Molecular Cell, Vol. 17, 405–416, February 4, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2004.12.024

Ribosome Stalling RegulatesIRES-Mediated Translation in Eukaryotes,a Parallel to Prokaryotic Attenuation

ribosomes either by themselves or with the assistance ofcellular proteins.

We recently showed that the mRNA encoding thecat-1 arginine/lysine transporter in the rat is translatedduring limited nutrient supply via an IRES within the 270

James Fernandez,1 Ibrahim Yaman,1 Charles Huang,1

Haiyan Liu,1 Alex B. Lopez,1 Anton A. Komar,2

Mark G. Caprara,3 William C. Merrick,2

Martin D. Snider,2 Randal J. Kaufman,4

Wouter H. Lamers,5 and Maria Hatzoglou1,*nt mRNA leader (Fernandez et al., 2002a). An important1Departments of Nutritionfeature of this leader is a 147 nt open reading frame2 Department of Biochemistry(uORF), whose translation is essential for regulation of3 Department of Molecular Biology and MicrobiologyIRES activity. We suggested that the cat-1 mRNA leaderCase Western Reserve University School of Medicineforms a stable structure that prevents translation of theCleveland, Ohio 44106cat-1 ORF, but favors translation of the uORF. Transla-4 Howard Hughes Medical Institutetion of the uORF operates like a zipper, causing transientUniversity of Michigan Medical Centerstructural remodeling of the mRNA leader. The RNAAnn Arbor, Michigan 48109structure functions as an IRES, leading to translation of5 Department of Pediatric Surgerythe downstream cat-1 ORF (Yaman et al., 2003). We alsoSophia Children’s Hospitalsuggested that formation of the active IRES is the resultErasmus Medical Centerof kinetic trapping of the transient RNA structure byRotterdamcellular proteins that are expressed during nutritionalThe Netherlandsstress (Yaman et al., 2003).

The zipper model of translational control is reminis-cent of attenuation mechanisms that control transcrip-

Summary tion and translation in prokaryotes. A well-studied exam-ple is the transcriptional control of the trp operon in

It was previously shown that the mRNA for the cat-1 E. coli. When cells are starved for Trp, a stalled ribosomeArg/Lys transporter is translated from an internal ribo- attempting translation of a leader peptide causes forma-some entry site (IRES) that is regulated by cellular tion of a transcription antiterminator, preventing tran-stress. Amino acid starvation stimulated cat-1 transla- scription termination (Yanofsky, 2001). A different mech-tion via a mechanism that requires translation of an anism regulates trp operon expression in B. subtillis.ORF in the mRNA leader and remodeling of the leader These cells have a repressor protein, TRAP, that regu-to form an active IRES (the “zipper model” of transla- lates transcription and translation. Translational controltional control). It is shown here that slowing of the involves a conformational change in the mRNA leader.leader peptide elongation rate, either by cyclohexi- Binding of the Trp-TRAP complex promotes RNA refold-mide or the introduction of rare codons, stimulated ing and inhibits translation initiation by sequesteringtranslation of the downstream ORF. These results sug- the Shine-Dalgarno (SD) sequence in an RNA hairpingest that ribosome stalling in the upstream ORF (Gollnick and Babitzke, 2002; Yanofsky, 2001). The SDcauses mRNA remodeling and formation of an active sequence is a stretch of 4–6 nucleotides in prokaryoticIRES. This control is reminiscent of translation attenu- mRNAs that participates in 30S ribosome binding andation in prokaryotic operons, where inhibition of trans- initiation of translation (Hershey and Merrick, 2000).lation elongation can regulate both mRNA translation More relevant to the current work, translation of theand gene transcription by altering mRNA structure. B. subtilis ermC mRNA can be regulated by ribosome

stalling in a uORF that encodes a 19 residue peptide(Bechhofer, 1990; Narayanan and Dubnau, 1987).Introduction

These and other examples suggest that ribosomestalling within a uORF can alter the RNA structure andTranslation of most eukaryotic mRNAs is initiated viafacilitate translation of a downstream ORF (Chen andribosome scanning. This mechanism involves recogni-Yanofsky, 2004; Valbuzzi and Yanofsky, 2001). We hy-tion of the 7-methyl guanosine cap at the 5� end ofpothesized that translation of the eukaryotic cat-1mRNAs by the translation initiation complex, eIF4F (Her-mRNA may be regulated in a similar manner: ribosome

shey and Merrick, 2000). This is followed by recruitmentstalling in the uORF will prolong the lifetime of the tran-

of the 43S preinitiation complex and scanning in a 5� tosient RNA secondary structure that can function as an

3� direction until an AUG in the appropriate context is IRES, promoting translation of the downstream ORF.encountered. An alternative mechanism involves initia- In order to test this hypothesis, we examined the effecttion at an internal ribosome entry site (IRES) indepen- of cycloheximide (Cx) on translation of an mRNA con-dent of the 5� end of the mRNA. IRES-mediated transla- taining the cat-1 mRNA leader. Cx is a glutarimide antibi-tion has been described for viral RNAs and mammalian otic that inhibits protein synthesis by associating withmRNAs, often under conditions when cap-dependent the 60S ribosome and inhibiting translocation along theinitiation is compromised. Mammalian IRESs are com- mRNA (Weissbach and Pestka, 1977). It is shown hereposed of regulatory structures in the mRNA that can bind that Cx increased cat-1 IRES-mediated translation even

though global protein synthesis was inhibited by �90%,suggesting that Cx causes an increase in IRES-mediated*Correspondence: mxh8@po.cwru.edu

Molecular Cell406

Figure 1. Cx Induces cat-1 IRES-Mediated Translation in a Manner that Requires Interactions within the RNA Leader

(A and D) Schematic representation of mRNAs transcribed from the bicistronic (A) and monocistronic (D) expression vectors. Bicistronicvectors contain chloramphenicol acetyltransferase (CAT) as the first and firefly luciferase (LUC) as the second cistron. Monocistronic vectorscontain only the LUC cistron. Sequences from the cat-1 UTR were introduced into the intercistronic region (A) or 5�-UTR (D) (indicated by thedotted lines). A detailed description of the cat-1 5�-UTR was previously published (Fernandez et al., 2002a). Construct M4 (D) contains thecat-1 gene promoter and the entire 270 nt UTR (Fernandez et al., 2003).(B and C) C6 rat glioma cells were transiently transfected with B1 (B) or the indicated constructs (C).

