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J. Biol. Chem., Vol. 275, Issue 30, 22663-22669, July 28, 2000
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From the Departments of § Biochemistry and Biophysics
and
Received for publication, December 28, 1999, and in revised form, May 26, 2000
Post-transcriptional editing of apolipoprotein B
(apoB) mRNA is regulated in hepatic cells to achieve a steady state
proportion of edited and unedited RNA molecules. This activity is
catalyzed by APOBEC-1 (apoB mRNA editing
catalytic subunit 1) in what has been widely accepted as
nuclear event occurring during or after mRNA splicing. Introns
impair the efficiency of editing within an adjacent exon in a
distance-dependent manner in reporter RNAs. We show here
that this inhibition can be overcome by overexpressing APOBEC-1 and
that the enhanced editing efficiency on these reporter RNAs occurred
after splicing on cytoplasmic transcripts. Given the absolute
requirement of auxiliary proteins in apoB mRNA editing, the data
suggested that auxiliary proteins were distributed with APOBEC-1 in
both the nucleus and cytoplasm of McArdle cells. In fact,
immunolocalization of one such auxiliary protein, APOBEC-1
complementation factor (ACF) demonstrated a nuclear and cytoplasmic distribution. We also demonstrate that in the
absence of alterations in APOBEC-1 expression, changes in edited apoB
RNA induced by ethanol arise through the stimulation of nuclear editing
activity. The finding that apoB mRNA editing can occur in the
cytoplasm but normally does not suggests that under biological
conditions, restricting editing activity to the nucleus must be an
important step in regulating the proportion of the edited apoB mRNAs.
Apolipoprotein B (apoB)1
mRNA editing involves a site-specific deamination reaction wherein
a cytidine at nucleotide 6666 is changed to a uridine thereby placing
an in frame UAA stop codon within the 14-kilobase apoB mRNA
(1, 2). Translation of the unedited and edited variants of apoB
mRNA generates two isoforms of apoB proteins, apoB100 and apoB48,
which behave differently in terms of lipoprotein secretion and uptake
by peripheral cells (3). A tripartite RNA sequence motif, consisting of
an 11-nucleotide mooring sequence, a spacer, and a regulatory element,
is required for site-specific RNA editing (4-8). Recently, a stem-loop
model of secondary structure involving the essential sequence required for editing has been proposed (9, 10). These cis-acting elements are required for the assembly of an editing complex, the C/U
editosome (5, 11), and site-specific editing activity. APOBEC-1,
the cytidine deaminase responsible for editing (12), is the
catalytic subunit of the editosome (9) and may function in this
capacity as a dimer (13, 14).
APOBEC-1 alone cannot edit apoB mRNA and requires multiple
yet-to-be characterized proteins referred to collectively as the auxiliary proteins (15-20). Auxiliary proteins are broadly expressed in mammalian cell lines and tissues, independent of the expression of
APOBEC-1 and apoB mRNA (12, 16-18, 21). Recently,
APOBEC-1 complementation factor
(ACF) has been cloned (40). ACF is 64.3-kDa RNA binding protein, it
binds to apoB mRNA in vitro and in vivo. ACF
and APOBEC-1 comprise the minimal protein requirements for specific and
efficient editing of apoB mRNA in vitro. Other candidate auxiliary proteins such as an hnRNP A/B homolog ABBP-1, hnRNP D, and
hnRNP C (22-24), mooring sequence-selective RNA-binding proteins of
100 and 55 kDa (15, 20, 25-27), and general RNA-binding proteins
40-44 kDa (15, 20, 25, 28) have been identified through their affinity
for APOBEC-1 or apoB RNA. A complex of proteins (referred to as AUX240
for the 240-kDa antigenic protein it contains) identified with
monoclonal antibodies raised against in vitro assembled 27 S
editing complexes (editosomes) (29) has also been proposed to contain
auxiliary proteins. The specific functional role that each candidate
auxiliary protein may play in apoB mRNA editing remains to be
further clarified.
A direct demonstration of the intracellular site of editing activity
through immunolocalization of APOBEC-1 has not been possible due to the
very low level of expression of the enzyme in cells and tissues.
Evidence for a nuclear localization of the editing activity was first
provided through the demonstration of editing on polyadenylated but
unspliced apoB mRNA and that the level of editing of spliced
nuclear apoB mRNA was equivalent to that of cytoplasmic or total
cellular mRNA (30, 31). These findings were corroborated in studies
in which chimeric splicing/editing RNA substrates were used to
demonstrate that the close proximity of introns to the editing site
within apoB encoding exon inhibited editing activity in McArdle cells
until splicing had occurred (32). Taken together, the data suggested
that editing of apoB mRNA took place in the cell nucleus; however,
the possibility of cytoplasmic editing has not been ruled out.
The localization of APOBEC-1 has been evaluated by indirect
immunofluorescence microscopy of HA-tagged APOBEC-1 in transiently transfected McArdle cells and HepG2 human hepatoma cells (33, 34). The
enzyme was distributed in both the nucleus and cytoplasm of these cells
but was localized in the cytoplasm of cells that did not support
editing activity (Chinese hamster ovary and COS-7 cells). The nuclear
distribution of APOBEC-1 required an N-terminal sequence (residues
13-35) similar to a bipartite nuclear localization signal (NLS). This
sequence, however, was not sufficient to direct chimeric proteins to
the nucleus and hence did not satisfy the strict definition of an NLS
(33). The data suggested that auxiliary proteins may play an important
role in determining the intracellular distribution of APOBEC-1.
