1 From the Department of Biochemistry and Biophysics, University of Rochester
School of Medicine and Dentistry, Rochester, New York 14642, USA
3 From the Department of Pathology, University of Rochester School of Medicine
and Dentistry, Rochester, New York 14642, USA
2 From the Department of the Environmental Health Sciences, University of
Rochester School of Medicine and Dentistry, Rochester, New York 14642,
USA
4 From the Department of Cancer Centers, University of Rochester School of
Medicine and Dentistry, Rochester, New York 14642, USA
* Present address: Prairie View A & M University, Prairie View, TX 77446,
USA
Author for correspondence (e-mail:
harold_smith{at}urmc.rochester.edu
)
Accepted 4 December 2001
![]() |
Summary |
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Key words: APOBEC-1, Editing, Ethanol, Insulin, Nucleus
![]() |
Introduction |
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Hepatic apoB lipoprotein synthesis, secretion and apoB mRNA editing are
regulated during development, by hormones and by metabolic factors
(Davidson, 1993;
Chan et al., 1997
;
Smith et al., 1997
). The
cis-acting RNA sequence requirements for site-specific apoB mRNA editing have
been delineated as a tripartite motif encompassing the edited cytidine at
nucleotide 6666 (Smith, 1993
;
Sowden et al., 1998
). The
mooring sequence is the most 3' element in the motif and is necessary
and sufficient for editing. The minimal protein complement for editing in
vitro in the human system consisted of ApoB mRNA editing catalytic subunit 1
(APOBEC-1), a 27 kDa cytidine deaminase responsible for base modification
(Teng et al., 1993
) and
APOBEC-1 complementation factor (ACF), a 65 kDa protein with mooring
sequence-selective RNA binding capacity
(Mehta et al., 2000
). APOBEC-1
has been proposed to bind to RNA at the editing site through its catalytic
domain (Anant et al., 1995
;
MacGinnitie et al., 1995
) and
function as a head-to-tail homodimer (Lau
et al., 1994
; Oka et al.,
1997
; Navaratnam et al.,
1998
). APOBEC-1 alone cannot functionally interact with the
editing site but rather acquires appropriate positioning at the editing site
through its ability to bind to ACF, which in turn binds to the mooring
sequence.
Human ASP (APOBEC-1 stimulating protein) was discovered in the same time
frame as ACF and is identical to ACF, with the exception of an eight amino
acid insertion at position 371 (Lellek et
al., 2000). The ability of ASP to fully complement APOBEC-1
editing was dependent on its association with KSRP, previously identified as a
KH-type splicing factor involved in alternative mRNA splicing of c-src
(Min et al., 1997
;
Chou et al., 1999
). The
relative role(s) of ASP-KSRP-APOBEC-1 and ACF-APOBEC-1 as minimal editosomes
in apoB mRNA editing is not understood. The simplicity suggested for the
editosome assembled under defined in vitro conditions contrasts significantly
with the aggregate size of 27S (predicted to be
500 kDa) observed for
editosomes assembled in extracts (Smith et
al., 1991
; Harris et al.,
1993
; Yang et al.,
1997a
). In this regard, multiple proteins have been proposed to
interact with APOBEC-1 and modulate its editing activity
(Lau et al., 1990
;
Harris et al., 1993
;
Navaratnam et al., 1993
;
Schock et al., 1996
;
Lau et al., 1997
;
Yang et al., 1997a
;
Greeve et al., 1998
;
Blanc et al., 2001
). The
composition and functional organization of the regulated apoB mRNA editosome
therefore remains an open question.
ApoB mRNA editing occurred during or immediately after pre-mRNA splicing
(Lau et al., 1991;
Sowden et al., 1996
) and
metabolic stimulation of editing activity has been shown to occur in the
nucleus (Yang et al., 2000
).
These data showed that apoB mRNA editing is a nuclear event. The intracellular
distribution of APOBEC-1 and ACF have only been studied in transfected cells
(Yang et al., 1997b
;
Yang et al., 2000
;
Blanc et al., 2001
). These
studies showed that recombinant APOBEC-1 and ACF were distributed in both the
nucleus and cytoplasm of hepatoma cells. Editing normally does not occur in
the cytoplasm. However, overexpression of APOBEC-1 in transfected cells
induced editing of cytoplasmic apoB mRNA
(Yang et al., 2000
), and
cytoplasmic extracts from normal hepatocytes supported apoB mRNA editing under
the conditions of the in vitro assay
(Harris et al., 1993
). Taken
together, the data suggested that the apoB mRNA editing factors are
distributed in both the nucleus and cytoplasm of hepatocytes but that these
populations may not be functionally or structurally equivalent, or both.
We report the molecular cloning of the rat homolog to ACF (p66/ACF) and
show that it is p66, the mooring sequence-selective RNA-binding protein
proposed to be involved in editing site recognition and editosome assembly
(Harris et al., 1993;
Navaratnam et al., 1993
).
Cytoplasmic p66/ACF was associated with the exterior surface of the
endoplasmic reticulum and localized to heterochromatic regions in the nucleus.
Sedimentation analysis revealed p66/ACF-APOBEC-1 as cytoplasmic 60S complexes
and nuclear 27S editosomes. These studies suggest that the observed
heterogeneity in editosome higher-order structure is subcellular-compartment
specific and hence functionally significant.
