From the Solange Gauthier Karsh Molecular Genetics Laboratory, Children's Hospital of Eastern Ontario, and Department of Pediatrics, University of Ottawa, Ottawa, Ontario K1H 8L1, Canada
Received for publication, July 8, 2002, and in revised form, November 20, 2002
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ABSTRACT |
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Many cellular stresses lead to the inhibition of
protein synthesis. Despite this, some cellular mRNAs are
selectively translated under these conditions. It was suggested that
the presence of internal ribosome entry site (IRES) sequences in the
5'-untranslated regions allow these mRNAs to be actively translated
despite the overall cessation of protein synthesis. Here we tested the
hypothesis that the IRES elements of genes that are involved in the
control of cell survival are distinctly regulated by cellular stresses. We show that the transient conditions of cellular stress favor the
translation of pro-survival IRES, while the severe apoptotic conditions
support translation of pro-death IRES elements. Furthermore, activation
of pro-death IRES during the etoposide-induced apoptosis is
caspase-dependent and correlates with the expression of
apoptotic fragments of two members of the eIF4G translation initiation
factor family, p97/DAP5/NAT1 and eIF4GI. Our results suggest that the regulation of IRES translation during stress contributes to the fine-tuning of cell fate.
Many chemotherapeutic agents and irradiation induce apoptosis
in human cancers. Induction of apoptosis leads to the selective cleavage of translation initiation factors that results in an inhibition of cap-dependent protein synthesis (1). Despite this, resistant tumor cells arise that up-regulate proteins
promoting their survival. Therefore, it is critical to identify
the proteins and the mechanism promoting their preferential translation.
Translational control is a final regulatory step in gene expression.
Cellular mRNAs are translated by the so-called ribosome scanning
mechanism (2). This mechanism involves the specific recognition of the
5'-end m7G structure by the cap-binding protein eIF4E. The
eIF4E is a part of the larger cap-binding protein complex eIF4F that
consists of eIF4A, eIF4G, and eIF4E. The binding of eIF4F to mRNA
further recruits other initiation factors as well as the 40 S ribosomal subunit. This complex is then thought to proceed in the 3' direction until an AUG initiation codon in a favorable context is encountered, and protein synthesis is initiated.
A broad range of cellular stresses lead to the inhibition of
translation. This is accomplished by (i) the phosphorylation of some
initiation factors and/or their regulators (3) or (ii) by the
proteolytic cleavage of several initiation factors (1). The rapid
inhibition of protein synthesis is believed to function as a protective
homeostatic mechanism. In this context, it is noteworthy that mRNAs
encoding several oncogenes, survival factors, and proteins critically
involved in apoptosis are preferentially translated by a poorly
understood cap-independent mechanism under conditions of compromised
translation initiation (4). These mRNAs contain
IRES1 (internal ribosome
entry sequence) elements in their respective 5'-UTR and were shown to
be translated during apoptosis (5, 6), cell cycle (7, 8), development
(9), amino acid availability (10), and endoplasmic reticulum stress
(11). Cellular IRES elements are found in a limited but growing number of mRNAs (12). Interestingly, they are found preferentially in the
mRNAs of genes involved in the control of cellular proliferation, survival, and death (e.g. FGF2 (13), PDGF (14), VEGF
(15), IGFII (16), c-Myc (17), c-Jun (18), PITSLRE (8)) XIAP (5),
DAP5 (6), Apaf-1 (19), and bag-1 (20). It was therefore suggested that
IRES-mediated translation plays a critical role in the regulation of
cell fate (4). It is not clear, however, how cellular IRES facilitate
translation or how this translation mechanism is regulated.