Attenuation Regulates IRES-Mediated Translation407

initiation that is large enough to offset the inhibition of are required for the response. In order to answer thefirst question, we examined the effect of Cx on IRES-elongation. As a further test of our hypothesis, a cluster

of rare codons was introduced into the uORF to slow mediated translation from vectors with mutations in thecat-1 mRNA leader. A series of bicistronic mRNA ex-its translation. These rare codons increased translation

of a downstream ORF, supporting the idea that transla- pression vectors (Figure 1A) were transfected into cells,and LUC and CAT activities were measured in controltion attenuation is responsible for this translational

control. and Cx-treated cells. The LUC/CAT ratios let us con-clude that induction of the cat-1 IRES by Cx has thefollowing similarities to induction by nutritional stressResults(Figure 1C): (1) nucleotides �225 to �1 of the leadercontain all the elements of the IRES (Figure 1C, compareCycloheximide Induces cat-1

IRES-Mediated Translation constructs B1 and B3); (2) in constructs with the com-plete uORF, translation of the uORF is an absolute re-We hypothesized that a slowly translating ribosome in

the cat-1 uORF will activate the IRES by prolonging quirement for induction by Cx (Figure 1C, compare con-structs B1 and B2; B3 and B4); (3) induction requiresthe lifetime of the RNA in the active conformation. This

hypothesis was tested by studying the effect of Cx on long-range RNA interactions because constructs withonly the 5� or 3� parts of the leader were inactive (com-cat-1 IRES-mediated translation using vectors that ex-

press bicistronic mRNAs with two reporter ORFs (Figure pare constructs B1 with B7 and B8); and (4) the stemG�182CUAGC(N166)G�10CUCAGC is an important com-1A). The CAT ORF is at the 5� end, and its translation

is initiated by a cap-dependent mechanism. LUC is at ponent for induction by Cx. Mutagenesis of either sideof the stem abolished induction (data not shown). How-the 3� end and is translated only if the intergenic region

contains an IRES. By analyzing the LUC/CAT ratios, ever a compensatory mutant (B5) which replacedthe G�182CUAGC(N166)G�10CUCAGC stem with the stemvariations due to transfection efficiency can be elimi-

nated. G�182CAUCAG�10(N166)CUGACU showed an inductionsimilar to the wild-type leader (Figure 1C, compare con-Treatment of cells with Cx (10–100 �g/ml) increased

IRES-mediated translation from construct B1, which structs B1 and B5). This induction was dependent onuORF translation (Figure 1C, compare constructs B5contains the 270 nt leader, after 4–6 hr of treatment

(Figure 1B), with a 12-fold increase in activity seen with and B6).However, not all constructs responded similarly to6 hr of 100 �g/ml Cx. This increase was seen even

though 50 �g/ml Cx inhibited protein synthesis by �95% nutritional stress and Cx. Bicistronic vectors with lead-ers beginning at �182, �185, and �188 (B9–B11) wereas shown by metabolic labeling of cells with 35S-Met

(Figures 4C and 4D). However, CAT activity only showed not induced by Cx, even though they were induced bycellular stress (Yaman et al., 2003). These data suggesta 10% decrease. Because CAT has a half-life of 50 hr

(Thompson et al., 1991), inhibiting synthesis should that translation of the uORF is absolutely required forinduction of IRES activity by Cx. In contrast, nutritionalcause only a small decrease in protein level. The LUC/

CAT ratio showed a large increase, reflecting the Cx- stress can induce the IRES in constructs that begin at�182. Truncations farther into the leader abolish induc-induced accumulation of LUC activity. This remarkable

result suggests that Cx increases translation initiation tion by nutritional stress; these constructs were also notinduced by Cx (Figure 1C, constructs B12 and B13).at the cat-1 IRES and that this stimulation exceeds the

inhibitory effect of Cx on translation elongation. Be- To further support the idea that the cat-1 IRES re-sponds to Cx, we tested the effect of Cx on monocis-cause similar results were seen for 50 and 100 �g/ml

Cx (data not shown), cells were treated with 50 �g/ml tronic RNAs. The vectors are shown in Figure 1D. Thedata in Figure 1E demonstrate the following: (1) transla-Cx in subsequent experiments.

We previously showed that nutritional stress induces tion of the LUC mRNA from the cat-1 leader is stimulatedby Cx, and only nucleotides �225 to �1 are requiredcat-1 IRES activity in a process that requires structural

remodeling of the 270 nt leader and eIF2� phosphoryla- for this effect (Figure 1E, construct M2); (2) Cx-mediatedtranslation is uORF dependent (Figure 1E, compare con-tion (Fernandez et al., 2002b; Yaman et al., 2003). The

active IRES is formed by the 182 nucleotides at the 3� structs M2 and M3); (3) Cx also caused increased LUClevels when the cat-1 gene promoter/UTR region wasend of the leader, following unwinding of the 5� end by

translation of the uORF (residues �224 to �80 relative to used to express constructs that also contained the cat-1leader (Figure 1E, construct M4); and (4) Cx caused athe cat-1 ORF initiation codon). These 182 3� nucleotides

can also form the stress-inducible IRES in truncated decrease in LUC expression from an mRNA with a 94nt leader that did not contain any cat-1 sequences,constructs that do not have a functional uORF. We there-

fore wondered (1) if the induction of IRES activity by Cx showing that non-IRES-mediated translation is inhibitedby Cx (Figure 1E, construct M1). Based on the data fromalso involves a uORF-dependent conformational change

of the mRNA, and (2) which nucleotides in the leader the mono- and bicistronic constructs, we conclude that

(E) Monocistronic expression vectors were cotransfected with a �-galactosidase-expressing plasmid.In all experiments, 48 hr after transfection, cells were incubated with or without the indicated concentrations of Cx (B) or 50 �g/ml Cx (C andE). Cell lysates were prepared and analyzed for LUC, CAT, and �-galactosidase activities at the indicated times (B) or 4 hr (C and E). Activity/�g protein values were normalized to the values for B1 in cells without Cx (B and C). Values for monocistronic mRNAs are expressed as LUC/�Gal (E). Values are mean � standard error of three independent experiments.

