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INTRODUCTION |
There is increasing evidence that many eukaryotic mRNAs
have 3'-untranslated regions
(3'-UTRs)1 that
contain important control sequences. These regulate gene expression at the post-transcriptional level through control of mRNA stability, regulation of translation efficiency, and altering the coding capacity of mRNAs. More recently, sequence elements within 3'-UTRs have been discovered that are able to localize mRNAs
to particular regions of the cytoplasm (1-3). In the early development
of Drosophila and Xenopus, this mRNA
localization is important for establishing morphological gradients,
apparently leading to local protein synthesis (4). mRNA
localization also occurs in somatic mammalian cells such as fibroblasts
where 3'-UTR sequences and the cytoskeleton have been shown to be
involved in targeting mRNAs to the cell periphery and to the
perinuclear cytoplasm (5-8).
The c-myc mRNA has been shown to contain a localization
signal within the 3'-UTR (5, 8); this directs a reporter transcript to
the perinuclear cytoplasm and to polysomes bound to the cytoskeleton (5). The perinuclear localization of c-myc mRNA and its
association with the cytoskeleton has been suggested to provide local
synthesis of the protein that promotes efficient import of the newly
synthesized protein into the nucleus. This could be important in the
case of unstable transcription factors; however, it is not clear if mRNAs coding for other transcription factors are also localized by
a similar mechanism.
It is well established that the c-fos proto-oncogene encodes
a transcription factor that is a very unstable protein, exhibiting rapid nuclear turnover (9) and a biphasic half-life. The equally unstable messenger RNA has been studied in some detail (10, 11) and is
subject to both transcriptional and post-transcriptional control. Much
evidence indicates that both the coding region and the 3'-UTR contain
the information required for RNA destabilization. The c-fos
3'-UTR is capable of destabilizing
-globin mRNA and furthermore,
AU-rich regions (ARE) within the 3'-UTR have been identified as
important in instability (11-14). In particular, the conserved motif
AUUUA has been found in a variety of unstable mRNAs coding for
proto-oncogenic transcription factors and cytokines (15). The
cytoplasmic lifespan of c-fos mRNA is dramatically increased by the addition of protein synthesis inhibitors, the so
called "superinduction phenomenon" (9, 10, 13, 16). Using chimeric
-globin-fos 3'-UTR gene constructs in fibroblast cell lines,
reporter stability was found to increase severalfold when the
translation of the reporter was decreased (17, 18). This indicated some
link between translation and ARE-dependent mRNA
degradation of the c-fos mRNA; however, the mechanisms
require further clarification.
c-fos mRNA has been shown to be enriched in
cytoskeletal-bound polysomes released from the cell matrix following
addition of cytochalasin D (19, 20), implying that c-fos
mRNA is translated on cytoskeletal-bound polysomes and is
associated with the cytoskeleton. However, it is not known if the
c-fos mRNA is localized or if this association with the
cytoskeleton involves a signal within the 3'-UTR.
The aims of the present study were 3-fold: first, to determine whether
the 3'-UTR of c-fos proto-oncogene contains a localization signal capable of directing a reporter construct to a distinct subcellular compartment or to the cytoskeleton; second, to study whether this localization is linked to mRNA translation/stability; and last, to determine whether the elements that regulate these aspects
of mRNA fate are distinct. Cell fractionation and in
situ hybridization analyses indicate that a specific 145-nt region of the c-fos 3'-UTR, distinct from the instability element,
is sufficient to localize a reporter transcript. Furthermore,
initiation of translation of the transcript is required for the
mRNA to be localized.
