoB were M51 at
C6666 (5'-ATCATAACTACTTTTAATATACTG-3'), PE6738R at
C6738 (5'-ATTTTTAATTTTTCCATGAT-3'), PE6743R at
C6743 (5'-GTTCATCAAGAATTTTTAAT-3'), PE6762 at
C6762 (5'-TAGATCAAATCATGGAAAAA-3'), PE6782/3 at
C6782 and C6783 (5'-AATTCTTGATGAACGTTATC-3'),
PE6802 at C6802 (5'-TATCATATCCGTGCACATTT-3'), and PEMOORL65
(5'-AATTTCATACGCTTTAATATACTG-3') for C6666 in the
apoB/N-myc chimeric RNAs containing the apoB editing
cassette (designated MOOR). The primer used to detect editing in
N-myc was MPE6914R at C6914
(5'-TACGAAAATATAAGTATCAA-3'). The percentage of editing is given as the
mean plus and minus the deviation of n experiments.
 |
RESULTS |
To investigate the influence of distal flanking sequences on
editing of C6666 and hyperediting of C6802 in
the apoB mRNA, we used an in vitro editing assay of
small apoB RNAs. The RNAs were produced by in vitro
transcription and incubated in the presence of rabbit liver extract (as
a source of auxiliary proteins) and an excess of recombinant APOBEC-1. The percentage of editing of a specific cytidine was then determined by
primer extension analysis.
Editing at C6666--
To determine the impact of
downstream elements on physiological editing of C6666, we
first generated RNAs with 3' sequences of different lengths (Fig.
1). Editing at C6666 was
determined in an in vitro editing assay after 1, 2, 4, and 16 h. The parental RNA B6507 (354 nt) was highly edited (82%) after 16 h (Fig. 1). Editing of smaller RNAs was similar; even B6629
-77 (119 nt) was edited to 77% after 16 h of incubation. However, removal of an additional 31 nt at the 3' end (6717-6747) decreased editing by almost half. We termed this 31-nt spanning region
located 51-81 nt downstream of C6666 the "3' efficiency
element." That the loss of editing efficiency was not simply due to
the length of the RNA was shown by using the N-myc chimeric
RNA MS-M (197 nt) and the P1 chimeric RNA MS-P1 (196 nt) in which
sequences 3' of the mooring sequence were replaced with
N-myc or P1 sequences. The AU-rich apoB/N-myc RNA
and B6629
-118 were edited to a similar extent. The GC-rich apoB/P1
RNA was marginally edited (MS-P1, Fig. 1).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 1.
In vitro editing time course experiment
of apoB RNAs to map the influence of distal 3' sequences. RNAs
were incubated for 1, 2, 4, or 16 h in an in vitro
editing assay, and the editing of C6666 was determined by
primer extension analysis. A, B6507 is a 354-nt apoB RNA
used as a control containing the mooring sequence (black
bar) and several mooring-like motifs downstream. One of the
mooring-like motifs (6809-6819) is marked by a striped bar.
The - refers to the number of nucleotides deleted compared with the
parental apoB RNA B6629 (e.g. B6629 -77 lacks the terminal
77 nt of B6629). MS-M is a chimeric RNA consisting of apoB
(bar) and N-myc RNA (line). MS-P1
consists of apoB and of P1 RNA (dotted line). MS consists of
58 nt of apoB sequences. The numbers in the top
part of the figure refer to the apoB cDNA position.
B, primer extension analysis of C6666 in two
apoB RNAs is shown. The top band represents the deamination
of C6666 to a uridine (U at 6666). The
middle band represents the unmodified cytidine (C at
6666). The extent of editing was determined with a radioanalytic
imaging system.
|
|
To investigate the influence of upstream flanking regions on editing of
C6666, we transcribed small RNAs lacking the 3' efficiency
element. RNAs starting at nt 6609 (e.g. B6609 (78 nt) and
B6609L (98 nt)) were edited with the same efficiency as the parental
construct B6507, resulting in 80-90% editing within 4 h of
incubation (Fig. 2). However, deletion of
20 nt in the 5' end resulted in a 50% decrease in editing after
16 h of incubation (Fig. 2, B6629
-108 (88 nt) and
B6629
-118 (78 nt)). The size of the RNA was not
responsible for the difference in editing, because B6629
-118 and
B6609 are the same length (78 nt). Thus, the difference in editing
efficiency was due to the RNA element 6609-6628, located 38-57 nt
upstream of C6666, which we termed the "5' efficiency
element." Because RNAs lacking the 5' efficiency element were still
edited to about 50% of the wild-type level after 16 h, we deleted
more 5' sequences to define closer regions that promote editing at
C6666. Additional deletion of 12 nt from the 5' end
resulted in an RNA, B6641 (46 nt), that was edited only to 7%
(n = 3) after 16 h of incubation (data not shown),
indicating the importance of a second region 26-37 nt upstream of
C6666 for efficient editing at C6666. This
second region is A-rich (AAAUGAAAAA), and mutations in this A-rich
region (6629-6640) revealed that three A
U and three A
G
substitutions decreased editing at C6666 (data not shown).
