Research Article
4521
The Chironomus tentans translation initiation factor
eIF4H is present in the nucleus but does not bind to
mRNA until the mRNA reaches the cytoplasmic
perinuclear region
Petra Björk1, Göran Baurén2, Birgitta Gelius3, Örjan Wrange3 and Lars Wieslander1,*
1Department of Molecular Biology and Functional Genomics, Stockholm University, SE-106 91 Stockholm, Sweden
2Global Genomics, Tomtebodavägen 21, SE-171 77 Stockholm, Sweden
3Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, SE-171 77 Stockholm, Sweden
*Author for correspondence (e-mail: [email protected])
Accepted 11 July 2003
Journal of Cell Science 116, 4521-4532 © 2003 The Company of Biologists Ltd
doi:10.1242/jcs.00766
Summary
In the cell nucleus, precursors to mRNA, pre-mRNAs,
associate with a large number of proteins and are processed
to mRNA-protein complexes, mRNPs. The mRNPs are then
exported to the cytoplasm and the mRNAs are translated
into proteins. The mRNAs containing in-frame premature
stop codons are recognized and degraded in the nonsensemediated mRNA decay process. This mRNA surveillence
may also occur in the nucleus and presumably involves
components of the translation machinery. Several
translation factors have been detected in the nucleus, but
their functional relationship to the dynamic protein
composition of pre-mRNPs and mRNPs in the nucleus is
still unclear.
Here, we have identified and characterized the
Introduction
In the nucleus, pre-mRNAs – precursors to mRNA – associate
with a large number of different proteins. These proteins are
essential for intranuclear steps in gene expression, such as
packaging, processing and export of pre-mRNAs. Many of
these proteins – for example, hnRNP proteins (Dreyfuss et al.,
1993; Krecic and Swansson, 1999; Dreyfuss et al., 2002) and
splicing factors (Neubauer et al., 1998; Will and Lührmann,
2001) bind to the pre-mRNAs co-transcriptionally, resulting in
the formation of pre-mRNA–protein complexes, pre-mRNPs.
The composition of individual pre-mRNPs is dynamic. For
example, splicing changes the protein composition of the premRNP (Luo and Reed, 1999; Kataoka et al., 2000; Le Hir et
al., 2000), and specific proteins are known to bind to premRNP during transcription and to dissociate from the premRNP before nucleocytoplasmic export through the nuclear
pores complexes (NPCs) (Daneholt, 2001). Certain proteins
that bind to pre-mRNA in the nucleus accompany the mRNA
to the cytoplasm and determine the cytoplasmic fate of the
mRNA (for a review, see Wilkinson and Shyu, 2001). For
example, some proteins influence the cytoplasmic localization
of mRNA (Hachet and Ephrussi, 2001; Johnstone and Lasko,
2001), and in oocytes, Y-box proteins bind to mRNA in the
translation initiation factor eIF4H in the dipteran
Chironomus tentans. In the cytoplasm, Ct-eIF4H is
associated with poly(A+) RNA in polysomes. We show that
a minor fraction of Ct-eIF4H enters the nucleus. This
fraction is independent on the level of transcription. CteIF4H could not be detected in gene-specific pre-mRNPs
or mRNPs, nor in bulk mRNPs in the nucleus. Our
immunoelectron microscopy data suggest that Ct-eIF4H
associates with mRNP in the cytoplasmic perinuclear
region, immediately as the mRNP exits from the nuclear
pore complex.
Key words: Gene expression, Pre-mRNA processing, Translation
nucleus; these proteins are important during translation
inactivation of the mRNAs in the cytoplasm (Matsumoto and
Wolffe, 1998).
Spliced mRNAs associate with a protein complex, the exonexon junction complex (EJC). This complex, which contains
REF/Aly, Y14, DEK, SRm160 and RNPS1, binds upstream of
exon-exon junctions (Le Hir et al., 2000; Blencowe et al., 1998;
McGarvey et al., 2000; Mayeda et al., 1999; Zhou et al., 2000;
Kataoka et al., 2000; Kataoka et al., 2001; Kim et al., 2001;
Lykke-Andersen et al., 2001). Some of the EJC components,
as well as EJC-associated proteins such as Mago (Palacios,
2002) and the nonsense-mediated decay factor hUpf3 (Kim et
al., 2001), accompany the mRNP to the cytoplasm. It has been
suggested that such proteins are involved in discriminating
between normal mRNAs and mRNAs containing in-frame stop
codons, in a process called nonsense-mediated decay (NMD)
surveillance (reviewed by Li and Wilkinson, 1998; Hentze and
Kulozik, 1999; Hilleren and Parker, 1999; Kataoka et al., 2000;
Lykke-Andersen et al., 2001; Maquat and Carmichael, 2001;
Wilusz et al., 2001). NMD requires translation and can take
place in the cytoplasm (Frischmeyer and Dietz, 1999; Zhang
et al., 1997).
In the nucleus, the m7 G-cap-structure of the pre-mRNA
4522
Journal of Cell Science 116 (22)
binds the cap-binding complex, CBC, which contains the two
cap-binding proteins CBP20 and CBP80. At least one of these,
CBP20, binds co-transcriptionally (Visa et al., 1996a). The
CBC stimulates pre-mRNA processing (Colot et al., 1996;
Lewis et al., 1996; Fortes et al., 1999), but a direct role in
nucleocytoplasmic mRNA export has not been established. In
the cytoplasm, the CBC is replaced by the translation factor
eIF4E at the cap structure, probably after the initial association
with eIF4G (Fortes et al., 2000). The heterotrimeric complex
between eIF4E, eIF4G and eIF4A, called eIF4F, together with
eIF4B is believed to unfold the 5′-UTR (untranslated region)
of the mRNA, an important step during the initiation of
translation (Hershey and Merrick, 2000). The recently
discovered eIF4H (Richter-Cook et al., 1998), is also involved
at this stage of translation and has similar functions as eIF4B
(Richter et al., 1999; Rogers et al., 1999; Rogers et al., 2001).