In contrast, residues 174-180 in the C terminus of APOBEC-1 behaved as
a strong nuclear export signal (NES)/cytoplasmic retention signal (CRS)
and were sufficient in directing chimeric proteins to the cytoplasm
(33). These findings further suggested that the nuclear and cytoplasm
distribution may be due to the combined activities of interactions
involving APOBEC-1's NLS and NES/CRS signals.
We report here that apoB mRNA editing can be experimentally induced
in the cytoplasm of McArdle cells, suggesting that auxiliary proteins
as well as APOBEC-1 are distributed in both the nucleus and cytoplasm
of liver cells. Biological alterations in the proportion of edited apoB
mRNA appear, however, to result primarily or exclusively from
changes in nuclear editing activity.
Plasmid Constructions--
The construction of plasmids encoding
His6-HA-APOBEC-CMPK,
His6-HA-APOBEC-1, and
His6-HA-CMPK hence referred to as APOBEC-CMPK, APOBEC-1, and CMPK, respectively (33), and the splicing/editing substrate IVS-apoB (32) has been described previously. Human ACF
(40) was amplified from human total liver RNA by RT-PCR methodology
with the following primers 5'-CTCGATATCATGGAATCAAATCACAAATCCGG and
5'-CTCTCTAGATCAGAAGGTGCCATATCCATC and subcloned via EcoRV and XbaI sites downstream of HA and His6
epitope tags in a modified pcDNA3 vector (33).
Tissue Culture--
The rat hepatoma McArdle RH7777 cells were
obtained from ATCC (Manassas, VA). Cells were transfected as
reported previously (32). Stable cell lines transfected with
apobec-cmpk were obtained by limiting dilution under 500 µg/ml G418 (Life Technologies, Inc.) selection 48 h after
transfection. Cells were treated with ethanol by rapidly mixing an
appropriated aliquot of ethyl alcohol (AAPER Alcohol and Chemical
Co.) into media as described previously (39).
Preparation of S-100 Extracts and Recombinant
Protein--
McArdle cells were processed into S-100 extracts as
described previously (29). Recombinant proteins were expressed in
Escherichia coli and purified by nickel-nitrilotriacetic
acid (Qiagen) metal-chelating chromatography as described
previously (19).
Isolation of Total, Cytoplasmic, and Nuclear RNA--
Total
cellular RNA was isolated from approximately 2 × 106
cells with Tri-Reagent (Molecular Research Center) according to the manufacturer's recommendations 24 h after transfection.
Differential isolation of nuclear and cytoplasmic RNA was achieved by
using NE-PERTM Nuclear and Cytoplasmic Extraction Kit
(Pierce) with the following modifications to the manufacturer's
protocols. After cells were scraped from the tissue culture plates and
processed through CER-I, RNase inhibitor (Eppendorf Scientific,
Inc.) was added before the cells were lysed in CER-II buffer.
The cytoplasmic fraction that was collected following centrifugation
was transferred immediately to a fresh tube and mixed with 2 volumes of
Tri-Reagent. The pellet containing partially purified nuclei (as
evaluated by phase microscopy) was resuspended with Buffer B (50 mM Tris-HCl, pH 8.0, 5 mM MgCl2) and purified by sedimentation through Buffer C (1 M
sucrose, 50 mM Tris-HCl, pH 8.0, 5 mM
MgCl2) and centrifuged (150,000 × g for 15 min) at 7 °C. The purity of nuclei was assessed by microscopy, and
nuclear RNA was extracted with 2 volumes of Tri-Reagent.
Purified RNAs were digested with RQ-DNase I (Promega, Madison, WI) and RsaI or EcoRI to ensure the removal of
copurifying endogenous rat or transfected human apoB DNA, respectively,
prior to RT-PCR. Differential RT-PCR amplification of RNA transcripts
at different stages in RNA processing was performed using primer pairs
MS1/MS2 to generate PCR products from the prespliced IVS-apoB message, whereas MS3/MS2 was used to generate PCR product from the apoB exon (Fig. 1A). Since the ratio of unspliced apoB mRNA
versus completely processed apoB mRNA in total cellular
RNA pool is small, the PCR products generated from apoB-specific primer
pairs MS3/MS2 are more likely to represent mature apoB mRNA.
Editing Assays--
The selective amplification of endogenous
rat apoB mRNA or of human apoB mRNA in the reporter RNAs was
performed using oligo(dT)-primed first strand cDNA synthesis
coupled with RT-PCR as described previously (32). For in
vitro apoB RNA editing activity, 50-µl editing reactions
containing 20 fmol of in vitro transcribed apoB RNA, 50 µg
of S-100 extract from wild type McArdle cells and recombinant APOBEC-CMPK proteins were incubated at 30 °C for 3 h under
conditions described previously (19, 20, 33). Editing efficiency was evaluated by poisoned primer extension analysis, and the primer extension products were resolved on a 10% denaturing polyacrylamide gel and quantified by laser densitometric scanning (PhosphorImager 425E, Molecular Dynamics) as described previously (20, 32-34).