However, introduction of a mooring sequence (UGAUCAGUAUA) returned
editing efficiency to high levels (65% after 16 h (data not
shown)).

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 2.
In vitro editing time course experiment
with apoB RNAs to investigate the influence of 5' sequences on editing
of C6666. RNAs were incubated for 1, 2, 4, or 16 h in an in vitro editing assay, and the editing of
C6666 was determined by primer extension analysis.
A, B6609 (78 nt) and B6609L (98 nt) start at position 6609 of the apoB cDNA. The three RNAs B6629 -108 (88 nt), B6629 -118
(78 nt), and MS (58 nt) start 20 nt farther downstream at position
6629. The numbers in the top part of the figure
refer to the apoB cDNA position. The apoB mooring sequence is
marked (black bar). B, primer extension analysis
of C6666 in four apoB RNAs is shown. The top
band represents the deamination of C6666 to a uridine
(U at 6666). The middle band represents the
unmodified cytidine (C at 6666). The extent of editing was
determined with a radioanalytic imaging system.
|
|
To distinguish further whether the 5' and 3' efficiency elements are
part of the minimal flanking requirement for efficient physiological
editing at C6666 or whether they are specific enhancer
elements for physiological editing, we made chimeric
apoB/N-myc RNAs (Fig. 3).
Previous deletion and mutagenesis experiments indicated that the 26-nt
apoB editing cassette, including C6666 and the mooring
sequence, is sufficient for low level editing in certain heterologous
RNA contexts (10, 12). We therefore exchanged sequences flanking this
apoB-editing cassette with N-myc sequences, resulting in
20 ± 14% (M-MOOR-M) editing at C6666 after 16 h. This represents about 25% of the editing activity detected in the
parental apoB RNA (B6609C). However, chimeric RNAs containing the apoB
5' efficiency element were edited to wild-type levels (EFF-MOOR-M and
EFF-MOOR-EFF), emphasizing the importance of the 5' efficiency element
(6609-6628) for efficient mooring-dependent editing.
Adding the 3' efficiency element (6717-6747) did not further increase
editing efficiency when the 5' efficiency element was present
(EFF-MOOR-EFF), but in the absence of the 5' efficiency element, the 3'
efficiency element increased editing 2-fold (M-MOOR-EFF). These data
indicate that in combination with the apoB editing cassette, the 5'
efficiency element and to a lesser extend the 3' efficiency element
restored editing at C6666 to wild-type levels.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3.
The apoB 5' and 3' efficiency elements
increase mooring-dependent editing at C6666.
RNAs were incubated for 16 h in an in vitro editing
assay, and the editing of C6666 was determined by primer
extension analysis (n = 3). A, B6609C is the
parental apoB RNA. M-MOOR-M consists of the 26-nt apoB editing cassette
including the mooring sequence (black bar), flanked by
N-myc RNA (line). EFF-MOOR-M consists of the apoB 5'
efficiency element and the apoB editing cassette flanked by
N-myc RNA. EFF-MOOR-EFF has the apoB 5' efficiency element,
the apoB editing cassette, and the 3' efficiency element linked by
N-myc RNA. M-MOOR-EFF has the apoB editing cassette and the
apoB 3' efficiency element flanked by N-myc RNA. The
numbers in the top part of the figure refer to
the apoB cDNA position. ApoB RNA is drawn as a bar.
B, primer extension analysis of C6666 in the
apoB RNA is shown. The top one or two bands
represent the deamination of C6666 to a uridine (U at
6666). The second band from the bottom represents the
unmodified cytidine (C at 6666). The extent of editing was
determined with a radioanalytic imaging system.
|
|
Our data indicate that efficient physiological editing at
C6666 is enhanced by both the 5' efficiency element (38-57
nt upstream) and by the 3' efficiency element (51-81 nt downstream).
If these apoB elements enhance editing at C6666, they may
promote hyperediting in N-myc RNA containing a mooring-like motif. To test this possibility, we exchanged the 26-nt apoB editing cassette for the equivalent N-myc sequences (Fig.
