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Inefficient Reinitiation Is Responsible for Upstream Open Reading Frame –Mediated Translational Repression of the Maize R GeneLiangjiang Wanga and Susan R. Wessleraa Departments of Botany and Genetics, University of Georgia, Athens, Georgia 30602 Correspondence to: Susan R. Wessler, sue{at}dogwood.botany.uga.edu (E-mail), 706-542-3910 (fax).
Maize R genes encode a small family of transcriptional activators of several structural genes in the anthocyanin biosynthetic pathway. The 5' leader region of most R genes contains a 38-codon upstream open reading frame (uORF) that previously was shown to be responsible for the repression of downstream gene expression in a transient transformation assay. In this study, we report that the 5' leader also can repress translation of the downstream luciferase gene both in the rabbit reticulocyte translation system and in transgenic rice plants. The ability to visualize the uORF peptide after in vitro translation permits quantification of both products of dicistronic mRNAs. Similarly, the construction of transgenic rice plants expressing wild-type and mutant constructs permits the quantification and correlation of steady state mRNA levels and reporter gene activities. Using these assays, we demonstrate directly that translation of the uORF is required for repression, that increasing translation of the uORF peptide decreases downstream gene expression, and that repression is unaffected by either subtle or gross changes in the uORF peptide. Rather, we find that ribosomes that translate the uORF reinitiate inefficiently and that the intercistronic sequence downstream of the uORF mediates this effect.
The initiation of translation of most eukaryotic mRNAs proceeds according to the ribosome scanning model (reviewed in
One prediction of the scanning ribosome model is that AUGs upstream of the main open reading frame (ORF) can repress downstream translation if these upstream AUGs are in a good sequence context. Although <10% of all eukaryotic mRNAs contain upstream AUGs, this frequency is much higher for regulatory genes, including those that control growth and development (
Upstream AUGs initiate ORFs that either overlap the main ORF or terminate before the downstream ORF. In the latter instance, ribosomes translating the upstream ORF (uORF) may resume scanning when translation is terminated. In these cases, the level of downstream translation is determined, in part, by the reinitiation frequency that has been shown to be influenced by factors such as uORF length (
In a few instances, the amino acid or nucleotide sequence of the uORF is involved in repressing downstream translation. For the uORFs encoded by the mammalian AdoMetDC gene (
It was reported previously that the 5' leader of the maize Lc gene inhibits downstream translation (
The 235-nucleotide 5' leader of Lc mRNA contains three in-frame AUG codons. The first, located 60 nucleotides from the 5' end, is in the best sequence context based on a comparison with both mammalian and plant initiation codons ( The conclusions of the previous study had to be tempered by the fact that translation repression was measured as an indirect effect of transcriptional activation and because translation of the uORF could not be verified. These limitations were overcome in this study in three ways. First, the 5' leader was fused directly to the luciferase reporter so that translation repression could be measured directly. Second, we demonstrated that repression could be assayed in an in vitro translation system as could the synthesis of the uORF peptide, which is stable after in vitro translation. Third, wild-type and mutant constructs were transformed into rice, with both steady state mRNA levels and luciferase activities being quantified and correlated.
Repression of Reporter Gene Translation by the Lc Leader in Vitro and in Vivo
The results presented in Figure 2A show that the Lc leader represses downstream luciferase translation both in vitro and in maize aleurone cells. The presence of the wild-type 5' leader in LUCWT represses translation 2.18-fold when compared with LUCm123, in which the three upstream AUG codons have been eliminated by point mutations. A similar result also was obtained with the wheat germ in vitro translation system (data not shown). When the same constructs were tested after bombardment into maize aleurone cells, LUCm123 encoded 17.9-fold more luciferase than did LUCWT.
The most prominent feature of the 5' leader is a 38-codon uORF. To determine whether translation of uORF was responsible for the observed repression, we tested whether increasing translation of uORF would affect downstream expression. To this end, the sequence surrounding the first AUG codon of the uORF (uAUG) was changed to match the eukaryotic consensus (
The Lc uORF Is Translated in Vitro Quantification of the 4.6-kD uORF peptide and the 61-kD luciferase protein encoded by LUCWT and LUCcontxt, respectively, confirmed that more uORF peptide and less luciferase were encoded by the latter construct (Figure 2B and Table 1). This was accomplished by measuring relative amounts of the in vitro translation products on gels by using a PhosphorImager and adjusting these values for the number of radioactive methionine residues present. For LUCWT and LUCcontxt, the relative amount of the 61-kD protein agreed well with the luciferase activity measurements (Table 1 and Figure 2A). Furthermore, the context improvement resulted in more than a twofold increase in uORF peptide with an almost twofold decrease in the 61-kD protein.