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EXPERIMENTAL PROCEDURES |
Gene Constructs--
Gene constructs pIREglo·fos,
pIREglo·fos
, and pIREglo·glo were made as previously described
(17). Briefly, the BamHI genomic DNA fragment containing
most of the mouse c-fos gene was recovered from p19/1 and
cloned into the pJ6
vector downstream from the rat
-actin
promoter (21). The fos
construct was obtained by excising the ARE
contained in the 3'-UTR with NstI and MstI
(respectively nucleotides 3701 and 3831 from the cap site) and
re-ligation of the blunt ends. The IRE from the human ferritin gene was
introduced into the constructs by cloning the following 43-base pair
double-stranded oligonucleotide cloned into the XbaI
site of pJ6
: (5'-CTAGGGATTCCTGCTTCAACAGTGTTGGACGGATCCCTCTAGA-3'. This created the constructs, pIREglo·glo, pIREglo·fos and
pIREglo·fos
. Further deletions in the fos 3'-UTR were
generated by polymerase chain reaction with the following
oligonucleotides, combining GDDS with successively the reverse primers
GDD2 (to produce
2), GDD3 (
3), GDD4 (
4), and GDD5
poly(A)).
The polymerase chain reaction products (GDDS, AGGGCAGCTGCTGCTTACAC;
GDD2, ACCATTCAGACCACCTCGAC; GDD3, GATACAATCCAGCACCAGGT; GDD4,
GGAACAACACACTCCATGCG; GDD5, TCCACATGTCGAAAGACCTC) were cloned
directly into pcDNA3.1/V5/his-TOPO (Invitrogen). All constructs
were verified by sequencing, and they are shown schematically in
Fig. 1.

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Fig. 1.
Details of gene constructs with variations in
the 3'-untranslated region. The coding region of -globin was
used as a reporter transcript. In one set of constructs, it was linked
both to the IRE from the ferritin gene (in a 5' position) and either
its own 3'-UTR (pIREglo·glo), the whole 3'-UTR from c-fos
(pIREglo·fos), or the c-fos 3'-UTR with a the instability
region deleted (pIREglo·fos , removing bases 589 715); these three
constructs used the fos polyadenylation signal (poly(A)). A
second set of constructs lacked the IRE and consisted of the -globin
coding region linked to the whole fos 3'-UTR, the 3'-UTR
without the instability region (pcDNA3.1poly(A) glo·fos ), or
the 3'-UTR with increasingly large deletions so that 2, 3, and
4 contained respectively, bases 0-646, 0-403, and 0-260 of the
fos 3'-UTR. These constructs utilized the vector
polyadenylation signal (from bovine growth hormone, BGH). A
final construct contained globin coding sequences, the whole
fos 3'-UTR, and the BGH polyadenylation
site.
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Cell Culture and Transfection--
Cells were grown in 90-mm
Petri dishes for cell fractionation or RNA extraction and in glass
chamber slides for in situ hybridization. LTK
fibroblasts were grown in Dulbecco's minimal Eagle's medium (Life Technologies, Inc.) and CHO cells in Ham's F12 modified with
L-glutamine (ICN Biomedicals Inc), both supplemented with
10% fetal calf serum and in an atmosphere of 5%
CO2.
Transfection of LTK
fibroblasts and Chinese hamster ovary
cells was carried out using LipofectAMINE (Life Technologies, Inc.). Cells were cotransfected with both the plasmid DNA of interest and a
pcDNA3 (Invitrogen) plasmid carrying neomycin resistance. Stable
transfectants were selected by culture in the presence of 1 mg/ml G418.
For transient transfection, cells were transfected 2 days after
subculture, the medium was changed after 24 h, and in
situ hybridization was carried out after a further 24 h.
In Situ Hybridization--
Comparison of mRNA distribution
was carried out in cells grown in multiwell chamber slides so that the
different cell lines and different treatments could be studied under
identical conditions; in this way mRNA distribution and its
quantification was directly comparable. Fixation, hybridization,
detection, and analysis was carried out as described previously (5, 8);
a digoxigenin-labeled antisense riboprobe was used followed by alkaline
phosphatase detection. The probe was generated using T7 polymerase from
a 511-base pair XbaI-BamHI fragment containing
most of the first two exons of the rabbit
-globin gene using a DIG
RNA labeling kit (Roche Molecular Biochemicals). Controls were either
hybridized with a digoxigenin-labeled sense probe generated from the
same fragment using SP6 polymerase or incubated with hybridization mix
containing no probe. Bound probe was detected by incubation with
alkaline phosphate-linked anti-digoxigenin and incubation with 4-nitro
blue tetrazolium for 16 h or with HNPP detection kit (Roche
Molecular Biochemicals). The staining produced by the alkaline
phosphatase activity was quantified using an image analysis system in
which the images were captured using a Pulnix camera and
Fenestra/Cyclops software (Kinetic Imaging Ltd., Liverpool, UK).