Immunoassays--
Cells fixed with 2% paraformaldehyde and
permeabilized with 0.4% Triton X-100 were incubated with anti-HA
monoclonal antibody for 1 h at room temperature (Babco, Berkeley,
CA; 1:500 in phosphate-buffered saline containing 3% bovine serum
albumin), followed by incubation for 1 h at room temperature with
fluorescein-conjugated goat anti-mouse antibody (ICN/Cappel; 1:25 in
3% bovine serum albumin/phosphate-buffered saline). Coverslips were
mounted onto DAPI containing mounting medium. Slides were
observed under an Olympus BH-2 fluorescence microscope using a 40×
Olympus objective.
Protein extracts were made from approximately equal number of
transfected cells and subsequently resolved and transferred to
nitrocellulose membrane, probed with anti-HA monoclonal antibody as
described previously (33).
The Inhibitory Effect of Introns on ApoB mRNA Editing Is
Reduced by Overexpressing APOBEC-1 in McArdle Cells--
When the
mRNA splicing cassette from the adenovirus late leader sequence was
placed either 5' or 3' of a 492-nucleotide apoB exon containing the
editing site, editing efficiency was markedly reduced in wild type
McArdle cells (32). In these studies, the number of edited apoB
mRNA molecules at steady state was demonstrated to be dependent on
the level of APOBEC-1 expression and was independent of the abundance
of apoB mRNA or the presence of competing RNA editing substrates
(32, 34). Taken together, the data suggested the "gating
hypothesis" wherein editing was proposed to occur at a temporal
and/or a spatial point (a gate) through which all apoB pre-mRNAs
pass during their maturation. The number of apoB mRNA molecules
that are edited at the gate was proposed to be determined by the
probability of assembling functional editosomes on each mRNA, which
in turn is determined by the number of APOBEC-1 molecules available for
assembly into these editosomes. As a further test of the gating
hypothesis, wild type McArdle cells and a stable transfected McArdle
cell line expressing APOBEC-1 (McAPOBEC cells, Ref. 20) were
transfected with plasmids encoding a splicing-editing RNA (IVS-apoB
RNA) and the editing efficiency evaluated by poisoned primer extension
analysis of RT-PCR product as described under "Experimental Procedures."
As determined previously (32), the presence of an intron severely
impaired editing efficiency of IVS-apoB RNA (Fig.
1A) in wild type McArdle cells
2.7% (n = 3, S.E. = 1.0%) (Fig. 1B, first lane). Overexpression of APOBEC-1 in McAPOBEC cells
markedly stimulated the editing of IVS-apoB 44% (n = 3, S.E. = 1.0%) (second lane). Additional editing of
C6661 due to overexpression of APOBEC-1 (promiscuous
editing Refs. 34-37) was also observed.
To evaluate whether the regulation of editing activity had occurred at
a "gate" localized within the cell nucleus, the proportion of
edited IVS-apoB RNA was determined in subcellular fractions of wild
type McArdle and McAPOBEC cells. The quality of the nuclear preparation
was evaluated by phase microscopy (Fig. 1C), the gross morphological integrity of the isolated nuclei, and the virtual absence
of cytoplasmic materials was apparent.
The amount of edited nuclear IVS-apoB was low in wild type McArdle
cells 2.1% (n = 3, S.E. = 0.5%) but may have been
slightly higher than that determined in the cytoplasm 1.7%
(n = 3, S.E. = 0.3%) (compare the first two
lanes of Fig. 1D). In contrast, an elevated proportion
of edited IVS-apoB RNA was more apparent in the cytoplasm 48%
(n = 3, S.E. = 2.3%) of McAPOBEC cells compared with
that measured on nuclear IVS-apoB RNA 7.8% (n = 3, S.E. = 4.4%, the last two lanes of Fig. 1D). The
markedly higher proportion of edited RNA in the cytoplasm of McAPOBEC
cells compared with that found in the nucleus suggested the
unprecedented possibility that additional editing of spliced IVS-apoB
RNA had occurred in the cytoplasm. To evaluate this possibility, an
APOBEC-1 chimera was designed that would retain the enzyme in the
cytoplasm through the addition of full-length CMPK (chicken muscle
pyruvate kinase, a well characterized neutral cargo protein in
the nuclear-cytoplasmic trafficking field) as bulk protein
sequence (33).
APOBEC-CMPK Fusion Protein Edits ApoB RNA in Vitro--
To
evaluate whether editing could occur in the cytoplasm, we targeted
APOBEC-1 to the cytoplasm by taking advantage of our previous finding
that the addition of protein mass to APOBEC-1 impaired the ability of
the chimeric enzyme to localize within the nucleus (33, 34). The
chimeric enzyme retained editing activity as evident from the ability
of HA- and His6-tagged APOBEC-CMPK fusion protein,
purified from E. coli. to stimulate in vitro apoB editing in a concentration-dependent manner when added to
wild type McArdle cell extracts (Fig.
2).