Repression Is Not Mediated by the Amino Acid Sequence of the uORF
Because single codon mutations had no effect on downstream expression, we tested whether a dramatically different uORF sequence could still mediate repression. Twenty-nine of the 38 uORF codons were replaced without changing the length, position, or flanking uORF sequences by deletion of an A three nucleotides downstream from the uAUG and the addition of an A 15 nucleotides upstream of the uORF stop codon (Figure 3A and Figure 3C). The fact that this construct (LUCfs) still encoded repressed levels of luciferase both in vitro and in maize aleurone cells indicates that neither the uORF codons nor the derived amino acids are responsible for the observed repression.
Translation of the Downstream ORF Is Largely Dependent on Ribosome Reinitiation in Maize Cells but Not in Vitro When translated in vitro, LUCWTovlp encoded 60% of the activity of LUCWT (Figure 4A), indicating that only 40% of the luciferase encoded by LUCWT is due to ribosomes that reinitiate after translating uORF. Luciferase activity was only 8% of LUCWT in the double mutant LUCcontxtovlp, which contains the context change shown previously to increase translation of uORF in vitro (Figure 2). Thus, when the uAUG was efficiently recognized, the contribution of reinitiating ribosomes to total luciferase translation increased from 40 to 85% (46 of 54), whereas the contribution of leaky scanners decreased from 60 to 15% (eight of 54). Independent evidence for these data was obtained by visualizing the products of translation after SDS-PAGE and autoradiography. Due to the stability of the extended uORF peptide, it could be seen that the context change led to an increase in peptide synthesis and a dramatic decrease in the synthesis of the 61-kD (luciferase) protein (Figure 4B).
Cap-independent translation has been demonstrated in vitro ( When assayed after bombardment into maize cells, LUCWTovlp and LUCcontxtovlp encoded only 7 and 1% of the activity of LUCWT (Figure 4A). This result provides additional evidence that the uAUG of LUCWT is recognized efficiently in maize cells and indicates that almost all of the observed luciferase activity is due to reinitiation in this assay system.
Quantitative Analysis of the in Vitro Translation Products
Involvement of Intercistronic Sequence in Inefficient Reinitiation
Previous studies also have demonstrated a role for a particular intercistronic sequence in the ability of ribosomes to reinitiate ( Visualization and quantitation of the translation products after SDS-PAGE again permitted a determination of the reinitiation frequency. As expected, LUCnew1 encoded the same amount of uORF peptide as did LUCWT (Table 1). Furthermore, the increase in luciferase protein (100 versus 169) was almost the same as the 1.85-fold value determined from luciferase activity measurements (Figure 5A). Of these 169 molecules, 60 could be attributed to scanning ribosomes, whereas the remaining 109 were due to reinitiation. Thus, 109 of 128 or 85% of the ribosomes that translated the uORF reinitiated downstream. LUCnew2 contains a 59-nucleotide intercistronic sequence that, like LUCWT, is AU rich downstream of the uORF stop codon (Figure 5c). When tested in vitro, LUCnew2 showed a level of luciferase activity comparable to that of LUCnew1 (Figure 5A). Furthermore, the reinitiation frequency of this construct was determined to be 93%, almost threefold greater than that of LUCWT (Figure 5B and Table 1). More significantly, when the sequence context of the uAUG of LUCnew2 was improved to the eukaryotic consensus, the double mutant, LUCnew2contxt, showed increased synthesis of both the uORF peptide and luciferase protein (Figure 5B and Table 1). This result was markedly different from that of LUCcontxt, in which the same context improvement increased production of the uORF peptide but reduced luciferase translation when compared with LUCWT (Table 1). The reinitiation frequency of LUCnew2contxt was determined to be 82%, ~3.5-fold greater than that of LUCcontxt (Figure 5B and Table 1).
