From the
Expression of the platelet-derived growth factor 2/c- sis gene is highly restricted and controlled at multiple levels. Its
structured mRNA leader, which is unusually long (1022 nucleotides),
serves as a potent translational inhibitor. One of the sites of PDGF2
synthesis is megakaryocytes, implying that PDGF2 translation efficiency
is modulated during megakaryocytic differentiation. To study the role
of the mRNA leader as a translational cis-modulator, the
hybrid T7/vaccinia cytoplasmic expression system was used to disconnect
between determinants controlling transcription, alternative splicing,
and mRNA stability from those controlling translation. Chimeric
transcripts in which the human PDGF2/c- sis mRNA leader
positioned in frame upstream of a reporter gene were used to determine
whether the mRNA leader can confer variable translational efficiencies
during differentiation. It is demonstrated that there is a time window
during megakaryocytic differentiation of K562 cells in which the strong
translational inhibition by PDGF2/c- sis mRNA leader is
relieved. The time course of the translational repression relief is
similar to that of PDGF2/c- sis transcriptional induction
during the differentiation process. A 179-nucleotides CG-rich fragment
immediately upstream of the initiator AUG codon is necessary for
coffering stringent modulation of the translational efficiency. In
NIH3T3 overexpressing translation initiation factor eIF4E, the
inhibitory effect of the mRNA leader of c- sis is not relieved,
suggesting that the changes in the translational machinery during
megakaryocytic differentiation are beyond eIF4E activity. The possible
involvement of a 5`-end-independent translational mechanism is
discussed.
Platelet-derived growth factor (PDGF)
Numerous eukaryotic
cis-elements controlling transcription have been thoroughly
studied. However, very little is known about the contribution of
specific cis-elements to the translational control component
of gene expression. It is accepted that genes encoding cell growth
regulatory proteins are designed to be expressed at low levels to
prevent harmful consequences due to their overexpression. This is
achieved by transcription and post-transcriptional control mechanisms,
one of which is inefficient initiation of translation. It is known that
the efficiency of translation initiation is influenced by the sequence
context in the vicinity of the AUG codon, the number of upstream AUG
codons, and the length and secondary structure of the mRNA leader (for
review, see Refs. 15 and 16). Leader sequences of most vertebrate mRNA
fall in the size range of 20-100 nucleotides, and an upstream AUG
codon occurs in fewer than 10% of the mRNAs
(17) .
Interestingly, genes for growth factors, cytokine receptors, and
proto-oncogenes often produce transcripts with unusually long,
structured, upstream AUG-burdened mRNA leaders. Such 5`-UTRs impose a
major difficulty to the conventional ribosomal scanning
(18) .
Coinciding with other mRNAs that encode critical regulatory proteins,
the exceptionally long 5`-UTR of c- sis mRNA has three upstream
mini open reading frames and high G+C content predicted by
computer analysis to form stable secondary structures. Such structures
have a calculated free energy that ranges from -100 to -250
kcal mol
According to the scanning
model for translational initiation, the translation machinery binds to
the cap structure at the 5`-end of the mRNA followed by a
one-dimensional, ATP-dependent migration along the leader to find a
start codon. The initial binding is mediated through eukaryotic
initiation factor 4E (eIF4E), which is a subunit of eIF4F complex that
functions to unwind secondary structure in the mRNA leader to
facilitate ribosomal scanning
(20) . Phosphorylated eIF4E is
believed to be the rate-limiting component of eIF4F necessary for mRNA
association with the 48 S initiation complex
(21) . Increased
translation levels have been directly correlated with increased
phosphorylation levels of eIF4E in response to a variety of growth
stimuli (Refs. 22-24 and references therein). As eIF4E
overexpression leads to cellular transformation of NIH3T3 cells
(25) , a model was proposed predicting that overexpression of
active eIF4E can facilitate translation of cellular mRNAs with lengthy
and structured 5`-UTRs. Many of such genes, including
PDGF2/c- sis, encode for cell growth regulatory proteins, some
of which are known proto-oncogenes. However, during cell growth or
differentiation, it is hard to determine whether enhanced expression of
a specific gene is a result of translation perse that becomes more efficient, since various process such as
transcriptional stimulation and mRNA stabilization take place as well.