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Materials and Methods |
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Molecular cloning of p66/ACF
A lambda gt11 rat liver cDNA library (Clontech, Palo Alto, CA) was screened
under moderate stringency with random hexamer primed, radiolabeled full-length
human ACF (huACF) cDNA. Two clones were identified after tertiary level
screening that corresponded to the 5' 600 bp and 3' 650 bp of ACF.
To isolate the middle portion of rat ACF, 3' RACE was performed on
Marathon rat liver cDNA (Clontech) according to the manufacturer's
recommendations. Three identical 1.1 kb clones were isolated that overlapped
both 5' and 3' lambda clones. The DNA sequence of rat ACF was
verified by sequencing of RT-PCR products from rat liver poly A+ RNA. P66/ACF
cDNA was subcloned into a modified pcDNAIII vector such that it was
expressed with a hexa-histidine motif and tagged with haemagglutinin (HA) and
V5 epitopes at the 5' and 3' termini, respectively. The rat
p66/ACF cDNA encodes 586 amino acids.
To produce recombinant protein, p66/ACF cDNA encoding a C-terminal 6His and HA-tag was subcloned into the yeast vector pYES 2.0 and under galactose-inducible control. Pelleted yeast were resuspended in an equal volume of 50 mM Tris, pH 8.0, 250 mM KCl, 10% glycerol, 5 mM MgSO4, 2 mM ß-mercaptoethanol, 1 mM PMSF, 5 µg aprotinin/ml (USB, Cleveland, OH), 5 µg leupeptin/ml (USB), 0.5 µg pepstatin (USB), 2 mM benzamidine and 1 g of alumina Type 305 powder (Sigma) per 5 ml yeast suspension added before freezing in liquid nitrogen.
Frozen yeast suspension was ground extensively in a porcelain mortar and pestle under liquid nitrogen, thawed and digested with 5 µg/ml DNase I (37°C, 20 minutes). Extracts were adjusted to 300 mM NaCl and cleared supernatants adsorbed to nickel affinity resin (Qiagen, Valencia, CA) according to the manufacturer's protocol. Adsorbed resins were washed sequentially with 1 M NaCl, 0.4% Triton X-100 and 50 mM Tris, pH 8.0, 50 mM NaCl, 10% glycerol and eluted in the later buffer containing 300 mM imidazole. P66/ACF was concentrated by dialysis against 20% polyethylene glycol (8,000 MW), and then dialyzed against and 50 mM Tris, pH 8.0, 50 mM NaCl, 10% glycerol and stored at -25°C until use.
Northern blot analysis
A Poly A+ northern blot (Clontech) was probed with a full-length p66/ACF
cDNA radiolabeled with 32P[dCTP] using the RTS RadPrime DNA
labeling system (GibcoBRL) according to the manufacturer's protocol. Blots
were hybridized to probe (1x106 cpm/ml) in ExpressHyb
(Clontech) and washed according to the manufacturer's recommendations.
Preparation of peptide-specific antibodies against ACF and
immunoassays
An N-terminal domain in human ACF corresponding to residues 4-19 was
identified by Mehta et al. (Mehta et al.,
2000) as an accessible epitope for antibody production. This
peptide was synthesized, analyzed by mass spectroscopy, conjugated to keyhole
limpet haemocyanin and used to immunize rabbits (Bethyl Laboratories, Inc.
Montgomery, TX). The resultant IgG was affinity purified.
Proteins from whole extracts or glycerol gradient fractions were resolved
by SDS/10.5% PAGE, transferred to BA85 nitrocellulose membrane (Schleicher
& Schuell, NH), probed with anti-ACF (1:5000 dilution) and a secondary
peroxidase-conjugated goat antimouse IgG (Zymed Laboratories, San Francisco,
CA) (1:3000 dilution) as described previously
(Yang et al., 1997a) and
visualized by chemiluminescence (Renaissance, NEN Life Science Products Inc.,
Boston, MA). Alternatively, the primary antibody (200 µg) was adsorbed to
50 µl of packed Protein A-conjugated sepharose beads (Calbiochem, La Jolla,
CA) and, following washes with 50 mM Tris, pH 8.0, 50 mM NaCl, used as an
immunoprecipitation reagent (see below).
Immunofluorescence and immunohistochemistry
McArdle cells, grown on glass slides, were fixed with 2% paraformaldehyde,
permeabilized with 0.4% Triton X100, blocked with 1% BSA and reacted with
affinity-purified anti-ACF and affinity-purified FITC-conjugated goat
anti-rabbit secondary antibody (Organon Teknika, West Chester, PA), each at
1:1000 dilution. Slides were observed and photographed under an Olympus BH-2
fluorescence microscope using a 100x Olympus oil objective.
For immunohistochemistry, rat livers were perfused in situ with 20 ml of
ice-cold 0.33 M STM (0.33 M sucrose, 50 mM Tris, pH 6.8, 5 mM MgCl2
10 mM sodium fluoride) containing 2 mM EGTA, 1 mM PMSF, 5 µg aprotinin/ml,
5 µg leupeptin/ml and 3,000 U of SUPERase-In RNase inhibitor (Ambion Inc.,
Austin, TX). Liver sections were fixed in 10% neutralized formalin (Fisher
HealthCare, Swedesboro, NJ) for 6-8 hours followed by washes with PBS,
paraffin embedding and cutting of 5 µm sections. Endogenous biotin and
peroxidase were blocked in the sections using avidin (kit SP-2001, Vector
Laboratories Burlingame, CA) and 3% H2O2, respectively.