Translational Repression in Transgenic Rice Plants Seven independent transgenic lines for each construct were obtained (from the International Laboratory for Tropical Agricultural Biotechnology, Scripps Institute, La Jolla, CA) and found to contain between one and six copies of the transgene after DNA gel blot analysis (data not shown). RNA gel blot analysis of all of the transgenic plants led to the detection of luciferase mRNA in only eight lines (Figure 6). For each of these plants, luciferase mRNA levels were quantified and standardized using an internal actin control. Similarly, luciferase activities were quantified and correlated with steady state mRNA levels. These results, presented in Table 2, indicate that elimination of the uAUGs increases translation efficiency by ~20-fold (LUCWT versus LUCm123). This value is very close to that obtained in the transient bombardment assays (Figure 2A). When the wild-type intercistronic sequence was replaced, translational efficiency was also increased ~15-fold (LUCWT versus LUCnew1). These data indicate that the increased luciferase activity encoded by LUCm123 and LUCnew1 is due to more efficient translation of their mRNAs in contrast to LUCWT rather than increased mRNA stability.
In this study, we used three different assay systems to determine how the Lc uORF inhibits downstream translation. The stability of the Lc uORF peptide in the cell-free translation system permitted the quantitation of both uORF peptide synthesis and downstream translation. However, because cell-free translation systems do not always faithfully reproduce the in vivo condition, constructs also were assayed after bombardment into maize aleurone cells and when stably integrated into the rice genome.
A Model for uORF-Mediated Repression
The major difference in the results obtained from plant cells (either transiently [maize aleurone cells] or stably [rice plants] transformed or in the wheat germ translation system) and from an animal cell-free system was the magnitude of repression: ~15- to 20-fold in plants (also referred to as in vivo) versus approximately twofold in the animal in vitro system. In the model, it is proposed that this difference is due, in part, to more efficient recognition of the uAUG in plants than in animals. Support for this claim comes from the bombardment of LUCWTovlp, which encodes only 7% of the luciferase levels of LUCWT (Figure 4A), indicating that >90% of ribosomes initiate translation at the uAUG in vivo. In contrast, LUCWTovlp encodes almost 60% of LUCWT level in vitro. Improvement of the context of the uAUG in LUCcontxtovlp reduces this value to 8%, making it more like the in vivo situation (Figure 4A). The stability of both the uORF peptide of LUCWT and the extended uORF peptide of LUCWTovlp allowed us to confirm that the context change did, in fact, increase the synthesis of both peptides (Figure 4B and Table 1). One prediction of this model is that increasing the translation of the uORF will decrease downstream translation. Evidence for this prediction comes from the comparison between LUCWT and LUCcontxt, in which it is shown directly that LUCcontxt encodes ~1.6-fold more uORF peptide (133 versus 212 molecules) but approximately half as much luciferase as does LUCWT (Figure 2A and Table 1). Thus, improving the uAUG context makes the in vitro system more like the in vivo condition, in which the uAUG is more efficiently recognized. The context change increases the magnitude of repression from 2.18-fold (LUCm123 versus LUCWT) to more than fourfold (LUCm123 versus LUCcontxt).
The second component of the model is that the act of translating the Lc uORF, but not what is translated, is repressive. Evidence for this claim is that the level of repression is unaffected by either single amino acid substitutions throughout the uORF peptide or the substitution of most of the uORF codons. Replacement of 29 of the 38 uORF codons in LUCfs (Figure 3C) has virtually no effect on repression either in vitro or in vivo (Figure 3A). For two other repressive uORFs, from the mammalian AdoMetDC and viral gp48 genes, the amino acid sequence at the C terminus was required for uORF-mediated repression (
The results of a previous study suggested that the uORF codons mediated repression in an in vivo bombardment assay (
The third component of the proposed model is that ribosomes translating uORF reinitiate inefficiently. Again, the stability of the peptide in vitro facilitated this analysis by permitting a calculation of reinitiation frequencies once the fraction of luciferase translation due to reinitiation was known (from analysis of the overlap constructs illustrated in Figure 4). For LUCWT and LUCcontxt, we found this value to be very similar at 30 and 24%, respectively (Table 1). The length of intercistronic sequences was shown in many systems to influence reinitiation frequencies both in vitro and in vivo ( The efficiency of reinitiation cannot be calculated in bombarded kernels or transgenic rice plants because uORF peptide cannot be detected in these systems (data not shown). Despite this limitation, the data presented support the conclusion that inefficient reinitiation is responsible primarily for repression in maize aleurone cells. First, the use of overlapping constructs demonstrates that uAUG is recognized efficiently in vivo and that reinitiating ribosomes account for ~90% of downstream translation (Figure 3 and Table 1). Second, mutations in the uORF codons have no effect on the magnitude of repression (Figure 3). Third, like the in vitro results, increasing the intercistronic length has virtually no effect on downstream repression, but replacement of the intercistronic sequence leads to a dramatic increase in luciferase activity (Figure 5 and Table 2). Under certain circumstances, sequence changes may alter mRNA stability and/or the secondary structure of the 5' leader region, both of which can influence the efficiency of translation initiation. However, it is unlikely that the observed repression and derepression can be explained in terms of mRNA stability or secondary structure. First, RNAs were stable during the course of the in vitro translation reactions, as assayed by RNA gel blots for all constructs, and luciferase activity showed a linear increase during at least the first 30 min of incubation (data not shown). Second, replacement of the intercistronic sequence led to a 15-fold increase in luciferase expression in transgenic rice plants (Table 2). This increase resulted directly from more efficient translation of mutant mRNAs rather than differences in mRNA stability.