4). The N-myc RNA (MY) was not
hyperedited, although it contains mooring-like motifs and its AU
content is equivalent to that of apoB RNA. When the apoB 5' and 3'
efficiency elements were added to the N-myc RNA, no
hyperediting was detected (Fig. 4, EFF-M-EFF). Thus, the apoB 5' and 3' efficiency elements promote editing in combination with
the apoB editing cassette (Fig. 3). However, providing the entire apoB
upstream flanking region (6609-6661), including the 5' efficiency
element (F-M) or the entire apoB downstream flanking region
(6688-6747), including the 3' efficiency element (M-F), resulted in
hyperediting of one (C6914) or two cytidines
(C6914 and C6909) in the N-myc RNA.
The flanking regions of the editing cassette in the apoB RNA therefore
act as recognition elements for hyperediting of a cytidine in the
N-myc RNA context. Because we hypothesized that several
elements in the apoB mRNA could function as recognition elements
and could support mooring-dependent editing (22), we examined the influence of the more downstream apoB RNA element, B6747,
on hyperediting of the N-myc RNA (Fig. 4). The apoB sequence applied in these chimeric RNAs does not cover sequences relevant for
efficient editing of C6666, but B6747 starts directly
downstream of the 3' efficiency element mapped above. Again, this
longer N-myc RNA (M) was not hyperedited (Fig. 4). In
M-B6747, however, the substitution of 78 nt of apoB RNA (B6747) in the
N-myc RNA caused editing of two cytidines 5' of the
N-myc mooring-like motif (Fig. 4). These results indicate that an element in the apoB RNA more than 81 nt downstream of C6666 influences editing.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 4.
Independent 5'- and 3'-flanking regions of
the apoB RNA promote hyperediting of a previously unedited cytidine in
chimeric RNAs. RNAs were incubated for 16 h in an in
vitro editing assay, and the hyperediting of C6914 in
the N-myc RNA was determined by primer extension analysis
(n = 3). MY consists of 138 nt of N-myc RNA
(line), including C6914 and the mooring-like
motif (UGAUACUUAUA, small box). EFF-M-EFF consists of the
apoB 5' efficiency element fused to N-myc RNA and to the
apoB 3' efficiency element. M-F has N-myc sequences fused to
the apoB 3'-flanking region. F-M-F consists of the apoB 5'-flanking
region fused to the 26-nt N-myc hyperediting cassette and
the apoB 3'-flanking region. F-M has the apoB 5'-flanking region fused
to N-myc RNA. M consists of 196 nt of N-myc RNA.
M-B6747 has the 3' last 78 nt of N-myc RNA replaced with the
78-nt apoB sequence, B6747, that includes the apoB mooring-like motif
(striped bar). The numbers in the top
part of the figure refer to the apoB cDNA position.
|
|
Hyperediting--
Our data indicate that both upstream and
downstream flanking regions contribute to editing of C6666.
To compare these findings to hyperediting, we first mapped in vitro the minimal upstream sequence required for hyperediting of
C6802 (Fig. 5). B6629
includes C6666, the mooring sequence downstream, and the
region with the clustered cytidines that are hyperedited in the apoB
mRNA from transgenic mice overexpressing APOBEC-1. All cytidines
investigated in B6629 (196 nt) were hyperedited in vitro in
a pattern similar to that observed in vivo (21). At
C6802 the level of hyperediting was 4.9 ± 1.7%
(n = 3). Deletion of the mooring sequence (Fig. 5,
B6687, 138 nt) abolished hyperediting at C6743
and increased hyperediting 2-fold at C6802 (11.8 ± 4%, n = 3). Deletion of an additional 60 nt of 5'
sequences (Fig. 5, B6747, 78 nt) resulted in the loss of
combined hyperediting of C6782 and C6783
(C6782/3), but C6802 was still hyperedited
(4.2 ± 1.5%, n = 3). However, deletion of an
additional 13 nt at the 5' end abolished hyperediting at C6802 (Fig. 5, B6760, 65 nt), indicating that
sequences 42-55 nt upstream are essential for hyperediting at this
site.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5.
In vitro hyperediting of short apoB
RNAs. RNAs were incubated for 16 h in an in vitro
editing assay, and the editing of each cytidine was determined by
primer extension analysis. B6629 (196 nt) is the parental RNA
containing the mooring sequence (dark bar) and several
mooring-like motifs downstream. One of these mooring-like motifs
(6809-6819) is marked by a striped bar. B6687 (138 nt),
B6747 (78 nt), B6760 (65 nt), and B6779 (46 nt) lack the mooring
sequence and have 5' sequences progressively deleted. The name of the
RNA refers to the first nucleotide of the RNA according to the apoB
cDNA position (e.g. 6629 starts at nt 6629). The
numbers in the top part of the figure refer to
the apoB cDNA. The cytidines investigated for editing and
hyperediting are indicated at the bottom of the
figure.
