From the Departments of Nutrition and
§ Biochemistry, Case Western Reserve University School of
Medicine, Cleveland, Ohio, 44106 and the ¶ Academic Medical
Centre, University of Amsterdam, Amsterdam, The Netherlands
Received for publication, October 24, 2000, and in revised form, December 12, 2000
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ABSTRACT |
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The cationic amino acid transporter,
Cat-1, facilitates the uptake of the essential amino acids arginine and
lysine. Amino acid starvation causes accumulation and increased
translation of cat-1 mRNA, resulting in a 58-fold increase in
protein levels and increased arginine uptake. A bicistronic mRNA
expression system was used to demonstrate the presence of an internal
ribosomal entry sequence (IRES) within the 5'-untranslated region of
the cat-1 mRNA. This study shows that IRES-mediated translation of the cat-1 mRNA is regulated by amino acid availability. This IRES causes an increase in translation under conditions of amino acid starvation. In contrast, cap-dependent protein synthesis is
inhibited during amino acid starvation, which is well correlated with
decreased phosphorylation of the cap-binding protein, eIF4E. These
findings reveal a new aspect of mammalian gene expression and
regulation that provides a cellular stress response; when the nutrient
supply is limited, the activation of IRES-mediated translation of
mammalian mRNAs results in the synthesis of proteins essential for
cell survival.
Amino acids are essential nutrients for cell growth and
maintenance. Mammalian cells have developed an adaptive response to changes in amino acid availability (1). When the amino acid supply is
limited, protein synthesis decreases, and there are increases in
catabolism of cellular proteins, amino acid biosynthesis, and amino
acid transport across the plasma membrane. Together these responses
provide cells with amino acids needed for survival. A significant part
of this adaptive response is the increased expression of the
cat-1 gene, which encodes the transporter for the essential
cationic amino acids, lysine and arginine (2). We have shown that the
level of the Cat-1 protein and the transport of cationic amino acids
increase in amino acid-depleted cells (3). Because amino acid
starvation inhibits cap-dependent initiation of protein
synthesis (4), we hypothesized that the cat-1 mRNA is translated in
amino acid-depleted cells through a cap-independent mechanism. This
translation would involve an internal ribosomal entry sequence
(IRES1) within the
5'-untranslated region (5'-UTR). IRES sequences have been implicated in
the translation of viral mRNAs in infected cells, where
cap-dependent translation of cellular mRNAs is
inhibited (5). These sequences are also important in the translation of
several mammalian mRNAs encoding regulatory proteins involved in
growth and differentiation (6-11). Furthermore, a role of IRES sequences in cell cycle-dependent translation of mammalian
mRNAs has been demonstrated recently (6, 12-14).
This study provides support for our hypothesis by demonstrating that
the 5'-UTR of the cat-1 mRNA contains an IRES sequence. Moreover,
translation from this IRES is stimulated in amino acid-starved cells,
when cap-dependent translation is decreased. These findings suggest a mechanism for synthesis of proteins required for amino acid
accumulation in starved cells when total protein synthesis is inhibited.
Cloning of the cat-1 cDNA 5'-UTR--
The 5'-end of the
cat-1 mRNA was cloned using the RACE technique (15). RACE was
performed using polyadenylated RNA from FTO2B rat hepatoma cells. Five
cDNA clones were isolated, with the largest containing an insert of
224 nucleotides of 5'UTR (cat1-224). The cat1-224 cDNA was
amplified by polymerase chain reaction using the primers in Fig.