With regard to secondary structure, the most stable secondary structure of the Lc 5' leader has predicted free energy (
uORFs Exhibit a Variety of Repressive Mechanisms
The precise manner in which a uORF peptide represses downstream translation has not been demonstrated as yet. However, for gp48 and AdoMetDC, the available evidence is consistent with a model in which the peptide interacts with the translating ribosome as it encounters the termination codon and causes the ribosome to stall, thus blocking the flow of ribosomes to the downstream cistron. In both of these cases, repression is abolished if the uORF is extended by a single codon, suggesting that translation termination is a key step in the repression mechanism (
There are several other differences between our results and those of the uORFs mentioned above. For both AdoMetDC and gp48, the sequences both upstream and downstream of the uORF do not effect repression (
Like the Lc uORF, the yeast GCN4 gene is regulated in part by the frequency of reinitiation. The 5' leader of GCN4 has four very short uORFs. Ribosomes that translate uORF2, uORF3, or uORF4 inhibit downstream translation at GCN4 because they reinitiate inefficiently. For the best-characterized uORF4, inefficient reinitiation is due to the high G+C content of the last uORF codon and the 10 nucleotides downstream of the uORF stop codon ( Although both the Lc uORF and uORF4 of GCN4 promote inefficient reinitiation, it is unlikely that the same mechanism accounts for both phenomena. The sequences immediately downstream of the Lc uORF stop codon are AU rich (Figure 5c), with eight of the 10 downstream nucleotides either an A or U. Furthermore, replacing the Lc intercistronic region with a relatively GC-rich sequence in LUCnew1 (in which five of the 10 downstream nucleotides are G or C) increases the frequency of reinitiation.
The most prominent feature of the Lc intercistronic sequence is the multiple stop codons downstream of the uORF (Figure 5c). Besides the uORF stop codon, three of the next six in-frame triplets are also stop codons. These stop codons may, in some manner, act to prevent newly dissociated 40S subunits from resuming a scanning mode. The process of translation termination in eukaryotes is poorly understood. It is thought that a single release factor (RF) recognizes all three termination codons and catalyzes the release of the peptide chain from the ribosome (
Plant Material
Plasmid Constructions
Additional mutations were created by first subcloning the HindIII-EcoRI fragment of LUCWT (Figure 1) into the pAlter-1 vector (Promega). Site-directed mutagenesis was performed using the Altered Sites II kit (Promega) with the following mutagenic oligonucleotides: 5'-TCTCTACCCT TACCATGGAAGT TC-3' for LUCcontxt; 5'-ACC-CT TCGCATGGAGT TCT TGCAT TGCT-3' and 5'-CTGATCATCAGTACGATGCCTCGTG-3' for LUCfs; 5'-GGCGTCT TCCAT T TGGAT-CTCTACTGATCATCAGACGATG-3' for LUCWTovlp and LUCcontxtovlp; 5'-GCAAGAACTGCCATGCGA-3' for E-2
To construct LUCWTovlp and LUCWTcontxtovlp, the sequence from the uORF stop codon to the first nucleotide of the BamHI site in the intercistronic region was deleted (Figure 1). The extended uORF is out of frame with the downstream luciferase ORF with an overlapping region of 83 nucleotides. LUCWTsl was generated by inserting a 102-bp stem–loop structure into the HindIII site of LUCWT (Figure 1). The 102-bp fragment is an inverted repeat of the pUC19 polylinker (the EcoRI-HindIII fragment; LUCint102 was created by inserting 48 bp into the BamHI site of LUCWT (Figure 1). The inserted sequence contains the BamHI-EcoRI fragment of the pCR2.1 polylinker (Invitrogen, Carlsbad, CA) and the BamHI-EcoRI fragment of the pUC19 polylinker with a 4-bp deletion at the KpnI site. The sequence was generated by first subcloning the XbaI-EcoRI fragment of the pUC19 polylinker into the corresponding restriction sites of pCR2.1, followed by BamHI digestion. The BamHI fragment is 52 bp, with an SpeI site near one end (from pCR2.1) and a KpnI site near the other (from pUC119). For further subcloning, the KpnI site was removed by KpnI digestion followed by treatment with the Klenow fragment of DNA polymerase I. Thus, the insert present in LUCint102 is 48 bp and is oriented such that the SpeI site lies near the uORF. LUCnew1 was derived from LUCint102 by deleting the 3' flanking sequence of the uORF. This was done by amplifying the uORF with primers 5'-GTCGAAGCT TGCGCGGAGGAGAGCTCC-TCCG-3' (HindIII site underlined) and 5'-CGGACTAGT TCGCGACTA-TCTACTGATCATCAGACGATGCCTCGTG-3' (SpeI site underlined). The PCR product was digested with HindIII and SpeI and used to replace the HindIII-SpeI fragment in LUCint102. The new intercistronic sequence of LUCnew1 transcript is 56 nucleotides, and the uORF stop codon is UAG (Figure 5c).