Sections were incubated in 1/100 anti-ACF overnight at 4°C, visualized by
streptavidin-biotin technique (Grumbach
and Veh, 1995) and counterstained with hematoxylin.
Immunoelectron microscopy
Rat livers were perfused as described above, diced into 5 mm cubes and
fixed in phosphate-buffered, 4.0% paraformaldehyde for 6 hours, rinsed in
Sorensen's phosphate buffer overnight, embedded into agarose and vibratomed
into 50-80 µm sections. Sections were blocked overnight at 4°C as
described by Brandstatter et al.
(Brandstatter et al., 1997) and
incubated with anti-ACF antibody (diluted 1:100 in PBS containing 1.0% normal
goat or horse serum), 0.8% BSA and 0.1% fish gelatin at 4°C for 5 days.
Sections were extensively rinsed in PBS at room temperature, incubated with a
1/200 dilution of a biotinylated secondary antibody and incubated overnight at
4°C. Sections were rinsed in PBS, reacted with a 1/300 dilution of
extrAvidin (Sigma) developed with diaminobenzedine (DAB) and fixed in 2.0%
gluteraldehyde (Brandstatter et al.,
1997
). DAB-labeled sections were silver enhanced, gold toned and
post fixed in 1.0% osmium tetroxide
(Brandstatter et al., 1997
).
Dehydrated sections were embedded in Spurr epoxy overnight, sectioned (80 nm),
stained with uranyl acetate and lead citrate and examined using a Hitachi 7100
electron microscope.
Analysis of editing complexes, RNA-binding proteins and editing
activity
S100 extracts were sedimented through 10%-50% glycerol gradients and the
relative S value determined from the sedimentation of size markers in parallel
gradients (Harris et al.,
1993). Gradients were fractionated from the top, and an equal
aliquot of each fraction subjected to western blot analysis and in vitro
editing assays.
RNA-protein interactions were evaluated by UV cross-linking as described
previously (Harris et al.,
1993; Smith,
1998
). Briefly, a 498 nt 32P radiolabeled apoB RNA
substrate was incubated with the 27S glycerol gradient fraction of rat liver
nuclear S100 extracts to assemble editosomes. The reaction was exposed to 254
nm UV light for 5 minutes, RNase A and T1 digested, resolved by SDS
PAGE and the radiolabeled proteins visualized by autoradiography.
Alternatively, samples prepared by the UV cross-linking assay were incubated with 50 µl of anti-huACF-Protein A-sepharose beads on ice for 1 hour, washed with buffer containing 1 M NaCl followed by 0.4% Triton X100 and eluted into TriReagent (for RNA analysis) or 3 M sodium thiocyante and acetone precipitated (for protein analysis).
In vitro editing activity was determined in 100 µl reactions by
incubating whole S100 extracts or glycerol gradient fractions with apoB RNA
(Harris et al., 1993). In vivo
editing activity was determined on total cellular RNA amplified by RT-PCR
using primers specific for apoB sequences encompassing the editing site at
C6666 (Van Mater et al., 1998
;
Yang et al., 2000
). The
proportion of unedited (CAA) and edited (UAA) RT-PCR products were determined
by the poisoned primer extension assay and denaturing gel analysis
(Smith et al., 1991
).
Subcellular fractionation and extract preparation
Nuclei and cytoplasm were prepared from primary hepatocytes using NE-PER
kit protocol (Pierce, Rockford, IL) and modifications as described recently
(Yang et al., 2000).
Proteinase, RNase and phosphatase inhibitors were added to all buffers as
described for 0.33 M STM perfusion buffer. Eight 100 mm plates of rat primary
hepatocytes, either with or without treatment with ethanol or insulin, were
scraped into CER-I buffer. Nuclei were sedimented through 2.2 M sucrose
containing STM, resuspended in 0.33 M STM and examined for purity by phase
microscopy as described previously (Yang
et al., 2000
). All procedures were performed at 4-7°C.
Nuclei and cytoplasm were isolated from whole rat liver for S100 editing
extract preparation as described previously
(Smith et al., 1991), with the
following modifications. Rat livers were perfused in situ as described above
and nuclei were purified through 2.2 M sucrose in STM
(Smith and Berezney, 1983
).
Nuclei were resuspended in extract buffer (EB, 50 mM Tris, pH 8.0, 150 mM
NaCl, 5 mM MgCl2 10 mM sodium fluoride, 0.2 mM DTT, 2 mM EGTA, 1 mM
PMSF, aprotinin (5 µg/ml), leupeptin (5 µg /ml), brought to 250 mM NaCl,
homogenized and incubated on ice for 20 minutes. S100 nuclear extracts were
obtained by microcentrifugation at 14,000 g for 20 minutes.
S100 nuclear and cytoplasmic extracts were dialyzed against EB minus
inhibitors for 4 hours at 7°C and stored at -20°C.