Molecular Cell408

Cx stimulates cat-1 IRES-mediated LUC expression de-spite decreased elongation rates caused by Cx, mostlikely by slowing the rate of uORF translation. We alsoconclude that some aspects of this induction must bedifferent than the induction by nutritional stress becauseCx does not induce some constructs that are inducedby nutritional stress.

An alternative explanation for these results is that Cxcauses accumulation of alternate mRNAs that expressLUC more efficiently. To test this possibility, we usedRT-PCR to determine whether RNAs with alterations inthe cat-1 5�-UTR sequences in the bicistronic mRNAwere induced by Cx treatment (see Supplemental FigureS1A at http://www.molecule.org/cgi/content/full/17/3/405/DC1/). All the RT-PCR products had the expectedsizes, and similar amounts were seen in control andCx-treated cells, suggesting that alternative splicing ordeletions do not occur in this region. Similar resultswere obtained with the M4 monocistronic RNA (data notshown). Second, we tested whether Cx caused mRNAaccumulation. Northern blot analysis of RNA from M4-transfected cells showed that Cx treatment did notchange RNA size or abundance (Supplemental FigureS1B). Therefore it is unlikely that Cx-induced expressionof altered mRNAs accounts for the increased LUC ex-pression in Cx-treated cells.

Cx Stimulation of the cat-1 IRES Requires TranslationInitiation at the LUC AUG Initiation CodonThe results in Figure 1 suggest that Cx slows translationof the cat-1 uORF, allowing the mRNA to assume aconformation that promotes initiation at the downstream

Figure 2. Cx Induction of Translation from the cat-1 IRES RequiresLUC ORF. To demonstrate that LUC translation initiatesTranslation Initiation at the Downstream AUG Initiation Codon and

at the correct codon, we mutagenized A�1UG to UUG Is Independent of eIF2� Phosphorylationin the bicistronic vector. As expected, there was no

C6 (A), MEF S/S, and MEF A/A (B) cells were transiently transfectedinitiation from this construct in the absence or presence with the indicated vectors. After transfection (48 hr), cells were incu-of Cx (Figures 1A and 2A, construct B14). In contrast, bated with or without 50 �g/ml Cx for 4 hr. Cell lysates were prepared

and analyzed for LUC and CAT activities. The LUC/CAT values werewhen an AUG was introduced at position �6, regulationnormalized to the value for B1 in cells without Cx. Values are mean �by Cx was restored (Figures 1A and 2A, compare B14standard error of three independent experiments.and B15). It is therefore concluded that translation initia-

tion at A�1UG is required for increased LUC activityduring Cx treatment. Because repositioning of the AUG

result and the data in Figure 1 demonstrate that theredownstream of its original position restored Cx-inducedare differences in the mechanisms by which Cx andIRES activity, the mechanism of cat-1 IRES-mediatedamino acid starvation induce the cat-1 IRES.translation may involve some ribosome scanning.

Cx Stimulation of the cat-1 IRES Is Independent The Position of the uORF Stop Codon Is Importantfor Cx-Induced cat-1 IRES Activityof eIF2� Phosphorylation

We previously showed that formation of the active IRES Translation of the uORF is required for cat-1 IRES activa-tion by Cx (Figure 1C), suggesting that Cx-induced ribo-during amino acid starvation depends on phosphoryla-

tion of eIF2� on Ser51 (Fernandez et al., 2002a). Because some stalling within the uORF forms an RNA structurethat is an active IRES. In order to map the region whereCx treatment causes an increase in the eIF2�-P/eIF2�

ratio (data not shown; Jiang et al., 2003), we tested if ribosome stalling can induce IRES activation, we placedthe uORF stop codon at different positions in bicistronicCx induces IRES activity of the full-length leader in cells

which are homozygous for a mutation of eIF2� Ser51 to expression vectors (Figure 3A) and changed the originalstop codon to U�80UG (Leu). These vectors were trans-Ala (Scheuner et al., 2001). We previously showed that

amino acid starvation does not induce the activity of fected in C6 cells, and the effect of Cx treatment wasdetermined. The following conclusions are drawn: (1)the cat-1 IRES in these cells (Yaman et al., 2003). Cx

induced cat-1 IRES activity in both wild-type (S/S) and When the stop codon was between �224 and �179, Cxdid not activate the IRES. This suggests that unwindingmutant (A/A) cells (Figure 2B). In contrast, construct B11,

which has nucleotides �182 to �1 of the cat-1 leader, of the region upstream of the G�182CUAGC/G�10CUCAGC stem is not sufficient to stabilize the active IRESis not induced by Cx (Figure 2B), despite the fact that

it is induced by amino acid starvation in S/S cells. This in the remaining 182 nt of the leader. This is in agreement

Attenuation Regulates IRES-Mediated Translation409

Figure 3. The Position of the cat-1 uORF Stop Codon Is Important for Cx-Induced cat-1 IRES-Mediated Translation

(A) C6 cells were transfected with either the B1 bicistronic mRNA expression vector (stop codon at position �80) or mutants with stop codonsat positions �209 to �11 (D). All mutants had U�80GA mutagenized to U�80UA. Constructs were transfected into C6 cells, and the effects ofCx were analyzed as in Figure 2. The LUC/CAT values were normalized to untreated cells transfected with B1. Values are mean � standarderror of three independent experiments. (B–D) Nuclease and chemical sensitivity mapping of an RNA containing nucleotides �270 to �1 ofthe cat-1 leader was performed as described in the Experimental Procedures using RNase T1 (T1), RNases T1 and A (T1/A), RNase V1 (V1),or DEPC/aniline (DEPC). The table in (B) shows a summary of cleavages of the region A�224 to A�6. A model of nucleotides �224 to �1 of thecat-1 mRNA leader with the cleavage sites is shown in (C). (D) shows the position of the stop codon mutants used in (A).