APOBEC-CMPK Is Localized in the Cytoplasm--
Having demonstrated
that APOBEC-CMPK was functional in apoB mRNA editing, McArdle cells
were transfected with plasmid encoding APOBEC-CMPK and evaluated for
the intracellular distribution of this chimeric protein using
antibodies specific for the HA tag in indirect immunofluorescence
analysis. The data suggested that most, if not all, of APOBEC-CMPK was
localized in the cytoplasm of transiently transfected McArdle cells
(Fig. 3).
APOBEC-CMPK Expression in McArdle Cells Increases Editing
Activity--
If apoB mRNA editing was primarily a nuclear event,
overexpression of APOBEC-CMPK should cause little or no change in the editing activity of McArdle cells due to its cytoplasmic distribution. To evaluate editing of the endogenous, McArdle cell apoB mRNA, cells were transiently transfected with plasmids encoding either APOBEC-1, CMPK, or APOBEC-CMPK cDNAs, and total cellular RNAs were
isolated and analyzed. The expression of full-length wild type and
chimeric proteins was evident on Western blots of cell extracts from an
equivalent number of transfected cells (~105) that were
reacted with HA-specific antibody (Fig.
4A). APOBEC-1 expression
elevated apoB mRNA editing in McArdle cells to 48% (n = 3, S.E. = 3.9%), whereas overexpression of CMPK
as a negative control did not significantly change editing activity
from that observed in untransfected cells, 14% (n = 3, S.E. = 1.9%) (Fig. 4B). In contrast, overexpression of
APOBEC-CMPK increased apoB mRNA editing efficiency to 50%
(n = 3, S.E. = 4.6%).
To evaluate whether the editing efficiency observed in cells expressing
APOBEC-CMPK was dependent on the expression level of the enzyme,
McArdle cell lines were established by clonal selection in the presence
of G418, and cell lines expressing low, medium, and high levels of
APOBEC-CMPK fusion protein were identified by Western blotting (Fig.
4C). Primer extension analysis of RT-PCR product specific to
endogenous McArdle cell apoB mRNA demonstrated that the editing
efficiency in three cell lines expressing low, medium, or high levels
of APOBEC-CMPK was 22% (n = 3, S.E. = 1.2%), 50%
(n = 3, S.E. = 1.8%), and 93%
(n = 3, S.E. = 2.4%), respectively (Fig.
4D). The data demonstrated that the level of APOBEC-CMPK expression determined the proportion of edited apoB mRNA despite the enzyme's distribution in the cytoplasm and thereby support the
possibility of cytoplasmic editing.
The Editing of IVS-ApoB RNA Is Restored by APOBEC-CMPK--
The
data raised the question of whether cytoplasmic APOBEC-CMPK was active
on cytoplasmic apoB mRNA or whether a small amount of APOBEC-CMPK,
which was below the detection limit of immunofluorescence microscopy,
was in the nucleus for a period of time long enough to edit nuclear
apoB. If APOBEC-CMPK's editing activity was cytoplasmic, then the
proportion of edited apoB should not be inhibited by the placement of
introns adjacent to the editing site, and the proportion of edited apoB
RNAs in the cytoplasm should be substantially higher than that observed
with nuclear apoB RNA. In contrast, if APOBEC-CMPK editing activity
occurred exclusively or predominantly in the nucleus, then nuclear apoB
RNAs should reflect elevated levels of editing activity equivalent to
that determined on total cellular apoB RNAs.
The cellular site of editing was evaluated in the APOBEC-CMPK high
level expression McArdle cell line and wild type McArdle cells as
negative control following transiently transfection of a plasmid
encoding IVS-apoB. The proportion of edited apoB RNA was assayed
24 h after transfection to ensure an early enough time point where
maximal detection of nuclear editing would theoretically occur before a
steady state cytoplasmic pool of edited RNA had been achieved. RT-PCR
amplification of IVS-apoB RNA transcripts at different stages in RNA
processing was performed using the indicated primer pairs (Fig.
1A) as described under "Experimental Procedures." The
data demonstrated that IVS-apoB was poorly edited in McArdle cells
regardless of the stage of splicing, unspliced IVS-apoB RNA, 2.4%
(n = 3, S.E. = 0.5%) and apoB exon RNA-specific, 2.7%
(n = 3, S.E. = 1.0%) (Fig.
5, first two lanes). Unspliced IVS-apoB RNA was marginally edited 3% (n = 3, S.E. = 0.7%) in the APOBEC-1-CMPK high expression cell line; however, editing of IVS-apoB after splicing was markedly elevated 64%
(n = 3, S.E. = 3.9%) (Fig. 5, the last two
lanes), and promiscuous editing was also observed.
To further confirm the occurrence of cytoplasmic editing, nuclear and
cytoplasmic fractions were prepared from APOBEC-1-CMPK high expression
cell line transiently transfected with IVS-apoB. The exon corresponding
to human apoB sequence in IVS-apoB RNA was specifically amplifies from
RNA in both cellular subfractions using MS2/MS3 amplimers.