Recently, experimental data have been presented that
suggest that translation takes place in the nucleus (Iborra et
al., 2001; Brogna et al., 2002). A possible rationale for nuclear
translation is that it is part of nuclear NMD (Dahlberg et al.,
2003). If translation does occur in the nucleus, it has to be
initiated, either by the known translation initiation factors or
by a modified initiation process. It has been reported that the
translation initiation factors eIF4E (Lejbkowicz et al., 1992;
Lang et al., 1994; Dostie et al., 2000a), eIF4G (Etchinson and
Etchinson, 1987; McKendrick et al., 2001) and eIF2 (Lobo et
al., 1997) are present in the cell nucleus in significant
amounts. Also, eIF4H has been reported to be present in the
nucleus (Martindale et al., 2000). There are several possible
explanations for the presence of these translation factors in the
nucleus. One is that they are in fact involved in a nuclear
translation process (Iborra et al., 2001; Brogna et al., 2002),
possibly as part of nuclear NMD (Bühler et al., 2002). If so,
there might be specific requirements for the initiation of
nuclear translation (Fortes et al., 2000; Ishigaki et al., 2001).
A second possibility is that they have so far unknown
functions in pre-mRNA processing or export of mRNA. This
possibility could be exemplified by eEF1A, which is present
in the nucleus under certain conditions (Gangwani et al., 1998;
Grosshans et al., 2000) and by eIF4E. In the case of eIF4E,
this factor piggy-backs into the nucleus on 4E-T, a recently
identified nucleocytoplasmic shuttling protein (Dostie et al.,
2000b). eIF4E has been implicated in nuclear export of cyclin
D1 mRNA (Rosenwald et al., 1995; Rousseau et al., 1996), a
function that is dependent on cap binding (Cohen et al., 2001)
and which is influenced by the promyelocytic leukemia
protein PML (Topisirovic et al., 2002). A third possibility is
that some translation initiation factors could bind to mRNAs
already in the nucleus and accompany them to the cytoplasm,
as a step in facilitating normal translation initiation. Finally,
translation factors could simply be misplaced inside the
nucleus without having a function there. It is important to
investigate which translation factors are present in the nucleus
and the functional relationship between these factors and
synthesis, processing, transport and possible translation of
mRNAs in the nucleus.
Here, we have isolated the gene encoding eIF4H from the
dipteran Chironomus tentans, Ct-eIF4H. We have analyzed its
cellular location and its relation to transcription and translation.
We have especially used the experimental possibilities offered
by the Balbiani ring (BR) genes and their large BR mRNP
particles to test the hypothesis that Ct-eIF4H is bound to premRNA/mRNA in the nucleus. Although we can show that CteIF4H is indeed present in the nucleus in small amounts, our
data suggest that this translation initiation factor is not bound
to pre-mRNA or mRNA in the nucleus. Instead, our data
propose that Ct-eIF4H associates with mRNA in the
cytoplasmic perinuclear region, immediately as the mRNA
enters the cytoplasm.
Materials and Methods
Biological material
Animals and cells
C. tentans was cultured as previously described (Meyer et al., 1983).
A C. tentans embryonic epithelial cell line was cultured as described
(Wyss, 1982). Defolliculated stage VI Xenopus laevis oocytes were
prepared by collagenase treatment (Almouzni and Wolffe, 1993).
Before microinjection, the oocytes were incubated over night at 1819°C in OR2 medium containing 1 mM CaCl2 (Colman, 1984).
Antibodies
The coding part of the Ct-eIF4H gene was cloned into the pET-15b
expression vector (Novagen) and expressed in Escherichia coli. Histagged Ct-eIF4H was purified by Ni-NTA affinity chromatography
(Qiagen) and used for immunization of both rabbits and hens.
Anti-Ct-eIF4H polyclonal antibodies were affinity purified by
chromatography on CNBr-activated Sepharose 4B (Amersham
Biosciences AB) to which the protein had been coupled. Anti-hrp45
(Kiseleva et al., 1994), anti-hrp36 (Wurtz et al., 1996) and anti-hrp23
(Sun et al., 1998) monoclonal antibodies were a gift from B. Daneholt
(Karolinska Institute, Stockholm, Sweden) and anti-CBP20 antibodies
were a gift from I. W. Mattaj (EMBL, Heidelberg, Germany). The
secondary antibodies used for immunocytology were: swine antirabbit Ig FITC (DAKO), diluted 1:100. The secondary antibodies used
for western blots were: swine anti-rabbit Ig horse radish peroxidase
(HRP), goat anti-mouse Ig HRP (DAKO) diluted 1:3000 and rabbit
anti-chicken IgY HRP (SIGMA) diluted 1:20.000. Anti-rabbit
immunoglobulin antibodies conjugated with 6 nm gold particles
(Jackson ImmunoResearch Labs) were used as secondary antibodies
for immunoelectron microscopy.
Cloning procedures
Total RNA was extracted from C. tentans salivary gland cells. In all,
400 salivary glands were dissected, fixed in 70% ethanol at 4°C and
stored in glycerol:ethanol (1:1 by volume) at –20°C. RNA was
extracted for 30 minutes at room temperature in 1 mM EDTA, 20 mM
Tris-HCl pH 7.4, containing 0.5% SDS and 0.1 mg/ml proteinase-K,
followed by phenol extraction and ethanol precipitation. Poly(A+)
RNA was purified by binding to oligo(dT) cellulose (Poly(A) Quick,
Stratagene). For cDNA synthesis, 5 µg of poly(A+) RNA was
annealed with oligo(dT) at 70°C for 5 minutes. Reverse transcriptase
(Superscript, BRL GIBCO) was added and synthesis was allowed to
proceed for 1 hour at 42°C. Degenerate PCR primers, corresponding
to the conserved RNP-1 and RNP-2 regions of the consensus-RBD
present in many RNA-binding proteins, were used for amplification
of corresponding regions in the cDNA, essentially as described (Kim
and Baker, 1993). PCR products were analyzed by agarose gel
electrophoresis. PCR products that were approximately 140 bp or 400
bp long were eluted from the agarose gel, cleaved with EcoRI and
BamHI and cloned into a plasmid vector (Bluescript, Stratagene).
cDNAs from individual clones were sequenced, and those exhibiting
sequence homology to consensus-RBDs were selected. Full-length
cDNA sequences were obtained by a combination of screening of a
C. tentans tissue culture cDNA library, constructed in the lambda Zap
eIF4H binds mRNA in the cytoplasmic perinuclear region
vector (Stratagene), and direct isolation of fragments extending in the
5′ or 3′ direction (Marathon, Clontech). The genomic sequence
organization was determined by analysis of several genomic PCR
products, which together covered the entire gene. All exon-intron
borders were sequenced and most introns were sequenced entirely.