We have described previously that the IRES element of a key intrinsic
inhibitor of apoptosis, XIAP, is actively translated during serum
starvation and low dose Cell Culture and Reagents--
Human embryonic kidney (293T),
human cervical carcinoma (HeLa), human bladder carcinoma (T24), human
glioblastoma (SF539), Chinese hamster ovary (CHO), and mouse fibroblast
(NIH3T3) cell lines were cultured in standard conditions in Dulbecco's
modified Eagle's medium (293T, HeLa, NIH3T3, SF539), F-12
(CHO), or McCoy's 5A (T24) medium supplemented with 10% fetal
calf serum, glutamate, and antibiotics. Transient DNA transfections
were done using LipofectAMINE Plus (NIH3T3 and T24 cells) or
LipofectAMINE 2000 (293T, HeLa, CHO, and SF539 cells) and the protocol
provided by the manufacturer (Invitrogen). Briefly, cells were seeded
at a density of 3 × 105 cells/ml in six-well plates
and were transfected 24 h later in serum-free Opti-MEM medium
(Invitrogen) with 2 µg of DNA. The transfection mixture was replaced
3 h later with fresh DMEM supplemented with 10% fetal calf serum
(LipofectAMINE Plus) or left on the cells overnight (LipofectAMINE
2000). Cells were collected for analysis 24 h post-transfection.
For the cellular stress experiments 293T cells were seeded at a density
of 3 × 105 cells/ml in six-well plates coated with
poly-D-lysine (5 µg/ml) along with transfection mixture
(12 µl of LipofectAMINE 2000 + 2 µg DNA in 500 µl of Opti-MEM per
well). 24 h after seeding and transfection the cells were exposed
to either anoxia (90% N2, 5% H2, 5%
CO2 in a MACS-VA500 microaerophilic work station), heat stress (42 °C), etoposide (200 µM), or zVAD.fmk (100 µM) for indicated time and then collected for analysis.
Co-transfections of 293T cells were done as described above except that
1 µg of bicistronic plasmid was combined with 1 µg of indicated
expression plasmid. When monocistronic plasmids were used, 0.5 µg of
pCI-lacZ reporter plasmid was included in each transfection to
normalize for transfection efficiency. The bicistronic vectors
p Western Blot Analysis--
Cells were harvested in ice-cold
phosphate-buffered saline, lysed in RIPA buffer (1% Nonidet P-40, 1%
sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.2, 2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride) for 30 min at
4 °C followed by centrifugation at 14,000 × g for
10 min. Protein concentration was assayed by protein assay kit
(Bradford Assay; Bio-Rad), and equal amounts of protein samples were
separated by 10% SDS-PAGE. Samples were analyzed by Western blotting
using mouse monoclonal anti-FLAG M2 (Stratagene), mouse monoclonal
HIF-1 Ribonuclease Protection Assay--
Total RNA was isolated from
transiently transfected cells with the TRIzol reagent
(Invitrogen) and treated with 1 unit of DNase I. The
32P-labeled antisense RNA probes were synthesized using the
MaxiScript T7 transcription kit (Ambion) and gel-purified. The RNA
probes span the entire Apaf-1 or DAP5 5'-UTRs and overlap with
additional 47 nucleotides of the CAT gene and 30 nucleotides of the
Cellular IRES Elements Exhibit Variable Strength and Cell Line
Specificity--
To allow direct comparison of translation mediated by
various IRES elements we used the previously described bicistronic
plasmid p Distinct Regulation of IRES Translation during Cellular
Stress--
It has been proposed that IRES-mediated translation is
preferentially utilized during cellular stress (4), because it employs different regulatory mechanisms than cap-dependent
translation. In addition, IRES-mediated translation may enable
selective regulation of IRES-driven genes in response to divergent
cellular stress. Indeed, we have shown that translation of XIAP IRES is
enhanced during serum starvation or low dose Etoposide-induced Activation of IRES Translation Is
Caspase-dependent--
We and others have suggested
previously that caspase-mediated proteolytic fragments of the eIF4G
family of initiation factors could specifically modulate IRES
translation of selected IRES elements following the induction of
apoptosis (1, 4, 29). We wished to test whether the observed activation
of Apaf-1 and DAP5 IRES elements during etoposide-induced apoptosis
could be mediated by such mechanism. To test this hypothesis the 293T
cells transfected with either Apaf-1 or DAP5 IRES containing
bicistronic plasmid were pretreated with broad-range caspase inhibitor
zVAD.fmk and then treated with etoposide for 48 h. The
pretreatment of cells with caspase inhibitor blocked etoposide-induced
cell death (data not shown). More importantly it also prevented the
induction of translation of both Apaf-1 and DAP5 IRES elements,
suggesting that the activation of caspases is required for the
stimulation of IRES activity (Fig.