|
|
Intriguingly, the sequence 42-55 nt upstream of C6802 is
AU-rich (AAAAAUUAAAA), similar to the A-rich region (AAAUGAAAAA) that contributes to physiological editing of C6666. Furthermore,
the AU-rich sequence contains a cryptic polyadenylation signal
(AUUAAA). We tested the possibility that the cryptic polyadenylation motif (6752-6757) influences hyperediting of C6802,
because we and others have shown a link between editing and activation
of this cryptic polyadenylation signal in the apoB mRNA (5, 6, 15,
27). The 78-nt apoB RNA, B6747, contains the cryptic polyadenylation
signal (Fig. 6). Mutation of the cryptic polyadenylation signal to AUUAUA by the substitution of a U for A did
not diminish hyperediting of C6802 in UB6747 (Fig. 6).
Moreover, in the MB6747 RNA, the replacement of the cryptic
polyadenylation signal with a mooring sequence (UGAUCAGUAUA) had no
effect on hyperediting of C6802. Therefore, the cryptic
polyadenylation signal (AUUAAA) itself is not necessary for
hyperediting at C6802. However, three A
G substitutions
in this region abolished hyperediting at C6802 (Fig. 6,
GB6747). Thus, there appears to be a preference, if not a
necessity, for an AU-rich sequence in this 5'-flanking region 42-55 nt
upstream of C6802.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 6.
Scrambling the cryptic polyadenylation site
(AUUAAA) in the 78-nt apoB RNA B6747 did not decrease hyperediting of
C6802. RNAs were incubated for 16 h in an
in vitro editing assay, and the editing of C6802
was determined by primer extension analysis. B6747 (n = 3) is a 78-nt parental apoB RNA used for the mutagenesis studies
including the mooring-like motif at 6809-6819 (striped
bar). UB6747 has U A substitutions in the region of the
cryptic polyadenylation site (U). MB6747 has the exact 11-nt
mooring sequence introduced at the same site (dark bar), and
GB6747 has G substituted for A (G). Underlined
letters indicate substituted nucleotides. The numbers
in the top part of the figure refer to the apoB cDNA
position. The percentage of editing is given as the mean plus or minus
the deviation.
|
|
To investigate the importance of the mooring-like motif (6809-6819)
downstream of C6802, we used the constructs shown in Fig.
7. This mooring-like motif is located
five nucleotides from the 3' end of all short apoB RNAs investigated
and is the only mooring-like motif downstream of C6802. In
rabbit apoB RNA, this mooring-like motif matches only six out of the 11 nucleotides in the mooring sequence and has a 6-nt instead of a 4-nt
spacer region (Fig. 7). In the 138-nt B6687 RNA template, all of the
cytidines investigated were hyperedited except for C6743
(Fig. 7). Scrambling the mooring-like motif at 6809-6819 (B6687S) decreased editing at C6802 to 33% of the level of the
parental RNA (B6687). Furthermore, scrambling of the mooring-like motif
(B6687S) and deletion of this region (B6687
-16) abolished
hyperediting of C6782/3. In contrast, both mutations only
slightly decreased hyperediting at C6762 and at other
hyperedited cytidines. For example, C6762 was hyperedited
to 9-15% in the mutated RNAs, which is slightly less than in the
parental RNA (19%). Therefore, the mooring-like motif at 6809-6819
influenced hyperediting of cytidines up to 27 nt upstream, but
hyperediting of C6762, located 47 nt upstream, was
unaffected by this mooring-like motif.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 7.
Scrambling the mooring-like motif at
6809-6819 decreased hyperediting of C6802 and
C6782/3 but not other hyperedited sites. RNAs were
incubated for 16 h in an in vitro editing assay, and
the editing of each cytidine was determined by primer extension
analysis. B6687, which includes the mooring-like motif (striped
bar), was used as parental apoB RNA for mutagenesis studies.
B6687S has the mooring-like motif at 6809-6819 scrambled
(S). B6687 -16 lacks the last 16 nt of B6687, including
the mooring-like motif. The numbers in the top
part of the figure refer to the apoB cDNA position. The
cytidines investigated for hyperediting are indicated below.
Underlined letters indicate nucleotides that match the
mooring sequence. B6687 was hyperedited at C6802 (11.8 ± 4%, n = 3), C6782/3 (2 ± 0.1%,
n = 2), and C6762 (19%). B6687S was
hyperedited at C6802 (3.9%), C6782/3 (<1%),
and C6762 (9%). B6687 -16 was hyperedited at
C6782/3 (<1%) and C6762 (15%).
|
|
To investigate this mooring-like motif at 6809-6819 without the
influence of distal elements, we concentrated on the smallest apoB RNA
template, B6747, that was still hyperedited at C6802. In
contrast to the 138-nt B6687S RNA (Fig. 7), where scrambling of the
mooring-like motif decreased hyperediting of C6802, in the
smaller 78-nt RNA B6747S, the same mutations abolished hyperediting at
C6802 (Fig. 8). Changing the
mooring-like motif to an exact 11-nt mooring sequence (B6747M) motif
enhanced editing of C6802 2-fold. Furthermore, restoring
the pattern of the mooring-like motif with an extra A in the spacer
region (B6747R) between the mooring-like motif and C6802
had no effect on hyperediting of C6802. These results
indicate that an AU-rich sequence is not sufficient to support
hyperediting at C6802 unless it resembles the mooring
sequence.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 8.