The fundamental question we wished to address in this study was
whether the encumbered 5`-UTR serves invariably as a translational
inhibitor or alternatively whether it ``senses'' cellular
changes and thus serves as a translational modulator. The later
predicts that certain transient changes in the cellular milieu may
create supportive conditions for translation of mRNAs that are poorly
translated otherwise. Such mechanism might allow for a more efficient
translation of c- sis in the right location and timing,
i.e. during megakaryocytic differentiation when PDGF is
synthesized and stored in
To determine whether differentiation is accompanied
by enhanced cellular capacity to translate mRNA harboring certain
5`-UTRs, two plasmids were constructed: pOS14, which produces CAT mRNA
with a short leader of 37 nucleotides, and pPD1, which contains the
entire 1022 bp of c- sis 5`-UTR joined in-frame with CAT (see
Fig. 5a). Differentiation of K562 cells toward the
megakaryocytic lineage was induced by treatment with 5 nM TPA
for 40 h and was associated with arrest of proliferation, typical
morphological changes, and induction of c- sis transcription
(not shown). CAT activity as well as CAT mRNA level was determined 24 h
following liposome-mediated transfection of each plasmid into
vTF7-3-infected K562 cells. Either megakaryocytic-differentiated
or control K562 cells were used for the infection/transfection
experiments. Fig. 1 a demonstrates the integrity of CAT
mRNA made by the vaccinia/T7 cytoplasmic transcription system in both
cell types. CAT activity was measured by phase extraction radioassay
and normalized against CAT mRNA amount as quantified by laser
densitometry. The variations in CAT activity/RNA unit reflect
differences in translational efficiencies. To compare the effect of the
5`-UTR in the different cellular environments, the CAT activity/RNA
unit of each plasmid in the TPA-treated cells was divided by its value
in the control K562 cells. Fig. 1 b demonstrates that the
translation efficiency of pOS14-derived CAT was enhanced 1.05 ±
0.07-fold in the TPA-treated compared with the control K562 cells,
whereas megakaryocytic differentiation led to 8.46 ± 1.57-fold
enhancement of pPD1-derived CAT mRNA translation.
One of the fascinating aspects of gene expression is the
demand for regulation to guarantee correct level, location, and timing.
The regulation of c- sis expression is most likely the result
of multiple mechanisms. In this study, we have analyzed the effect of
c- sis 5`-UTR on translational efficiency during megakaryocytic
differentiation of human K562 cells upon TPA treatment. To disconnect
between determinants controlling transcription quality/quantity,
splicing and RNA stability from those controlling translation, we have
employed the prokaryotic bacteriophage T7 transcription machinery in
the cytoplasm of K562 cells. The T7/vaccinia is a most efficient
transient expression system as 90-100% of the vaccinia-infected
cells express the transfected plasmid
(44) . The broad host
range of vaccinia virus eliminates the technical difficulties of gene
transfer into differentiated K562 cells. Such hybrid T7/vaccinia system
enabled us to evaluate the 5`-UTR contribution to translation
efficiency in different cellular environments, i.e. during
megakaryocytic differentiation. In agreement with its performance as
inhibitor in COS-1, bovine endothelial cells
(14) , and HOS
cells
(5) , c- sis 5`-UTR operated as a strong
translational inhibitor in nondifferentiated K562 cells. However,
during the differentiation process, the inhibitory effect of the
cumbersome 5`-UTR was strikingly relieved (Fig. 1). This
observation shows that the mRNA leader of c- sis is not a
translational inhibitor but rather a translational modulator. A 179-bp
CG-rich fragment immediately upstream of the initiator AUG codon was
found to be necessary for coffering stringent modulation of translation
efficiency to heterologous mRNA throughout the differentiation process
(Fig. 5).
Among the TPA-triggered cellular changes that
eventually lead to differentiation of K562 cells is the induction of
c- sis transcription. The accumulation of c- sis mRNA
during the course of differentiation has also been observed by others
(4, 26, 45) . The present study shows that the
5`-UTR-stimulatory effect on translation of heterologous mRNA has a
similar time frame pattern (Fig. 4). It seems that the c- sis gene acquires in parallel both the transcription and translation
abilities for a transient period of time during the course of
megakaryocytic differentiation. This finding fits a rational of proper
timing of events during differentiation, i.e. the
translatability of certain mRNA matches the time interval of its
maximal abundance. It is unknown whether these phenomena are coupled.
However, such a double-safety control mechanism guarantees that no
protein will be synthesized from c- sis mRNA in case it is
accidentally present in the wrong tissue or at the wrong timing. We
have further confirmed that in other cell lines that cannot undergo
megakaryocytic differentiation and do not express PDGF such as Hela and
J774A, the cis-stimulatory effect on translation is not
conferred by c- sis 5`-UTR upon 5 nM TPA treatment
(not shown).
One of the global translational control mechanisms is
based on the phosphorylation state of the cap binding protein eIF4E.