For each cell, staining intensity was measured in three small areas of
identical size (8). First, a measurement of staining was taken from the
perinuclear region; this was defined as a region of cytoplasm close to
the nucleus but not including any of it. A second measurement was taken
in an adjacent area of the cytoplasm but in the cellular periphery,
near the cell membrane, and finally a cell blank was taken out with the
cell. Following subtraction of the blank reading, a
perinuclear/peripheral staining ratio was calculated to obtain a
quantitative measure of the extent of the localization in each case.
3-4 measurements were made in each cell, and 30 individual cells
selected at random were analyzed for each cell line. The experiments
and analyses were repeated at least three times.
The staining pattern produced using the fluorogenic substrate HNPP was
examined by confocal laser scanning microscopy (CLSM). CLSM was
performed using a Bio-Rad confocal laser with a krypton-ion laser and
an emission line at 568 nm. Cells were examined using a × 60 objective lens and optically sectioned at 0.5-µm slice thickness
parallel to the substratum. Images were directly transferred to an
optical disc and subsequently analyzed using Confocal Assistant software. Images were converted to TIFF format, and figures were assembled using Freehand version 8.
Cell Fractionation, RNA Extraction, and Northern
Hybridization--
For analysis of endogenous fos
expression, LTK
cells were serum-starved for 20 h
prior to treatment with cyclohexamide (10 µg/ml) and serum (10%
fetal calf serum) for 30 min prior to extraction of polysomes; this
treatment elevates endogenous fos mRNA expression (9,
22). Cells stably transfected with pIREglo·glo, pIREglo·fos, and
pIREglo·fos
did not require serum induction or cyclohexamide treatment prior to fractionation. Free cytoskeletal-bound and membrane-bound polysomes were isolated using a sequential
detergent/salt extraction procedure followed by centrifugation at
32,000 × g for 16 h through a 10-ml cushion of
40% sucrose, as described previously (5, 23, 24).
Total RNA was extracted by the method of Chomczynski and Sacchi (25),
and the RNA species were then separated by electrophoresis through a
denaturing 2.2 M formaldehyde, 1.2% agarose gel (26) and
transferred to a nylon membrane (Genescreen from PerkinElmer Life
Sciences) by capillary blotting. RNA was fixed to the membrane by
exposure to UV light, and membranes were prehybridised overnight at
42 °C with 0.1 gm/ml denatured salmon sperm DNA in 50% formamide, 10% dextran sulfate, 0.2% bovine serum albumin, 0.2%
polyvinlypyrrilidone, 0.2% ficoll, 0.1% sodium pyrophosphate, 1%
SDS, and 50 mM Tris-HCl, pH 7.5.
The
-globin probe corresponded to the
XbaI-XhoI fragment previously used for Northern
analysis (5). The c-fos probe was produced from a
1.3-kilobase pair PstI fragment of the v-fos gene in the pfos-1 vector (27); and c-myc from a 1.8-kilobase
pair HindIII fragment from the mouse pT7-2 vector (5). A
50-100-ng sample of each DNA probe was labeled with
[32P]dCTP by random priming (Amersham Pharmacia Biotech)
and the labeled DNA was then separated from free nucleotides by gel
filtration on Sephadex G-50; probe-specific activities were
~109 cpm/µg DNA. The labeled probes were added to the
prehybridization mix and hybridized at 42 °C for 24 h. The
membranes were then washed to remove nonspecific hybridizations twice
in 2× SSC at room temperature for 5 min, followed either by 0.75×
SSC, 1% SDS (globin); 0.5× SSC, 1% SDS (fos and
myc) or 0.2× SSC, 1% SDS (18 S) at 65 °C for 1 h
(twice). Specific hybridization was then detected and quantified using
Canberra Packard Instantimager. After stripping, membranes were
rehybridised with a 1.4-kilobase probe for 18 S rRNA (28) to quantify
RNA loading.
