IRES-mediated translation of a mammalian mRNA is ...2000/12/12 · Results The previously cloned cat-1 cDNA contained only 80 bp of 5’-UTR sequence (19). To test our hypothesis

IRES-mediated translation of a mammalian mRNA is regulated by amino acidavailability

James Fernandez* , Ibrahim Yaman*, Rangnath Mishra* , William C. Merrick†, Martin

D. Snider†, Wouter H. Lamers‡ and Maria Hatzoglou*§

Departments of *Nutrition and †Biochemistry, Case Western Reserve University School

of Medicine, Cleveland, Ohio, 44106. ‡Academic Medical Centre, University of

Amsterdam, The Netherlands

§To whom correspondence should be sent: Email [email protected]

10900 Euclid AvenueCase Western Reserve UniversityDepartment of NutritionClevelandOhio 44106-4906Tel 216-368-3012Fax 216-368-6644

Running Title: Nutritional control of IRES-mediated translation

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Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on December 12, 2000 as Manuscript M009714200 by guest on June 30, 2020

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Summary

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.

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Introduction

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.

Experimental Procedures

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.

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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 PCR using the primers in Fig. 1A and cloned into

the SalI/NcoI site of the pSVCAT/BiP/LUC plasmid (16) by replacing the BiP IRES. 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 Drosophila Antennapedia cDNA (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 (CMV) 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 PCR-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 DMEM/F12 medium supplemented with 10% FBS. Fed cells were incubated in

DMEM/F12 supplemented with FBS dialyzed against phosphate-buffered saline. Starved cells were

incubated in KRB supplemented with dialyzed FBS (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 DMEM/F12 medium (2). Treatments were performed by culturing cells (5x105 cells/35 mm dish)

for 48 h in growth medium followed by culture under fed or starved conditions for the appropriate

times.

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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 Biorad assay. To measure [3H]arginine transport, cells were plated at

2x105 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). eIF2α was detected using a rabbit polyclonal antibody specific for

eIF2α and the phospho-eIF2α was detected using a rabbit polyclonal anti-eIF2α[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).

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Results

The previously cloned cat-1 cDNA contained only 80 bp 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 bp 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).

The ability of the cat-1 5’-UTR sequence to mediate internal translation initiation was tested using

vectors developed by Sarnow’s laboratory which 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 intercistronic

spacer (ICS) region (Fig. 1B). 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.

In order 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

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a stable RNA hairpin upstream of the CAT ORF (Fig. 1 B, 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 to CAT/cat1-224/LUC (Fig. 2B 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.

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

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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. 3B, 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. 3B 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.

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), while 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), while 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).

Amino acid starvation is also known to cause increased phosphorylation of the α subunit of the

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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 eIF2α during

a time course of amino acid starvation because it has been reported that eIF2α is phosphorylated

transiently in response to different stimuli by specific kinases (24). An increase in phospho-eIF2α

levels occurred the first hour of amino acid starvation (3.2-fold) followed by a gradual decrease

thereafter (Fig 4 B). The increased phospho-eIF2α levels can either be due to increased total eIF2α or

increased phosphorylation. To determine the degree of eIF2α phosphorylation, we determined the

ratio of phospho-eIF2α/total eIF2α during the time course of amino acid starvation (Fig 4B). The

ratio increased by two fold during the first hour of amino acid starvation and then decreased in a

pattern similar to the changes in phospho-eIF2α levels (Fig 4B), We therefore conclude that eIF2α

phosphorylation transiently increased in amino acid depleted cells. Because eIF2α phosphorylation rises

and then declines before any increase in LUC activity is seen, we propose that phosphorylation of

eIF2α 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), while mRNA levels increased by only 16-fold (not shown, 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.

The nucleotide sequence of the 5’UTR of the cat-1 mRNA revealed the presence of a 49 amino acid

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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 CMV promoter (3). C6 cells

were transiently transfected with one of the expression vectors along with a vector expressing the β↑

galactosidase gene in order to normalize for transfection efficiency. Cell extracts were prepared 48 h

after transfection and the LUC and β↑galalactosidase activities were measured and expressed as the

LUC/β↑gal 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.

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

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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 plays 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 (Figs 6A and 6C).

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

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 folder 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 than 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

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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 α-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 which

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 or not 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

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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 3 C). Is the ORF translated within the bicistronic mRNA?

Translation of the 49 AA 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, while cap-

dependent translation decreased. The increased phosphorylation levels of eIF2α 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

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(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, 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.

This work was supported by grant R01 DK53307-01 (to M. H.) and 5T32 DK07319 (to J. F.). We

would like to 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 us with the RACE.

Thanks to Dr P. Sarnow for providing the bicistronic mRNA vectors.

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Footnotes

1The abbreviations used are: 5’-UTR, 5’-untranslated region; IRES, internal ribosome entry site;

CAT, chloramphenicol acetyltransferase ; cat-1, cationic amino acid transporter; FBS, fetal bovine

serum; KRB, Krebs Ringer bicarbonate buffer; LUC, firefly luciferase; ORF, open reading frame.