Scrambling the mooring-like motif at
6809-6819 in the 78-nt apoB RNA B6747 abolished hyperediting at
C6802. RNAs were incubated for 16 h in an
in vitro editing assay, and the editing of C6802
was determined by primer extension analysis (n = 2).
The smallest apoB RNA that was hyperedited at C6802, B6747,
was used for a scrambling study of the mooring-like motif at 6809-6819
(striped bar). B6747S has the 6 nt that match the mooring
sequence scrambled (S). B6747M contains the exact 11-nt
mooring sequence (dark bar), and B6747R has a reconstituted
mooring-like motif in a different frame (R).
Underlined letters indicate nucleotides that match the
mooring sequence. The numbers in the top part of
the figure refer to the apoB cDNA position.
|
|
An AU-rich element and the mooring-like motif support hyperediting of
C6802, but downstream elements also influence hyperediting.
B6760 (65 nt) was not hyperedited at C6802 because it lacks
the AU-rich element (Fig. 9). When
constructs were generated that transcribed the B6760 RNA with either 28 or 58 nt of additional 3' sequences, only the longer transcript was hyperedited at C6802. Thus, if the AU-rich element is not
present, hyperediting at C6802 requires that the
3'-flanking region include sequences 51-80 nt downstream (6853-6882)
of the hyperedited site.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 9.
Flanking sequences downstream of
C6802 support hyperediting. RNAs were incubated
for 16 h in an in vitro editing assay, and the editing
of C6802 was determined by primer extension analysis
(n = 3). B6760 lacks the 5' AU-rich region. B6760L and
B6760LE have 28 and 58 nt, respectively, of additional sequences 3' to
B6760. The numbers in the top part of the figure
refer to the apoB cDNA position. The mooring-like motif at
6809-6819 is marked (striped bar).
|
|
 |
DISCUSSION |
Previous studies indicated that several sequence elements are
needed for efficient editing of apoB mRNA. For example, small apoB
RNAs and chimeric RNAs with only 26-63 nt of specific apoB sequence
were edited inefficiently (11, 14, 15, 28). Specifically, in the
GC-rich apoE RNA, 63 nt of specific apoB mRNA were not edited (15).
In contrast, when 354 nt of specific apoB sequence were translocated
into the same locus of the apoE mRNA, wild-type levels of editing
were detected, indicating that all sequence requirements for efficient
editing at C6666 are present within this segment (15). In
this in vitro study, we have defined essential RNA sequence
elements in the apoB mRNA necessary for its physiological editing
and for its aberrant hyperediting.
Physiological Editing of ApoB mRNA--
We found that for
normal physiological editing, an apoB RNA fragment of 139 nt consisting
of the upstream 5' efficiency element (6609-6628), an upstream 5'
A-rich element (6629-6640), the proximal efficiency sequence defined
by Driscoll et al. (6648-6661) (11), the mooring sequence
(6671-6681) (10-12), and the 3' efficiency element (6717-6747) were
sufficient for editing with an efficiency equal to that of full-length
apoB mRNA. We also found evidence that sequences even further
downstream (6747-6824) contributed to editing at C6666
(Fig. 10).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 10.
Schematic of elements that influence
physiological editing at C6666 and hyperediting at
C6802. The apoB mRNA encompassing
C6666 and C6802 is drawn linearly, and the
positions according to the cDNA are given below. Elements and RNA
regions that influence efficient physiological editing and
hyperediting in vitro are marked (shaded
bars). The distal downstream apoB region is marked as a
hatched bar.
|
|
The importance of the 5' efficiency element for physiological editing
was demonstrated by using short apoB RNA constructs in which the 5'
efficiency element was present or absent (Fig. 2) and by testing
chimeric apoB/N-myc RNAs (Fig. 3). In these chimeric RNAs,
the presence of the apoB 5' efficiency element with the apoB editing
cassette (6662-6687) was sufficient to give wild-type levels of
editing at C6666. How the two elements increased editing of
C6666 to wild-type levels is unknown. One possibility is
the formation of Watson-Crick base pairing between the two elements.