The DNA and protein sequences were analyzed by programs in the
GCG package (Devereaux et al., 1984) and Biology Workbench
package (San Diego Supercomputer Center, University of California,
San Diego).
Protein preparation and western blotting
Nuclear and cytoplasmic extracts of C. tentans tissue culture cells
were prepared essentially as described (Wurtz et al., 1996). To check
for cytoplasmic contamination of the nuclear fraction, we used a
cytoplasmic protein of unknown function. Proteins to be analyzed by
western blotting were boiled in sample buffer (62.5 mM Tris-HCl pH
6.8, containing 10% glycerol, 2.3% SDS, 5% mercaptoethanol and
0.02% bromophenol blue) for 3 minutes and separated on 12% SDSpolyacrylamide gels. The separated proteins were transferred to
PVDF-filters by semi-dry electrophoresis. HRP-labeled secondary
antibodies were detected by the ECL method (Amersham Biosciences
AB). Quantification of the bands was performed with a FUJIFILM
LAS-1000 camera using the software Image Gauge V 3.45. Presented
values are averages from three independent experiments.
Analysis of polysomes
C. tentans tissue culture cells were homogenized in 10 mM Tris-HCl
pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 2 mM DTT and centrifuged at
20,000 g. The supernatant was centrifuged at 100,000 g in a AH-650
rotor (Sorvall) for 3 hours at 4°C to pellet polysomes. The pellet was
washed and resuspended in 250 mM sucrose, 1 mM DTT, 0.1 mM
EDTA, 20 mM Tris-HCl pH 7.5. KCl was added to a final
concentration of 0.5 M and ribosomes were repelleted at 100,000 g
for 4 hours at 4°C. The supernatant and the pellet was analyzed by
western blotting.
Immunocytological localization
Cultured C. tentans diploid cells were prepared and stained with antiCt-eIF4H antibodies essentially as described (Baurén et al., 1996).
Chromosomes were isolated from salivary glands of C. tentans fourth
instar larvae and probed with anti-Ct-eIF4H antibodies, essentially as
described (Kiseleva et al., 1994). Anti-hrp45 antibodies were used for
parallel preparations. These antibodies stain hrp45 bound to nascent
BR pre-mRNPs (Baurén et al., 1996) and served as a positive control
(data not shown). The secondary antibody alone and pre-immune
serum did not give any significant labeling (data not shown). All
preparations were mounted in antifade medium (VectaShield, Vector
Laboratories) and viewed and photographed in an Axioplan II
microscope (Zeiss).
Immunoelectron microscopy
Immunoelectron microscopy of ultrathin cryosections of C. tentans
salivary gland cells was performed according to Tokuyasu (Tokuyasu,
1980) as described (Visa et al., 1996b). The specimens were examined
and photographed in a Zeiss CEM 902 electron microscope at 80 kV.
Negative controls with only secondary antibodies and positive
controls with anti-hrp45 antibodies were performed in parallel (data
not shown). For quantification of Ct-eIF4H labeling, all gold particles
present in 500 nm wide regions on both sides of the nuclear membrane
were counted. In a 150 nm cytoplasmic perinuclear region, the
location of all gold particles were analyzed and it was determined if
they were associated with NPCs or located between NPCs. In the
digitized micrographs, the gold particles were enlarged to improve
visualization.
4523
Expression of Ct-eIF4H in Xenopus oocytes and analysis of its
cellular location
The coding part of Ct-eIF4H was cloned into the plasmid vector
pβGFP/RN3P (Zernika-Goetz et al., 1996). Capped RNA was
transcribed in vitro (Ambion) and injected into the cytoplasm of
Xenopus oocytes, using a Nanoliter 2000TM (World precision
instruments) as described before (Belikov et al., 2000). After 1-2 days,
cytoplasm and nuclei were manually dissected and the presence of CteIF4H was detected in western blots using anti-Ct-eIF4H antibodies.
The antibodies detected a few endogenous Xenopus proteins in both
injected and uninjected cells. In certain oocytes, the nuclear
membrane was manually removed from isolated nuclei and the
nuclear membrane and the nucleoplasm were analyzed separately. To
assay for the influence of increased transcription, Ct-eIF4H mRNA
was co-injected with in vitro transcribed mRNA for the glucocorticoid
receptor and a plasmid containing the herpes simplex thymidine
kinase gene (HS VTK), driven by the MMTV promoter. Nuclei and
cytoplasm were analyzed in oocytes treated or not treated with 1 µM
of the synthetic glucocorticoid triamcinolone acetonide (TA) for 8
hours to induce transcription from the MMTV promoter. As a
reference for the amounts of material loaded in each lane in the
western blots, the blots were probed with an anti-BRG-1 antibody,
thus detecting endogenous BRG-1 (Östlund-Farrants et al., 1997). The
intensity of the bands were quantified as explained above.
Analysis of poly(A+) RNA
C. tentans tissue culture cells were UV-irradiated and poly(A+) RNA
was isolated from cytoplasm using Oligotex according to the
manufacturer (Qiagen), and from nuclei essentially as described
(Pinol-Roma et al., 1989). After elution of the RNA from the
oligo(dT) cellulose and RNase treatment, the co-purified proteins
were analyzed by western blotting.
Immunoprecipitation of pre-mRNP and mRNP complexes
Pre-mRNP and mRNP complexes were immunoprecipitated from
nuclear extract of C. tentans tissue culture cells essentially as
described (Sun et al., 1998). In brief, monoclonal anti-hrp23 antibody
or polyclonal anti-Ct-eIF4H antibodies were added to nuclear extracts
in the presence of 0.1% NP-40, and incubated for 90 minutes at 4°C
with gentle rotation. Rabbit anti-mouse immunoglobulins (DAKO),
covalently coupled to protein G-Sepharose beads (Zymed
Laboratories) or protein G-Sepharose beads were added and the
rotation was continued for 90 minutes at 4°C. The sepharose beads
were washed three times with PBS containing 0.1% NP-40 and once
with PBS. The immunoprecipitated complexes were eluted with 0.5%
SDS at room temperature. The proteins were precipitated with acetone
and subsequently analyzed by western blotting.