4A). Since the naturally
occurring cellular mRNAs exist as monocistronic mRNAs, we next
wished to test whether the enhanced translation of Apaf-1 and DAP5 IRES
is also true in the monocistronic context. Treatment of transiently
transfected cells with etoposide and/or caspase inhibitor zVAD resulted
in the enhancement of Apaf-1 and DAP5 IRES-containing monocistronic reporter constructs to levels similar to those observed with the bicistronic constructs (Fig. 4A).
Apaf-1 and DAP5 IRES Are Activated by Caspase-mediated Fragments of
the eIF4G Family of Translation Initiation Factors--
Since
induction of apoptosis leads to many intracellular changes, including
activation of nucleases, we wished to determine whether the changes in
Apaf-1 and DAP5 IRES activity could be due to activation of nucleases
with subsequent production of functionally monocistronic reporter
mRNAs. However, no differences in the integrity or the amount of
the bicistronic RNA were observed between etoposide-treated and control
cells excluding this possibility (Fig. 4B). Induction of
apoptosis leads to the caspase-mediated cleavage eIF4G family members
(eIF4GI, eIF4GII, and p97/DAP5/NAT1), which in turn inhibits translation (6, 25). It was suggested that the apoptotic fragments of
the eIF4G proteins would allow preferential translation of
IRES-containing mRNAs (1, 4, 29). Indeed, the initiation factor
p97/DAP5/NAT1 is processed by caspases to produce a truncated form
DAP5/p86, which can stimulate the translation of its own IRES thus
establishing a positive feedback loop (6). Similarly, both eIF4GI and
eIF4GII are cleaved by caspases to produce distinct fragments, although
their ability to support IRES translation has not been determined (25,
30, 31). While the caspase cleavage of eIF4GII produces several
fragments that do not persist in the apoptotic cell (32), the middle
part of the caspase-cleaved eIF4GI, termed M-FAG/p76, retains the core
region capable of binding eIF3 and eIF4A that supports translation
initiation (33) and is associated with ribosomes in apoptotic cells
(25). Therefore, we wished to test whether apoptotic fragments of the
eIF4G family could be responsible for the induction of cellular IRES
elements, particularly Apaf-1 and DAP5, in our experimental system.
293T cells were co-transfected with IRES bicistronic plasmids and
either GFP (pGFP), p97/DAP5/NAT1 (p97), DAP5/p86 (p86), N-FAG/p76,
or M-FAG expression plasmids, and the relative activity of IRES
translation was determined (Fig. 4, C and D, and
Table II). We observed that the
overexpression of p86/DAP5 resulted in a significant increase in the
activity of Apaf-1 and DAP5, but not the other IRES elements. The
overexpression of p97/DAP5/NAT1 had no effect on the translation of
either IRES element tested. Similarly, the expression of M-FAG/p76 stimulated translation of Apaf-1 and DAP5 IRES elements, while the
expression of the N-terminal fragment of eIF4GI, N-FAG, had no effect
on IRES translation. It should be noted, however, that the p86/DAP5
fragment had a stronger effect on the IRES translation than
M-FAG/p76.
In this study we have shown that distinct cellular IRES elements are
regulated by different cellular mechanisms. We tested the hypothesis
that IRES elements of cellular mRNAs involved in the control of
cell survival are differentially regulated by physiological stress. It
was anticipated that physiological conditions favoring translation of
pro-death mRNAs such as DAP5, Apaf-1, or c-Myc would suppress
translation of pro-survival molecules such as XIAP and vice versa.