with findings that the sequences �182 to �1 can only stalling of ribosomes on the mRNA between �168 and�80 can stabilize the active IRES, we studied the struc-form the active IRES during the response to stress (Ya-

man et al., 2003). (2) Cx did not activate the IRES in ture of the 270 nt leader as described earlier (Yaman etal., 2003). Three enzymatic probes were used. RNaseconstructs with the stop codon between �80 and �11,

suggesting that either the primary RNA sequences in T1 cleaves unpaired Gs, RNase A cleaves unpaired Csand Us, and RNase V1 cleaves double-stranded regions.this region are important or disruption of RNA structure

in this region by translation inhibits IRES activity. This We also used chemical cleavage of DEPC-treated RNAs,which results in cleavage at unpaired A residues. Theis consistent with our finding that sequences between

�80 and �20 are essential for IRES activation by amino results are summarized in Figures 3B–3D. Based on thecleavage patterns and on structures predicted by mfoldacid starvation (data not shown). (3) Cx activated IRES

activity in constructs with stop codons between �168 (Zuker, 2003) and KineFold (http://kinefold.u-strasbg.fr/)software, we propose a structure of the uORF-con-and �80 (Figure 3B), with the greatest activation

(�7-fold) when the stop codon was at the natural posi- taining cat-1 mRNA leader (Figure 3C). It was previouslyshown that residues �224 to �150 and �11 to �1 aretion (�80).

In order to better evaluate the conclusion that the involved in a sophisticated network of interactions that

Molecular Cell410

traps the leader in an inactive conformation (Yaman etal., 2003). It is suggested here that sequences �150 to�80 and �80 to �20 fold independently.

Introduction of Rare Codons within the cat-1 uORFIncreases both IRES Activity and Sensitivity to CxAs an independent test of the hypothesis that ribosomestalling in the uORF stabilizes the active IRES, we exam-ined the effects of introducing rare codons into theuORF. Ribosome pausing occurs at these codons be-cause they are recognized by tRNAs that are underrep-resented in the cellular pool (Smith and McNamara,1971). In order to test our hypothesis, we replacedC�149UCUACACAGCU with four rare codons: three con-secutive CGA codons (Arg) and one ACA codon (Thr) inconstruct B19 (Figure 4A). This sequence was chosenbecause it is present in the c-myc mRNA, where itcauses ribosome stalling in rat cells in vivo and in therabbit reticulocyte lysate (RRL) cell-free translation sys-tem (Lemm and Ross, 2002). As a control, this sequencewas replaced with four Met (AUG) codons in constructB21. These bicistronic vectors were transfected in C6cells and analyzed at different concentrations of Cx.

The LUC/CAT ratio from the construct with the rarecodons (B19) was 2-fold higher than the wild-type inthe absence of Cx (Figure 4B). Introduction of the Metcodons had no effect on the basal activity of the IRES(Figure 4B, construct B21). These data suggest that ribo-some pausing during translation of the rare Arg and Thrcodons in the uORF of B19 RNA may stabilize the RNAstructure that forms the active cat-1 IRES. Both mutantconstructs were induced only when they contained afunctional uORF (Figure 4B, compare B19–B22). Thesensitivity of these constructs to Cx was also examined.LUC expression from the construct with rare codons(B19) was activated similarly to wild-type (B1). In con-trast, higher concentrations of Cx were required for IRESactivation in the construct with Met codons (B21), sug-gesting that the codons in this region may affect Cx-induced ribosome stalling. For both mutant constructs,the maximum level of activation by Cx was the sameas wild-type, consistent with the idea that ribosomepausing in the uORF, caused by either rare codons orCx, activates the IRES through a stabilization of interme-diate RNA structures.

It is possible that the effects of introducing rare co-

(A) Schematic representation of mRNAs transcribed from bicistronicexpression vectors B1 and B19–B22.(B) Vectors were transfected into C6 cells, and the effect of theindicated concentrations of Cx were determined as described inFigure 2. The LUC/CAT ratios were normalized to the B1 controlvalue.(C) Effect of Cx on the incorporation of radioactivity by C6 cellsincubated with 35S-Met for 2 hr. Incorporation of 35S-Met, expressedas percent of Cx-free control, is plotted against the Cx concentra-tion. Mean � standard error from triplicate determinations is shown.(D) C6 cells were incubated in 50 �g/ml Cx for the indicated times,incubated in Met-free medium for 15 min, and then labeled with 35S-Met for 15 min in the presence of Cx. Mean � standard error fromtriplicate determinations is shown.

Figure 4. Introduction of Rare Codons into the cat-1 uORF En- (E) Vectors were transfected into C6 cells, and the effects of Cx (4hances Basal Activity of the cat-1 IRES in a Manner Dependent on hr, 50 �g/ml) or amino acid starvation (9 hr) were determined. TheuORF Translation LUC/CAT ratios are shown.

Attenuation Regulates IRES-Mediated Translation411

dons are due to a change in the uORF peptide. However, tions, Cx inhibited translation of the mRNAs. However,translation of a cat-1/IRES-LUC RNA was inhibited lessthis is unlikely because no effects were seen for con-

struct B21. In addition, LUC expression was also in- efficiently than a LUC RNA with an unstructured 5�leader. At 0.08 �g/ml Cx, LUC activity, expressed asduced by Cx in a construct with a frame-shift mutation

in the second uORF codon that completely changed the Cx/control, was three times higher for the cat-1/IRES-LUC RNA than for the control RNA (data not shown).peptide sequence (Figure 4E, B16). It is also possible

that the observed effects are due to changes in the Consequently, Cx may have similar effects in vivo andin vitro.mRNA sequence, rather than the introduction of rare

codons. More experiments comparing constructs with Our analysis of 35S-Met-labeled translation productsshows that the uORF peptide was detected with therare and abundant Arg and Thr codons will be required

to address this issue. bicistronic B21 RNA, but not with B1 or B19 RNAs (Figure5B). This difference is probably explained by the pres-We previously suggested that amino acid starvation

induces cat-1 IRES activity by a mechanism that in- ence of five Met residues in the B21 peptide but onlyone N-terminal residue in B1 and B19. Until this study,volves stabilization of intermediate RNA structures by

stress-induced proteins (Yaman et al., 2003). Do amino the uORF peptide was not detected in cell-free transla-tion assays. The ability to detect the peptide will allowacid starvation and Cx treatment lead to formation of

the same intermediate RNA structure? To answer this us to study the mechanism of uORF translation directlyin future studies.question, we determined the effect of amino acid starva-

tion on the cat-1 IRES constructs with rare codons. It The LUC cistron was translated very inefficientlyin vitro relative to the CAT cistron in the bicistronicis shown that, similar to Cx treatment, the activity of the

cat-1 IRES was increased by amino acid starvation in mRNAs (Figure 5B). To estimate translation efficiency,we compared LUC activity from (1) a bicistronic RNAa manner dependent on uORF translation (Figure 4E,