Results
Cloning and characterization of Ct-eIF4H
In a screen for proteins that are associated with mRNAs, we
took advantage of the fact that such proteins often contain a
conserved RNA-binding domain, RBD (Varani and Nagai,
1998). Messenger RNA was purified from C. tentans salivary
gland cells and cDNA was obtained by using oligo (dT)
primers. Two different degenerate primers, corresponding to
the conserved RNP-1 and RNP-2 amino acid sequences present
in the RBD in RNA-binding proteins, were used for PCR. The
PCR fragments were cloned into a plasmid vector and
individual cDNAs were sequenced. One of the gene fragments
that coded for part of an RBD was used to obtain the entire
coding region of the corresponding gene from a C. tentans
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Journal of Cell Science 116 (22)
cDNA library. The 1460 bp long cDNA sequence encoded a
protein consisting of 316 amino acids. On the basis of sequence
similarities and properties described below, we concluded that
this protein is C. tentans eIF4H (Ct-eIF4H).
In Fig. 1, Ct-eIF4H is compared to its closest sequence
homologues in Drosophila melanogaster (41% identity) and
Homo sapiens (32% identity). In Fig. 1A it is evident that
eIF4H in the three species have a very similar RBD in the Nterminus and also a highly conserved C-terminus. The
greatest difference between the proteins is found in the
central region. Here, the proteins differ in length and
sequence. The human eIF4H is considerably shorter than
eIF4H in dipterans, and the size difference is localized to the
central region. Two splice variants of the human eIF4H have
been detected that differ by 20 amino acid residues, and this
splice variation is present in the central region. Also, the
dipteran proteins in the central region are different in each of
the species mentioned. In all three species, the central region
is rich in glycine residues and Ct-eIF4H contains six RGG
motifs. Such motifs can contribute to RNA binding (Kiledjian
and Dreyfuss, 1992) and protein-protein interactions (Gary
and Clarke, 1998).
By in situ hybridization, the Ct-eIF4H gene was located to
chromosome I, region 16C (data not shown). The exon-intron
structure of the Ct-eIF4H gene was deduced by comparing
genomic and cDNA PCR fragments. Five introns were
identified and the exon-intron borders were sequenced. The
introns are between 67 bp and 233 bp long, and their positions
are indicated in Fig. 1B. The eIF4H gene in D. melanogaster
contains four introns. The introns 1, 2, 3 and 4/5 are in very
similar positions in C. tentans and D. melanogaster. The intron
corresponding to intron 4 in the Ct-eIF4H gene is lacking in
the D. melanogaster gene. In the human gene, there are six
introns. Only intron 6 is in the same position as the most 3′
located intron in the dipteran genes. Exon 5 in humans is
known to be subject to exon skipping/inclusion to produce two
splice variants.
Ct-eIF4H is present in the cytoplasm and bound to
mRNA in polysomes
Polyclonal antibodies were raised in rabbits and in hens against
the recombinant Ct-eIF4H. In both cases the antibodies
specifically detected one single
protein with the relative mobility of 36
kDa in western blots of C. tentans cell
extracts (Fig. 2A). When cytoplasm
and nuclei in C. tentans tissue culture
cells were biochemically separated,
approximately 95% of Ct-eIF4H were
detected in the cytoplasmic fraction
and approximately 5% in the nuclear
fraction (Fig. 2A, lanes 1 and 2).
In agreement, immunofluorescence
localization of Ct-eIF4H in C. tentans
tissue culture cells showed that CteIF4H is present throughout the
cytoplasm (Fig. 2B).
Translation factors belonging to the
eIF4 group are co-purified with
polysomes in a salt-dependent manner
(Merrick,
1979).
We
isolated
polysomes from C. tentans tissue
culture cells. In Fig. 3A (lane 1), we
showed that Ct-eIF4H co-sediments
with polysomes. We also showed that
when isolated polysomes were treated with 0.5 M KCl, CteIF4H dissociated from the polysomes (Fig. 3A, lanes 2 and 3).
Fig. 1. Sequence variation in eIF4H between species.
(A) Comparison of amino acid sequences for eIF4H in H. sapiens
(accession number Q15056), D. melanogaster (accession number
CG4429) and C. tentans (accession number AJ511864). The Nterminus, with its RNA-binding domain, and the C-terminus are
similar in the three species. The largest variation is in the central part
of the protein, where all proteins are rich in glycine residues.
Conserved residues are highlighted: black indicates identical
residues; grey indicates related residues. (B). The intron positions
(indicated by the arrows) in eIF4H in H. sapiens, D. melanogaster
and C. tentans are shown. Gaps (larger than two amino acid residues)
have been introduced in the proteins in accordance with the
alignment in Fig. 1A.
eIF4H binds mRNA in the cytoplasmic perinuclear region
Fig. 2. Localization of Ct-eIF4H. (A) Western blot analysis of
cytoplasmic and nuclear fractions of C. tentans diploid cells. The cells
were biochemically fractionated into a cytoplasmic (C) and a nuclear
(N) fraction. The fractions were analyzed by western blotting, using
anti-Ct-eIF4H antibodies. The majority (about 95%) of Ct-eIF4H is
present in the cytoplasmic fraction, but a small amount (about 5%) is
found in the nuclear fraction. The extra band in the cytoplasmic
fraction (approximately 3% of the signal in this lane) was specific for
the antibody, but we do not know the identity of this band. In lanes 3
and 4, the fractions were probed with an antibody specific for a
cytoplasmic protein to check for contamination of cytoplasm in the
nuclear extract. (B) C. tentans diploid cells were stained with anti-CteIF4H antibodies and detected by immunofluorescence (left panel). A
predominant cytoplasmic staining was seen. Consistently, a weak
granular staining was detected in the nucleus. DNA, in the same cell,
stained with DAPI (right panel). Bar, 1 µm.
To further analyze the association of Ct-eIF4H with polysomes,
we UV-irradiated C. tentans tissue culture cells and purified
cytoplasmic poly(A+) RNA. We then found that Ct-eIF4H was
co-purified with the poly(A+) RNA and that this co-purification
was dependent on UV-irradiation (Fig. 3B). The hrp36 protein,
known to be bound to mRNA in the cytoplasm, served as a
positive control (Fig. 3B). The Ct-RBD-1 protein, known to be
present in the cytoplasm and partly associated with 40S
ribosomal subunits served as a negative control (data not shown).
In summary, Ct-eIF4H is present throughout the cytoplasm, and
is probably associated with poly(A+) RNA in polysomes.