![]() |
Results |
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|
To confirm the functional relationship between rat p66 and huACF, a
peptide-specific antibody was prepared against a conserved N-terminal 15 amino
acid sequence (Fig. 1).
Glycerol gradient fractions of the rat liver 27S editosome were reacted with
radiolabeled apoB RNA under editosome assembly conditions and subjected to UV
cross-linking. Subsequent to SDS PAGE and western transfer, membranes were
autoradiographed to identify cross-linked proteins and then reacted with
anti-p66/ACF antibodies. The result (Fig.
2) showed the well characterized p66 and p44 apoB RNA-binding
proteins present in rat liver editosomes
(Harris et al., 1993;
Navaratnam et al., 1993
;
Yang et al., 1977b
;
Smith, 1998
;
Steinburg et al., 1999
) and
immunoreactivity with anti-p66/ACF to a single protein band of 66 kDa,
migrating coincident with the center of the UV cross-linking signal
(Fig. 2). The data supported
the possibility that the mooring-sequence-selective p66 RNA-binding protein is
the rat homolog to huACF.
|
To validate that the immunoreactive rat liver protein was a homolog of
huACF, in vitro editing reactions containing rat liver extract were evaluated
by immunoprecipitation. Reactions containing either radiolabeled apoB RNA or a
radiolabeled control RNA (WT-1, which lacked mooring sequences)
(Yang et al., 1997a), were
immunoprecipitated with the peptide-specific antibody against huACF and the
amount of radiolabeled RNA recovered was quantified by liquid scintillation
counting. Significant radiolabeled RNA only was immunoprecipitated from
reactions containing radiolabeled apoB RNA
(Fig. 3A). The yield of
immunoprecipitated apoB RNA increased over the first 30 minutes of the
reaction, consistent with the previously described kinetics of editosome
assembly (Smith et al., 1991
).
The amount of immunoprecipitated WT-1 RNA remained low throughout the 60
minute reaction.
|
The RNA-binding protein responsible for the selective recovery of apoB RNA
in the immunoprecipitates was evaluated by PAGE and autoradiography
(Fig. 3B). To demonstrate the
effectiveness of the immunoprecipitation conditions, recombinant huACF was
reacted with radiolabeled apoB RNA in the presence of 1000-fold molar excess
of competing unlabeled WT-1 RNA or apoB RNA and subjected to
immunoprecipitation. A radiolabeled band corresponding to huACF was observed
under these conditions but was significantly reduced when unlabeled apoB RNA
was used as competitor. Reactions containing rat liver nuclear extract,
radiolabeled apoB RNA and 1000-fold molar excess of competing unlabeled WT-1
also yielded a radiolabeled protein of approximately 66 kDa. By contrast, when
the reactions were carried out in the presence of excess cold apoB RNA,
virtually no radiolabeled protein could be detected. RNA excess competition
analyses have been used previously to demonstrate the selectivity of p66 UV
cross-linking to apoB mRNA (Harris et al.,
1993, Yang et al.,
1997a
). The current data demonstrated therefore that an apoB
RNA-selective binding protein of the same size as huACF and cross-reactive
with anti-huACF antibodies was present in rat liver extracts. Taken together,
these data strongly suggested that p66 is the rat homolog of huACF. The rat
homolog will be referred to as p66/ACF (Genbank accession number,
AF290984).
P66/ACF mRNA and protein expression
Northern blot and RT-PCR analyses showed that huACF was expressed
predominantly in liver and to a lesser extent in other tissues
(Mehta et al., 2000). Northern
blot analysis of rat poly A+ mRNA showed p66/ACF expression in liver and
kidney as 3.0 kb and 9.5 kb transcripts
(Fig. 4A). Expression of the
smaller transcript was also observed in spleen, lung and testis. Probing of
the same blot with GAPDH revealed approximate equal loading of RNAs from all
tissues.
|
Protein-expression levels were evaluated in cell lines commonly used to study apoB mRNA editing. P66/ACF was prominent in McArdle rat hepatoma cells, HepG2 human hepatoma cells and Caco-2 human colorectal carcinoma cells (Fig. 4B). Far less p66/ACF was detectable in monkey kidney cells (COS-7). Chinese hamster ovary cells (CHO) expressed a cross-reacting protein of approximately 52 kDa but p66/ACF expression was below detection limits in human cervical carcinoma cells (HeLa).
P66/ACF is in nuclear and cytoplasmic liver extracts
Editing activity has been studied in this laboratory using liver
homogenate, post-nuclear S100 extracts. Given the preponderance of evidence
supporting the nuclear localization of editing activity
(Lau et al., 1990;
Lau et al., 1991
;
Sowden et al., 1996
;
Yang et al., 2000
), we
evaluated editing activity and the organization of editing factors in S100
extracts from both rat liver cytoplasm and from purified nuclei. On a per
microgram protein basis, nuclear extracts were more than twice as efficient in
editing exogenous apoB RNA substrates than cytoplasmic extracts in the
standard in vitro editing assay (Fig.
5A). Western blots containing identical amounts of nuclear and
cytoplasmic proteins that were reacted with anti-ACF showed p66/ACF in both
fractions (Fig. 5B).