B19–B22). These data suggest that the RNA sequence (Figure 5C), (2) a capped monocistronic mRNA with thecat-1 5�-UTR, (3) the same RNA with an A cap, whichin the region �149 to �138 does not affect regulation of

IRES activity by amino acid starvation. This was further reduces the efficiency of cap-dependent initiation, and(4) a capped monocistronic RNA with a 25 nt unstruc-supported by a different mutation in the same region

that was also induced by amino acid starvation in a tured 5�-UTR. RNA 4 was translated 20 times more effi-ciently than RNA 2, demonstrating that the cat-1 5�-UTRuORF-dependent manner (Figures 1A and 4E, compare

B17 and B18). Furthermore, the activities of B17 and inhibits ribosome scanning. Furthermore, RNA 2 wastranslated twice as efficiently as RNA 3, showing thatthe mutant with Met codons (B21) were similar in the

absence of treatments; only the mutant with rare codons some of the expression from the monocistronic RNAsis due to cap-dependent initiation. Finally RNA 3 was(B19) had elevated activity. This suggests that the ele-

vated activity in B19 may be due to ribosome stalling translated three times more efficiently than RNA 1, dem-onstrating that the cat-1 IRES drives LUC expression atin this region rather than changes in RNA sequence

or structure. similar levels in mono- and bicistronic RNAs.There are reports of IRES-mediated translation during

apoptosis (Bushell et al., 2004; Holcik et al., 2000). Be-Regulation of cat-1 IRES-Mediated Translationcause Cx induces apoptosis in some cell lines, we deter-by Ribosome Stalling In Vitromined if Cx has this effect in C6 cells by studying theWe further examined the effects of rare codons in thecleavage of poly-ADP-ribose polymerase (PARP), ancat-1 uORF by studying the translation of bicistronicearly event in apoptosis. PARP cleavage was not in-constructs in the RRL cell-free system. This system wasduced by 4 hr of Cx treatment, although cleavage wasused to show translational pausing at the rare codonsinduced by etoposide, a topoisomerase inhibitor thatof the c-myc mRNA (Lemm and Ross, 2002). Bicistronicinduces apoptosis (Supplemental Figure S2A).mRNAs from vectors B1 (control), B19 (Arg3Thr rare co-

To further support the positive effect of Cx on cat-1dons), and B21 (Met4) were translated, and the activitiesprotein synthesis in C6 cells, we determined cat-1 pro-of the LUC and CAT products were measured in enzymetein levels in cells treated with a lower concentration ofassays or by incorporation of 35S-Met (Figures 5A andCx (10 �g/ml). cat-1 protein levels increased during 65B). As expected, CAT protein, which is translated byhr of treatment (Supplemental Figure S3). In contrast,a 5� cap-dependent mechanism, was synthesized atCx caused a decrease in cat-1 protein levels in cellssimilar levels from all three mRNAs (Figures 5A and 5B).where expression had been previously induced byIn contrast, the amount of LUC was 5-fold greater fromamino acid starvation (Supplemental Figure S3). TheseB19 RNA than from B1, consistent with the in vivo stimu-data are in agreement with previous reports showinglation of LUC expression by rare codons in the cat-1that Cx induces translation of mRNAs that are translateduORF. A 3-fold increase in LUC expression was alsoinefficiently, but decreases translation of mRNAs thatseen from B21 RNA. This may be due to the fact thatare translated efficiently (Walden and Thach, 1986).the content of Met elongator tRNAs in RRL is half that

of the Leu, Tyr, Thr, and Ala codons that were replaced(Smith and McNamara, 1971). These results provide Discussionstrong support for our conclusion that ribosome pausingwithin the uORF of the cat-1 leader results in increased An unusual finding is reported in this study: translation

attenuation within the leader peptide of the Arg/LysIRES-mediated translation at a downstream ORF.The effect of Cx on translation from monocistronic transporter cat-1 mRNA results in increased translation

initiation at the cat-1 AUG. Stimulation of translation bymRNAs in vitro was also examined. At all concentra-

Molecular Cell412

Figure 5. Introduction of Rare Codons into the cat-1 uORF Modulates cat-1 IRES-Mediated Translation In Vitro

(A) Capped bicistronic RNAs were translated in RRL as described in the Experimental Procedures. LUC and CAT activities were measuredand normalized to the values for B1 RNA.(B) RNAs were translated in RRL in the presence of 35S-Met, and labeled products were analyzed on an SDS-PAGE gel. The positions of LUCand CAT proteins (arrows) and globin (*) are shown. The bands between the CAT and LUC proteins are aborted translation products becausethey were not seen when RNA was omitted (data not shown). The two lanes on the right are an overexposed sample to clearly show theuORF peptide.(C) The indicated mono- and bicistronic RNAs were translated in RRL. LUC activities from triplicate determinations (mean � standard error)are shown. The graph shows results with the constructs on the right from top (1) to bottom (4).