Ct-eIF4H is present in the nucleus, but not associated
with pre-mRNA or mRNA
In the cellular fractionation experiments (Fig. 2A, lane 2) and
4525
Fig. 3. Ct-eIF4H is present in polysomes and can be UV-cross-linked
to cytoplasmic poly(A+) RNA. (A) Polysomes from C. tentans tissue
culture cells were pelleted by centrifugation. Ct-eIF4H, detected by
western blotting, was present in the pelleted polysomes (lane 1). The
polysomes were resuspended, treated with 0.5 M KCl and repelleted.
Ct-eIF4H was then released from the polysomes (lane 2) and found
in the supernatant (lane 3). (B) C. tentans tissue culture cells were
UV-irradiated, and poly(A+) RNA was isolated from cytoplasmic
extracts. After elution of the RNA from the oligo (dT) cellulose and
RNase treatment, the co-purified proteins were analyzed by western
blotting. Ct-eIF4H (lane 1) and hrp36 (lane 3) co-purifies with
poly(A+) RNA after UV-cross-linking but not when UV-crosslinking was left out (lanes 2 and 4, respectively).
in the immunostaining of the tissue culture cells (Fig. 2B, left
panel), we detected Ct-eIF4H not only in the cytoplasm, but
also to a minor extent in the nucleus. In the biochemical
fractionations, we detected no contamination from the
cytoplasm in the nuclear fraction, using a control antibody
specific for a cytoplasmic protein (Fig. 2A, lanes 3 and 4). The
immunofluorescence staining of the nuclei, although weak,
was significant compared with controls and had a granular
pattern throughout the nucleus.
To further analyze the nuclear localization of Ct-eIF4H, we
used Xenopus oocytes, C. tentans salivary gland cells and C.
tentans diploid cells. Xenopus oocytes allow experimental
expression of Ct-eIF4H, combined with efficient isolation of
nuclei, and analyses of the relation between the level of
transcription and nuclear content of Ct-eIF4H. Ct-eIF4H was
expressed in Xenopus oocytes by the injection of in vitro
transcribed RNA. Subsequently, we analyzed the cellular
location of the protein. Fig. 4A (lanes 1-4) shows that Ct-eIF4H
was specifically detected both in the cytoplasm and in the
nucleus. It also shows that Ct-eIF4H was present inside the
nucleus and not solely bound to the outside of the nuclear
membrane (Fig. 4A, lanes 5 and 6). To address whether the
nuclear content of Ct-eIF4H is influenced by transcription
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Journal of Cell Science 116 (22)
Fig. 5. Ct-eIF4H is not bound to nascent pre-mRNAs. (A) Isolated C.
tentans polytene chromosome IV was probed with anti-Ct-eIF4H
antibodies. No staining of the transcriptionally active BR gene loci
(BR1, BR2 and BR3), or of other gene loci could be detected. Bar,
10 µm. (B) The same chromosome viewed in phase contrast.
Fig. 4. Analysis of the localization of Ct-eIF4H in Xenopus oocytes.
(A) Nuclei (N) and cytoplasm (C) were dissected from injected and
uninjected Xenopus oocytes. The presence of Ct-eIF4H was analyzed
by western blotting (lanes 1-4). The nuclei from injected oocytes
were further dissected and the nucleoplasm (lane 5) and the nuclear
membrane (lane 6) were analyzed separately. The position of CteIF4H is indicated. The additional bands represent endogenous
Xenopus proteins that were present in both uninjected and injected
oocytes. (B) Hormone-induced transcription of the HS VTK gene did
not increase the nuclear content of Ct-eIF4H. Oocytes, injected with
Ct-eIF4H mRNA and the HS VTK gene construct, were dissected
and the content of Ct-eIF4H in nuclei (N) and cytoplasm (C) was
analyzed by western blotting in the absence (lanes 3 and 4) or in the
presence (lanes 5 and 6) of hormone. Uninjected oocytes were
analyzed as a control (lanes 1 and 2). As an internal control for the
amount of material loaded in each lane, the endogenous BRG-1
protein on the same filter was detected with a specific antibody.
Quantification of the signals showed that the amount of material
loaded in lane 5 was approximately 1.3 times the amount loaded in
lane 3, which explains the apparent difference in strength of the CteIF4H band in these two lanes.
activity we took advantage of the glucocorticoid hormone
inducible MMTV promoter. Hormone induction of this
promoter after injection into Xenopus oocytes results in a
strong induction of transcriptional activity and a concomitant
chromatin remodeling of all injected copies, while the
uninduced promoter remains virtually silent (Belikov et al.,
2000). By injecting 10 ng of the 10.25 kb construct pMTV:M13
(Belikov et al., 2000) as ssDNA we achieved about 1-2×109
copies of the gene in each oocyte. The hormone induction
resulted in a massive increase in MMTV promoter-driven
transcription (data not shown). However, this did not result in
any significant change in the distribution of Ct-eIF4H, which
was coexpressed by RNA injection into these oocytes (Fig.
4B).
C. tentans salivary gland cells allow a detailed analysis of
the nuclear distribution of Ct-eIF4H in relation to specific gene
loci and gene-specific mRNA-protein complexes (mRNPs). We
stained isolated polytene chromosomes from C. tentans
salivary gland cells with the anti-Ct-eIF4H antibodies. As seen
in Fig. 5, we could not detect any significant staining of the
transcriptionally highly active Balbiani ring (BR) gene loci.
Furthermore, we could not detect any staining of other gene
loci on the polytene chromosomes. This finding strongly
suggests that Ct-eIF4H is not associated with nascent premRNAs at the gene loci.
To investigate whether Ct-eIF4H binds to mRNPs in the
interchromatin after release of the processed mRNPs from the
gene loci, we used immunoelectron microscopy. In C. tentans
salivary gland cells, transcripts from the BR1 and BR2 genes
form 7 nm thick RNA-protein fibers which fold into mRNP
particles with a diameter of 50 nm (Lönnroth et al., 1992).
These mRNP particles are morphologically distinguishable as
they are synthesized on the gene and as they subsequently are
transported through the interchromatin. In the electron
microscope, we could not detect any Ct-eIF4H labeling of
the nascent BR pre-mRNP, in agreement with the lack of
immunostaining of isolated chromosomes. We detected
specific gold particles, indirectly labeling Ct-eIF4H,
throughout the interchromatin, but no labeling of BR mRNP
particles could be detected (Fig. 8A).