1A and cloned into the SalI/NcoI site
of the pSVCAT/BiP/LUC plasmid (16) by replacing the IRES from the BiP mRNA. The resulting vector was named pSVCAT/cat1-224/LUC. The plasmid
pSVhpCAT/cat1-224/LUC was constructed by replacing the BiP IRES within
the pSVhpCAT/BiP/LUC (16) vector, at the
NcoI/SalI sites. The pSVCAT/ICS/LUC vector
contains 400 nucleotides of antisense antenapedia cDNA of
D. Melanogaster (16) in the ICS region and was used as
a negative control for IRES-mediated translation. The
pUHD10-3cat1-224/LUC, pUHD10-3cat1-224mut/LUC, and pUHD10-3con/LUC expression vectors were generated by cloning the chimeric LUC cDNAs
into the pUD10-3 expression vector cleaved at the XbaI site 3' to the promoter region and the EcoRI site 5' to the
polyadenylation signal (3). This vector contains a minimal
cytomegalovirus promoter with very low promoter activity and the SV40
polyadenylation signal. The cat1-224/LUC DNA contained 224 nucleotides
upstream of the LUC ORF. The cat1-224mut/LUC contained the same
sequence as cat1-224/LUC, but the 49-amino acid ORF was eliminated by
mutating the initiating ATG to TTG using polymerase chain
reaction-based mutagenesis. The con/LUC DNA contained 25 nucleotides of
linker sequence upstream of the LUC ORF. The mRNAs transcribed from
these expression vectors would contain 5'-UTR sequences of 249 bases for cat1-224 and cat1-224mut and 50 bases for the con.
Cells and Cell Culture--
Plasmid DNA was transfected into C6
rat glioma cells using the calcium phosphate technique (2). Stable
mass-culture cell lines were generated by cotransfecting an expression
vector containing the neo gene and selecting the transfectants in 0.1%
G418. All cells were maintained in Dulbecco's modified Eagle's
medium/F12 medium supplemented with 10% fetal bovine serum. Fed cells
were incubated in Dulbecco's modified Eagle's medium/F12 supplemented with fetal bovine serum dialyzed against phosphate-buffered saline. Starved cells were incubated in KRB supplemented with dialyzed fetal
bovine serum (2, 3). No difference in the regulation of the cat-1 gene
by amino acid starvation was observed when KRB containing all amino
acids was used in place of Dulbecco's modified Eagle's medium/F12
medium (2). Treatments were performed by culturing cells (5 × 105 cells/35-mm dish) for 48 h in growth medium
followed by culture under fed or starved conditions for the appropriate times.
Enzyme and Transport Assays--
Cell extracts were prepared and
analyzed for LUC and CAT activities as described previously (17). The
activities were normalized to the protein content of the cell extracts,
which was measured using the Bio-Rad assay. To measure
[3H]arginine transport, cells were plated at 2 × 105 cells/18-mm plate for 48 h. Transport in amino
acid-fed and -starved cells was measured as described previously (2,
3). Because system y+ amino acid transporters carry out the
exchange of amino acids, 0.5 mM lysine was added to the
amino acid-deficient medium, permitting the detection of maximum
transport activity. The induction of expression from the endogenous
cat-1 gene and the bicistronic mRNA vectors in amino acid-free KRB
and KRB containing 0.5 mM lysine were identical (not shown).
Western Blot Analysis--
Western blot analysis was carried out
as previously described (2). Cat-1 was detected using a
polyclonal antibody against a Cat-1 peptide (3) that was
immunoaffinity-purified using the antigen. Asparagine synthase was
detected using 3G6, a monoclonal antibody against human asparagine
synthase (18). eIF2 The previously cloned cat-1 cDNA contained only 80 nucleotides
of 5'-UTR sequence (19). To test our hypothesis that the cat-1 protein
is translated using a cap-independent mechanism, the 5'-UTR of the
cat-1 mRNA was isolated using RACE (15). The longest of the clones
contained 224 base pairs of the cat-1 5'-UTR. This sequence contains an
open reading frame of 49 amino acids, beginning 223 bases upstream from
the cat-1 ORF (Fig. 1A).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was detected using a rabbit polyclonal antibody
specific for eIF2
, and the phospho-eIF2
was detected using a
rabbit polyclonal anti-eIF2
[pS51] phosphospecific
antibody (generous gifts from T. Dever). Total and phosphorylated
(Ser-209) eIF4E were detected using polyclonal antibodies for eIF4E and
phospho-eIF4E, respectively (Cell Signaling).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The 5'-UTR of the cat-1 mRNA and
bicistronic expression vectors. A, sequence of the rat
cDNA clone, cat1-224, containing 224 nucleotides of the cat-1
5'-UTR. The ORF within the UTR is shaded, and the beginning
of the cat-1 ORF is marked with an arrow. Polymerase
chain reaction primers used to amplify the 5'-UTR for subcloning are
shown. B, the pSVCAT/cat1-224/LUC and
pSVhpCAT/cat1-224/LUC expression vectors and the expected bicistronic
mRNAs.