However, according to RNA folding data obtained with the Mfold program (29, 30), no secondary structures are favored between the 5' efficiency
element and the editing cassette (data not shown).
Our data also indicate that a second apoB RNA element supported
physiological editing of C6666. This A-rich element is
located at position 6629-6640, directly downstream of the 5'
efficiency element. Deletion and mutagenesis studies in this A-rich
element influenced editing at C6666 severalfold (data not
shown). Furthermore, a third proximal efficiency sequence at positions
6648-6661 was reported to promote editing in combination with the
mooring sequence (11).
Independent of other sequences, the 3' efficiency element of the apoB
mRNA increased editing at C6666. A previous study
indicated that 3'-flanking sequences of the apoB mRNA increase
editing at C6666 (14), but no efficiency element was
defined. Our results indicate that the 3' efficiency element located
within a 31-nt fragment (6717-6747) was necessary for efficient
physiological editing in the apoB RNA (Fig. 1). It also increased
editing in combination with the apoB editing cassette (Fig. 3),
although to a lesser extent than did the 5' efficiency element.
Furthermore, in the presence of the 5' efficiency element, the 3'
efficiency element did not further increase editing (Fig. 3),
indicating that the 5' efficiency element had a stronger impact on
editing of C6666 in small apoB RNA at least in
vitro.
Physiological editing at C6666 may also be enhanced by
elements more than 81 nt from the editing site. Although these
sequences were not required for efficient editing, the apoB RNA region
encompassing C6802 may function as a recognition element
for the editing complex (Figs. 4 and 10). According to our two-step
model for apoB mRNA editing, such recognition could bring the
editing complex into closer proximity to the editing site at
C6666 (22). In a second step, the enzyme complex may then
bind with a much higher affinity to the editing site at
C6666 than to the sequence elements that compose the
binding site for the C6802 (Fig. 5).
Hyperediting of ApoB mRNA--
When APOBEC-1 is overexpressed,
multiple cytidines are hyperedited (21-23). Detailed examination of
the editing of one of these cytidines, C6802, in small apoB
RNAs showed that sequences similar to those important in physiological
apoB mRNA editing are also involved in the hyperediting of
C6802. Hyperediting at C6802 was supported by
three RNA regions in the apoB mRNA: a proximal 5'-flanking region
(6747-6759), a mooring-like motif (6809-6819), and a 3'-flanking
region (6853-6882) (Fig. 10).
The 5'-flanking region has a greater influence than the 3'-flanking
region on the hyperediting of C6802. A proximal 5'-flanking
region (6747-6759) and 5' sequences upstream from that element plus
the mooring-like motif were all that was necessary for high level
(11.8%) hyperediting of C6802 (Fig. 5). Like the 5' A-rich
element that enhanced physiological apoB mRNA editing, the proximal
5'-flanking region (6747-6759) that enhanced the hyperediting of
C6802 is AU-rich (Fig. 6). Three A
G substitutions in
this element abolished hyperediting (Fig. 6). The importance of the
3'-flanking region for the hyperediting of C6802 was
demonstrated in the experiment shown in Fig. 9. An apoB RNA substrate
lacking the proximal 5'-flanking region was not hyperedited at
C6802 until the 3'-flanking region (6853-6882) was added.
Intriguingly, the minimal size of flanking regions required for
hyperediting at C6802 was comparable to that required for
efficient physiological editing at C6666. Hyperediting and
physiological editing required 55-57 nt of upstream flanking sequences
and approximately 80 nt of downstream flanking sequences (Fig. 10). The
similar size of the required flanking sequences and the importance of
the mooring-like motif for hyperediting at C6802 (Fig. 8)
suggest that hyperediting depends on loose recognition of RNA features
that define the physiological editing site at C6666.
Besides the flanking sequences, a mooring-like motif supports
hyperediting at C6802. Several mutagenesis studies
demonstrated that physiological apoB mRNA editing depends on the
highly conserved mooring sequence and the spacer region (10-13).
Single substitution mutations in the mooring sequence either
drastically decreased or abolished editing (10, 12), and alterations in
the 4-nt spacer decreased editing severalfold (10, 13). However, the
mooring-like motif downstream of C6802 has only 6 of the 11 nt in the mooring sequence and has a spacer region of 6 rather than 4 nt. Furthermore, it consists of seven Us and four As and lacks the TGAT
motif previously suggested to support hyperediting (23). Nevertheless,
this mooring-like motif supported hyperediting of C6802,
and scrambling the mooring-like pattern abolished hyperediting of
C6802 in short apoB RNAs and reduced hyperediting in longer
apoB RNAs (Figs. 7 and 8). Furthermore, the introduction of an exact
mooring motif (B6747M) increased hyperediting at this site 2-fold (Fig. 8). These results emphasize that the mooring-like pattern is essential for hyperediting at C6802 and that not every AU-rich
sequence supports hyperediting at C6802, although APOBEC-1
has been shown to bind to AU-rich RNA (31, 32).