To further analyze whether Ct-eIF4H is associated with
mRNAs in general in the nucleus, we isolated poly(A+) RNA
from nuclear preparations of C. tentans diploid tissue culture
cells after UV-irradiation. We could not detect Ct-eIF4H UVcross-linked to nuclear poly(A+) RNA (Fig. 6). The hnRNP
protein hrp36 served as a positive control and could be
detected. Even though hrp36 is likely to be bound in several
copies to each mRNA, a comparison of Fig. 6 and Fig. 3B
shows that relative to hrp36, Ct-eIF4H associated to poly(A+)
RNA can be detected after UV-cross-linking in the cytoplasm,
but in the nucleus it is below the level of detection.
We also addressed whether the nuclear Ct-eIF4H could be
detected in pre-mRNP or mRNP complexes in C. tentans
diploid cells. It has previously been shown that the SR protein,
eIF4H binds mRNA in the cytoplasmic perinuclear region
4527
Fig. 6. Ct-eIF4H is not bound to nuclear poly(A+) RNA. C. tentans
tissue culture cells were or were not UV-irradiated and poly(A+)
RNA was isolated from nuclear extracts. The co-purified proteins
were analyzed by western blotting. Ct-eIF4H did not co-purify with
poly(A+) RNA after UV-cross-linking (lanes 1 and 2), whereas hrp36
did (lanes 3 and 4).
hrp45 (Alzhanova-Ericsson et al., 1996) and the SR related
protein hrp23 (Sun et al., 1998) (Björk et al., in preparation)
are restricted to the nucleus and that they are both present
in pre-mRNP and mRNP complexes. We used anti-hrp23
antibodies in immunoprecipitations of nuclear extracts. We
then asked whether we could detect Ct-eIF4H in the
precipitated complexes. We found that Ct-eIF4H could not be
co-immunoprecipitated with hrp23 (Fig. 7B). By contrast, we
could co-immunoprecipitate hrp45 and CBP20 (Fig. 7B). We
also used anti-Ct-eIF4H antibodies for immunoprecipitation.
In this case we could only precipitate Ct-eIF4H, but not hrp23,
hrp45 or CBP20 (Fig. 7A). These results suggest that Ct-eIF4H
is not generally present in pre-mRNP or mRNP complexes in
the nucleus.
Ct-eIF4H is present in the cytoplasmic perinuclear
region, preferentially close to nuclear pore complexes
If Ct-eIF4H does not bind to mRNA in the nucleus, the
question is where in the cell this association takes place. To
attempt to answer this question, we used immunoelectron
microscopy to study the location of Ct-eIF4H in the cytoplasm
and in the interchromatin, especially in relation to the NPC
in the nuclear membrane and to the export of BR mRNP.
We detected Ct-eIF4H throughout the cytoplasm in close
connection to the rough endoplasmic reticulum (data not
shown), in agreement with our immunofluorescence data (Fig.
2B). The labeling was evenly distributed in the cytoplasm all
the way up to the outer nuclear membrane. The number of gold
particles specific for Ct-eIF4H was approximately the same in
a 500 nm region closest to the outer nuclear membrane
compared to the proceeding 500 nm cytoplasmic region. In the
nucleus, specific immunolabeling was observed throughout the
Fig. 7. Ct-eIF4H cannot be immunoprecipitated together with
proteins present in pre-mRNP and mRNP complexes in the nucleus.
Nuclear extracts (NE) from C. tentans tissue culture cells were
immunoprecipitated with antibodies directed against either Ct-eIF4H
(A) or the nuclear mRNP protein hrp23 (B). Immunoprecipitated (IP)
complexes were analyzed by western blotting. Anti-Ct-eIF4H
antibodies precipitated Ct-eIF4H, but not hrp23 and hrp45 (both are
known to be associated with pre-mRNPs in the nucleus) or CBP20.
Anti-hrp23 antibodies precipitated hrp23, hrp45 and CBP20 but not
Ct-eIF4H.
entire interchromatin. We detected no labeling of the chromatin
(data not shown), in agreement with our imunofluorescence
data (Fig. 5). To compare the distribution of specific gold
particles on both sides of the nuclear membrane, we counted
all the gold particles also in two 500 nm regions on the nuclear
side of the membrane. The number of gold particles in the
nuclear 500 nm region closest to the inner nuclear membrane
was about ten times lower than on the corresponding
cytoplasmic region (Table 1). The same was true for the
proceeding 500 nm nuclear region. Even though the
interchromatin was specifically labeled, we could not detect
any labeling of BR mRNP particles (Skoglund et al., 1986) in
the interchromatin, close to the nuclear membrane or docked
at the basket structure of the NPCs (Fig. 8A).
In the cytoplasm, we focused on the Ct-eIF4H-specific
labeling in the cytoplasmic perinuclear region, defined as a 150
nm region closest to the nuclear membrane. Here, labeling was
about as frequent as within the proceeding 350 nm from
the nuclear membrane (Table 1). When we analyzed the
distribution along the nuclear membrane, we found that almost
80% of the gold particles in the 150 nm perinuclear region were
Table 1. Distribution of Ct-eIF4H-specific gold particles on the nuclear and cytoplasmic sides of the nuclear membrane
500 nm cytoplasmic region,
closest to the nuclear membrane
Location
Number of
gold particles
500 nm proceeding
interchromatin
500 nm interchromatin,
closest to the nuclear membrane
150 nm cytoplasmic
perinuclear region
350 nm
500 nm proceeding
cytoplasmic region
63
68
100
340
369
All gold particles in two consecutive 500 nm regions on the cytoplasmic side of the nuclear membrane and in two consecutive 500 nm regions on the nuclear
side of the nuclear membrane were counted. The distribution of gold particles within the 500 nm cytoplasmic region closest to the nuclear membrane was
analyzed by comparing the number of gold particles within the 150 nm cytoplasmic perinuclear region with the number of gold particles within the following 350
nm region.
4528
Journal of Cell Science 116 (22)
associated with NPCs (Fig. 8A,B), whereas about 20% of the
gold particles were located between the NPCs. This suggests
that the majority of Ct-eIF4H present in the cytoplasmic
perinuclear region is not bound to mRNAs associated with the
polysomes that are bound to the outer nuclear membrane.