The half-life of transcripts derived from appropriate constructs was
estimated in stable transfected cell lines by inhibition of
transcription with actinomycin D (5 µg/ml) and subsequent RNA extraction after 2, 4, 6, and 10 h, and estimation of transcript abundance by Northern hybridization.
Statistical Analysis--
Data from quantification of cell
fractionation or in situ hybridization were expressed as
logarithms of the ratio of cytoskeletal/free polysomes or
perinuclear/peripheral staining and analyzed using the Student's
t test.
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RESULTS |
Endogenous c-fos mRNA Is Enriched in Cytoskeletal-bound
Polysomes--
It has previously been reported that c-fos
mRNA (along with other cytoskeletal-bound mRNAs) can be
released from the cell matrix by treatment with cytochalasins (19). A
more recent sequential detergent and salt extraction releases
cytoskeletal-bound polysomes from the cell matrix (29); a nonionic
detergent soluble fraction contains free polysomes (F), a
salt extract contains cytoskeletal-bound polysomes (C), and
a deoxycholate-solubilized fraction contains membrane bound polysomes
(M). As shown in Fig.
2A, c-fos mRNA was found enriched in the C fraction as has been observed previously for c-myc mRNA (5). This was confirmed by quantification
of the Northern analysis; the C/F ratio of c-fos mRNA
abundance was 1.8 (data not shown), indicating enrichment of
c-fos mRNA in cytoskeletal-bound polysomes.

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Fig. 2.
Distribution of c-fos,
c-myc, and -globin
transcripts between free, cytoskeletal-bound, and membrane-bound
polysomes in LTK fibroblasts. LTK
cells were subjected to sequential detergent and salt extraction to
release free (F), cytoskeletal-bound (C), and
membrane-bound polysomes (M). RNA was extracted from the
polysomes and analyzed by Northern hybridization using
32P-labeled DNA probes. Results are shown for 10 µg of
total RNA, and each hybridization was carried out sequentially on the
same nylon membrane. A, samples from untransfected cells
were analyzed successively for c-fos mRNA,
c-myc mRNA, and 18 S rRNA (to control for any variation
in loading). The c-fos and c-myc mRNAs were
found at highest abundance in the C fraction. B,
RNA from transfected cells was analyzed for globin, c-myc,
and 18 S. Globin transcripts with the endogenous 3'-UTR (glo·glo)
were found at highest abundance in the F fraction with
considerable amounts in the C. In contrast, the transcripts
with globin linked to the whole fos 3'-UTR (glo·fos) or
the fos 3'-UTR with the ARE removed (glo·fos ) showed
highest abundance in the C fraction, in parallel to
c-myc distribution. C, quantification of Northern
blots from four experiments confirmed that addition of the
fos 3'-UTR (or fos ) increased association of globin
transcripts with the cytoskeleton. Data were expressed as mRNA
abundance per unit 18 S rRNA, and then the ratio of abundance in
C fraction compared with that in the F fraction
was calculated.
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c-fos 3'-UTR Localizes a Reporter mRNA Sequence to
Cytoskeletal-bound Polysomes and the Perinuclear Cytoplasm--
To
study the localization functions of the fos 3'-UTR, stable
transfected cell lines were made that express chimeric gene constructs
in which the fos 3'-UTR, fos
3'-UTR, or globin
3'-UTR were linked to the
-globin coding region as a reporter.
Addition of either the c-fos 3'-UTR or c-fos
3'-UTR to the
-globin coding region reporter led to the globin
transcripts being recovered at highest abundance in the
cytoskeletal-bound polysome fraction, which was not the case in the
-globin control (Fig. 2B). In all three cell lines
(glo·glo, glo·fos, and glo·fos
) the c-myc mRNA was enriched in the C fraction (Fig. 2B). The ratio of
transcript abundance (per unit 18 S rRNA) in cytoskeletal-bound
polysomes/free polysomes was significantly higher (p < 0.05) in both the glo·fos and glo·fos
cells compared with the
glo·glo control cells (Fig. 2C).
In situ hybridization was used to detect the subcellular
localization of the globin transcripts. In LTK
cells
transfected with a control construct containing the whole of the
-globin gene there was no specific subcellular localization of the
globin reporter transcript (Fig.