The mooring-like motif at C6802 supports hyperediting of Cs
located up to 27 nt upstream. When the mooring-like motif downstream of
C6802 was mutated, hyperediting of C6802 was
diminished or abolished (Figs. 7 and 8). Furthermore, both mutations
abolished hyperediting at C6782/3 (Fig. 7), indicating that
hyperediting of C6782/3 depends also on the mooring-like
motif located 26-27 nt farther downstream. Hence, hyperediting has a
relaxed spacer constraint compared with physiological editing, which
was abolished by increasing the spacer to 12 nt (10, 11, 33).
Our in vitro study shows that the essential elements for
physiological editing at C6666 of the apoB mRNA
encompass 139 nt, consisting of defined upstream efficiency elements,
the mooring sequence, and the 3' efficiency element. Further
investigations on the secondary structure of the apoB mRNA editing
locus are needed to define the interaction of these elements. Our study
also shows that similar RNA features are necessary for the hyperediting
of cytidines when APOBEC-1 is overexpressed.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Shinya Yamanaka for sharing
technical experience and for the many helpful discussions; Drs. Robert
Pitas and John Taylor for valuable comments on the manuscript; Amy
Corder and Stephen Gonzales for photography; John Carroll for graphics; Gary Howard and Stephen Ordway for editorial support; and September Plumlee for manuscript preparation.
 |
FOOTNOTES |
*
This work was supported in part by the National Institutes
of Health Program Project Grant HL47660 and by Boehringer Ingelheim International, GmbH, as well as by fellowships from the Swiss National
Science Foundation and Cancer Research Switzerland (to M. H.).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.
To whom correspondence should be addressed: Gladstone Inst. of
Cardiovascular Disease, P.O. Box 419100, San Francisco, CA 94141-9100. Tel.: 415-826-7500; Fax: 415-285-5632; E-mail:
martin_hers berger{at}quickmail.ucsf.edu.
1
The abbreviations used are: apoB, apolipoprotein
B; nt, nucleotide(s); PCR, polymerase chain reaction.
 |
REFERENCES |
-
Boström, K.,
Garcia, Z.,
Poksay, K. S.,
Johnson, D. F.,
Lusis, A. J.,
and Innerarity, T. L.
(1990)
J. Biol. Chem.
265,
22446-22452[Abstract/Free Full Text]
-
Hodges, P. E.,
Navaratnam, N.,
Greeve, J. C.,
and Scott, J.
(1991)
Nucleic Acids Res.
19,
1197-1201[Abstract]
-
Johnson, D. F.,
Poksay, K. S.,
and Innerarity, T. L.
(1993)
Biochem. Biophys. Res. Commun.
195,
1204-1210[CrossRef][Medline]
[Order article via Infotrieve]
-
Navaratnam, N.,
Morrison, J. R.,
Bhattacharya, S.,
Patel, D.,
Funahashi, T.,
Giannoni, F.,
Teng, B.-B.,
Davidson, N. O.,
and Scott, J.
(1993)
J. Biol. Chem.
268,
20709-20712[Abstract/Free Full Text]
-
Chen, S.-H.,
Habib, G.,
Yang, C.-Y.,
Gu, Z.-W.,
Lee, B. R.,
Weng, S.-A.,
Silberman, S. R.,
Cai, S.-J.,
Deslypere, J. P.,
Rosseneu, M.,
Gotto, A. M., Jr.,
Li, W.-H.,
and Chan, L.
(1987)
Science
238,
363-366[Medline]
[Order article via Infotrieve]
-
Powell, L. M.,
Wallis, S. C.,
Pease, R. J.,
Edwards, Y. H.,
Knott, T. J.,
and Scott, J.
(1987)
Cell
50,
831-840[Medline]
[Order article via Infotrieve]
-
Chan, L.,
Chang, B. H.-J.,
Nakamuta, M.,
Li, W.-H.,
and Smith, L. C.
(1997)
Biochim. Biophys. Acta
1345,
11-26[Medline]
[Order article via Infotrieve]
-
Innerarity, T. L.,
Borén, J.,
Yamanaka, S.,
and Olofsson, S.-O.
(1996)
J. Biol. Chem.
271,
2353-2356[Free Full Text]
-
Navaratnam, N.,
Patel, D.,
Shah, R. R.,
Greeve, J. C.,
Powell, L. M.,
Knott, T. J.,
and Scott, J.
(1991)
Nucleic Acids Res.