Cx has previously been observed in mouse fibroblasts, that the 270 nt cat-1 mRNA leader contains an IRES thatis induced by physiological stresses such as amino acidwhere translation of a small number of mRNAs increases

in the presence of low levels of Cx (Walden and Thach, starvation and endoplasmic reticulum stress (Fernandezet al., 2002b). Our findings suggested that the leader1986). It was suggested that Cx induces translation of

mRNAs that are inefficiently translated because they forms a stable structure that has poor IRES activity butfavors uORF translation and that remodeling of thecompete poorly with other mRNAs for translation factors

(Godefroy-Colburn and Thach, 1981). Cx could increase mRNA leader during uORF translation is required forinduction (Yaman et al., 2003). This translation perturbsthe translation of these mRNAs because factors are

available due to the global decrease in protein synthesis. the leader’s conformation, causing formation of interme-diate metastable structures. During stress, the phos-Among these mRNAs is the ferritin mRNA, whose trans-

lation is stimulated by iron. Translation is induced by phorylation of eIF2� results in the production of a fac-tor(s) that stabilizes one of these intermediate RNACx in iron-poor, but not in iron-rich, media, suggesting

that Cx stimulates translation of the message under structures, thus forming an active IRES. The synthesisof this factor was proposed because starvation activatesconditions when it is poorly translated (Walden and

Thach, 1986). Cx has similar effects on the regulation the IRES with a long lag time (6–9 hr) and activationpersists after eIF2� phosphorylation levels have re-of the cat-1 IRES. mRNAs that contain the cat-1 mRNA

leader are translated poorly in vivo and in vitro (Fernan- turned to baseline (Fernandez et al., 2002a). The activeIRES is formed by the 182 nucleotides at the 3� end ofdez et al., 2001; Yaman et al., 2003), and translation of

these mRNAs can be activated by Cx or by stress. In the leader, after unfolding of the sequences �270 to�182; an active IRES can be formed from the entireaddition, levels of endogenous cat-1 protein were in-

duced by Cx in amino acid-fed cells, but not in starved uORF-containing leader or from a mutant that containsnucleotides �182 to �1, which does not have a func-cells (Supplemental Figure S3).

The mechanism of Cx activation of the cat-1 IRES tional uORF.The mechanism of Cx-induced cat-1 IRES activity haswas investigated in light of our previous findings on the

translational control of the cat-1 mRNA. It was shown similarities with the induction by stress. In both cases,

Attenuation Regulates IRES-Mediated Translation413

IRES induction probably involves kinetic trapping of an mutations could change RNA sequences that are re-quired for IRES function. (2) The mutations could changeunstable RNA structure. It is proposed here that Cx and

physiological stresses result in formation of the same RNA secondary structure that is required for IRES func-tion. (3) Unfolding of sequences upstream of �80 couldRNA structure that functions as an active IRES. This

proposal is supported by the finding that the stem be required for IRES function, and these could refoldafter uORF translation.G�182CUAGC(N166)G�10CUCAGC is required in both cases

(Yaman et al., 2003). Moreover, the structure of the stem, What is the structure of the cat-1 mRNA leader andhow do uORF translation and ribosome stalling remodelrather than its sequence, is critical for IRES activity.

However, we cannot exclude the possibility that the this structure to form the active IRES? We previouslyshowed by structure probing and mutagenesis that se-mRNA structures induced by amino acid starvation and

Cx are different. Extensive mutagenesis studies of the quences �224 to �182 are involved in a sophisticatednetwork of pseudoknots (Yaman et al., 2003). Thesecat-1 5�-UTR will be required to address this issue.

Although the active conformation of the cat-1 leader interactions keep the mRNA in the inactive conformationand are disrupted by uORF translation. The nucleotidesis probably similar during induction by amino acid star-

vation and by Cx, the mechanisms that form and stabilize �182 to �1 form the active IRES, because deletion ofsequences �177 to �27 within the 270 nt leader abol-the structure are different. IRES induction by starvation

requires eIF2� phosphorylation. Moreover truncated con- ished induction of the IRES by both stress and Cx (datanot shown). Moreover, it was previously shown that thestructs that do not have a functional uORF are activated.

In contrast, several lines of evidence suggest that ribo- stem G�182CUAGC(N166)G�10CUCAGC is important forIRES induction. In an attempt to model the RNA structuresome stalling creates the active mRNA structure by a

different mechanism: (1) Induction by Cx requires a func- between �182 and �10, we probed the interactions inthis region. As a working hypothesis, it is proposed thattional uORF; mRNAs beginning at �182 were not in-

duced. (2) Introduction of rare codons into the uORF sequences �145 to �80 and �80 to �20 fold indepen-dently (Figure 3B). Interestingly, residues �83 to �19increased cat-1 IRES activity in a uORF-dependent man-

ner. (3) Only uORFs terminating between �168 and �80 form a tRNA-like structure. Ribosome stalling may stabi-lize the tRNA-like structure with C�83UGUG/U�GCAG19were effective, suggesting that attenuation in a defined

region is required for IRES induction. (4) eIF2� phos- resembling the aminoacyl stem. A tRNA-like structurehas also been proposed for the hepatitis C virus IRES,phorylation was not required for IRES induction by Cx

even though this treatment stimulates eIF2� phosphory- which may bind the E site of the ribosome, thus allowingribosome recruitment (Lyons and Robertson, 2003).lation in vivo (Jiang et al., 2003).

The rare codons for arginine (CGA) and threonine The proposed model of translational control for thecat-1 IRES is reminiscent of control by translational at-(ACA) that were used in this study to cause ribosome

pausing within the uORF are in the coding region of the tenuation in prokaryotes. One example is the transla-tional control of the ermC methylase in Bacillus subtilisc-myc mRNA, where they regulate mRNA decay (Lemm

and Ross, 2002). The protein CRD-BP has been shown (Bechhofer, 1990). ermC has two open reading frames,one that encodes the methylase and a uORF within theto bind the coding region of the c-myc mRNA and pro-

tect it from degradation. Two consecutive rare CGA ACA 141 nt leader that encodes a 19 residue peptide. EachORF has a SD sequence upstream of the translationcodons upstream of the CRD-BP binding site cause

ribosome stalling, thus allowing binding of the protein initiation codon. Synthesis of the methylase is regulatedby changes in the secondary structure within the mRNAdownstream in a ribosome-free region (Lemm and Ross,

2002). This model is similar to our model for the stress- leader so that the methylase SD sequence is either ex-posed (efficient translation) or sequestered (inefficientinduced activation of the cat-1 IRES. A slowly translated

uORF (due to a stable RNA structure) allows binding of translation). Translation of the leader peptide unfoldsthe sequestered methylase SD sequence, but this isa protein(s) during stress, leading to IRES stabilization.