Nuclear editing of IVS-apoB in APOBEC-1-CMPK high expression cell line
was low, 8.0% (n = 3, S.E. = 2.9%) compared with 69%
(n = 3, S.E. = 4.6%) editing in the cytoplasmic
fraction (Fig. 6). These data strongly
suggest that although it is possible that a small amount of nuclear
editing activity had occurred in the APOBEC-CMPK overexpressing cell
line after splicing but before RNA export (3% versus 8%,
compare Figs. 5 and 6), most of the editing activity had to have taken
place in the cytoplasm. Taken together, the data indicated that the
interference from adjacent introns on editing can be overcome if the
editing enzyme was specifically targeted to the cytoplasm as in the
case of APOBEC-CMPK cell line or when APOBEC-1 itself is
overexpressed.
ACF Is Localized in Both Nuclear and Cytoplasmic Compartments of
McArdle Cells--
We have taken advantage of the recent findings by
Mehta et al. (40) that ACF and APOBEC-1 comprise the minimal
protein requirements for apoB mRNA editing in vitro to
probe the intracellular distribution of an auxiliary protein in McArdle
cells (Fig. 7). Given ACF's important
role in apoB mRNA editing, the subcellular distribution of ACF
would provide more complete proof for the subcellular localization of
the editing activity. To this end, cells were stained 16 h after
transfection with full-length HA-tagged ACF to ensure minimal level of
expression and avoid potential artifacts due to overexpression and
saturation of nuclear transport process. Indirect immunofluorescence microscopy revealed that HA-tagged ACF had both a nuclear and cytoplasmic distribution in McArdle cells, thus supporting the data
suggesting that editosomes could be assembled in the cytoplasm.
Ethanol Stimulates Predominantly Nuclear ApoB RNA Editing in
McArdle Cells--
The ability of experimentally manipulated cells to
support cytoplasmic mRNA editing suggests that constraints must be
present in wild type cells that regulate the proportion of edited
mRNAs such that only a fraction of the total cytoplasmic apoB
mRNA is edited for any given metabolic condition (3). To evaluate
this possibility in the context of wild type cells, we have taken
advantage of the ability of ethanol to rapidly (39) stimulate editing activity in liver cells without inducing the expression of APOBEC-1 (38). It is anticipated that if ethanol increased editing by the
induction of the cytoplasmic editing, the increase in editing would
only be observed in the cytoplasm. In contrast, a rapid induction of
nuclear editing by ethanol followed by an increase in edited
cytoplasmic apoB mRNA would suggest that under these conditions
alterations in edited cytoplasmic mRNA are secondary to those in
the nucleus and the result of the nuclear export of mRNA to the cytoplasm.
McArdle cells were treated with ethanol as described previously (39)
and subsequently fractionated into nuclei and cytoplasm at the
indicated time points after treatment, and the proportion of edited
apoB mRNA was determined as described above (Fig.
8A). The editing efficiency
over the course of treatment is summarized (Fig. 8B), and
the ratio of edited apoB mRNA in treated versus untreated cell fractions is plotted (Fig. 8C). The
relative proportion of edited RNA in the nuclear compartment of
ethanol-treated cells increased rapidly, showing a 1.8-fold increase
over that in untreated cells within 15 min. Following slight
fluctuations at 30-min and 1-h time points, the proportion of edited
nuclear apoB mRNA remained increased 1.4-fold in ethanol-treated
cells relative to that in untreated cells up to 14 h after
treatment. Increased edited apoB mRNA was not apparent in the
cytoplasmic compartment until 30 min after ethanol treatment, after
which point it followed the trend of the nuclear compartment and
achieved a 1.4-fold increase over that measured in untreated cells by
14 h. The increase in editing efficiency in the cytoplasm lagged
that observed in the nucleus, suggesting that ethanol increased editing
of nuclear apoB mRNAs, which were subsequently exported to the
cytoplasm, where they contributed to an increase in the proportion of
edited cytoplasmic RNA at a later time point. Taken together, the data suggest that ethanol increased apoB mRNA editing through the
selective induction of nuclear editing activity.
This is the first description of cytoplasmic editing of apoB
mRNA. The expression of APOBEC-1 in rat liver or McArdle cells is
below the detection limit of most conventional immunoassays, and hence
localization studies have had to rely on epitope-tagged proteins to
study the enzyme's subcellular distribution. In these studies,
immunofluorescence microscopy demonstrated that HA-tagged or green
fluorescent protein-tagged APOBEC-1 was distributed in both the nucleus
and cytoplasm of transiently or stably transfected McArdle cells (33,
34). Recent studies with a variety of epitope-tagged proteins involved
in RNA processing demonstrated biologically relevant localization of
these proteins even under conditions of their overexpression (41). The
dual intracellular distribution has been proposed to be biologically
significant as it was observed regardless of the level of APOBEC-1
overexpression (33, 34) and could be converted into 100% nuclear
distribution, even at the highest level of APOBEC-1 overexpression, by
deleting residues 174-180 encoding APOBEC-1's CRS/NES (33).