The ability of the cat-1 5'-UTR sequence to mediate internal translation initiation was tested using vectors developed by the Sarnow laboratory that encode a bicistronic mRNA (16). The CAT enzyme is translated from the first cistron by a cap-dependent scanning mechanism. The second cistron, encoding the firefly LUC enzyme, is translated only if it is preceded by an IRES in the ICS region (Fig. 1B). The following three vectors were tested for IRES-mediated translation of the LUC gene: CAT/ICS/LUC as a negative control, CAT/BiP/LUC containing the BiP 5'-UTR as a positive control (16), and CAT/cat1-224/LUC containing the cat-1 5'-UTR sequence instead of the BiP sequence.
The plasmids were transfected into C6 rat glioma cells, and
48 h later cell extracts were prepared and assayed for LUC and CAT
activities. The data are expressed as the ratio of LUC and CAT
activities to normalize for differences in transfection efficiency (Fig. 2). The normalized LUC activity for
CAT/cat1-224/LUC was 35 times higher than the CAT/ICS/LUC negative
control and similar to CAT/BiP/LUC (Fig. 2), suggesting that the 5'-UTR
of the cat-1 mRNA contains an IRES element. Northern blot analysis
of transfected cells demonstrated a single transcript hybridizing to
both LUC and CAT hybridization probes (Fig. 2, inset),
supporting the idea that the LUC and CAT activities derive from
translation of the bicistronic mRNAs.
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To demonstrate that expression of the LUC gene does not result from readthrough of ribosomes from the CAT cistron, we constructed a vector encoding a bicistronic CAT/cat1-224/LUC mRNA with a stable RNA hairpin upstream of the CAT ORF (see Fig. 1B and Ref. 16). RNA hairpins in the 5'-UTR of mRNAs have been shown to inhibit translation initiation at a downstream AUG (20). hpCAT/cat1-224/LUC gave normalized LUC activity that was 15-fold higher than CAT/cat1-224/LUC (Fig. 2A). This increase was due to decreased CAT and unchanged LUC activities in hpCAT/cat1-224/LUC compared with CAT/cat1-224/LUC (Fig. 2, B and C). These results are consistent with the cap-dependent initiation of CAT and the cap-independent initiation of LUC translation from an IRES in the cat-1 5'-UTR. As previously shown (16), the relative LUC activity from hpCAT/BiP/LUC was 5-fold higher than the activity from CAT/BiP/LUC (Fig. 2A).
To further demonstrate that the cat-1 5'-UTR supports IRES-mediated translation under conditions where the cap-dependent translation of cellular mRNAs is inhibited, the normalized LUC activity was measured in cells treated with rapamycin. This compound partially inhibits cap-dependent translation by promoting the dephosphorylation and activation of 4E-BP1, a repressor of the cap-binding protein 4E (21). Rapamycin treatment caused an 80% decrease of CAT expression from CAT/cat1-224/LUC, but LUC activity did not change (Table I). The normalized LUC activity increased 5.1-fold (Table I), supporting internal translation initiation of LUC from the cat-1 5'-UTR. As expected (16), a 5.6-fold increase in the normalized LUC activity was caused by rapamycin treatment of cells transfected with CAT/BiP/LUC as a positive control (not shown). These data support the conclusion that the 5'-UTR of the cat-1 mRNA contains an IRES element that has high activity when cellular cap-dependent translation is inhibited.