However, when the cat-1 IRES is activated by Cx or by transient and does not allow efficient methylase transla-tion. However, in the presence of erythromycin (it bindsa cluster of rare codons, the active structure is probably

stabilized by the stalled ribosome rather than by a bind- the 50S ribosomal subunit), a ribosome stalls at codons9–10 of the uORF, thus stabilizing the RNA structure,ing protein(s).

Our results provide information on the site in the uORF exposing the methylase SD sequence, and allowing syn-thesis of the methylase. A model of this regulation iswhere ribosome stalling leads to IRES activation. The

effectiveness of the rare codons at �148 to �139 sug- given in Figure 6. We propose that regulation of thecat-1 IRES by Cx has similarities to the mechanism usedgests that stalling in this region causes RNA remodeling

and that primary sequences or secondary structure in by ermC methylase. In both cases, ribosome stalling ina uORF causes conformational changes in the mRNAthis region are not required for IRES function. It was

also observed that Cx only activates the IRES in con- and stimulates translation initiation at a downstreamORF.structs with the uORF stop codon between �168 and

�80. The size of this region raises the possibility that The translational control mechanism described in thisstudy introduces a new twist in our thinking on mamma-stalling may occur at several sites rather than in a single

location. The lack of activation by uORFs with a stop lian gene expression: modulation of translation elonga-tion rates may be a regulatory mechanism. Interestinglycodon upstream of �168 suggests that unwinding of

these upstream sequences is not able to remodel the Cx was shown to induce synthesis of the �Ca2�/calmod-ulin-dependent protein kinase II (�CamK II) in neuronsleader to form the active IRES. uORFs terminating down-

stream of �80 were also not activated by Cx. There are (Scheetz et al., 2000). �CamK II is involved in synapticplasticity and is an important modulator of dendriticseveral possible explanations for this finding: (1) The

Molecular Cell414

Figure 6. A Model of Ribosome Stalling as aRegulator of Translation Initiation in Eukary-otes and Prokaryotes

Translational control for two mRNAs withsmall uORFs are shown: (1) In prokaryotes,ermC methylase mRNA translation is regu-lated by structural remodeling of the leaderthat hides or exposes the methylase SD se-quence and initiator AUG. Erythromycin-induced ribosome stalling within the 19 resi-due leader peptide exposes these sequences,promoting methylase translation. (2) In eu-karyotes, cat-1 arginine/lysine transportermRNA translation is regulated by structuralremodeling that mediates formation of an ac-tive IRES. This remodeling is promoted byribosome stalling within the uORF of themRNA leader.

protein synthesis during synaptogenesis. Activation of mechanism proposed in this study. This mechanism canadjust the rate of cat-1 translation quickly during tran-the NMDA receptor inhibits total protein synthesis in

synaptosomes, but causes an increase in �CamK II. sient changes in intracellular amino acid levels. Long-term stress would induce the adaptive phase, with cat-1Cx caused a similar increase in �CamK II synthesis in

synaptosomes even though protein synthesis de- translation regulated via the stress-induced factor.We conclude that the studies in this report suggest acreased by 60%. It is intriguing that the �CamK II mRNA

leader has an IRES that contains a uORF (Pinkstaff et mechanism of translational control that extends beyondthe cat-1 transporter. Translation attenuation withinal., 2001). Future studies will determine if the �CamK II

and cat-1 mRNAs are translated via similar mechanisms. small ORFs of mammalian mRNAs can be viewed as aregulatory switch in gene expression. The use of IRES-Ribosome pausing in the cat-1 uORF may be an im-

portant regulatory event. Limited availability of charged mediated translation in mammalian cells may facilitatetranslation of small ORFs in coding or noncoding regionstRNAs caused by amino acid starvation could regulate

the rate of uORF translation by the mechanism de- of mRNAs (Meijer and Thomas, 2002; Morris and Geb-alle, 2000) for fine-tuning of gene expression. In mostscribed for Cx. The uORF contains codons for many

essential amino acids, including five Arg and two Lys, cases, translation attenuation within an ORF is inhibi-tory. Our studies suggest that it may be stimulatory forwhich are the transporter’s substrates (Palacin et al.,

1998). Although none of these codons is rare, it is possi- some mRNAs.ble that amino acid depletion causes ribosome stalling

Experimental Procedureswithin the uORF via a decrease in charged Arg and LystRNAs. This would result in increased translation of the

Materials and Cell Culturecat-1 transporter protein.The LUC-P98 expression vector was obtained from M.W. Hentze.

Cells also have an adaptive response to a decrease All other eukaryotic bicistronic vectors were constructed by in-in the charged tRNA pool that involves eIF2� phosphory- serting cat-1 5�-UTR fragments into the intercistronic region of the

pSVCAT/LUC vector using the SalI and NcoI restriction sites (Ma-lation and increased cellular protein breakdown (Tal-cejak and Sarnow, 1991). Mutations were generated by PCR andloczy et al., 2002). In the case of the cat-1 IRES, thisverified by DNA sequencing. DNA vectors for in vitro transcriptionresponse requires several hours of deprivation. There-were generated by introducing the appropriate bicistronic CAT/cat1-fore, the cellular response to amino acid limitation is aUTR/LUC fragments into the Hind III/Not I site of Bluescript KS�

multistep process that involves preadaptive and adap- (Stratagene). A stretch of 98 As was cloned at the 3� end of thetive phases. In the preadaptive phase, cat-1 protein syn- LUC ORF and upstream of the NotI site (Bergamini et al., 2000).

