JXB Advance Access originally published online on February 14, 2005
Journal of Experimental Botany 2005 56(413):1039-1047; doi:10.1093/jxb/eri097
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RESEARCH PAPER |
Expression of AtMHX, an Arabidopsis vacuolar metal transporter, is repressed by the 5' untranslated region of its gene
Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel
To whom correspondence should be addressed. Fax: +972 3 5351824. E-mail: orsha{at}mail.biu.ac.il
Received 26 August 2004; Accepted 3 December 2004
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Abstract |
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AtMHX is an Arabidopsis tonoplast transporter that can exchange protons with Mg2+ and Zn2+ ions. This transporter, which may play a role in ion homeostasis of plants, is encoded by a single gene in Arabidopsis. The molecular mechanisms that regulate the expression of this transporter are practically unknown. While AtMHX transcript can be easily visualized, expression of the corresponding protein is apparently low. To understand whether AtMHX expression is repressed at the translational level, the 5' untranslated region (5' UTR) of this gene was fused to reporter genes. In vitro analyses showed that the 5' UTR of AtMHX can repress the translation of downstream coding sequences. The major cause of the repression was efficient initiation at an upstream open-reading-frame (uORF) included in the 5' UTR. Although the sequence context of the upstream AUG (uAUG) codon was highly unfavourable, it was recognized by over 90% of the scanning ribosomes both in vitro and in vivo. The inhibitory effect of the uORF was mediated by imposing the need for reinitiation and not by ribosome stalling, as the inhibition was not dependent on the amino-acid sequence of the uORF peptide. The efficiency of reinitiation was low. The in vivo studies, carried out with transiently transformed tobacco plants, indicated that alternations in the Mg2+ or Zn2+ levels did not affect the rate of translation. These data suggest that AtMHX expression is repressed by the 5' UTR of its gene.
Key words: Arabidopsis, AtMHX, leader, magnesium, translational repression, uORF, 5' UTR, vacuole, zinc
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Introduction |
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The vacuole, which occupies most of the plant cell volume, plays a major role in the regulation of ion homeostasis in the cell and in detoxification of the cytosol (Marschner, 1995
). This regulatory role is fulfilled through the action of various transport proteins (reviewed by Marschner, 1995; Allen and Sanders, 1997; Martinoia et al., 2000; Maeshima, 2001). AtMHX is an Arabidopsis thaliana tonoplast transporter that can exchange protons with Mg2+ and Zn2+ ions, and can thus participate in balancing the levels of these ions between the cytosol and the vacuole (Shaul et al., 1999). This function is important, as both an excess and a deficiency of Mg2+ or Zn2+ in the cytosol can seriously impair cellular function (Marschner, 1995; Shaul, 2002). In situ hybridization showed that a large proportion of AtMHX transcript is located in the vascular region (Shaul et al., 1999). AtMHX is encoded by a single gene in Arabidopsis. Practically nothing is known to date about the molecular mechanisms that regulate the expression of this transporter.
AtMHX transcript can easily be visualized by northern blot hybridizations of Arabidopsis plants (Shaul et al., 1999). However, only faint bands corresponding to the AtMHX protein can be observed in western blot analyses of total microsomal membranes extracted from various Arabidopsis organs (data not shown). The signal is improved in vacuolar-enriched membrane fractions (Shaul et al., 1999). These analyses were carried out with anti-peptide AtMHX antibodies, which easily recognize this protein in transformed tobacco plants overexpressing this transporter (Shaul et al., 1999). Although these observations can be explained in different ways, the question arose as to whether the expression of AtMHX is regulated or repressed at the translational level.
The efficiency of mRNA translation is dependent, among other factors, on the 5'-untranslated region (5' UTR). Highly structured 5' UTRs, as well as upstream open-reading frames (uORFs), may inhibit translation (Kozak, 1989; Damiani and Wessler, 1993; Lukaszewicz et al., 1998; Chang et al., 2000; Morris and Geballe, 2000; Wang and Wessler, 2001; Kwak and Lee, 2001; Gaba et al., 2001; Kozak, 2002; Meijer and Thomas, 2002; Locatelli et al., 2002; Ng et al., 2004). Upstream ORFs repress translation either by a relatively rare mechanism in which the uORF peptide arrests the ribosome (Geballe and Morris, 1994; Cao and Geballe, 1996), or by imposing the need for reinitiation. Reinitiation is usually inefficient, as the 40S ribosomal subunit must reacquire the initiation factors that have been released following initiation at the uAUG (Kozak, 1987b). Some ribosomes may bypass the uAUG codon and initiate translation at the main ORF, in a process referred to as leaky-scanning. Recognition of an AUG codon by the scanning ribosome is dependent on its context. Purines (A or G) at positions –3 and +4 are the most important nucleotides for AUG recognition in higher eukaryotes (Kozak, 1987a; Joshi et al., 1997). When an AUG codon resides in a very weak context, which lacks purines at both of these positions, most ribosomes skip it and continue scanning (Kozak, 2002). For example, the 5' UTR of the maize Lc gene includes three uAUG codons and substantially represses Lc expression (Damiani and Wessler, 1993). Eliminating the first uAUG fully derepressed the expression, even though the second and third uAUGs were intact. While the first uAUG of Lc had a purine at the +4 position, the other two uAUGs had pyrimidines at both the –3 and +4 positions and, consequently, were not recognized in transiently-transformed maize tissue.