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The regulation of CAT/cat1-224/LUC mRNA translation by amino acid
starvation was studied next. Rat C6 glioma cells were transfected either with CAT/cat1-224/LUC or CAT/BiP/LUC, along with a vector expressing the neo gene, and two stable mass-culture lines,
C6/pSVcat1-224 and C6/pSVBiP, were generated, each containing ~70
clones. These cell lines were cultured either in amino acid-containing
or amino acid-free media, and the effects of starvation on CAT and LUC expression were examined. As expected, the cells expressed a single mRNA transcript containing both the CAT and LUC cistrons (not shown). Amino acid starvation of C6/pSVcat1-224 cells increased the
normalized LUC activity after 6 h, reaching a 7-fold increase by
12 h (Fig. 3A, left
panel). This increase was due to increased LUC and decreased CAT
activities (Fig. 3, B and C, left
panels). No further increase in LUC activity was observed when
amino acid starvation exceeded 12 h (not shown). These data
support the conclusion that cat1-224/IRES-mediated translation of the
LUC cistron increased during amino acid starvation. Is this
translational regulation specific for the cat1-224 IRES? This was
tested by examining the effect of amino acid starvation on C6/pSVBiP
cells. In contrast to C6/pSVcat1-224 cells, both CAT and LUC
activities decreased in amino acid-starved C6/pSVBiP cells (Fig. 3,
B and C, right panel). The normalized
LUC activity increased by only 1.7-fold (Fig. 3A),
suggesting that the BiP/IRES-mediated translation of the LUC
cistron is more efficient than the cap-dependent
translation of the CAT cistron, but four times less efficient than the
cat1-224 IRES, under conditions of amino acid starvation.
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A striking result in these studies was that following 12 h of
amino acid starvation, cap-dependent translation of the CAT cistron decreased by 70% (Fig. 3), whereas IRES-mediated translation of the LUC cistron increased by 3.5-fold. What caused the dramatic decrease of CAP-dependent translation? It is well known
that heat shock, serum deprivation, and viral infection inhibit
cap-dependent translation by regulating the phosphorylation
state of the cap-binding protein, eIF4E (22). Reduced phosphorylation
of eIF4E correlates with inhibition of cap-dependent
protein synthesis (23). We therefore compared the phosphorylation state
of eIF4E in amino acid-fed and -starved cells. As a negative control,
we analyzed rapamycin-treated cells, because this drug does not change
eIF4E phosphorylation (23). Amino acid starvation induced
dephosphorylation of eIF4E (75% decrease), whereas total eIF4E levels
remained the same (Fig. 4). As expected,
rapamycin had no effect (Fig. 4). The 75% decrease in eIF4E
phosphorylation paralleled the 75% decrease in
cap-dependent CAT expression from the CAT/cat1-224/LUC
bicistronic mRNA following 12 h of amino acid starvation
(compare Figs. 3 and 4). This is consistent with the idea that
phosphorylated eIF4E is a key part of the eIF4F cap-binding complex
that is required for the cap-dependent initiation of
translation (22).
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Amino acid starvation is also known to cause increased phosphorylation
of the subunit of the initiation factor eIF2 (4). This
phosphorylation results in decreased formation of ternary complexes
(initiator Met-tRNA, eIF-2, and GTP). We therefore tested the
phosphorylation state of eIF2
during a time course of amino acid
starvation, because it has been reported that eIF2
is phosphorylated transiently in response to different stimuli by specific kinases (24).
An increase in phospho-eIF2
levels occurred during the first
hour of amino acid starvation (3.2-fold) followed by a gradual decrease
thereafter (Fig. 4B). The increased phospho-eIF2
levels can be due to either increased total eIF2
or increased
phosphorylation. To determine the degree of eIF2
phosphorylation, we
determined the ratio of phospho-eIF2
/total eIF2
during the time
course of amino acid starvation (Fig. 4B). The ratio
increased by 2-fold during the first hour of amino acid starvation and
then decreased in a pattern similar to the changes in phospho-eIF2
levels (Fig. 4B). We therefore conclude that eIF2
phosphorylation transiently increased in amino acid-depleted cells.
Because eIF2
phosphorylation rises and then declines before any
increase in LUC activity is seen, we propose that phosphorylation of
eIF2
does not directly regulate cat-1/IRES activity.
The IRES-mediated translation of cat-1 mRNA during amino acid
starvation could enable cells to increase Cat-1 protein levels under
conditions where global protein synthesis is inhibited. Asparagine
synthase protein levels, analyzed as a positive control, were increased
by amino acid starvation (Fig.
5A). Cat-1 protein expressed
from the endogenous gene increased by 58-fold at 12 h of amino
acid starvation (Fig. 5A), whereas mRNA levels increased by only 16-fold (not shown; see Ref. 3). The fact that the Cat-1
protein was induced to a greater extent than the mRNA supports the
translational regulation of cat-1 gene expression. Cat-1
protein expressed from the endogenous gene and LUC expressed from
pSVCAT/cat1-224/LUC increased with similar time courses (compare Figs.