Although we do not know whether or not natively expressed APOBEC-1 has
a cytoplasmic and nuclear distribution, the data suggest that auxiliary
proteins are distributed in both cellular compartments. Given the
absolute requirement of auxiliary proteins for APOBEC-1 mRNA
editing activity, the observation that McArdle cells could carry out
editing in the nucleus and cytoplasm suggests the novel concept that
auxiliary proteins may also be distributed in both cellular
compartments. We cannot rule out the trivial explanation that nuclear
auxiliary factors were recruited to the cytoplasm by APOBEC-1 upon its
overexpression. This argument in favor of a bipartite distribution of
auxiliary factors was strongly supported by the data showing a nuclear
and cytoplasmic distribution of HA-tagged ACF, a 64.3-kDa apoB
RNA-binding auxiliary protein (40) in McArdle cells.
Our findings clarify a long standing question of why nuclear and whole
cell S100 extracts both contain editing activity. Previous studies have
demonstrated that efficient in vitro RNA editing activity
can be assayed with both nuclear and cytoplasmic extracts from rat
liver (30). Although an analysis of apoB mRNA editing at various
stages of apoB mRNA maturation suggested that apoB mRNA editing
occurred coincident or immediately subsequent to pre-mRNA splicing,
the authors reasoned that at least part of the cytosolic editing
activity in vitro could have resulted from leakage from the
nuclear compartment during the preparation of extracts. A biological
role for cytoplasmic apoB mRNA editing has been suggested from
mathematical modeling of steady state proportion of edited apoB RNA in
the cell and its regulation (31).
Wild type McArdle cells edit 8-20% of the endogenous apoB
mRNA that they transcribe (32, 33, 36, 42). The proportion of RNA
that is edited is related to the level of enzyme activity in the cell
and is not affected by the number of total RNAs in the cell or the
expression of competing RNA editing substrates. Therefore the inability
of wild type McArdle cells to efficiently edit IVS-apoB RNA is not due
to competition with the endogenous apoB mRNA substrate, but rather,
the data suggested that the intron in IVS-apoB had impaired editing. As
the only opportunity for this to occur is with the nucleus, the data
suggested that IVS-apoB mRNA editing had to occur in the nucleus
and that there was no further opportunity for editing to take place
once spliced IVS-apoB entered the cytoplasm. Taken together with the
data showing that ethanol altered the proportion of edited apoB
mRNA by predominantly increasing nuclear editing activity, we
propose that under biological conditions apoB mRNA editing may be
restricted to the nucleus.
Our findings raise an important question concerning the regulation of
the proportion of edited mRNAs in cells. If all the factors
required for editing have a nuclear and cytoplasmic distribution, why
does additional editing not take place on cytoplasmic mRNAs under
biological conditions? If there are regulatory molecules in the
cytoplasm, then overexpression of APOBEC-1 may have induced cytoplasmic
editing by exceeding the capacity of these factors to block the
enzyme's activity in the cytoplasm. Proteins have been identified
through a variety of affinity approaches that impair in
vitro editing activity and may therefore serve this regulatory
role in cells. For example, hnRNP C1 protein (24) and variants of hnRNP
D protein (23) inhibit APOBEC-1 editing activity in vitro.
Cytosolic extracts have been shown to contain auxiliary factors and
APOBEC-1 as 60 S aggregates (15). These aggregates could be
biochemically disaggregated in vitro to form 27 S complexes
with high levels of editing activity (15). Auxiliary proteins have been
identified in the 60 S complexes association with a 240-kDa antigen
(29). Whether the 60 S complexes or AUX240 play a role in
down-regulating editing activity in the cytoplasm remains to be determined.
APOBEC-1 abundance is maintained at very low levels in wild type cells.
Regulation of APOBEC-1 expression occurs under some forms of stimuli
(42). Our data demonstrated that experimental overexpression of
APOBEC-1 increased both nuclear and cytoplasmic editing. Very high
levels of enzyme must, however, be induced experimentally to affect
relatively small increases in cellular editing activity (34). We
interpreted our data therefore as suggesting that in wild type cells,
interactions between APOBEC-1 and auxiliary proteins in the cytoplasm
may be kinetically unfavorable. Such a situation could arise if
cytoplasmic auxiliary proteins did not have appropriate
post-translational modifications and/or were involved in more favorable
interactions with other cytoplasmic proteins. Overexpression of
APOBEC-1 or APOBEC-CMPK may have provided sufficient number of enzyme
molecules to overcome these kinetic barriers and to allow functional
editosomes to assemble on cytoplasmic apoB mRNA.
Finally, it was of great interest to see if cytoplasmic editing can be
induced by physiological/pharmacological stimuli when APOBEC-1 is not
overexpressed. Ethanol feeding was chosen to test this possibility,
because ethanol has been shown to up-regulate apoB mRNA editing
without increasing apobec-1 mRNA levels (38). Our
previous data showed that in rat primary hepatocytes, editing efficiency increased rapidly after treatment with 0.2-2.5% ethanol. The induction occurred rapidly and reached maximum levels within 2 h and remained elevated for 18 h after treatment (39). In the
present study, the kinetics of ethanol stimulated editing activity in
McArdle cells was evaluated at the level of which subcellular
compartment was maximally affected. The abundance of edited apoB
mRNA in the cytoplasm was slightly higher than that observed in the
nucleus in all cells regardless of time or ethanol treatment. Similar
difference in editing efficiency between completely processed nuclear
apoB mRNA and polysomal apoB mRNA has been reported (30).