Indeed, we observed that different conditions favored distinct IRES
elements. Although heat stress reduced activity of all IRES elements
studied, XIAP, c-Myc, and BiP were affected to a lesser extent than
Apaf-1 or DAP5. Conversely, following 48 h of anoxia the activity
of XIAP IRES was enhanced, while the activity of Apaf-1 and DAP5 IRES
were reduced. The most dramatic difference was seen following the
treatment of cells with the apoptosis-inducing agent etoposide. This
resulted in the significant increase in Apaf-1 and DAP5 IRES activity,
while XIAP, BiP, and c-Myc IRES were repressed. Importantly, these
results suggest that the cellular IRES elements may belong to separate
and distinct classes that will likely share either common regulatory
mechanisms or trans-acting factors. For example, both Apaf-1 and DAP5
IRES were severely inhibited by heat stress and induced by the
etoposide treatment, while other IRES were not. Similarly, XIAP and
EMCV elements were the only two IRES induced by anoxia. The
identification of the shared elements among various IRES elements
awaits further experiments.
To further understand the mechanisms of regulation of IRES translation
during cellular stress, we investigated the etoposide-induced enhancement of IRES translation in more detail. We found that the
activation of Apaf-1 and DAP5 IRES elements is
caspase-dependent, since the pretreatment of cells with
caspase inhibitor zVAD.fmk prevented IRES translation enhancement.
Significantly, we found that this specific activation of Apaf-1 and
DAP5 IRES elements correlated with the expression of apoptotic
fragments of the eIF4G family of initiation factors p97/DAP5/NAT1 and
eIF4GI. This observation is perhaps not surprising considering that
DAP5/p86 and M-FAG/p76 share 32% sequence homology. Furthermore, both
fragments retain the portion of the full-length proteins that interacts
with eIF3 and eIF4A and are therefore considered sufficient to maintain translation initiation (33). Interestingly, however, while both DAP5/p86 and M-FAG/p76 induced translation of Apaf-1 and DAP5 IRES
elements, the effect was much stronger with DAP5/p86, although the
relative levels of both proteins were comparable. This suggests that
these two apoptotic fragments may be involved in the regulation of IRES
translation under different cellular circumstances. It should be noted,
however, that in our experimental system the apoptotic fragments of
p97/DAP5/NAT1 and eIF4GI were expressed transiently in cells that were
not undergoing apoptosis and therefore contained endogenous full-length
p97/DAP5/NAT1 and eIF4GI. The presence of the endogeneous proteins may
have therefore attenuated the stimulatory effect of the apoptotic
fragments on IRES translation. It was suggested previously that the
DAP5/p86 fragment could function as a molecular switch that would
enable IRES-mediated translation during the conditions of cellular
stress (4, 6). The data presented here suggest that both DAP5/p86 and
M-FAG/p76 can fulfill this function. Significantly, however, both
fragments support translation of "pro-death" IRES elements,
suggesting that they participate in the acceleration of the cell death.
Recently, an independent study showed that the DAP5/p86 fragment can
enhance translation of XIAP, Apaf-1, c-Myc, and DAP5 IRES elements
(34). However, in contrast we did not observe any effect of
overexpression of DAP5/p86 on the translation of XIAP and c-Myc IRES
elements. Furthermore, in our experiments we observed 6-fold and
21-fold induction of Apaf-1 and DAP5 IRES, respectively, while
Henis-Korenblit et al. (34) reported modest 2- and
3-fold increases in the activity of the same IRES elements. In
addition, our results oppose the finding of Henis-Korenblit et
al. (34) that the overexpression of M-FAG/p76 has no effect on IRES translation. While it is difficult to determine the reason for
these discrepancies, it is possible that the use of different reporter
systems and/or the levels of overexpressed proteins could contribute to
these differences. However, in our study the in vivo
caspase-dependent induction of Apaf-1 and DAP5 IRES
elements by etoposide parallels the induction seen by overexpression of either DAP5/p86 or N-FAG/p76. This suggests that only these two IRES
elements are physiologically inducible by the apoptotic fragments of
eIF4G family of proteins. Furthermore, the p97/DAP5/NAT1 is a promoter
of apoptosis (6), and therefore it would be expected to enhance
translation of pro-death (Apaf-1 and DAP5) but not the pro-survival
(XIAP) IRES elements.