3 and 5A), indicating a parallel regulation of translation.
Furthermore, a 4.5-fold increase in high affinity arginine transport
was observed after 12 h of amino acid starvation (Fig.
5B), demonstrating that cells depleted of amino acids induce
Cat-1 protein synthesis to support cationic amino acid transport once
amino acids become available.
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The nucleotide sequence of the 5'UTR of the cat-1 mRNA revealed the
presence of a 49-amino acid ORF (Fig. 1A). Because ORFs within the 5'-UTR of mRNAs are almost always associated with
translational control of the mRNA (25), we studied the role of the
ORF in the translation of the cat-1 mRNA. We studied two
monocistronic mRNAs. One (cat1-224/LUC) had the cat-1 5'UTR
upstream of the LUC coding sequence. The other (cat1-224mut/LUC) had
the initiating AUG of the upstream ORF mutated to UUG. Both mRNAs
were expressed from the minimal cytomegalovirus promoter (3).
C6 cells were transiently transfected with one of the
expression vectors along with a vector expressing the -galactosidase
gene to normalize for transfection efficiency. Cell extracts were
prepared 48 h after transfection, and the LUC and
-galactosidase activities were measured and expressed as the
LUC/
-galactosidase ratio. In amino acid-fed cells, the
cat1-224mut/LUC mRNA was translated 2.8 times more efficiently
than the cat1-224/LUC mRNA (Fig.
6A). These data suggest that
the upstream ORF is translated in amino acid-fed cells and inhibits
translation from the downstream ORF.
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The studies with the bicistronic vectors show that the cat-1 5'-UTR contains an IRES that participates in the regulation of translation by amino acid availability. Is there similar regulation by the cat-1 5'-UTR in a monocistronic mRNA? To answer this question, we generated C6 cell lines stably transfected with cat1-224/LUC, cat1-224mut/LUC, or a control vector containing a 5'-UTR that will result in cap-dependent translation (con/LUC). These cell lines were cultured either in amino acid-containing or amino acid-free media, and the effects of starvation on LUC expression were examined. As expected, the cells expressed a single LUC-containing mRNA. The level of cat1-224mut/LUC mNRA increased in amino acid-starved cells but only by 30% (Fig. 6B).
Amino acid starvation of C6/cat1-224/LUC cells increased LUC activity after 6 h (1.9-fold), reaching a 5.4-fold increase by 18 h (Fig. 6C, left panel). This demonstrates that the cat-1 5'-UTR regulates translation of a downstream ORF in a monocistronic mRNA. No further increase in LUC activity was observed when amino acid starvation exceeded 18 h (not shown). We next determined the effect of amino acid starvation on the translational efficiency of the cat1-224mut/LUC mRNA. Starvation of C6/cat1-224mut/LUC cells increased LUC activity by 6 h (2-fold), reaching a maximum increase (2.7-fold) by 18 h (Fig. 6C, right panel), suggesting that the ORF is not absolutely required for the regulation of translation by amino acid starvation. This increase was lower than the 5.4-fold increase observed with the cat1-224/LUC mRNA. The 2-fold difference in the LUC activity between the wild-type and mutant 5'-UTRs suggests that the upstream ORF does contribute to the increased translation of the cat-1 mRNA during amino acid starvation. The smaller increase in translation of cat1-224mut/LUC mRNA may be due to the higher level of translation in fed cells (Fig. 6, A and C).
Finally, we compared the translation of the mRNAs containing the
cat-1 5'-UTR to the control mRNA (con/LUC). Amino acid starvation of the C6/con/LUC cells caused a 40% decrease in LUC activity, consistent with translation of this mRNA via a
cap-dependent scanning mechanism. LUC expression from this
mRNA was at least 50-fold higher than from the mRNAs with the
cat-1 5'-UTR. This is consistent with inefficient initiation of
translation caused by the secondary structure of the cat-1 5'-UTR
mRNA. This low level of expression was seen with both the
cat1-224/LUC and cat1-224mut/LUC mRNAs. Consequently, the
inefficient translation initiation from these mRNAs cannot be due
solely to the presence of the ORF in the 5'-UTR. This is in agreement
with the inhibitory effects of IRESs within the 5'-UTR of mRNAs on
cap-dependent translation initiation (26).