Occasionally, a morphologically identifiable BR mRNP
particle can be seen exiting an NPC at the cytoplasmic side as
an extended mRNP fiber. Such mRNP fibers exit with their 5′
end first. The fibers have been morphologically described as
being connected to particles, which have the dimensions of
ribosomes (Mehlin et al., 1992). We detected such electrondense fibers extending from the NPCs and associated with CteIF4H-specific gold particles (Fig. 8C,D). This suggests that
Ct-eIF4H becomes bound to BR mRNP fibers immediately as
these are exposed in the cytoplasm during translocation
through the NPCs.
In summary, our immunoelectron microscopy data show that
Ct-eIF4H is not bound to BR mRNP particles as these are
docking at the nuclear side of the NPC. In the cytoplasmic
perinuclear region, Ct-eIF4H is preferentially located close to
the NPCs. Our data suggest that Ct-eIF4H associates with BR
mRNP particles as these exit the NPCs, even before the entire
mRNP has been translocated, and then accompanies the BR
mRNP to the endoplasmic reticulum.
Discussion
It is important to analyze when and where mRNA interacts
with the translation machinery. Translation and most probably
surveillance of mRNA translatability are dependent on correct
association between the mRNA and the 40S ribosomal subunit.
Therefore, special attention should be given to factors that are
involved in the initiation of translation.
Fig. 8. Electron micrographs of anti-Ct-eIF4H antibody labeling in
the interchromatin and in the cytoplasmic perinuclear region in C.
tentans salivary gland cells. A schematic drawing of each micrograph
is shown to the right to simplify interpretation. The nuclear
membrane is represented by the dashed lines and the gaps indicate
the positions of NPCs as shown in the schematic drawing of B. In
each micrograph, the nucleus (Nuc) and cytoplasm (Cyt) are
indicated. Gold particles are denoted by black dots. (A) BR mRNP
particles (circles marked BR in the schematic drawing) in the
interchromatin and docking at NPCs (arrow) were not labeled by the
anti-Ct-eIF4H antibodies. (B) A Ct-eIF4H-specific gold particle
associated with the cytoplasmic side of a NPC. This is also seen for
the two gold particles in the cytoplasmic perinuclear region in A.
(C,D) Ct-eIF4H-specific gold particles associated with electrondense fibers extending from NPCs into the cytoplasmic perinuclear
region. These fibers represent extended BR mRNP particles during
passage through the NPCs. In the schematic drawing these fibers are
marked BR. Bar, 200 nm.
The eIF4B/eIF4H proteins
We have studied the translation initiation factor eIF4H in C.
tentans. This translation factor has been discovered only
recently (Richter-Cook et al., 1998). It is apparent from the
sequence alignment in Fig. 1 that there is substantial sequence
variation in eIF4H between species. In particular, a central
region, rich in glycine, arginine and asparagine residues, is
variable, both as to sequence and to length. In mammals, a part
of the central region interacts with a herpes simplex virus
nuclease as part of viral-induced degradation of mRNAs that
undergo cap-dependent translation (Feng et al., 2001; Everly
et al., 2002). The N-terminus, which contains the RBD, and
the C-terminus are conserved to a high extent. This is also true
for eIF4H sequence homologues in Anopheles gambiae and
Mus musculus. The D. melanogaster genome encodes an
additional eIF4H sequence homologue. This protein is longer
and has less sequence identity compared with human eIF4H.
However, it is more similar to eIF4H than to eIF4B.
We noted that the RBD and the C-terminus in eIF4H are
relatively well conserved compared with corresponding
regions in mammalian eIF4B. Like eIF4B, eIF4H stimulates
the ATPase and helicase activities of eIF4A (Richter et al.,
1999; Rogers et al., 2001). In these and other assays, eIF4B
and eIF4H exhibit an additive stimulatory behaviour, and it is
possible that the two proteins can complement each other
(Rogers et al., 2001). In mammals, eIF4B is considerably
longer than eIF4H. It extends approximately 60 amino acid
residues at the N-terminus and 230 residues at the C-terminus.
As for Ct-eIF4H, eIF4B has an extra region, approximately 70
amino acid residues long, after the RBD, compared with
mammalian eIF4H. This region of the eIF4B is part of the
eIF4H binds mRNA in the cytoplasmic perinuclear region
DRYG domain (Milburn et al., 1990; Naranda et al., 1994),
which is rich in aspartic acid, arginine, tyrosine and glycine
and is responsible for dimerization of eIF4B (Méthot et al.,
1996). There is no clear sequence similarity between the
DRYG domain and the glycine-rich central region in dipteran
eIF4H.
In both Saccharomyces cerevisiae (Tif3p) and
Schizosaccharomyces pombe (Sce3p), there is an eIF4B
homologue, but no eIF4H homologue has been detected.
If only one eIF4B/eIF4H protein is present in unicellular
eukaryotes and a variable number is present in D. melanogaster
and mammals, including pre-mRNA processing variants, it is
possible that evolution of eIF4H/eIF4B has allowed mRNAspecific variation in translation initiation. Tissue-specific
differences in mRNA levels for eIF4H and eIF4B have been
found, as well as functional variations in biochemical assays
(Richter et al., 1999).
Translation initiation factors in the nucleus
Several translation initiation factors are present not only in the
cytoplasm but also in the cell nucleus, including the eIF4
factors, eIF4E (Lejbkowicz et al., 1992; Lang et al., 1994;
Dostie et al., 2000a) and eIF4G (Etchinson and Etchinson,
1987; McKendrick et al., 2001). Our results show that CteIF4H is present in the nucleus in diptera and Xenopus. It has
also been reported that eIF4H can be present in mammalian
nuclei (Martindale et al., 2000). The function of these
translation initiation factors in the nucleus is unclear.