Our data support the hypothesis that IRES-mediated translation escapes
the control mechanisms that regulate cap-dependent translation during the conditions of cellular stress. In addition, conditions of transient cellular stress, such as anoxia, favor translation of pro-survival IRES, such as XIAP, while the severe apoptotic conditions result in the activation of pro-death IRES of
Apaf-1 and DAP5. While the exact molecular pathways that regulate IRES
translation in transient cellular stress need to be further investigated, our results indicate that the apoptotic fragments of
eIF4G translation initiation factor family mediate up-regulation of
pro-death IRES elements during apoptotic stress and may
contribute to the acceleration of cell death.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-irradiation (5, 21). Importantly, the
IRES-mediated translation of XIAP is critical for enhanced survival of
cells under acute, but transient conditions of cellular stress, thus
supporting the notion that IRES-mediated translation regulates cell
fate (5). In this study we wished to investigate the XIAP IRES
translation under different cellular stresses. Furthermore, we wished
to test the hypothesis that the IRES elements of other cellular
mRNAs that are involved in the cell survival are regulated by
physiological stress. We demonstrate that XIAP IRES is active in the
conditions of transient stress. In addition, we find that while
transient cellular stress favors the translation of pro-survival IRES,
severe apoptotic conditions support translation of pro-death IRES.
Importantly, the activation of Apaf-1 and DAP5 IRES elements during
etoposide-induced apoptosis is caspase-dependent and
correlates with the expression of apoptotic fragments of eIF4G
initiation factor family members eIF4GI and p97/DAP5/NAT1.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
gal/hUTR/CAT (containing the human XIAP IRES), p
gal/EMCV/CAT
(containing the IRES of EMCV), and p
gal/Apaf-1/CAT were described
previously (5, 22). The other IRES elements were reverse
transcriptase-PCR amplified from total RNA isolated from BJAB
cells using the following primers tagged with XhoI
restriction site: DAP5 (6) (forward:
5'-cagcagtgagtcggagctctatgg; reverse:
5'-ttggcgcttgacaacgaagaatcttc), BiP (23) (forward: 5'-caagcgtgggctgtacc; reverse: 5'-cgcggaggcttggggca), c-Myc (24) (forward: 5'-aattccagcgagaggcaga; reverse:
5'-gctcgcgggaggctgctgga). The reverse transcriptase-PCR
fragments were TOPO TA cloned (Invitrogen) and then inserted into the
linker region of the bicistronic vector p
gal/CAT (5). The correct
sequence and the orientation of all constructs were confirmed by
nucleotide sequencing. Monocistronic plasmids were constructed from
bicistronic construct by removing the
-gal reporter cassette.
Plasmids pCI-p97 and pCI-p86 were constructed by inserting the
FLAG-tagged p97/DAP5 and p86/DAP5 cDNAs (a generous gift from Adi
Kimchi) into the expression vector pCI (Invitrogen). Expression
plasmids pCI-N-FAG (containing the N-terminal fragment of eIF4GI, amino
acids 1-495 (25)), and pCI-M-FAG (containing the middle fragment of
eIF4GI, amino acids 494-1139 (25)) were constructed by PCR
amplification from Jurkat cells cDNA (Invitrogen) of the respective
fragments of eIF4GI into the expression vector pCI (Invitrogen). The
primers for PCR amplification were as follows: N-FAG forward,
5'-ctcgagaaatgaacacgccttctcag; reverse,
5'-tctagatcaatccagaaggtctccaacag; M-FAG forward,
5'-gtcgactggatgccttcaaggaggcgaac; reverse,
5'-tctagatcaatcaagccggtccccacggt. Both constructs also contained the
FLAG tag at their N terminus for Western blot analysis.
-Galactosidase and CAT Analysis--
Transiently transfected
cells were washed in cold phosphate-buffered saline and harvested in
the CAT ELISA kit lysis buffer (Roche Molecular Biochemicals)
and cell extracts were prepared using the protocol provided by the
manufacturer.