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DISCUSSION |
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Our studies have shown that the 5'-UTR of the cat-1 mRNA contains an IRES sequence. Moreover, we have shown that translation from this IRES is induced by amino acid starvation. To our knowledge, this is the first report of an IRES that shows this regulation. We propose that some mammalian mRNAs use IRES-mediated translation to provide cells with proteins essential for survival when the nutrient supply is limited. Among these proteins is the Cat-1 arginine/lysine transporter, which also provides cells with the substrate for NO synthesis (27). Expression of the cat-1 gene has been shown to be regulated at the transcriptional and post-transcriptional level by hormones (28, 29), nutrients (2, 3) and growth factors (30, 31). The translational regulation reported in this study further extends the complex regulatory mechanisms that control the cell's supply of arginine and lysine.
We have shown in this study that amino acid starvation caused a 50-fold increase in Cat-1 protein levels and only a 4-fold increase in y+ amino acid transport. This difference may be due to the fact that this transporter is stimulated by intracellular cationic amino acids, a property called trans-stimulation (32, 33). Arginine transport in this study was performed in cells starved of all amino acids but lysine. We speculate that the difference between cat-1 protein levels and y+ transport in starved cells is due to low cytoplasmic cationic amino acids that cause inadequate trans-stimulation. However, we cannot exclude the possibility that Cat-1 protein synthesized during amino acid starvation is not functional, because it is not properly folded or is not transported to the plasma membrane.
Translational regulation by amino acid availability has also been
demonstrated for the yeast transcription factor GCN4 (1). However, the
mechanism of this regulation is different from the one we observed for
cat-1 mRNA. Amino acid starvation increases translation of GCN4
mRNA by 8-fold by a mechanism involving cap-dependent initiation and reinitiation at the GCN4 ORF, following ribosome scanning of four small ORFs in the 5'-UTR (1). Removal of the ORFs
increased basal expression of the GCN4 mRNA by 80-fold and abolished translational control by amino acid starvation (34). Thus the
upstream ORFs inhibit basal translation and participate in the
starvation-induced stimulation. Another example of translation by
induced amino acid starvation was recently reported for the branched-chain -ketoacid dehydrogenase kinase, but the mechanism of
translational control was not studied (35).
This is the first report that nutrient supply can regulate expression of a mammalian mRNA via an IRES-mediated mechanism. What features of the cat-1 IRES enable it to recruit ribosomes and regulate translation? Computer analysis suggests that the cat-1 IRES is very structured (not shown), a general feature of other cellular and viral IRESs (26). However, studies of other IRESs have not revealed a consensus sequence (36) nor a specific RNA conformation important for activity (37). In fact, segments as short as 9 nucleotides may have independent IRES activity or affect the activity of the IRES in which they occur (37). The fact that amino acid depletion induced translation mediated by the cat1-224/IRES four times more than the BiP/IRES suggests that the cat1/IRES has special features that support enhancement of translation during starvation. The residues within the cat-1 IRES that are important in translational regulation will be the focus of future studies.
What is the role of the 49-amino acid ORF in the cat-1 5'-UTR? A general feature of IRESs is the presence of ORFs (26). It is believed that the ORFs are present within the IRES to inhibit cap-dependent translation. Our studies of the wild-type and mutant mRNAs have shown that the cat-1 ORF inhibits translation. This is in agreement with the very low levels of Cat-1 protein in amino acid-fed cells (Fig. 5). Amino acid starvation increased translation of monocistronic mRNAs containing the cat-1 5'-UTR whether they contained the 49-amino acid ORF, consistent with the idea that the regulation does not depend on the presence of the ORF. However, the extent of induction was 2-fold greater in the ORF-containing mRNA (Fig. 6C). This could reflect a change in the regulation by amino acid starvation. Alternatively, it could be due to the fact that there is greater cap-dependent translation from the mutant than from the wild-type 5'-UTR. This translation will be inhibited by amino acid-starvation, causing a decrease in the overall induction seen with this construct. This is in agreement with the 3.5-fold induction of the LUC activity in the bicistronic mRNA, where the LUC cistron was translated exclusively by the IRES. Future studies will determine the mechanism (cap-dependent or IRES-mediated) via which the cat-1 mRNA is translated in amino acid-fed and -starved cells.