LUCnew2 and LUCnew2contxt contain a new intercistronic sequence from the LacZ
For expression in maize cells and in transgenic rice plants, the HindIII-AvaI fragment of LUCWT or the mutant derivatives replaced the KpnI-AvaI fragment of pJD300 (
In Vitro Transcription and Translation Each RNA was translated in the rabbit reticulocyte lysate in vitro translation system (Promega) at a final RNA concentration of 4 µg/µL. Reactions were performed at 30°C for 30 min and then frozen on dry ice. Luciferase activity showed a linear increase during the first 30 min of incubation (data not shown). For SDS-PAGE analysis of the in vitro translation products, 35S-methionine (Amersham) was included in the translation reactions at a final concentration of 0.8 mCi/mL .
Immunoprecipitation
SDS-PAGE Analysis
Transient Expression in Maize Aleurone Cells
Rice Transformation
RNA Isolation and Gel Blot Analysis
Enzyme Assays
For the bombarded maize kernels, each half kernel was ground in 350 µL of 100 mM KPO4, pH 7.80, and 1 mM DT T at 4°C. After centrifugation, 100 and 25 µL of the supernatant were assayed for luciferase and CAT activity, respectively. CAT activity was expressed as ethyl acetate–soluble counts per minute (
For the transgenic rice plants, 0.5 g of leaf tissue was used for protein extraction, and luciferase expression levels were adjusted by the total protein concentration in the samples, as determined by using the
We thank Li-Li Chen and Dr. Claude Fauquet (International Laboratory for Tropical Agricultural Biotechnology, supported by the Rockefeller Foundation and ORSTOM) for rice transformation. We also thank Dr. Ronald Damiani for helping to set up the in vitro assay system and Drs. Lane Arthur, Michael Terns, and Claiborne Glover for critical reading of the manuscript. This work was supported by a grant from the U.S. Department of Energy to S.R.W. Received June 24, 1998; accepted August 11, 1998.
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ABSTRACT
INTRODUCTION
R, respectively. Five times more in vitro translation reactions were used for these lanes than for immunoprecipitation with the uORF peptide–specific antisera. MW stands for the protein molecular mass markers, with their sizes in kilodaltons indicated at left. The 61-kD product corresponds to the luciferase protein. The predicted size of the Lc uORF peptide is 4.6 kD.
G) of -18 kcal/mol (
coding region of pCR2.1 (bases 361 to 407) (Invitrogen). The sequence was amplified by primers 5'-CGGACTAGT-AT TACAAT TCACTGGCCGTCG-3' (SpeI site underlined) and 5'-GTG-GATCCT T T TCCCAGTCACGACGT TGTA-3' (BamHI site underlined). The uORF region of LUCWT and LUCcontxt was amplified using primers 5'-GTCGAAGCT TGCGCGGAGGAGAGCTCCTCCG-3' (HindIII site underlined) and 5'-CGGACTAGT TATCTACTGATCATCAGACGA-TGCCTCGTG-3' (SpeI site underlined). The HindIII-SpeI fragment was ligated with the sequence from pCR2.1 (an SpeI-BamHI fragment) by using the SpeI site and was used to replace the HindIII-BamHI fragment of pGEM–luc (Promega). 