-Galactosidase enzymatic activity in cell extracts was
determined by the spectrophotometric assay using
o-nitrophenyl-
-D-galactopyranoside (26), and
the CAT levels were determined using the CAT ELISA kit (Roche Molecular Biochemicals) and the protocol provided by the manufacturer. The relative IRES activity was determined as a ratio of CAT/
-gal in three independent experiments performed in triplicates.
(BD Transduction Laboratories), mouse monoclonal anti-Hsp70
(Stressgen), or rabbit cleaved caspase-3 (Cell Signaling Technology)
antibodies at the manufacturer's suggested dilutions followed by
secondary antibody (horseradish peroxidase-conjugated sheep anti-mouse
or anti-rabbit IgG; Amersham Biosciences). Antibody complexes
were detected using the ECL system (Amersham Biosciences).
-gal gene. Ribonuclease protection assay analysis was
performed using the ribonuclease protection assay III kit
(Ambion) and the protocol provided by the manufacturer. Briefly, 10 µg of total RNA was hybridized overnight at 42 °C with 5 × 104 cpm of high specific activity RNA probe and then
digested with RNase A/RNase T1 mix. The digested samples were
ethanol-precipitated and separated on a denaturing polyacrylamide gel.
The protected fragments were visualized by detection to x-ray film.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
gal/CAT that uses
-galactosidase and chloramphenicol
acetyltransferase as first and second cistrons, respectively (5). The
IRES elements of XIAP, DAP5, Apaf-1, c-Myc, BiP, and EMCV were
PCR-amplified and inserted into this construct (Fig.
1). Bicistronic plasmids were transfected
into 293T cells, and the activity of IRES-directed translation was
determined. Surprisingly, the activity of Apaf-1, DAP5, BiP, and c-Myc
IRES elements was only 3-4-fold above that of the control plasmid
(each IRES element in the antisense orientation). In contrast, the IRES
elements of XIAP and EMCV directed translation at ~40- and 80-fold,
respectively, above background (Fig. 2). The activity of all IRES elements in the antisense orientation was
indistinguishable and was the same as that of the empty bicistronic vector (data not shown). It has been suggested that cellular IRES elements may require specific protein factors for their function (27).
One reason why we observed very low levels of activity for Apaf-1,
DAP5, BiP, and c-Myc IRES could have been that these auxiliary factors vary among various cell lines and are not present or
are present at low concentration in 293T cells. We therefore transfected the IRES-containing plasmids into five different cell lines
(Fig. 2). In all cell lines tested the translation directed by IRES of
DAP5, Apaf-1, BiP, and c-Myc was very low when compared with EMCV and
XIAP IRES elements. The exception was the T24 bladder carcinoma cell
line where the translation of XIAP IRES was very low. In contrast,
translation of EMCV IRES was stronger in this cell line than in any
other cell line tested. These results suggest that the activity of
cellular IRES elements vary dramatically. In all cases the cellular
IRES elements were not as efficient in directing the translation of the
downstream cistron as the viral (EMCV) IRES. IRES-directed translation
is cell line-dependent, which likely reflects the
availability or status of trans-acting protein factors that are
involved in the facilitation of IRES translation. It is noteworthy that
although the cellular IRES elements used in this study originate from
human mRNAs they were able to direct translation in human as well
as rodent cell lines (CHO, NIH3T3), confirming that the mechanism of
IRES-dependent translation is evolutionary conserved.
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Fig. 1.
The schematic diagram of the bicistronic
constructs used in this study. The indicated IRES elements were
inserted into the linker region separating the -galactosidase and
CAT reporter open reading frames.
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Fig. 2.