Using the bicistronic mRNA system, we have shown that translation mediated by the ORF-containing cat-1/IRES increased 3.5-fold (Fig. 3C). Is the ORF translated within the bicistronic mRNA? Translation of the 49-amino acid ORF might occur either by reinitiation of cap-dependent translation of the first cistron or by initiation at the IRES at or before the AUG of the ORF. Using the hpCAT/cat1-224/LUC bicistronic mRNA, we have shown that the cat-1/IRES activity is independent of the cap-dependent translation of the first cistron (Fig. 2). Consequently if this 49-amino acid ORF is translated this must occur by initiation within the IRES. The structure of the cat-1 IRES and its role in the regulation of translation initiation by amino acid starvation will be the subject of future studies.
The cellular proteins that interact with the IRESs and modulate
translation are not known. We have shown that during amino acid
starvation, cat-1 IRES-mediated translation increased, whereas cap-dependent translation decreased. The increased
phosphorylation levels of eIF2 may contribute to the IRES-mediated
stimulation of cat-1 expression in starved cells. The
inhibition of cap-dependent translation may also be due to
changes in the activity of the cap binding complex, eIF4F. We showed
that amino acid starvation decreased the amount of phosphorylated
eIF4E, a component of eIF4F and a regulator of its activity (36).
However, eIF4E may not play a role in the increased translation from
the cat-1 IRES. We showed that rapamycin, which reduces the amount of
active eIF4E, caused an 80% decrease in cap-dependent
protein synthesis but did not induce IRES-mediated translation,
consistent with previous findings (38). It is therefore likely that the
increased activity of the cat-1 IRES in amino acid-starved cells is due
to the synthesis or activation of regulatory proteins. The 3-h lag in
the increase of cat1-224/IRES-mediated translation in amino
acid-deficient cells may represent the time required for synthesis or
modification of protein(s) that interact with the IRES and/or the
recruited ribosomes. In fact, cellular IRES-binding proteins have been
shown to control the functional state of viral IRESs (14). The
elucidation of these regulatory mechanisms will be of great importance
in our understanding of cell responses to nutritional stress.
Nuclear proteins have also been implicated in IRES-mediated translation
(36). A few nuclear RNA-binding proteins, such as the polypyrimidine
tract and poly(rC)-binding proteins, have been shown to enhance
IRES-mediated translation (36, 39). The cat-1 IRES contains three
pyrimidine tracts (47,
111, and
183, relative to the cat-1 ORF;
see Fig. 1) that may bind to pyrimidine tract-binding proteins.
IRES-mediated translation in mammals has also been described for viral
mRNAs (5) in infected cells where cap-dependent translation is inhibited. It is likely that these viral mRNAs are
translated using a normal cellular mechanism that is activated by
infection. Interestingly, the Cat-1 protein is the receptor for the
type C ecotropic retrovirus (40, 41), whose mRNAs are translated
via an IRES-dependent mechanism (42). Based on the common
regulatory elements in the mRNAs of the type C ecotropic retrovirus
and its receptor, Cat-1, it is intriguing to speculate on their coevolution.
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ACKNOWLEDGEMENTS |
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We thank Drs. B. Bode, W. Lamers, and T. W. Nilsen for useful discussions. Thanks to K. K. Kwikcers for immunopurifying the cat-1 antibody and D. Robinson for assisting with RACE. Thanks to Dr. P. Sarnow for providing the bicistronic mRNA vectors.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants R01 DK53307-01 (to M. H.) and 5T32 DK07319 (to J. F.).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.
To whom correspondence should be addressed: Dept. of
Nutrition, Case Western Reserve University, 10900 Euclid Ave.,
Cleveland, OH 44106-4906. Tel.: 216-368-3012; Fax: 216-368-6644;
E-mail: mxh8@ po.cwru.edu.
Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M009714200
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ABBREVIATIONS |
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The abbreviations used are: IRES, internal ribosome entry site; 5'-UTR, 5'-untranslated region; CAT, chloramphenicol acetyltransferase; KRB, Krebs-Ringer bicarbonate buffer; LUC, firefly luciferase; ORF, open reading frame; RACE, rapid amplification of cDNA ends; ICS, intercistronic spacer; mut, mutant; con, control.
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REFERENCES |
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