Maximal phosphorylation of eIF4E, reached a few hours following
exposure of cells to mitogenic agents, has been correlated with
increase in translation rates
(22, 23, 24, 39, 46) . It has
been suggested that mRNAs harboring structured 5`-UTRs are weak
competitors for eIF4F, therefore their translation is dependent upon
cellular levels of phosphorylated active eIF4E
(25) . This
notion was supported by the stimulated translation of the structured
5`-UTR bearing ornithine decarboxylase mRNA upon insulin- or phorbol
ester-treatment under conditions leading to eIF4E phosphorylation
(39) . The enhanced translation of mRNA harboring synthetic
leaders with stable secondary structures at the range of
The 5`-UTR complexity of a given mRNA determines its
translational level in both vertebrate and yeast systems
(18, 50) . Specific cis-elements in the mRNAs
of ferritin
(51) , ribosomal proteins
(52) , c- myc(53) , ornithine decarboxylase
(39) , transforming
growth factor
It is generally believed
(51, 52, 53, 54) that the relative
contribution of the cis-elements on translation is dictated by
temporal appropriate trans-factors. The present study
demonstrates that the 5`-UTR of c- sis confers in cis an ability to respond to modification in the translational
machinery during differentiation. Such modifications might facilitate
ribosomal scanning from the 5` end of the mRNA by activation of
existing translation initiation factors and/or by the induction of RNA
helicases and other transacting factors that facilitate the melting of
secondary structures. A novel model, distinct from the basic scanning
model, describes binding of the translation machinery to an internal
site within the mRNA leader independent of its 5` end. Members of
picornaviruses that contain exceptionally long, structured, uncapped
mRNA leaders utilize the cap-independent mechanism for efficient
translation initiation
(61) . This alternative mechanism
requires high order structures of the 5`-UTR and cellular RNA-binding
proteins, some of which have been already identified
(62, 63, 64) . It is believed that these
proteins mediate ribosomal binding to internal ribosomal entry site
within the leader. Interestingly, it has been demonstrated that few
cellular mRNAs, which harbor exceptional 5`-UTRs, such as
immunoglobulin heavy chain binding protein
(65) , fibroblast
growth factor-2
(66) , and the homeotic gene Antenopedia
(67) , accommodate an internal ribosomal entry site activity. We
would like to suggest that temporary appearing transacting factors
confer a differentiation-linked internal ribosomal entry site activity
to the 5`-UTR of c- sis. This property is anticipated to
intensify aspects of expression regulation with regards to timing and
cell-type specificity, which might have general implications in
cellular growth, differentiation, and development. Our current
experiments are designed to address this possibility.
We thank N. Sonenberg for 3T3-pMV7 and
3T3-pMV7-4E cells and for eIF4E probe and antibodies. We also
thank I. Fabian for K562 cells, B. Moss for vTF7-3, S. Aaronson
for c- sis cDNA, and O. Sella for helpful comments.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
is
known to be involved in the regulation of cell proliferation. This
growth factor is a potent mitogen of all cells of mesenchymal origin
and has a major role in wound healing and in cellular transformation.
It is a homo- or heterodimer of two related polypeptides, one of which,
PDGF2, has been shown to be the proto-oncogene c- sis.
Expression of PDGF is highly restricted. PDGF2/c- sis can
normally be found in blood platelets, activated monocytes, endothelial
cells, and placental trophoblasts. However, a large fraction of tumor
tissues as well as sarcoma and glioblastoma cell lines express the
c- sis mRNA (for review, see Refs. 1-3). PDGF2/c- sis obtained from purified platelets is believed to be synthesized
within bone marrow megakaryocytes, the platelet progenitor cells.
PDGF2/c- sis is equipped with regulatory elements controlling
its transcription and RNA stability
(4, 5, 6, 7, 8) . In addition,
one of the striking features of c- sis gene is its unusual
5`-untranslated region (5`-UTR) that is composed of 1022 nucleotides
and is highly conserved in evolution
(9, 10, 11, 12) . PDGF2/c- sis 5`-UTR functions as a potent translational inhibitor
(5, 13, 14) , apparently because it imposes a
barrier to ribosomal scanning.
(9, 14) , capable of
preventing the migration of 40 S ribosomes
(19) . The three mini
cistrons within the 5`-UTR were found to have no regulatory effect on
translation
(13, 14) .