Whole-band, densitometric quantification showed that there was 1.5-fold higher
p66/ACF immunoreactivity in the nuclear lane compared to that in the
cytoplasmic protein lane. However, correcting this ratio for the total amount
of protein recovered in each fraction relative to that loaded on the gel (see
Materials and Methods) revealed that, on average, 96% of the total cellular
p66/ACF was recovered in the cytoplasmic S100 extract
(Fig. 5C).
|
Immunolocalization of p66/ACF
We recently showed that exogenously expressed huAPOBEC-1 and huACF were
distributed in the nucleus and cytoplasm of transfected McArdle rat hepatoma
cells (Yang et al., 1997b;
Yang et al., 2000
). As these
cells expressed sufficient endogenous p66/ACF for western blot detection it
was possible to re-evaluate the intracellular distribution of p66/ACF
suggested by biochemical fractionation in the context of intact cells.
Indirect immunofluorescence using the peptide-specific antibody showed
homogeneous nuclear p66/ACF reactivity in McArdle cells together with
cytoplasmic staining in the form of a highly reactive array of aggregates
(speckling) varying in size (Fig.
6).
|
McArdle cells are an immortal cell line and therefore the distribution of proteins may not reflect that in normal hepatocytes. The histological distribution of p66/ACF was evaluated using formalin-fixed, rat liver sections. These did not reveal uniform staining, but rather, zones of hepatocytes were more intensely immunoreactive than others (Fig. 7). These may correspond to hepatocytes known to be actively synthesizing and secreting lipoproteins localized around the central veins that drain the liver (Tanikawa, 1979).
|
The ultrastructural distribution of normal hepatocyte p66/ACF was evaluated by immunoelectron microscopy. P66/ACF immunoreactivity was observed in both the cytoplasm and nucleus of in situ hepatocytes (Fig. 8A). Nuclear p66ACF was observed associated with heterochromatin (Fig. 8B; Fig. 9A,B). In peripheral heterochromatin (the chromatin associated with the nuclear lamina), p66/ACF tended to localize at the borders of heterochromatin and the interchromatin regions. The interchromatin domain itself and the nucleolus contained low or no immunoreactivity.
|
|
Cytoplasmic p66/ACF immunoreactivity was concentrated along regions of endoplasmic reticulum (Fig. 8A), predominantly at the outer surface of the endoplasmic reticulum membrane (Fig. 8B; Fig. 9A). The lumen of the endoplasmic reticulum had low or no immunoreactivity. Some immunoreactivity was observed along the surface of the Golgi apparatus (Fig. 9B) but mitochondria had no or trace reactivity (Figs 8, 9).
P66/ACF in 27S and 60S complexes
The finding that p66/ACF immunolocalized to both heterochromatin regions
and on the outer surface of the ER raised the question as to whether there
were distinguishing physical characteristics underlying its distribution. To
evaluate the aggregate size of complexes containing p66/ACF, and specifically
to determine whether 27S editosomes exist in rat liver, cytoplasmic and
nuclear extracts were prepared and resolved by glycerol gradient sedimentation
without prior in vitro incubations. As a control for both the selectivity of
the nuclear and cytoplasmic fractionation and possibility of aggregation of
RNA-protein complexes during extract preparation or sedimentation, blots from
the cytoplasmic and nuclear gradient fractions
(Fig. 10A and 10B,
respectively) were reacted with antibodies specific for KSRP, a protein
involved in alternative pre-mRNA splicing. Significant immunoreactivity was
only detected in nuclear fractions 1-4 with a peak in fraction 2 or 8S
(Fig. 10B), suggesting that
leaching of nuclear proteins and aggregation of RNA-binding proteins had not
occurred.
|
Cytoplasmic p66/ACF immunoreactivity sedimented as heterogeneous complexes
recovered in fractions 3-11 corresponding to 10S to 100S
(Fig. 10C). Native APOBEC-1
was expressed below the detection limit of antibodies currently available, and
therefore its presence could only be inferred from the ability of fractions to
support editing in vitro. Cytoplasmic APOBEC-1 was not active in editing apoB
mRNA within intact cells (Yang et al.,
2000
). However, it has been shown that the conditions of the in
vitro editing assay could disaggregate and activate these complexes
(Harris et al., 1993
). Using
these methods, cytoplasmic APOBEC-1 could be demonstrated to co-sediment with
p66/ACF in fractions 5-10 with a peak of activity in fractions 7-9,
corresponding to 60S (Fig.
10E).
Nuclear p66/ACF also sedimented in heterogeneous complexes but in contrast
to cytoplasmic extracts, nuclear p66/ACF immunoreactivity was recovered in
fractions 1-8, corresponding to 4S-60S
(Fig. 10D). Nuclear APOBEC-1
co-sedimented with p66/ACF in fractions 3-9 with a peak in fractions 4-6
centered on 27S (Fig. 10F).
This size is consistent with that characterized previously for the in vitro
assembled functional editosome (Smith et
al., 1991; Harris et al.,
1993
). Although strong p66/ACF immunoreactivity was also observed
in the 8S-11S region of this gradient
(Fig. 10D), little or no
editing activity could be detected in these fractions
(Fig. 10F), suggesting that
this region was devoid of functional APOBEC-1. A low level of editing in
complexes as large as 60S (Fig.