3A, as previously reported (8); this is indicated by staining throughout the cytoplasm of the
cell. However, cell lines transfected with constructs in which the
c-fos 3'-UTR was linked to a
-globin coding sequence, resulted in a marked perinuclear distribution, visible as rings of
staining around the nucleus, but with little or no staining toward the
cellular periphery (Fig. 3A). A similar perinuclear-staining pattern was seen in cells expressing the stable glo·fos
transcripts (Fig. 3A); the increased staining intensity in
these cells, compared with glo·fos cells, may be attributed to the
greater stability of the glo·fos
transcript. These differences in
transcript distribution were confirmed by image analysis (see
"Experimental Procedures" and Ref. 5); as shown in Fig.
3B, the glo·fos and glo·fos
transcripts showed an
enrichment of the perinuclear cytoplasm, which was significantly
(p < 0.01) greater than that of the glo·glo control.
The difference between the c-fos
construct and the globin-fos construct is that the instability element has been deleted from the
3'-UTR (17); indeed the glo·fos
transcript is not rapidly degraded
and has a half-life of greater than 10 h compared with 2 h
for glo·fos (Fig. 4). Because both the
rapidly degraded glo·fos and the stable glo·fos
transcripts are
localized, it is evident that not only is the fos 3'-UTR
capable of targeting the globin reporter to cytoskeletal-bound
polysomes and the perinuclear cytoplasm but that this targeting is
independent of message stability.

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Fig. 3.
In situ hybridization and
quantification of the distribution of globin transcripts showing the
effects of c-fos 3'-UTR on distribution of
reporter. A, LTK fibroblasts were stably
transfected with glo·glo, glo·fos, and glo·fos constructs.
Globin transcripts were detected by in situ hybridization
using a digoxigenin-labeled riboprobe specific for the globin coding
sequence and alkaline phosphatase-linked anti-digoxigenin antibody with
4-nitro blue tetrazolium as substrate. Using antisense probes, specific
transcript distribution was detected. In glo·glo cells
(a), there was distinct localization of transcripts, but in
both glo·fos (b) and glo·fos (c), rings of
staining were observed in the perinuclear cytoplasm with little or no
staining in the cell periphery. Bar represents 10 µm.
B, LTK fibroblasts stably transfected with
either glo·glo, glo·fos, or glo·fos . Globin transcripts were
detected by in situ hybridization using a
digoxigenin-labeled riboprobe specific for the globin coding sequence
and alkaline phosphatase-linked anti-digoxigenin antibody with 4-nitro
blue tetrazolium as substrate. Images were captured and staining was
quantified in the perinuclear and peripheral cytoplasm of at least 30 cells chosen at random. The data were collected from the same stable
cell lines illustrated in A.
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Fig. 4.
Stability of chimeric globin-fos 3'-UTR
transcripts in transfected CHO cells. mRNA stability was
assessed by measurement of transcript levels using Northern
hybridization. Total RNA was isolated from glo·fos ( ),
glo·fos ( ), glo·fos 2 ( ), glo·fos 3 ( ), and
glo·fos 4 ( ) cells grown in normal medium and 2, 4, 6, and
10 h after treatment with actinomycin D.
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Confocal Microscopy of CHO Cells Transfected with Globin Reporter
Linked to c-fos 3'-UTR--
CHO cells have a wider, broader morphology
and were used to confirm the results obtained in LTK
cells, coupled with the use of a fluorescent HNPP detection method and
confocal microscopy. Confocal microscopy allows analysis of z-sections through the cells, thereby reducing artifacts in
analysis because of differences in cellular thickness. Cells were
transiently transfected with each of the constructs, and fluorescent
in situ hybridization was used to visualize the distribution
of the reporter transcripts.
Cells transfected with the glo·glo construct (Fig.