Significantly, however, in ethanol-treated cells an increase in edited
apoB mRNA appeared first in the nucleus, followed by an increase in
the cytoplasm. The simplest explanation for these observations is that
ethanol increased editing activity on nuclear apoB mRNA, which then
exported to the cytoplasm, where it subsequently contributed to a
larger pool of edited mRNAs. Our data suggested that under
conditions where APOBEC-1 abundance is not altered, there is little or
no cytoplasmic editing of apoB mRNA. The data leave open the
possibility that alterations in edited apoB mRNA due to an
increased expression of APOBEC-1 may involve cytoplasmic editing activity.
In conclusion, we have demonstrated that apoB mRNA editing can
occur in the cytoplasm of McArdle cells when APOBEC-1 is overexpressed. The ability of McArdle cells to carry out apoB mRNA editing in both
nuclear and cytoplasmic locations suggests that the auxiliary proteins
are distributed along with APOBEC-1 in both compartments as further
demonstrated by the dual nuclear/cytoplasmic subcellular distribution
of ACF in McArdle cells. Ethanol treatment stimulates nuclear apoB
mRNA editing with rapid onset without affecting APOBEC-1 expression
level and does not induce cytoplasmic editing. The inability of wild
type or ethanol-treated McArdle cells to carry out cytoplasmic editing
suggests that negative regulatory mechanism(s) must be in place to
prevent it from occurring. Our results, therefore, have important
implications for how editing might be metabolically and developmentally
controlled and experimentally manipulated.
We thank Dr. Yi Yang for APOBEC-CMPK
construct. We are grateful to Jenny M. L. Smith for the
preparation of figures.
*
This work was supported in part by United States Public
Health Service Grant DK43739 and grants from The Council for Tobacco Research, The Alcoholic Beverage Medical Research Foundation, and the
Rochester Area Pepper Center for Research on Aging (to H. C. S.).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.
Published, JBC Papers in Press, May 31, 2000, DOI 10.1074/jbc.M910406199
The abbreviations used are:
apoB, apolipoprotein
B;
APOBEC-1, apoB mRNA editing catalytic subunit 1;
ACF, APOBEC-1
complementation factor;
CMPK, chicken muscle pyruvate kinase;
CRS, cytoplasmic retention signal, HA, hemagglutinin;
NES, nuclear export
signal, NLS, nuclear localization signal;
RT-PCR, reverse transcription
polymerase chain reaction;
DAPI, 4,6-diamidino-2-phenylindole.
Induction of Cytidine to Uridine Editing on Cytoplasmic
Apolipoprotein B mRNA by Overexpressing APOBEC-1*
,§, and§¶
Pathology and the ¶ Environmental Health
Sciences Center, University of Rochester School of Medicine and
Dentistry, Rochester, New York 14642
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (54K):
[in a new window]
Fig. 1.
The inhibitory effect of introns on apoB
mRNA editing is reduced by overexpressing APOBEC-1 in McArdle
cells. A, diagram of the chimeric apoB RNA
splicing/editing construct composed of the adenovirus-spliced
late-leader sequence and the human apoB sequence. The location of PCR
amplimer pairs for intron containing RNA (MS1/MS2) and specific to the
apoB exon (MS3/MS2) is shown. B, poisoned primer extension
assay of apoB RNA editing. The position of the radiolabeled primer and
primer extension products corresponding to unedited apoB RNA
(CAA) and edited apoB RNA (UAA) is indicated.
Editing efficiency was calculated as the PhosphorImage density of UAA
divided by the sum of UAA and CAA and multiplied by 100. Editing
efficiencies were determined on MS3/MS2 RT-PCR products as 2.7 ± 1.0% and 44 ± 1.0%, respectively. Values are means ± S.E.
from three independent experiments. C, phase contrast
micrographs demonstrating gross morphological integrity and purity of
the nuclei isolated from McArdle cells and used as a source of RNA. The
slide containing a drop of nuclear suspension was examined by an
Olympus inverted microscope with 40× objectives. The image was
captured by a Kodak DCS system (Eastman Kodak Co., Rochester, NY).
D, poisoned primer extension assays of apoB RNA editing on
MS3/MS2 RT-PCR products amplified from RNA isolated from nuclear and
cytoplasmic subfractions. The proportion of edited IVS-apoB RNA in the
nucleus and cytoplasm was: 2.1 ± 0.5% and 1.7 ± 0.3%,
respectively (n = 3) for wild type McArdle cells and
7.8 ± 4.4% and 48 ± 2.3%, respectively (n = 3), for McAPOBEC cells. The primer extension products corresponding
to promiscuously edited IVS-apoB mRNA (33) are indicated as
"2" and were figured into the calculation of editing
efficiency whenever observed.
View larger version (93K):
[in a new window]
Fig. 2.
In vitro reconstitution of editing
activity using recombinant APOBEC-CMPK and wild type McArdle cell S-100
extracts. Primer extension analyses of editing activity are shown
that resulted from in vitro reactions containing 50 µg of
McArdle cell extracts and increasing amounts of recombinant APOBEC-CMPK
(0.5-5 µg, indicated as a ramp). Similar results were
obtained from two independent recombinant protein and extract
preparations. The percent editing was calculated as described in the
legend to Fig. 1B.