10F) suggested that APOBEC-1-p66/ACF complexes similar in size to
those found in the cytoplasm (Fig.
10E) may be present as a minor component in the nucleus. The data
argue strongly that the size of the minimal functional editosome
(p66/ACF-APOBEC-1 complexes) in hepatocytes is 27S. Moreover, the data show
that the previously identified 60S complex of editing factors in rat liver
extracts is predominantly a cytoplasmic form, and therefore not functionally
engaged in editing.
Induction of editing activity increased the amount of nuclear
p66/ACF
The importance of the cell nucleus as the site of apoB mRNA editing led us
to consider whether the amount of p66/ACF in the nucleus might have to change
in response to metabolic conditions that stimulate editing activity. To
evaluate this hypothesis, insulin and ethanol were selected as agents known to
stimulate apoB mRNA editing activity. Insulin promoted enhanced editing
activity by inducing the expression of APOBEC-1 through transcription and
translation (Funahashi et al.,
1995; Thorngate et al.,
1994
; Phung et al.,
1996
; von Wrongski et al., 1998). Ethanol also stimulated hepatic
apoB mRNA editing but without inducing apobec-1 gene expression
(Lau et al., 1995
;
Van Mater et al., 1998
).
Following 6 hours of treatment, with either ethanol or insulin, an equivalent amount of cytoplasmic and nuclear proteins from control, ethanol-treated or insulin-treated rat primary hepatocytes were resolved by SDS PAGE and western blots reacted with anti-p66/ACF antibodies. RNA was also isolated from cultures treated in parallel for the analysis of apoB mRNA editing. As predicted, ethanol and insulin treatment stimulated apoB mRNA editing from control levels 53%±3% s.e.m. to 86%±4% s.e.m. and 75%±3% s.e.m., respectively (Fig. 11). Whole band densitometric quantification of the p66/ACF signals from western blots showed that there was a marked increase in the proportion of immunoreactivity in the nucleus relative to the cytoplasm following ethanol and insulin stimulation, although the calculated mass of total liver p66/ACF from densitometric quantification (as described in Materials and Methods) did not change. The ratio of nuclear to cytoplasmic immunoreactivity (N/C ratio) increased from 1.7 in control hepatocytes to 7 and 10 in ethanol- and insulin-treated cells, respectively (Fig. 11), corresponding to an increase in the proportion of total cellular p66/ACF in the nucleus from 4% in control liver to 23%±6% s.e.m. and 31%±4% s.e.m. (n=3), respectively. These data are consistent with a regulatory mechanism whereby p66/ACF-APOBEC-1 complexes may be rate limiting and therefore an increase in p66/ACF's abundance in the nucleus (by import or retention) may facilitate metabolic stimulation of apoB mRNA editing activity.
|
![]() |
Discussion |
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The subcellular distribution of native p66/ACF and APOBEC-1
To date, the expression of factors involved in apoB mRNA editing has been
inferred by RT-PCR of mRNA encoding APOBEC-1
(Teng et al., 1993;
Funahashi et al., 1995
;
Thorngate et al., 1994
;
Phung et al., 1996
; von
Wrongski et al., 1998), by UV cross-linking of auxiliary proteins in extracts
to apoB RNA (Harris et al.,
1993
; Navaratnam et al.,
1993
; Yang et al.,
1997a
; Lellek et al.,
2000
) and the induction of editing activity following expression
of recombinant factors in transfected cells
(Yang et al., 1997b
;
Siddiqui et al., 1999
;
Yang et al., 2000
;
Yang et al., 2001
;
Blanc et al., 2001
). In tissues
where editing naturally occurs, APOBEC-1 was not expressed to high enough
levels for detection with the currently available antibodies. The cellular
localization of native APOBEC-1 and other editing factors in various tissues
has therefore remained unknown.
It was somewhat surprising to find that recombinant APOBEC-1 and huACF
(Yang et al., 1997b;
Yang et al., 2000
) were
distributed in both the cytoplasm and nucleus when they were expressed in
transfected cells. Overexpression of proteins is subject to the criticism that
cellular transport mechanisms could become saturated and this could lead to
artifacts in their intracellular distribution. This study has established that
native p66/ACF was distributed in the nucleus and the cytoplasm of rat primary
hepatocytes in situ. By virtue of the colocalization of editing activity, the
data also suggested that some or all of native APOBEC-1 was also distributed
with p66/ACF in the nucleus and cytoplasm. Immunoelectromicroscopy further
showed that p66/ACF was concentrated with or near heterochromatin in the
nucleus and as regions of concentration on the surface of the endoplasmic
reticulum.
Only a fraction of the total cellular p66/ACF is in the nucleus
The size of the apoB mRNA editosome has been suggested to be 27S, based on
complexes assembled in vitro in extracts on apoB reporter RNAs
(Smith et al., 1991;
Harris et al., 1993
). This
study has proven that 27S complexes of native p66/ACF and APOBEC-1 (the
minimal functional components of editosomes) were the predominant form of
biologically active, nuclear editosomes in rat primary hepatocytes. Support
for the specificity of these complexes comes from the finding that although
the sedimentation profile of p66/ACF was broad, APOBEC-1 and p66/ACF
functional interactions were only observed within a discrete size
distribution.