5a) again showed no particular
localization of transcripts, as indicated by fluorescent staining
present throughout the cytoplasm. In contrast, as shown in Fig. 5,
b and c, cells expressing either glo·fos or glo·fos
transcripts both showed a clear localization of staining in the perinuclear region. The differences in staining pattern were
confirmed by image analysis. Profiles of staining intensity showed that
the intensity of staining remains high throughout the cytoplasm (even
toward the extremities of the cells) in cells transfected with the
glo·glo construct, but that the cells expressing the glo·fos and
glo·fos
constructs exhibited a peak of intensity in the
perinuclear region (results not shown). Combined, these data indicate
that the differences in globin reporter distribution on addition of
c-fos 3'-UTR sequences represent a true mRNA
redistribution and cannot be accounted for by differences in cellular
thickness.

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Fig. 5.
Confocal microscopy showing
-globin transcript distribution in transiently
transfected CHO cell lines. CHO cells were transiently transfected
with the glo·glo, glo·fos, and glo·fos constructs. Globin
transcripts were detected by in situ hybridization using a
digoxigenin-labeled riboprobe specific for the globin coding sequence
and alkaline phosphatase-linked anti-digoxigenin antibody with the
fluorogenic HNPP as substrate. The distribution of the fluorescent
product was detected by confocal microscopy and 0.5 µm
z-series sections that were collected through the cells. The
sections shown are taken through the middle of the cell. There was no
localization of the -globin in the cells transfected with glo·glo
(a), but those transfected with either glo·fos
(b) or glo·fos (c) showed distinct
perinuclear localization of the globin transcripts. Note the
perinuclear ring present in b and c but not in
a. The data confirm that the c-fos 3'-UTR is
capable of targeting a globin reporter to the perinuclear cytoplasm.
Bar represents 10 µm.
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Effect of Altering Translation on Reporter Transcript
Distribution--
The glo·fos and glo·fos
constructs contain an
IRE in a position 5' to the globin reporter. The presence of this IRE
makes translation of the reporter transcript sensitive to iron
concentration in the culture media (17). Under conditions where iron is
abundant (such as cells treated with ferric ammonium citrate) the IRE
promotes mRNA translation, whereas under conditions where iron is
scarce (such as after treatment with chelator desferrioxamine),
translation is prevented and the RNA is found in free mRNPs. This
effect has been found to be specific for transcripts containing an IRE,
and global protein synthesis is not affected by iron availability. When
cells expressing either glo·fos or glo·fos
transcripts were cultured in elevated iron concentrations, (conditions in which the
reporter transcript is actively translated), the perinuclear localization of the transcripts was maintained (Fig.
6, b and d), as
indicated by a strong ring of staining surrounding the nucleus.
Conversely, addition of desferrioxamine for 16-20 h (which reduces the
translation of the chimeric transcripts), led to a less marked ring of
perinuclear staining in both cell lines (Fig. 6, a and
c). Treatment with ferric ammonium citrate or
desferrioxamine had no effect on cell morphology (Fig.
7).

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Fig. 6.
In situ hybridization showing the
effects of modulating iron concentration on globin transcript
distribution in transfected CHO cells. CHO cells transfected with
pIREglobin·fos and pIRE globin·fos were cultured in medium
either supplemented with ferric ammonium citrate (10 µM)
or treated with desferrioxamine (100 µM) for 16 h
prior to fixation and in situ hybridization using an
antisense riboprobe specific for the globin coding sequences. Specific
labeling was detected using an alkaline phosphatase-linked
anti-digoxigenin antibody and 4-nitro blue tetrazolium as substrate.
Cells transfected with either the IREglo·fos (b) and
IREglo·fos (d) showed distinct perinuclear distribution
when the cells were grown in medium supplemented with iron
(IRE-containing transcripts translated). In contrast, both IREglo·fos
(a) treated with desferrioxamine (to reduce iron levels and
so prevent translation of transcripts containing the IRE) and
IREglo·fos (c) cells treated with desferrioxamine
showed no localization of the transcripts. In the latter, there was no
ring of perinuclear staining, and staining was found throughout the
cytoplasm into the cell periphery. Bar represents 10 µm.
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Fig. 7.
Cellular morphology after modulating iron
concentration in the media of CHO glo·fos cells. CHO cells
transfected with IREglo·fos were cultured in either normal medium
(a), in medium supplemented with 10 µM ferric
ammonium citrate (b) or treated with 100 µM
desferrioxamine (c) for 16 h prior to analysis. No
change in cellular morphology was observed following either
experimental treatment.