Antibodies for Western blot analysis were anti-PARP (#9542, Cellthesis may be sustained or increased via the attenuation

Attenuation Regulates IRES-Mediated Translation415

Signaling), anti-cat-1 (Fernandez et al., 2001), and anti-ribosomal coding region that regulate translation initiation for the anti-TRAPprotein of B. subtilis. Mol. Cell 13, 703–711.protein S5 (raised in rabbits against the peptide N-Cys-MTEWETAA

PAVAETPD-COOH). Fernandez, J., Yaman, I., Mishra, R., Merrick, W.C., Snider, M.D.,C6 cells were grown in DMEM supplemented with 5% fetal bovine Lamers, W.H., and Hatzoglou, M. (2001). Internal ribosome entry

serum and 5% calf serum. eIF2� S/S and A/A mouse embryonic site-mediated translation of a mammalian mRNA is regulated byfibroblasts were cultured in DMEM supplemented with 10% fetal amino acid availability. J. Biol. Chem. 276, 12285–12291.bovine serum (FBS) and both essential and nonessential amino acids

Fernandez, J.M., Yaman, I., Merrick, W.C., Koromilas, A.E., Wek,(1). Cells were starved for amino acids by culturing in Krebs-Ringer

R.C., Sood, R., Hensold, J.O., and Hatzoglou, M. (2002a). Regulationbicarbonate buffer with 10% dialyzed FBS. Transfections, enzymatic

of internal ribosome entry site-mediated translation by eukaryoticassays of LUC, CAT, and �-Gal activities, and protein determinations

initiation factor-2alpha phosphorylation and translation of a smallwere performed as previously described (Fernandez et al., 2001).

upstream open reading frame. J. Biol. Chem. 277, 2050–2058.C6 cells were metabolically labeled by incubating 1.5 105 cells

Fernandez, J.M., Yaman, I., Sarnow, P., Snider, M.D., and Hatzoglou,for 2 hr at 37C with 100 �Ci/ml 35S-Met (Amersham) with the indi-M. (2002b). Regulation of internal ribosomal entry site-mediatedcated concentrations of Cx. Control incubations contained no Cx.translation by phosphorylation of the translation initiation factorCells were harvested and proteins were precipitated by trichloroace-eIF2alpha. J. Biol. Chem. 277, 19198–19205.tic acid onto Whatman glass fiber filters (GF/C). Incorporated 35S-MetFernandez, J., Lopez, A.B., Wang, C., Mishra, R., Zhou, L., Yaman,was determined by liquid scintillation counting.I., Snider, M.D., Hatzoglou, M., and Hatzolgou, M. (2003). Transcrip-tional control of the arginine/lysine transporter, cat-1, by physiologi-Cell-Free Translation Assayscal stress. J. Biol. Chem. 278, 50000–50009.DNAs were linearized by digesting with NotI, and capped RNAs

were synthesized using the mMessage mMachine T3 kit (Ambion). Godefroy-Colburn, T., and Thach, R.E. (1981). The role of mRNAThe purity and integrity of the RNA molecules was checked on competition in regulating translation. IV. Kinetic model. J. Biol.urea-polyacrylamide gels. In vitro translations were performed using Chem. 256, 11762–11773.Promega’s nuclease-treated rabbit reticulocyte lysate cell-free sys- Gollnick, P., and Babitzke, P. (2002). Transcription attenuation. Bio-tem in the presence or absence of 35S-Met (10 mCi/ml, Amersham) chim. Biophys. Acta 1577, 240–250.for 1.5 hr at 30C. The final concentration of template RNAs was

Hershey, J., and Merrick, W. (2000). The pathway and mechanism0.04 mg/ml. Translation products were analyzed on an SDS-poly-of initiation of protein synthesis. In Translational Control of Geneacrylamide gel, and products were detected by phosphorimagingExpression, J.W.B. Hershey, N. Sonenberg, and M.B. Mathews, eds.of fixed and dried gels.(Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press),pp. 33–88.

Structure ProbingHolcik, M., Sonenberg, N., and Korneluk, R.G. (2000). Internal ribo-Structure probing of the cat1(-270) leader RNA was performed assome initiation of translation and the control of cell death. Trendspreviously described (Yaman et al., 2003). Briefly, RNAs were tran-Genet. 16, 469–473.scribed and 5� end labeled using T4 polynucleotide kinase in theJiang, H.Y., Wek, S.A., McGrath, B.C., Scheuner, D., Kaufman, R.J.,presence of [�-32P]ATP. After purification on polyacrylamide gels,Cavener, D.R., and Wek, R.C. (2003). Phosphorylation of the alphathe RNAs were suspended in water, incubated in 10 mM HEPESsubunit of eukaryotic initiation factor 2 is required for activation of(pH 7.9), 100 mM KCl, 1 mM DTT, and 5 mM MgCl2 at 50C for 20NF-kappaB in response to diverse cellular stresses. Mol. Cell. Biol.min, and then annealed by cooling gradually to room temperature.23, 5651–5663.The RNAs (2–5 pmol) were incubated with either RNases T1 (0.5 U),

A1 (8 pg), or V1 (0.0016 U) in 30 �l for 5 min at room temperature, or Lemm, I., and Ross, J. (2002). Regulation of c-myc mRNA decay bydiethylpyrocarbonate (DEPC, 2 �l) followed by aniline. EDTA (20 mM) translational pausing in a coding region instability determinant. Mol.and tRNA (10 �g) were added followed by phenol/chloroform extrac- Cell. Biol. 22, 3959–3969.tion and ethanol precipitation. Samples were analyzed on 8% poly- Lyons, A.J., and Robertson, H.D. (2003). Detection of tRNA-likeacrylamide sequencing gels with 7 M urea. All experiments were structure through RNase P cleavage of viral internal ribosome entrydone at least three times. The analyses included mock-treated RNAs site RNAs near the AUG start triplet. J. Biol. Chem. 278, 26844–to evaluate background products and samples subjected to alkaline 26850.hydrolysis or RNase T1 treatment as size markers.

Macejak, D.G., and Sarnow, P. (1991). Internal initiation of translationmediated by the 5� leader of a cellular mRNA. Nature 353, 90–94.

AcknowledgmentsMeijer, H.A., and Thomas, A.A. (2002). Control of eukaryotic proteinsynthesis by upstream open reading frames in the 5�-untranslatedWe thank M.W. Hentze for providing the LUC-P98 vector. This workregion of an mRNA. Biochem. J. 367, 1–11.was supported by grants R01-DK60596, R01-DK53307, and 35200-Morris, D.R., and Geballe, A.P. (2000). Upstream open reading10639 from the National Research Initiative/U.S. Department of Agri-frames as regulators of mRNA translation. Mol. Cell. Biol. 20, 8635–culture (to M.H.) and Postgraduate Fellowship T32-DK07319 (to8642.A.B.L.).

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