View larger version (61K):
[in a new window]
Fig. 3.
Subcellular localization of APOBEC-CMPK in
transiently transfected McArdle cells. The DAPI and anti
HA-APOBEC-CMPK stained images of the same high magnification field are
shown (left and right panels).
Arrowheads indicate the cell nucleus (Nu). Note
that the cell in the bottom left of the field was not
transiently transfected and serves as a background control for antibody
reactivity and autofluorescence.
View larger version (41K):
[in a new window]
Fig. 4.
Expression of APOBEC-CMPK in McArdle cells
increases editing activity. A, proteins from an
equivalent number of McArdle cells transiently transfected with
plasmids encoding APOBEC-1, CMPK, or APOBEC-CMPK were analyzed for the
expression of full-length protein by Western blotting using anti-HA
monoclonal antibody. B, poisoned primer extension analyses
were performed upon RT-PCR-amplified apoB cDNA templates from each
transfection. The editing efficiency of the corresponding transfections
in "B" from left to right was:
48 ± 3.9%, 14 ± 1.9%, and 50 ± 4.6%
(n = 3). C, proteins from an equivalent
number of cells from three clonal McArdle cell lines, each expressing a
different level of APOBEC-CMPK, were analyzed by Western blotting and
reacted with anti-HA antibody. D, editing of apoB mRNA
isolated from the cell lines in "C" was analyzed by
poisoned primer extension and quantified as described in the legend to
Fig. 1B. Editing efficiency was: 22 ± 1.2%, 50 ± 1.8%, and 93 ± 2.4% (n = 3) for the low to
high APOBEC-CMPK-expressing cell lines.
View larger version (51K):
[in a new window]
Fig. 5.
The editing of IVS-apoB RNA is restored by
APOBEC-CMPK expression. Wild type McArdle cells and the stable
cell line in Fig. 4C expressing the highest level of
APOBEC-CMPK were transiently transfected with plasmids encoding
IVS-apoB and editing activity on the ectopically expressed RNA
determined on pre-mRNA ("intron") using the
intron-specific RT-PCR products generated with MS1/MS2 and on the
predominantly processed mRNA ("apoB") using the apoB
exon-specific RT-PCR products generated by MS3/MS2. Editing efficiency
of IVS-apoB RNA was: 2.4 ± 0.5% and 2.7 ± 1.0 for
intron and apoB in wild type McArdle cells
(n = 3) and 3 ± 0.7% and 64 ± 3.9% for
intron and apoB in McAPOBEC-CMPK cells (n = 3).
View larger version (27K):
[in a new window]
Fig. 6.
The editing of IVS-apoB RNA is primarily in
the cytoplasm. APOBEC-CMPK high expression cell line was
transiently transfected with plasmids encoding IVS-apoB. Twenty-four
hours after transfection, cells were harvested and fractionated into
nuclear and cytoplasmic fractions, and RNAs from both fractions were
extracted as described under "Experimental Procedures." MS2/MS3
amplimers were used to generate RT-PCR product specific to apoB exon
from both fractions. The editing efficiency of IVS-apoB RNA in the
nucleus (N) and cytoplasm (C) was: 8 ± 2.9% and 69 ± 4.6% (n = 3).
View larger version (34K):
[in a new window]
Fig. 7.
Subcellular localization of HA-tagged ACF in
transiently transfected McArdle cells. A, HA-tagged ACF
was localized in both the nucleus and cytoplasm of transiently
transfected McArdle cells. The DAPI- and anti-HA-stained images of the
same high magnification field are shown (left and
right panels). Arrowheads indicate the cell
nucleus (Nu). Note that the two cells in the bottom
left of the field were not transiently transfected and serve as a
background control for antibody reactivity and autofluorescence.
B, transfected cells were analyzed by Western blotting using
anti-HA monoclonal antibody to demonstrate the expression of
full-length HA-tagged ACF. The migration of molecular mass standard
proteins is indicated to the left.
View larger version (47K):
[in a new window]
Fig. 8.
Ethanol predominantly induces editing in the
nucleus. Wild type McArdle cells were treated with 2.5% ethanol
for 5 min, 15 min, 30 min, 1 h, or 14 h. After the indicated
duration of treatment, cells were harvested, fractionated as nuclei and
cytoplasm, and RNAs from both fractions were extracted. ND1/ND2
amplimer pairs were used to generate RT-PCR product specific for the
endogenous apoB mRNA isolated from ethanol-treated (+) and
untreated control McArdle cells ( 
) grown in parallel.
A, poisoned primer extension assays of apoB mRNA in
nuclear and cytoplasmic subfractions from each time point.
N, nuclear; C, cytoplasmic. B, the
editing efficiency assayed in "A" was calculated as
described in the legend to Fig. 1B, and the ratio of editing
efficiencies of treated versus untreated (+/)
determined for both the nuclear and cytoplasmic apoB mRNAs.
C, the ratio of edited apoB mRNA in treated
versus untreated cell fractions is plotted over the time
course of ethanol treatment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence and reprint requests should be
addressed. Tel.: 716-275-4267; Fax: 716-273-1027; E-mail:
harold_smith@urmc.rochester.edu.
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DISCUSSION
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