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A 145-Nucleotide Region of the c-fos 3'-UTR Is Required to Localize
a Reporter Construct--
The data from cells transfected with the
glo·fos
construct show that the region between bases 589 and 715 is not necessary for localization. The c-fos 3'-UTR is
highly conserved throughout evolution, and sequence alignments indicate
an 80% homology between species with certain regions showing high
conservation. It is possible that one of these other conserved regions
is involved in localization. To define further the 3'-UTR region
required for localization, three new deletions were made in which
different regions of highly conserved homology were removed. The
different 3'-UTR deletion products were linked to
-globin reporter
sequences (as in the previous constructs), resulting in the
-globin
linked to the c-fos 3'-UTR with increasingly large
deletions. During cloning of these constructs the endogenous poly(A)
site was removed and replaced with that of the bovine growth hormone
within the pcDNA3.1 mammalian expression vector. To ensure that
this had no effect on the localization of the mRNA, a further
construct was produced, which removed only the region containing the
endogenous c-fos poly(A) site (
poly(A)GF). The
replacement of the poly(A) site was found to have no effect on mRNA
of the reporter by the c-fos 3'-UTR (data not shown).
CHO cells were transfected with each of the
2,
3, and
4
constructs and were analyzed by in situ hybridization after
both transient and stable transfection. In each case, the
2 and
3 both showed a perinuclear distribution of globin transcripts (Fig. 8, a and b).
However, cells transfected with the
4 construct no longer exhibited
a perinuclear localization (Fig. 8c), with globin
transcripts distributed throughout the cell to the periphery. The
localization of the globin transcripts was quantified in both stable
and transient transfectants by scoring the percent cells with
transcript distribution localized in the perinuclear region. In each
group under study, at least 100 cells were analyzed and, as shown in
Table I, the results
showed that 64-85% of the cells transfected with
2 or
3
exhibited perinuclear localization, but only 14-22% of the cells
expressing
4 showed localization. This confirms the observations
illustrated in Fig. 8 and shows that removal of bases 3' to position
403 does not affect localization, but that removal of the 145-nt region
between bases 260 and 403 largely destroys the ability of
c-fos 3'-UTR sequences to localize globin transcripts to the
perinuclear cytoplasm. The stability of
2,
3, and
4
transcripts was assessed following actinomycin D treatment, and all
three were found not to be rapidly degraded but to be stable, similar
to the glo·fos
transcript; the half-lives were all similar and all
greater than 10 h (Fig. 4). The comparable stability of these
transcripts indicates that the differences in localization are
independent of stability. Overall, the data show that the 145-nt region
of the c-fos 3'-UTR between bases 260 and 403 is sufficient
to localize the globin reporter.

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Fig. 8.
Effect of deletion in the c-fos
3'-UTR on its ability to target -globin
transcripts to the perinuclear cytoplasm. -globin transcript
distribution was studied by in situ hybridization in
transiently transfected CHO cells. Globin transcripts were detected by
in situ hybridization using a digoxigenin-labeled riboprobe
specific for globin coding sequence and alkaline phosphatase-linked
anti-digoxigenin antibody with 4-nitro blue tetrazolium as substrate.
Cells transfected with glo·fos 2 (a) and glo·fos 3
(b) showed distinct perinuclear distribution of the globin
transcripts (note the perinuclear ring of staining), but cells
transfected with glo·fos 4 showed no localization of transcripts.
Cells reacted with sense riboprobe showed no staining (d).
Bar represents 10 µm.
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Table I
Effect of deletions in the c-fos 3'-untranslated region on its ability
to localise -globin reporter transcripts
The distribution of globin transcripts was studied in cells transfected
with a series of deletion constructs using in situ
hybridization with a digoxigenin-labeled riboprobe and alkaline
phosphatase detection. Both stable and transient transfectants of each
construct were studied, and in every case at least 100 cells were
scored for transcript localization. The data presented are from two
separate
experiments.