-granules prior to the final platelets
maturation. A system in which c- sis expression can be induced
makes it possible to determine the contribution of different
transcriptional, post-transcriptional, and translational regulatory
mechanisms during induction. The human hematopoietic stem cell line
K562 was chosen for this study since phorbol esters induce its
differentiation toward the megakaryocytic lineage with a concomitant
induction of c- sis transcription
(26) . Since the
5`-UTR of c- sis is known to be a strong translational
inhibitor, our working hypothesis was that during the megakaryocytic
differentiation process when PDGF2 protein synthesis occurs, an ability
to alleviate such inhibition is induced. In this study we have been
able to show that there is a time window during megakaryocytic
differentiation of K562 cells upon treatment with
12- O-tetradecanoylphorbol-13-acetate (TPA), in which the
strong translational inhibitory effect of the lengthy structured 5`-UTR
of c- sis is relieved. During the differentiation process, we
observed simultaneous induction of c- sis transcription and
relief of the 5`-UTR-mediated translational repression. In NIH3T3
overexpressing translation initiation factor eIF4E, the inhibitory
effect of the mRNA leader of c- sis was not relieved. This
finding suggests that the changes in the translational machinery during
megakaryocytic differentiation are beyond eIF4E activity.
Plasmids Constructions
Plasmid pOS14
(27) contains the Escherichia coli chloramphenicol
acetyltransferase (CAT) gene placed between the bacteriophage T7
promoter and transcription terminator. The unique NcoI site of
pOS14 is located downstream to the T7 promoter and harbors the
translational initiation codon ATG of the CAT coding sequence. Plasmids
pPD1 and pPD8 were constructed by inserting NcoI fragments of
1022 or 843 bp containing the complete or truncated PDGF2 5`-UTR,
respectively, into the NcoI site of pOS14 resulting in
placement of the 5`-UTR segments between T7 promoter and the CAT gene.
Both 5`-UTR fragments were generated by polymerase chain reaction using
pc-sis4.0
(14) as template and the oligonucleotide
primer 5`-CCCCCCATGGCGACTCCGGGCCCGGCCC-3`, which is homologous to the
most 3` end as well as to a sequence present 179 nucleotides upstream
to the 3` end of c- sis 5`-UTR and
5`-CCCCCCATGGTGGCAACTTCTCCTCCTGCA-3`, which is homologous to the most
5` end of the 5`-UTR. pPD9 was constructed by inserting klenow-treated,
blunt-ended 1.0-kilobase pair MluI- PvuII fragment of
pc-sis4.0 into the Klenow filled-in NcoI site of
pOS14.
Cell Culture and Megakaryocytic
Differentiation
The chronic myelogenous leukemia cell line K562
(28) was grown in RPMI 1640 supplemented with 50 units/ml of
penicillin, 50 µg/ml of streptomycin, and 10% fetal calf serum
(Biological Industries, Israel). Megakaryocytic differentiation was
induced by treatment of exponentially growing K562 cells with TPA
(Sigma) at a concentration of 5 nM. 3T3-pMV7 control cells and
3T3-pMV7-4E cells which overexpress eIF4E, were generous gifts
from Dr. N. Sonenberg (McGill University, Montreal, Quebec, Canada) and
were grown as described
(25) .
Virus Infections and Plasmid
Transfections
Transfections were carried out in a 6-well dish
using 5 µg of supercoiled plasmid DNA/well. In each well, a total
of 9 10
3T3-pMV7 or 3T3-pMV7-4E, 1.7
10
NIH3T3, or 10
control K562 or TPA-treated
K562 cells were used. 30 min prior to transfection, the cells were
infected with recombinant vaccinia virus vTF7-3
(29) at
multiplicity of infection of 1 plaque-forming unit/cell followed by
liposome-mediated transfection as described previously
(30) .
The purified vTF7-3 viral stock was prepared as described
(30) .
RNA Analysis and CAT Assay
CAT activity and
Northern blot assays for RNA were performed simultaneously on cells
infected with recombinant vaccinia vTF7-3 and transfected with
the various CAT-expressing plasmids. At 24 h following
infection/transfection, the cells were harvested for RNA preparation or
CAT activity assay. Total RNA was extracted by the guanidine/phenol
extraction procedure (RNAzol reagent, Biotex Laboratories, Inc.).
Poly(A) RNA was isolated from noninfected K562 cells
using the guanidine/phenol extraction procedure as above followed by
purification using oligo(dT) magnetic beads (Dynatech, Inc.). Total or
poly(A)
RNAs were subjected to Northern blot analysis
that was carried out according to standard procedures
(31) using
P-labeled cDNA probes specific for the
CAT gene
(32) , c- sis(33) , eIF-4E
(25) ,
-actin
(34) , or ribosomal RNA
(35) .