In addition to APOBEC-1 and p66/ACF there are likely to be other protein
components that contribute to the estimated mass of 1.5 mDa of the 27S
editosome. The alternative splicing factor KSRP was evaluated as a control for
the potential loss of nuclear proteins to the cytoplasm during subcellular
fractionation. However, sedimentation analysis suggested that a small fraction
of native KSRP co-sedimented with 27S editosomes. This suggested the
possibility that ASP may also be present in 27S complexes. However, formal
proof of this possibility awaits the development of antibodies capable of
distinguishing ACF from ASP.
RNA-binding proteins p100, p55 and p44 and I3 serum proteinase
inhibitor are candidate components of the 27S editosomes based on their
co-purification with 6His-tagged APOBEC-1 overexpressed in McArdle rat
hepatoma cells and affinity purified from cell extracts as functional editing
complexes (Yang et al.,
1997a
). GRYRBP (Blanc et al.,
2001
; Lau et al.,
2001
), hnRNP proteins C
(Greeve et al., 1998
) and
ABBP1 (Lau et al., 1997
) are
also candidate components of the 27S editosome: they all bind APOBEC-1, are
nuclear proteins and modulate editing activity. The biological role of all of
the proteins reported to associate directly or indirectly with APOBEC-1
remains to be shown. It is possible that there is heterogeneity in the
composition of the 27S editosome that results from the regulation of its
interaction with apoB mRNA and its catalytic activity.
Editing of ApoB mRNA may take place in association with
heterochromatin domains
Given the obligate nature of p66/ACF in apoB mRNA editing, our data suggest
that editosome assembly and perhaps editing might take place at the borders of
heterochromatin and the interchromatin domain, known to be the sites of
nascent transcript synthesis and ribonucleoprotein particle assembly
(Puvion and Moyne, 1978;
Fakan and Puvion, 1980
;
Shopland and Lawrence, 2000
).
Consistent with this possibility are data showing that apoB mRNA editing may
occur on some transcripts prior to polyadenylation and that the majority of
editing occurred coincident with or immediately after pre-mRNA splicing
(Lau et al., 1991
).
It is important to interpret these data cautiously, as we do not know
whether the p66/ACF epitope is equally accessible throughout the nucleus (i.e.
it may not be available to react with the antibody in the interchromatin
domain). Moreover, studies of pre-mRNA splicing factors have shown them to be
capable of intranuclear redistribution within cycles of splicing activity and
storage (Misteli et al., 1997;
Kauffman and O'Shea, 1999
;
Eils et al., 2000
). The sites
where nuclear proteins are maximally concentrated may or may not be the sites
where they exert their function in pre-mRNA processing. Only a fraction of
splicing factors would be expected to be engaged in splicing at any given
time, and therefore the immunological staining patterns must be due to bulk
splicing factors that are in the process of recycling, in reserve or otherwise
engaged.
By analogy, the bulk of nuclear p66/ACF sedimented more slowly than 27S,
leaving open the possibility that the localization of p66/ACF with
heterochromatin represents free p66/ACF or complexes of p66/ACF smaller than
27S. In this case, the role of heterochromatin-associated p66/ACF is nuclear,
although it is generally held in the field that auxiliary proteins have
additional roles in the cell (Schock et
al., 1996; Smith et al.,
1997
). We cannot rule out the alternative explanation that
editosomes are associated with heterochromatin but that nuclear p66/ACF
dissociated during biochemical fractionation, leading to its recovery in
fractions
27S.
The potential biological significance of two populations of editing
factors
This is the first report in which the 60S complex has been characterized as
a predominantly cytoplasmic assembly. The data suggested that editing factors
were maintained in an inactive state in the cytoplasm through their physical
associations as 60S complexes and are functional in the nucleus only as 27S
editosomes. Although the bulk of p66/ACF was cytoplasmic in resting liver, the
amount of functionally active nuclear editing factors could be increased in
response to agents that stimulate editing activity. This raises important new
questions of what regulates the proportion of cytoplasmic and nuclear p66/ACF
(and APOBEC-1) and whether the cytoplasmic and nuclear pools of editing
factors communicate through the exchange of proteins.
It is important to keep in mind that 60S complexes are not active in
editing apoB mRNA in vivo (Yang et al.,
2000) but can be activated under the dissociating conditions of
the in vitro editing assay (Harris et al.,
1993
). Under these conditions it was shown that isolated 60S
complexes dissociated to form functional 27S editosomes. In theory therefore,
the 60S complexes in the cytoplasm could dissociate under appropriate
metabolic stimuli to liberate editing factors of 27S editosomes for nuclear
import.
Given that editing does not occur in the cytoplasm, the 60S complexes must
maintain APBOEC-1 and p66/ACF in an inactive state through inhibitors or
post-translational modifications, or both. Candidates for this form of
regulation are proteins such as I3 serum proteinase inhibitor
(Schock et al., 1996
) that
sequester editing factors or GRY-RBP (Blanc
et al., 2001
) and hnRNP proteins
(Greeve et al., 1998
) that
inhibit APOBEC-1 catalytic activity.