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DISCUSSION |
Endogenous c-fos mRNA has been shown to be
associated with the cytoskeleton (19, 20). The present results confirm
this observation using a complementary method involving separation of
cytoskeletal-bound polysomes by salt treatment of the cell matrix (23).
Furthermore, the data show that the c-fos 3'-UTR is also
sufficient to target a chimeric reporter to cytoskeletal-bound polysomes and the perinuclear cytoplasm. This suggests that the c-fos 3'-UTR contains a perinuclear targeting signal.
It was formally possible that the perinuclear distribution of the
glo·fos transcript was caused by mRNA instability rather than a
mRNA targeting mechanism. However, although removal of the
instability element from the 3'-UTR (glo·fos
) produced transcripts with a significantly longer half-life it had no effect on the perinuclear localization of transcripts or association with the cytoskeleton induced by the c-fos 3'-UTR. In addition, a
further deletion in the 3'-UTR (
4) destroyed localization without
affecting mRNA stability. These data suggest that the observed
localization caused by c-fos 3'-UTR sequences is due to a
mRNA localization mechanism and not an effect on mRNA
instability. Furthermore, they indicate that the localization signal is
distinct from the ARE instability region, and that localization by the
c-fos 3'-UTR is not linked to the mRNA degradation pathway.
Previous studies using stable (17) and transient (18) transfected cell
lines expressing chimeric globin-fos 3'-UTR constructs containing a IRE
found that prevention of the initiation of translation (by addition of
an iron chelator) led to a significant increase in the half-life of the
reporter mRNA. Furthermore, removal of the ARE in the glo·fos
construct led to a stable mRNA that was insensitive to Fe
availability (10, 17, 30). These observations indicated that the
stability of transcripts due to control by the ARE within the
fos 3'-UTR is linked to translation. In contrast, although
the present results indicate no link between localization by the
fos 3'-UTR and mRNA stability, they do suggest a link
between translation and localization; alterations in Fe concentration in the medium not only prevent the initiation of translation of glo·fos and glo·fos
transcripts (17), but also affect their localization. Because desferrioxamine has no effect on degradation of
the more stable glo·fos
mRNA (17), its effect on transcript localization (Fig. 6) cannot be accounted for by altered transcript stability. Thus, the changes in glo·fos
distribution in response to reduced iron availability indicate that initiation of translation is
required for localization and that these effects do not reflect differences in mRNA stability.
Further deletion studies showed that removal of the fos
3'-UTR from 647 to the poly(A) signal and from 403 to 647 had no effect on localization. However, removal of bases 260-403 led to loss of
localization without any change in transcript stability, showing that
this 145-nt sequence contains the localization signal required for
perinuclear localization and association with the cytoskeleton. This
region has not been implicated in stability, is distinct from the ARE
region, but it is highly conserved between species. More detailed
investigation into the region of the c-fos 3'-UTR is
required to define the exact part of a 145-nt region that is responsible for localization and to determine the precise nature of the
perinuclear targeting signal.
The c-fos 3'-UTR is highly conserved between species, which
implies that it has an important functional role. This indeed appears
to be the case and overall, the present results support the view that
the c-fos 3'-UTR has a multifunctional role in mediating mRNA degradation and localization, and that these in turn are linked to translation. Furthermore, it is apparent that the elements within the 3'-UTR responsible for mRNA localization and instability are distinct. The presence of the instability element is well documented (11, 12, 14, 30, 31), but the present observation of a
localization function is novel.
The evidence presented here suggests that 3'-UTR sequences ensure that
c-fos mRNA is translated in the correct location, namely associated with the perinuclear cytoskeleton. Furthermore, perinuclear mRNA localization on the cytoskeleton promotes efficient
nuclear protein import of metallothionein-1 (32). Targeting of
mRNAs to the cytoskeleton may be a general mechanism to
promote efficient nuclear protein import, and this may be
particularly important for an unstable protein that is
required to be used as efficiently as possible (33).
Furthermore, such a mechanism could be a control point;
-actin mRNA localization responds to growth factor
stimulation (34), and if c-myc or
c-fos mRNA localization was modulated similarly, it could provide a mechanism to regulate nuclear
localization of the protein.