To quantify the RNA, the intensity of specific bands on the x-ray film
was determined by laser densitometry (Ultroscan, LKB), or
alternatively, a PhosphorImager (FUJIX BAS100, Fuji) was used. For CAT
activity assay, the cell pellet was lysed by three freeze-thaw cycles
in 0.1 M Tris, pH 8. CAT activity in the lysates of cells in
each well was determined by a phase-extraction assay, which quantitates
butyrylated
H-labeled chloramphenicol products by liquid
scintillation counting following xylene extraction (pCAT Reporter Gene
Systems, Promega). Total protein concentration in each sample was
determined by the Bradford assay
(36) .
Western Blot Analysis
A total of 9
10
control or vaccinia-infected 3T3-pMV7-4E cells
were harvested and lysed by 5 min incubation on ice with 50 µl of
lysis buffer containing 0.5% Nonidet P-40, 100 mM NaCl, 20
mM Tris-HCl, pH 7.5, 50 mM NaF, and 2 mM
phenylmethylsulfonyl fluoride, followed by a 10-min spin in a
microcentrifuge. 20 µg of protein of the supernatant as assayed by
Bradford
(36) was separated by SDS-polyacrylamide gel
electrophoresis on a 12% acrylamide gel. Following electrophoresis, the
proteins were transferred to a nitrocellulose membrane by semidry
blotting using a transfer buffer containing 25 mM Tris-HCl, pH
7.5, 190 mM glycin, and 20% methanol. The membrane was blocked
overnight with 5% lowfat dry milk in phosphate-buffered saline at 4
°C followed by a 2-h incubation with rabbit polyclonal antibody
against mouse eIF4E and a 1-h incubation with horseradish peroxidase
anti rabbit IgG (BioMakor, Israel). All incubations and postincubation
washes were done at room temperature using blocking solution. To
visualize bands, the membrane was developed using the ECL kit (Amersham
Corp.).
The Translational Inhibitory Effect of c-sis 5`-UTR Is
Relieved upon Megakaryocytic Differentiation
To study the role
of the unusually long c- sis 5`-UTR in mediating translation
efficiency during differentiation, it was required to disconnect
between determinants controlling transcription, alternative splicing,
and mRNA stability from those controlling translation. For this
purpose, we have used a vaccinia virus-based expression system.
Vaccinia virus, a member of the smallpox virus family, is a cytoplasmic
DNA virus encoding for its own transcription and RNA-modifying enzymes.
Vaccinia is widely used as a viral expression vector both because of
its wide host range (mammalian and avian cells) and because it leads to
high expression level of the target gene. The recombinant vaccinia
virus vTF7-3
(29) provides expression of the
bacteriophage T7 RNA polymerase, a prokaryotic enzyme that does not
contain a nuclear localization signal. The prokaryotic RNA polymerase
can transcribe any T7 promoter-containing DNA, which is introduced into
the cytoplasm of the infected cell. The T7 transcripts are then further
modified by the cytoplasmic vaccinia-encoded capping and
polyadenylation enzymes and subjected to the host translational
machinery. It has been previously shown that the cytoplasmic T7
transcripts generated by the T7/vaccinia system are relatively stable
and carry the 5` and 3` T7 promoter- and terminator-derived sequences
(37) . Furthermore, it has been shown that differences in
expression levels of a target gene represent variations in
translational efficiency of the mRNA due to different 5`-UTRs
(38) . In the present study, plasmids containing various 5`-UTRs
between a reporter gene and T7 promoter were used to transfect
TPA-treated or control K562 cells, which have been infected with the
vaccinia recombinant virus vTF7-3 that express the cytoplasmic T7
RNA polymerase.
Figure 5:
Translation efficiency of CAT mRNA
harboring truncated c- sis 5`-UTRs during the course of
megakaryocytic differentiation. a, schematic showing of the T7
transcription unit harbored by each plasmid. Lightboxes, c- sis 5`-UTR sequence; darkboxes, the CAT coding sequence; triangle, T7
promoter; circle, T7 terminator. b, log-phase K562
cells were treated with 5 nM TPA for 0-96 h as indicated
followed by vTF7-3 infection and transfection of either pOS14,
pPD1, pPD9, or pPD8 as described under ``Experimental
Procedures.'' 24 h later, CAT activity was determined by the phase
extraction radioassay. The presented relative cat activity values are
the CAT activity value (cpm/mg of protein) obtained from each plasmid
relative to the value obtained from pOS14 at every time point. The
bar values represent the average ± S.E. of three to
five independent experiments.