The relative translational efficiency of
distinct IRES elements is cell line-specific. Human embryonic
kidney (293T), human cervical carcinoma (HeLa), human bladder carcinoma
(T24), human glioblastoma (SF539), CHO, and mouse fibroblast (NIH3T3)
cell lines were transfected with indicated bicistronic plasmids as
described under "Experimental Procedures," and the cells were
collected for analysis 24 h post-transfection by washing in cold
phosphate-buffered saline and lysing in the CAT ELISA kit lysis buffer
(Roche Molecular Biochemicals), and cell extracts were prepared
using the protocol provided by the manufacturer. -Galactosidase
enzymatic activity in cell extracts was determined by the
spectrophotometric assay using
o-nitrophenyl-
-D-galactopyranoside (26), and
the CAT levels were determined using the CAT ELISA kit (Roche Molecular
Biochemicals) and the protocol provided by the manufacturer. The
relative IRES activity was determined as a ratio of CAT/
-gal. The
activity of each IRES construct in an antisense orientation was set as
1 in each experiment. The bars represent the average ± S.D. of three independent experiments performed in triplicates.
-irradiation (5, 21). Similarly, the IRES of c-Myc was shown to be maintained following genotoxic stress despite overall reduction of protein synthesis (28).
To further test this hypothesis, transiently transfected 293T cell were
subjected to three different types of cellular stress (heat stress,
anoxia, and etoposide), and the efficiency of cap-dependent
versus IRES-dependent translation was determined for each IRES element. While both heat stress and anoxia could be
considered as transient conditions, the treatment of cells with
etoposide will induce apoptosis and represents severe conditions of
cellular stress. As summarized in Fig. 3,
diverse treatments evoked distinct changes in the translation mediated
by different IRES elements. Heat stress resulted in significant
reduction of IRES translation. In particular, the translation mediated
by the IRES of Apaf-1 and DAP5 was completely abolished. The anoxic
conditions resulted in an initial reduction of IRES translation that
was followed by partial (DAP5) or almost full (BiP, c-Myc) restoration of IRES activity. Significantly, translation mediated by XIAP and EMCV
elements was enhanced following prolonged anoxic treatment. In
contrast, translation mediated by the IRES element of Apaf-1 was
completely repressed. The treatment of cells with etoposide resulted in
the induction of cell death with only 15-20% of surviving cells at
48 h (data not shown). Interestingly, the translation mediated by
XIAP, BiP, and c-Myc IRES elements was reduced, while the translation
of EMCV IRES remained largely unaffected. Significantly, however, the
activity of Apaf-1 and DAP5 IRES was substantially enhanced 48 h
following the addition of etoposide. This increase in IRES activity was
due to both the reduction of cap-dependent as well as
enhancement of cap-independent translation (Table
I). These results suggest that during
transient stress there exists a mechanism that blocks translation of
pro-apoptotic molecules such as Apaf-1 while allowing translation of
anti-apoptotic XIAP. Conversely, severe apoptotic conditions support
translation of pro-death molecules such as Apaf-1 and DAP5, while the
translation of anti-apoptotic XIAP remained reduced.
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Fig. 3.
IRES-mediated translation following cellular
stress. Transiently transfected 293T cells were exposed to either
heat stress (A) (42 °C), anoxia (B), or
etoposide (C) (200 µM) for 24 h
(gray bars) or 48 h (white bars) and then
collected for analysis as described under "Experimental
Procedures." The relative IRES activity in the untreated cells was
set as 100 for each IRES (black bars). The bars
represent the average ± S.D. of three independent experiments
performed in triplicates (*, p < 0.05, one-way ANOVA).
D, the stress conditions were verified by Western blot
analysis using antibodies against marker proteins Hsp70, HIF-1 , and
cleaved caspase-3.
Etoposide-induced apoptosis enhances Apaf-1 and DAP5
IRES-dependent but not cap-dependent
translation
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Fig. 4.
Translation of Apaf-1 and DAP5 IRES is
enhanced by the apoptotic fragments of eIF4G family members
p97/DAP5/NAT1 and eIF4GI. A, 293T cells were
transfected with indicated bi- or monocistronic plasmids. 24 h
following transfection the cells were pretreated with zVAD.fmk (100 µM) for 2 h and then treated with etoposide (200 µM) for 48 h. Cell lysates were prepared as
described under "Experimental Procedures," and the IRES activity
was determined. The relative IRES activity in the untreated cells was
set as 100 for each IRES (black bars). The bars
represent the average ± S.D. of three independent experiments
performed in triplicates (*, p < 0.05, one-way ANOVA).