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Figure Legends

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. PCR 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.

Fig. 2. The 5’-UTR of the cat-1 mRNA confers IRES-mediated translation. C6 cells were

independently transfected with pSVCAT/ICS/LUC (ICS), pSVCAT/cat1-224/LUC (cat1-224),

pSVhpCAT/cat1-224/LUC (hpcat1-224), pSVCAT/BiP/LUC (BiP) and pSVhpCAT/BiP/LUC

(hpBiP). The data are presented as the ratio (A), CAT (B) and LUC (C) activities measured in cell

extracts 48 h following transfection. The bars represent the average ± SE of three independent

experiments. The inset shows a Northern blot of total RNA (25 µg) from cells transfected with either

pSVCAT/BiP/LUC (BiP) or pSVCAT/cat1-224/LUC (cat1-224) expression vectors and hybridized to

a LUC cDNA probe. A single mRNA transcript, hybridizing to both LUC (shown) or CAT (not

shown) hybridization probes was expressed from both vectors.

Fig. 3. Cat-1 IRES-mediated translation increases in amino acid depleted cells. The stably

transfected cells, C6/pSVcat1-224 and C6/pSVBiP, were incubated under amino acid-fed conditions

for 12 h or under starved conditions for the indicated times. The LUC/CAT ratio (A), CAT (B) and

LUC (C) activities in cell extracts are expressed as arbitrary units. The bars represent the average ± SE

of four independent experiments.

Fig. 4. Amino acid starvation changes the levels of phosphorylated eIF4E and eIF2α. C6 cells were

incubated in amino acid fed (Fed), amino acid-starved (Starved) conditions or in the presence of

rapamycin (Rap, 50 ng/ml) for 12 h (A), or for the time indicated (B). Western blots of whole cell

lysates (15 µg protein) were probed for eIF2α, phospho-eIF2α, eIF4E, and phospho-eIF4E.

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Fig. 5. Cat-1 protein levels and high affinity [3H]arginine uptake increase in amino acid-depleted

cells. (A) C6 cells were incubated in either amino acid fed (Fed) or amino acid-starved (Starved)

conditions for the indicated times. Western blots of whole cell lysates (50 µg protein) were probed for

cat-1 and asparagine synthase proteins. The relative levels of the cat-1 protein levels are shown at the

bottom of the figure. (B) C6 cells were incubated under fed or starved conditions for the times

indicated and Y+ transport activity was measured as described previously. Values are the mean ±

standard deviation of five independent experiments.

Fig 6. Amino acid starvation induces translation of a monocistronic mRNA containing the cat-1/5’-

UTR. (A) C6 cells were transiently transfected with pUHD10-3cat1-224/LUC or pUHD10-3cat1-

224mut/LUC and the LUC activities were measured in cell extracts after 48 h. The bars represent the

average ± SE of three independent experiments. (B) The C6/cat1-224/LUC and C6/cat1-

224mut/LUC stable transfectants were incubated in amino acid fed (F) or amino acid starved media (S)

for the times indicated. A Northern blot of total RNA (25 µg) from these cells was hybridized to a

LUC cDNA probe. Ethidium bromide staining of the 18S ribosomal RNA is shown. (C and D) The

stable transfectants, C6/cat1-224/LUC, C6/cat1-224mut/LUC and C6/con/LUC, were incubated under

amino acid-fed conditions for 12 h or under starved conditions for the indicated times. The LUC

activities in cell extracts are expressed in arbitrary units. Mean ± SE of four independent experiments

is shown.

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Table I. Rapamycin does not inhibit translation from the cat-1 IRES

C6 cells were transfected with the pSVCAT/cat1-224/LUC plasmid and 36 h after transfection were

incubated without (control) or with 50 ng/ml rapamycin for 18 h. Cell extracts were analyzed for LUC

and CAT activities; the LUC and CAT activities (arbitrary units) and the LUC/CAT ratio are shown;

values represent the mean ± SE of three independent experiments.

CAT(units/µg protein)

LUC(units/µg protein)

LUC/CAT

Control 167 ± 6 509 ± 25 3.0 ± 0.1

Rapamycin 34.6 ± 0.5 537 ± 33 15.4 ± 0.8

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Snider, Wouter H. Lamers and Maria HatzoglouJames Fernandez, Ibrahim Yaman, Rangnath Mishra, William C. Merrick, Martin D.

availabilityIRES-mediated translation of a mammalian mRNA is regulated by amino acid

published online December 12, 2000J. Biol. Chem.

10.1074/jbc.M009714200Access the most updated version of this article at doi:

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IRES-mediated translation of a mammalian mRNA is ...2000/12/12 · Results The previously cloned cat-1 cDNA contained only 80 bp of 5’-UTR sequence (19). To test our hypothesis