Figure 1:
Effect of megakaryocytic
differentiation on c- sis 5`-UTR-mediated translation. Log
phase K562 cells were incubated with 5 nM TPA for 40 h. As
described under ``Experimental Procedures,'' the TPA-treated
and control K562 cells were infected/transfected with vTF7-3 and
pOS14 or pPD1. 24 h following infection/transfection, total RNA was
prepared or CAT activity was measured. a, Northern blot
analysis of 5 µg of RNA extracted from cells transfected with pOS14
( lanes1 and 3) or pPD1 ( lanes2 and 4) and probed with P-labeled
DNA specific for CAT. Band intensity was measured by laser
densitometry. b, CAT activity (cpm/ng of protein) was
determined by phase extraction radioassay and divided by the band
intensity value of CAT mRNA obtained in each infection/transfection
experiment. In each set of transfections, the CAT activity/RNA unit
value obtained for each plasmid in the TPA-treated cells was divided by
its value in the control K562 cells in order to calculate the effect of
megakaryocytic differentiation on the RNA translatability. The bar values represent the average ± S.E. of five independent
experiments.
Phorbol esters
through protein kinase C can lead to either growth stimulation or
differentiation depending on the conditions and cells used. Resting
cells exposed to high concentrations of phorbol ester undergo
proliferation. Under these conditions, the rapid phosphorylation of
several translation initiation factors including eIF4E have been
correlated with increase in translation rates
(24, 39) .
In contrast, exposure of certain log-phase cells to low concentration
of phorbol esters results in growth arrest and induction of a long term
differentiation process
(26, 40) . We wished to assess
the effect of the two different TPA treatments on the ability of the
cells to translate the pOS14- and pPD1-derived mRNA. NIH3T3 cells,
which cannot undergo differentiation but express the receptor for
protein kinase C, were used in addition to K562 cells. As shown in
Fig. 2
, the translation efficiency of pPD1-derived mRNA was
enhanced about 3-fold as a result of a 3-h incubation of either K562 or
NIH3T3 cells with 1600 nM TPA. This observation suggests that
the immediate events induced by exposure of resting cells to high
concentration of TPA might lead to a general phenomenon of enhanced
translation of mRNA that bears a cumbersome leader. However, exposure
of log-phase cells to the low concentration of TPA (5 nM)
normally used to induce differentiation of K562 cells, did not result
in a significant relief of the 5`-UTR-mediated translational repression
in NIH3T3 cells (Fig. 2 b). This observation suggests
that the effect observed in K562 cells (Fig. 1 b) is
associated with megakaryocytic differentiation. When K562 cells were
treated with hemin to induce their differentiation toward the
erythrocytic lineage
(41) , such enhanced translation effect was
not observed (not shown).
Figure 2:
Effect of TPA conditions. A total of
10 K562 cells ( a) or 1.7
10
NIH3T3 cells ( b)/well of a 6-well dish were treated with
5 nM or 1600 nM TPA for 3 or 48 h as indicated. The
cells were then infected with vTF7-3 and transfected with pOS14
( lightbars) or pPD1 ( darkbars).
24 h following infection/transfection, CAT activity was measured as
explained under ``Experimental Procedures.'' For each
experiment, the CAT activity value of pOS14 in the control untreated
cells was arbitrarily set as 1 in order to calculate the effect of
c- sis 5`-UTR and of the TPA treatment. The bar values
represent the average ± S.E. of three independent
experiments.
eIF4E Over-expression Does Not Relieve the Inhibitory
Effect of c-sis 5`-UTR
To clarify the role of the rate-limiting
initiation factor eIF4E on the translation efficiency of mRNA harboring
c- sis 5`-UTR, 3T3-pMV7-4E cells expressing high level of
eIF4E described by Lazaris-Karatzas et al.(25) were
used. Both 3T3-pMV7-4E and 3T3-pMV7 control cells were infected
with vaccinia recombinant vTF7-3 and transfected with either pPD1
or pOS14 followed by analysis of CAT mRNA level and CAT activity 24 h
later. To compare the effect of eIF4E overexpression on the
translatability of the different 5`-UTR-bearing mRNAs, the CAT
activity/RNA unit value obtained for each plasmid in 3T3-4E cells
was divided by its value in 3T3-pMV7 cells. Fig. 3 a demonstrates that the translation efficiency of pOS14-derived CAT
mRNA was enhanced 2.1 ± 0.24-fold in 3T3-4E compared with
3T3-pMV7 cells, whereas pPD1-derived CAT mRNA activity was enhanced
only 1.1 ± 0.04-fold by the eIF4E overexpression.