B, the integrity of the p gal/Apaf-1/CAT and
p
gal/DAP5/CAT bicistronic RNAs was examined in etoposide-treated and
control cells by ribonuclease protection assay analysis using
probes targeting either Apaf-1 (left) or DAP5
(right) 5'-UTRs. The position of the molecular weight
markers is indicated on the right. C, 293T cells
were co-transfected with 1 µg of the indicated bicistronic constructs
and 1 µg of either pGFP, pCI-p97, or pCI-p86, and cell lysates were
harvested 24 h post-transfection. The relative IRES activity of
pGFP-transfected cells was set as 100 for each IRES. The
bars represent the average ± S.D. of three independent
experiments performed in triplicates (*, p < 0.05, one-Way ANOVA). The levels of FLAG-tagged p97 and p86 overexpressed
proteins were assessed be Western blot analysis using anti-FLAG M2
antibody (Stratagene). D, 293T cells were co-transfected
with 1 µg of the indicated bicistronic constructs and 1 µg of
either pGFP, pCI-N-FAG, or pCI-M-FAG, and cell lysates were harvested
24 h post-transfection. The relative IRES activity of
pGFP-transfected cells was set as 100 for each IRES. The
bars represent the average ± S.D. of three independent
experiments performed in triplicates (*, p < 0.05, one-way ANOVA). The levels of FLAG-tagged N-FAG and M-FAG proteins were
assessed by Western blot analysis using anti-FLAG M2 antibody
(Stratagene). E, 293T cells were co-transfected with 1 µg
of the indicated mono-cistronic constructs, 0.5 µg of pCI-LacZ, and 1 µg of either pGFP, pCI-97, pCI-p86, pCI-N-FAG, or pCI-M-FAG, and cell
lysates were harvested 24 h post-transfection. The relative CAT
activity (normalized for
-gal) of pGFP-transfected cells was set as
100 for each construct. The bars represent the average ± S.D. of three independent experiments performed in triplicates (*,
p < 0.05, one-way ANOVA).
Apoptotic fragments of eIF4G and p97/DAP5/NAT1
enhance Apaf-1 and DAP5 IRES-dependent but not
cap-dependent translation
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ACKNOWLEDGEMENTS |
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We thank the members of our laboratory for stimulating discussions. We are grateful to Adi Kimchi for the gift of FLAG epitope tagged p97/DAP5/NAT1 and DAP5/p86 cDNAs.
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FOOTNOTES |
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* This work was supported by grants from the Canadian Institutes of Health Research (CIHR), the Canadian Networks of Centers of Excellence (NCE), and the Howard Hughes Medical Institute (HHMI).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Canadian Institutes of Health Research
Senior Scientist Award, a Fellow of the Royal Society of Canada, and a
HHMI International Research Scholar.
§ CIHR New Investigator. To whom correspondence should be addressed: Solange Gauthier Karsh Molecular Genetics Laboratory, Children's Hospital of Eastern Ontario Research Institute, 401 Smyth Rd., Rm. R.306 Ottawa, Ontario K1H 8L1, Canada. Tel.: 613-738-3207; Fax: 613-738-4833; E-mail: martin@mgcheo.med.uottawa.ca.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M206781200
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ABBREVIATIONS |
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The abbreviations used are:
IRES, internal ribosome entry site;
UTR, untranslated region;
XIAP, X-linked
inhibitor of apoptosis protein;
CHO, Chinese hamster ovary;
zVAD.fmk, z-Val-Ala-Asp-fluoromethylketon;
EMCV, encephalomyocarditis virus;
-gal, galactosidase;
CAT, chloramphenicol acetyltransferase;
ELISA, enzyme-linked immunosorbent
assay;
GFP, green fluorescent protein;
ANOVA, analysis of
variance.
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