19,
1741-1744[Abstract]
-
Backus, J. W.,
and Smith, H. C.
(1992)
Nucleic Acids Res.
20,
6007-6014[Abstract]
-
Driscoll, D. M.,
Lakhe-Reddy, S.,
Oleksa, L. M.,
and Martinez, D.
(1993)
Mol. Cell. Biol.
13,
7288-7294[Abstract]
-
Shah, R. R.,
Knott, T. J.,
Legros, J. E.,
Navaratnam, N.,
Greeve, J. C.,
and Scott, J.
(1991)
J. Biol. Chem.
266,
16301-16304[Abstract/Free Full Text]
-
Chen, S.-H.,
Li, X. X.,
Liao, W. S. L.,
Wu, J. H.,
and Chan, L.
(1990)
J. Biol. Chem.
265,
6811-6816[Abstract/Free Full Text]
-
Backus, J. W.,
and Smith, H. C.
(1994)
Biochim. Biophys. Acta
1217,
65-73[Medline]
[Order article via Infotrieve]
-
Boström, K.,
Lauer, S. J.,
Poksay, K. S.,
Garcia, Z.,
Taylor, J. M.,
and Innerarity, T. L.
(1989)
J. Biol. Chem.
264,
15701-15708[Abstract/Free Full Text]
-
Davidson, N. O.,
Innerarity, T. L.,
Scott, J.,
Smith, H.,
Driscoll, D. M.,
Teng, B.,
and Chan, L.
(1995)
RNA
1,
3[Medline]
[Order article via Infotrieve]
-
Driscoll, D. M.,
and Zhang, Q.
(1994)
J. Biol. Chem.
269,
19843-19847[Abstract/Free Full Text]
-
Teng, B.,
Burant, C. F.,
and Davidson, N. O.
(1993)
Science
260,
1816-1819[Medline]
[Order article via Infotrieve]
-
Yamanaka, S.,
Poksay, K. S.,
Balestra, M. E.,
Zeng, G.-Q.,
and Innerarity, T. L.
(1994)
J. Biol. Chem.
269,
21725-21734[Abstract/Free Full Text]
-
Yamanaka, S.,
Balestra, M. E.,
Ferrell, L. D.,
Fan, J.,
Arnold, K. S.,
Taylor, S.,
Taylor, J. M.,
and Innerarity, T. L.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
8483-8487[Abstract]
-
Yamanaka, S.,
Poksay, K. S.,
Driscoll, D. M.,
and Innerarity, T. L.
(1996)
J. Biol. Chem.
271,
11506-11510[Abstract/Free Full Text]
-
Yamanaka, S.,
Poksay, K. S.,
Arnold, K. S.,
and Innerarity, T. L.
(1997)
Genes Dev.
11,
321-333[Abstract]
-
Sowden, M.,
Hamm, J. K.,
and Smith, H. C.
(1996)
J. Biol. Chem.
271,
3011-3017[Abstract/Free Full Text]
-
Garcia, Z. C.,
Poksay, K. S.,
Boström, K.,
Johnson, D. F.,
Balestra, M. E.,
Shechter, I.,
and Innerarity, T. L.
(1992)
Arterioscler. Thromb.
12,
172-179[Abstract]
-
Higuchi, R.
(1990)
in
PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds), pp. 177-183, Academic Press, San Diego
-
Driscoll, D. M.,
Wynne, J. K.,
Wallis, S. C.,
and Scott, J.
(1989)
Cell
58,
519-525[Medline]
[Order article via Infotrieve]
-
Heinemann, T.,
Metzger, S.,
Fisher, E. A.,
Breslow, J. L.,
and Huang, L.-S.
(1994)
J. Lipid Res.
35,
2200-2211[Abstract]
-
Smith, H. C.
(1993)
Semin. Cell Biol.
4,
267-278[CrossRef][Medline]
[Order article via Infotrieve]
-
Zuker, M.
(1989)
Science
244,
48-52[Medline]
[Order article via Infotrieve]
-
Zuker, M.,
and Jacobson, A. B.
(1995)
Nucleic Acids Res.
23,
2791-2798[Abstract]
-
Anant, S.,
MacGinnitie, A. J.,
and Davidson, N. O.
(1995)
J. Biol. Chem.
270,
14762-14767[Abstract/Free Full Text]
-
Navaratnam, N.,
Bhattacharya, S.,
Fujino, T.,
Patel, D.,
Jarmuz, A. L.,
and Scott, J.
(1995)
Cell
81,
187-195[Medline]
[Order article via Infotrieve]
-
Greeve, J.,
Altkemper, I.,
Dieterich, J.-H.,
Greten, H.,
and Windler, E.
(1993)
J. Lipid Res.
34,
1367-1383[Abstract]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.