3T3-pMV7-4E cells did express an elevated amount of recombinant
eIF4E as confirmed by Northern blot analysis (not shown). To rule out
degradation of eIF4E in 3T3-pMV7-4E cells during the course of
the experiment, the cells were infected with vaccinia virus at various
multiplicities, and eIF4E level was determined by Western blot analysis
at various times after infection. Fig. 3 b shows that
eIF4E level in 3T3-pMVA-4E cells remained similar during the 24 h of
the experiment, even though a 10-fold excess of virus was used (compare
lanes1 and 5). Moreover, it has been
reported that during the course of the virus replication, there is
neither a significant decrease in the amount of eIF4E nor a significant
alteration in eIF4E phosphorylation
(42, 43) . According
to the observations presented in Fig. 3, it seems that eIF4E by
itself is not sufficient for the relief of the c- sis 5`-UTR-mediated translational repression, suggesting that other
factors are involved.
Figure 3:
Effect of eIF4E. a, 3T3-4E
or 3T3-pMV7 cells were infected with vTF7-3 and transfected with
pOS14 or pPD1 as described under ``Experimental Procedures.''
24 h following infection/transfection, total RNA was prepared or CAT
activity was determined. The extracted RNA was analyzed by Northern
blot analysis probed with P-labeled DNA specific for CAT
followed by
P-labeled DNA specific for ribosomal RNA. The
intensity of CAT- and ribosomal- specific bands was measured by laser
densitometry. CAT activity (cpm/ng of protein) was determined by phase
extraction radioassay and divided by the band intensity value of CAT/18
S RNA obtained in each infection/transfection experiment. In each set
of transfections, the CAT activity/RNA unit value obtained for each
plasmid in 3T3-4E cells was divided by its value in 3T3-pMV7
cells in order to calculate the effect of eIF4E overexpression on the
RNA translatability. The bar values represent the average
± St.E. of three independent experiments. b, a total of
9
10
NIH3T3-4E cells were infected with
vTF7-3 at multiplicity of infection of 1 or 10. At various times
postinfection, cells were harvested for detection of eIF4E levels by
Western blot analysis as described under ``Experimental
Procedures.''
The Relief of c-sis 5`-UTR-mediated Translational
Inhibition Parallels the Induction of Its Transcription during the
Course of Differentiation
The initial event occurring in
response to stimulation with phorbol esters is phosphorylation of key
proteins followed by a wide range of cellular consequences at accurate
timing. One of the TPA differentiation-induced events in K562 cells is
the induction of c- sis transcription
(4, 5, 6, 7) . We wished to analyze the
time course of both (i) the endogenous c- sis mRNA accumulation
and (ii) the translational efficiency of mRNA harboring c- sis 5`-UTR during the differentiation process. To measure the
endogenous c- sis mRNA steady state levels, poly(A) mRNA was extracted from K562 cells that had been treated with 5
nM TPA for 0-96 h and analyzed by Northern blot
hybridization with a DNA probe specific for c- sis. During the
course of differentiation, elevated steady-state c- sis mRNA
levels were observed followed by a decline at 96 h after the exposure
to TPA (Fig. 4 a). To examine the contribution of the
5`-UTR-mediated translational regulation to the control of c- sis expression during differentiation, the T7/vaccinia hybrid
expression system was utilized. CAT was expressed from pOS14 and pPD1
plasmids in K562 cells that had been pre-treated with 5 nM TPA
for 0-96 h. It was noted that pOS14-derived CAT activity was
hardly changed in the different cellular environment generated
throughout the 96 h of the experiment. In sharp contrast, pPD1-derived
CAT activity was markedly enhanced for a transient period of time.
Fig. 4b represents the CAT activity ratio of pPD1 to
pOS14 and thus reflects the time course of the 5`-UTR-mediated
translational enhancement during differentiation. The data presented in
Fig. 4
suggest that the megakaryocytic differentiation process
involves simultaneous induction of both c- sis mRNA
accumulation and of the capability to efficiently translate mRNA fused
to the inhibitory 5`-UTR of c- sis. This observation suggests
that when the cells do not contain any c- sis mRNA they also
lack the ability to efficiently translate it.
Figure 4:
Time course of c- sis mRNA
accumulation and of c- sis 5`-UTR-containing CAT mRNA
translation during differentiation. a shows a Northern blot of
4 µg poly(A) RNA from log phase K562 cells that
have been treated with 5 nM TPA for 0-96 h as indicated
and probed with a
P-labeled DNA specific for c- sis followed by
P-labeled DNA specific for
-actin.