The association of p66/ACF with the outer surface of the endoplasmic
reticulum (ER), but not significantly with other cytoplasmic organelles, is of
interest in terms of known protein trafficking mechanisms. There is ample
evidence for the intracellular redistribution of proteins active in the
nucleus from their sites of sequestration in the cytoplasm
(Li et al., 1994;
Rupp et al., 1994
;
Li et al., 1997
;
DeBose-Boyd et al., 1999
;
Görlich and
Kutay, 1999
; Haze et al.,
1999
; Kauffman and O'Shea,
1999
). The endoplasmic reticulum frequently serves as the site
where proteins are sequestered. Their release may be signaled by chaperones,
covalent modification and/or proteolysis.
In this regard, apoB mRNA may serve as a means of tethering p66/ACF to the
outer surface of the ER. Polysomes containing apoB mRNAs had an aberrantly
slow sedimentation in glycerol gradients that was dependent on the presence of
the mooring sequence (Chen et al.,
1993). It has been proposed that the aberrant sedimentation may
result from the persistent association of editing factors with the mooring
sequence. In fact, there are 15 mooring sequences proximal to C6666
(Smith, 1993
), and four of
these have been shown to have associated editosomes and hence bound p66/ACF
(Backus and Smith, 1991
;
Navaratnam et al., 1991
;
Backus et al., 1994
).
Consequently, there may be multiple p66/ACF bound to each apoB mRNA in the
cytoplasm. Furthermore, arrested apoB mRNA translation complexes are docked on
the ER of nonstimulated liver where they await appropriate metabolic
stimulation to resume synthesis of apoB and subsequent formation of
lipoprotein particles (Mitchell et al.,
1998
; Pariyarath et al.,
2001
).
Taken together, the immunolocalization of p66/ACF as clusters on the exterior surface of the ER could be a consequence of multiple p66/ACFs associated with apoB mRNA-ribosome translation complexes. This possibility is intriguing because release of p66/ACF for nuclear translocation might be accomplished through the resumption of translation, covalent modification of p66/ACF and/or release following ribonuclease activity. The mechanism(s) whereby p66/ACF accumulates in the nucleus following ethanol and insulin stimulation of editing is the subject of our ongoing research.
Trafficking, or the movement of proteins between intracellular compartments
in response to specific signals, may explain the increase in nuclear p66/ACF
in response to ethanol and insulin. The mechanism regulating p66/ACF nuclear
import might involve an increase in apoB mRNA synthesis that occurs coincident
with the stimulation of editing activity by insulin and ethanol
(Thorngate et al., 1994;
Funahashi et al., 1995
;
Lau et al., 1995
). The
expression of more mooring sequences in the nucleus could serve as a mechanism
for recruiting p66/ACF to the nucleus by providing additional intranuclear
binding sites. The abundance of unedited apoB mRNA in the nucleus, by itself,
is not likely to recruit p66/ACF.
Shuttling of some of the proteins involved in RNA processing has been
described as the movement of nuclear proteins to the cytoplasm in association
with exported RNAs and subsequent return to the nucleus via their own NLS
(nuclear localization signal) or in association with chaperones
(Kauffman and O'Shea, 1999;
Kim et al., 2001
). In other
words, the process may be continually ongoing and dynamic. Some splicing
factors shuttle by a carrier-mediated pathway independent of RNA trafficking
(Gama-Carvalho et al., 2001
).
Given that p66/ACF's association with apoB mRNA is mooring-sequence dependent
but is not influenced by whether the mRNA is edited or not
(Harris et al., 1993
), it
seems unlikely that the accumulation of nuclear p66/ACF in response to ethanol
or insulin stimulation could be due to an increased export to the cytoplasm of
edited apoB mRNA. It is possible that once established, an equilibrium of
cytoplasmic and nuclear p66/ACF (and APOBEC-1) could be maintained through
some form of shuttling.
Recent studies have suggested that APOBEC-1 requires a chaperone for its
nuclear localization (Yang et al.,
2001). The studies presented here suggested that APOBEC-1 is
associated with p66/ACF throughout the cell and therefore it may import to the
nucleus as an APOBEC-1-p66/ACF complex. A bipartite nuclear localization
signal is predicted in p66/ACF (and ASP). However, the role of p66/ACF or
other auxiliary proteins as a nuclear chaperone for APOBEC-1 awaits further
proof.
In summary, the molecular cloning of p66/ACF and the production of peptide-specific antibodies reactive with this protein on western blots and tissues has enabled several important advancements in the apoB mRNA editing field. Our data have simplified the concepts in the field by showing that ACF is the well-studied 66 kDa, mooring-sequence-selective, RNA-binding protein that interacts early with apoB mRNA during editosome assembly. We also showed that functional complexes of p66/ACF and APOBEC-1 in the nucleus have a higher order complexity of 27S, thereby validating the biological relevance of in vitro assembled 27S editosomes. Native APOBEC-1 and p66/ACF are shown to also be in the cytoplasm of hepatocytes, where their physical interactions as 60S complexes are proposed to lead to their inactivation. Importantly, apoB mRNA editing and the relative abundance of p66/ACF in the nucleus and cytoplasm hepatocytes were metabolically regulated. These findings suggest that the intracellular distribution of editing factors and their assembly as 27S or 60S complexes may be an important strategy in the metabolic regulation of apoB mRNA editing.
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