eIF4E is imported to the nucleus (Dostie et al., 2000b) where
it is concentrated in nuclear bodies, often associated with PML
bodies (Cohen et al., 2001; Topisirovic et al., 2002; Strudwick
and Borden, 2002). eIF4E is bound to at least some mRNAs in
the nucleus, presumably to the mRNA cap (Dostie et al.,
2000a; Lejeune et al., 2002). These mRNAs do not contain
introns and are not bound by the EJC or by the NMD factors
Upf2, Upf3 and Upf3X (Lejeune et al., 2002; Ishigaki et al.,
2001). For these reasons, eIF4E-bound nuclear mRNAs are
probably not the substrate for nuclear NMD. It has been
suggested that eIF4E is involved in exporting a subset of
mRNAs, including cyclin D1 mRNA (Rosenwald et al., 1995;
Rousseau et al., 1996). This function is dependent on cap
binding and influenced by the PML protein, which reduces the
affinity of eIF4E for the mRNA cap (Cohen et al., 2001;
Kentsis et al., 2001). eIF4G interacts with the nuclear mRNA
CBC and colocalizes with splicing factors in speckles. In vitro,
the protein is associated with spliceosomes, but it does not
appear to participate directly in splicing (McKendrick et al.,
2001). The CBC is known to influence pre-mRNA processing
(Lewis et al., 1996; Flaherty et al., 1997) and mRNA export
(Jarmolowski et al., 1994; Visa et al., 1996a) and to facilitate
CBC replacement by eIF4F (Fortes et al., 1999; McKendrick
et al., 2001). Because some translation initiation factors are
associated with the CBC, this raises the possibility that the
factors could influence any of these processes.
Several other translation components have also been located
in the nucleus (Brogna et al., 2002; Pederson and Politz, 2000;
Wilkinson and Shyu, 2002), and it has been reported that
translation takes place inside the nucleus (Iborra et al., 2001;
Brogna et al., 2002). Others have found that efficient nuclear
export occurs, in that several translation factors are present in
4529
the nucleus in very small amounts (Bohnsack et al., 2002;
Calado et al., 2002), making it unlikely that nuclear translation
takes place at any significant level. The functional significance
of possible nuclear translation is unclear, but it may be
connected to the mRNA quality assurance mechanism, NMD
(Maquat and Carmichael, 2001; Maquat, 2002). Messenger
RNAs are thought to be subjected to one initial round of
translation, to ensure that they do not contain premature inframe nonsense codons. This process is thought to occur before
the mRNAs are released into the cytoplasm (Hentze and
Kulozik, 1999; Maquat and Carmichael, 2001; Bühler et al.,
2002), but conclusive experimental data are not yet available
to rule out alternative possibilities (Dahlberg et al., 2003).
Ct-eIF4H binds to BR mRNA in the cytoplasmic
perinuclear region
We have shown that Ct-eIF4H is present in small amounts
in the cell nucleus. We have, in four different types of
experiments – UV-cross-linking to poly(A+) RNA (Fig. 6),
co-immunoprecipitation (Fig. 7), immunocytological staining
of isolated chromosomes (Fig. 5) and immunoelectron
microscopy (Fig. 8) – failed to detect an association between
eIF4H and pre-mRNA/mRNA. We therefore suggest that CteIF4H is not bound to gene-specific pre-mRNPs or mRNPs,
nor to nuclear mRNPs in general. By contrast, our data show
that Ct-eIF4H is bound to mRNAs in the cytoplasm, and our
biochemical and immunoelectron microscopy data suggest that
these mRNAs are present in ER-bound polysomes.
By studying the BR mRNP particles, which allow analysis
at the level of individual mRNPs, we showed that Ct-eIF4H
does not bind to these gene-specific mRNPs in the nucleus. The
most plausible explanation for Ct-eIF4H not binding to mRNA
in the nucleus is that other factors necessary for binding
are not present or that some factors prevent binding. It is
known that the BR mRNP particle changes morphology on
translocation through the NPC and that some proteins leave the
mRNP at this stage (Daneholt, 2001). In our immunoelectron
microscopy study, we could not detect Ct-eIF4H binding to BR
mRNP particles on the nuclear side of the NPCs. However, our
data show that Ct-eIF4H is present in close association with
the NPCs at their cytoplasmic side as the 5′ end of the BR
mRNP emerges from the NPC. Our data furthermore suggest
that Ct-eIF4H becomes associated with the BR mRNP very
close to the NPC, probably before the entire mRNP has been
translocated through the NPC.
Initiation of translation and NMD
We are unaware of any studies showing that eIF4B, the
functional partner to eIF4H, is present in the nucleus. Nor has
it been reported that eIF4A, a subunit of eIF4F, is present in
the nucleus. Several reports suggest that the initial round of
translation occurs via a mechanism that may involve a different
initiation complex compared with the subsequent steady-state
initiation complex. The initial round of translation may be part
of NMD and take place before CBP80-CBP20 are exchanged
at the mRNA cap structure (Fortes et al., 2000; Ishigaki et al.,
2001; Lejeune et al., 2002). eIF4F is normally engaged in the
binding of the mRNA to the 40S ribosomal subunit. It is
therefore possible that there are distinctly different translation
4530
Journal of Cell Science 116 (22)
initiation complexes in the cell. Perhaps only the steady-state
initiation complex in the cytoplasm includes all eIF4 factors,
thereby ensuring highly efficient translation initiation.
We have investigated the association between BR mRNP and
Ct-eIF4H. The BR mRNP is unfolded when it is translocated
through the NPC with its 5′ end first. It is not refolded on the
cytoplasmic side of the NPC (Mehlin et al., 1992). It is known
that newly synthesized BR mRNA rapidly form polysomes
(Daneholt et al., 1977) and becomes associated with the
endoplasmic reticulum (Lönn, 1977). It is therefore possible
that translation of BR mRNA is normally initiated in the
cytoplasmic perinuclear region and that the observed
association between Ct-eIF4H and BR mRNP reflects such
initiation events. If Ct-eIF4H is involved in the initiation of
translation coupled to NMD surveillance of the BR mRNAs,
our data suggest that NMD does not take place inside the
nucleus. Instead, it could occur in close connection to
nucleocytoplasmic export, in the cytoplasmic perinuclear
region. This is one possibility that could explain association of
NMD with the nucleus (Dahlberg et al., 2003). To obtain a
more complete picture, we need to further analyze the spatial
association of other translation initiation factors with mRNPs.
This requires highly specific antibodies to these factors.
We wish to thank Kerstin Bernholm and Inger Granell for excellent
technical assistance and Dr I. W. Mattaj for the anti-CBP20 antibodies
and Dr B. Daneholt for the anti-hrp45, anti-hrp36 and anti-hrp23
antibodies. We also thank Dr Neus Visa and Birgitta Björkroth for
valuable advice. This work was supported by the Swedish Research
Council (Natural and Engineering Sciences) and the Magnus
Bergvalls Stiftelse Swedish Cancer Foundation.
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