RNA bands were quantified by PhosphorImager, and arbitrary
units of c- sis/actin mRNA are presented. b, log-phase
K562 cells were treated with 5 nM TPA for 0-96 h as
indicated followed by infection with vTF7-3 and transfection of
pOS14 or pPD1 as explained under ``Experimental Procedures.''
24 h following infection/transfection, CAT activity was determined by
the phase extraction radioassay. The presented relative CAT activity
values are the CAT activity value (cpm/mg of protein) obtained from
pPD1 plasmid relative to the value obtained from pOS14 plasmid at each
time point. The bar values represent the average ± S.E.
of five independent experiments.
Effect of Truncations in c-sis 5`-UTR on Translation
Efficiency during Differentiation
A highly GC-rich 140-bp
sequence immediately preceding the c- sis initiator ATG codon
had been shown to be nearly as effective as the entire 5`-UTR in
translation inhibition in COS-1 and NIH3T3 cells
(14) . This
region contains a GC content of 82% implicating a highly stable
secondary structure. To examine the role of this sequence in mediating
the modulation of translation efficiency during megakaryocytic
differentiation, we have constructed plasmids lacking 28 or 179 bp
preceding the initiator ATG (pPD9 or pPD8, respectively)
(Fig. 5 a). As shown in Fig. 5 b, deletion
of 28 bp did not markedly change the translation efficiency pattern
conferred by the full-length c- sis 5`-UTR. However, larger
deletion, which produced a 843-bp 5`-UTR that lack the GC-rich sequence
resulted in less inhibition at zero time and in an extended duration of
the enhanced translation phase. The above means that the 5`-UTR with
the larger deletion (pPD8) was ``less sensitive'' to cellular
changes, suggesting that the CG-rich fragment immediately upstream of
the initiator AUG codon is necessary for coffering stringent modulation
of translational efficiency during the differentiation process.
G
= -17 to -81.7 kcal mol
in NIH3T3
cells overexpressing eIF-4E
(47) led to a proposed model to
explain the oncogenic effect
(25) of eIF4E. The model predicts
that the high cellular level of active eIF4E enables the productive
translation of proto oncogenes with massive mRNA leaders. Supporting
the model, heterologous mRNA harboring the 5`-UTR of ornithine
decarboxylase was translated more efficiently in 3T3-pMV7-4E
cells, which overexpress eIF4E
(48) . In contrast, the
translation efficiency of mRNA harboring the structured 5`-UTR of
S-adenosylmethionine decarboxylase was not affected by the
eIF4E overexpression
(48) . Moreover, eIF4E overexpression did
not relieve the translational repression of ribosomal protein mRNAs in
resting cells
(49) . In our hands, eIF4E overexpression did not
affect the translation of mRNA harboring the c- sis 5`-UTR that
contains secondary structures, of which the stability ranges from
G = -100 to -250 kcal mol
.
However, eIF4E overexpression did enhance the translation of the
control mRNA that contains a hairpin 5`-UTR of which the stability is
G = -12.4 kcal mol
(Fig. 3).
More experiments are needed to verify whether eIF4E phosphorylation is
required for the rapid increase in the translation of mRNA harboring
c- sis 5`-UTR under growth-stimulatory conditions by TPA
observed in Fig. 2. In view of the present study, we suggest that
during megakaryocytic differentiation of K562 cells eIF4E does not play
a key role in the induction of c- sis mRNA translation and that
other factors might be involved. It has been recently demonstrated that
in contrast to the growth stimulatory effect by mitogens, induction of
differentiation is not necessarily associated with enhanced eIF-4E
activity. Treatment of log phase HL60 cells with 10 nM phorbol
12-myristate 13-acetate did not result in any substantial shift of
eIF4E from free state to heavy subcellular fraction despite a rapid
down-regulation of protein synthesis rates.
(
)
In
addition, differentiation of P19 embryonal carcinoma cells with
retinoic acid does not involve phosphorylation of eIF4E.
(
)
1 and
3
(54, 55, 56, 57) , proenkephalin
(58) , and androgen receptor
(59) confer regulated
translation on their transcript. Apparently, the expression level of
the above genes is regulated at multiple levels. In this study we
demonstrate that activation of protein kinase C in K562 cells leads to
regulation of PDGF2 expression on both the mRNA steady state and
translational levels. In hematopoietic cell lines, protein kinase C
activation also exerts distinct levels of regulation on TGF
1
expression
(54, 60) . We show that the
cis-element modulating the translation level of c- sis is within its 5`-UTR. Sequences in the 5`-UTR were shown not to
affect the level of c- sis RNA
(5) . In addition,
translation regulation is not attributes to the minicistrons presence
in the 5`-UTR
(14) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.