The mature 5' UTR of AtMHX is relatively long, including 169 nt (Shaul et al., 1999). Its secondary structure has free energy of –46.3 kcal mol–1, as predicted by the Zuker Mfold algorithm (Zuker, 2003) (Fig. 1). This 5' UTR includes an uORF which, if translated, encodes a short peptide of 13 amino acids. The sequence flanking the uAUG of AtMHX includes pyrimidines at both the –3 and +4 positions, and thus seems to be highly unfavourable (Table 1). The capability of this uORF to affect gene expression was therefore obscure, and had to be determined experimentally. To study the role of the 5' UTR of AtMHX in the expression of this gene, it was fused to reporter genes, and expression of the chimeric genes was studied in vitro and in vivo. The results show that the 5' UTR of AtMHX can repress the translation of downstream coding sequences. The major causes of repression were efficient initiation at the uORF, despite its unfavourable context, and a low rate of reinitiation.
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Materials and methods |
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Plant material
Nicotiana benthamiana (tobacco) seeds were germinated in 1:1 mixture of sand and Perlite®, and watered in a modified Johnson solution (3 mM KNO3, 0.25 mM K2SO4, 0.25 mM K2HPO4, 0.5 mM MgSO4, 0.5 mM CaSO4, 5 µM Fe as a Fe-EDDHA chelate (Sequestrene® 138, Novartis), 12.5 µM KCl, 6.25 µM H3BO4, 1.25 µM MnSO4, 1 µM ZnSO4, 0.125 µM CuSO4, 0.125 µM NaMoO4, and 0.001 µM CoCl2). Five weeks after germination, plants were transferred into a hydroponic growth system consisting of 1.3 l boxes including the same basic mineral solution with constant aeration. Agroinfiltration was carried out about 2 weeks following transfer to the hydroponic growth system. Mineral treatments were applied by the addition of the indicated minerals to the basic growth solution. Plants were maintained in a climate-controlled growth chamber with a photoperiod of 16/8 h light/dark.
Construction of plasmids for in vitro transcription assays
Plasmid pbsZmCDC2AF (kindly provided by G Grafi) is a derivate of 
Z-AT-Cdc2 (Zhao and Grafi, 2000). In short, pbsZmCDC2AF is a pBluescript SK vector (Stratagene) in which the coding sequence of a mutated (T14/Y15 into A/F) Z. mays cdc2 kinase (Colasanti et al., 1991; accession no. M60526) was cloned between the BamHI and PstI sites of the vector (the 5' end is at the BamHI site, which is located downstream to the T3 promoter). The resulting plasmid had the following 59 nt between the site of T3 polymerase transcription initiation and the initiating AUG codon of the marker cdc2 protein: 5'-GGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCACC-3'. The BamHI site is underlined. The 5' UTR of AtMHX was directly fused 3' to this BamHI site, thus excluding the three ACC nt that follow this site. In this construct, called pSKWTcdc2, the initiating ATG codon of cdc2 (included in the NcoI site) replaced the initiating ATG codon of the main ORF of AtMHX. A point mutation (A into C) was made in the last (the 3') nucleotide of the 5' UTR of AtMHX to obtain an NcoI site that included the initiation ATG codon of the main ORF. For this, the 5' UTR of AtMHX was amplified by PCR using as a template plasmid p218, including the full length cDNA of AtMHX (Shaul et al., 1999), a sense primer including a BamHI site (underlined): 5'-GGGTCGACGGATCCAACGCTTGAC-3', and an antisense primer including an NcoI site (underlined): 5'-AGAATTGAGGCCATGGTAA-3'.
To fuse the 5' UTR of AtMHX directly to the T3 promoter, plasmid pSKWTcdc2 was amplified by PCR, using a sense primer that included the T3 promoter upstream to a BamHI site and to the initial nucleotides of the 5' UTR of AtMHX: 5'-AATTAACCCTCACTAAAGGGATCCAACGCT-3'. The site of transcription initiation is underlined. The antisense primer corresponded to the vector sequence downstream to the cdc2 coding sequence and to the Acc65I site of the vector: 5'-TGGGTACCGGGCCCCCCCTCGAGG-3'. The amplified fragment was cloned into the EcoRV site of pGEM5Zf+ (Promega) (the T3 promoter was arbitrarily oriented to the T7 promoter of the vector). An NcoI site in the sequence of the pGEM5Zf+ vector was eliminated by digestion with AatII and SacII, fill-in reaction, and self-ligation. The resulting plasmid (called pWT-cdc2) is the WT construct used in the in vitro reactions. All modifications and mutations were carried out by amplification of this plasmid using the ‘megaprimer’ PCR approach (Sarkar and Sommer, 1990), to yield the constructs described in the text. All amplification products were digested with BamHI and NcoI, and cloned into the corresponding sites of pWT-cdc2. A point mutation (AUG to AAG) eliminated the initiating uAUG codon to create pAUG-cdc2. In the missense construct (pMis-cdc2), a frameshift mutation was introduced into the uORF by including an extra G nucleotide immediately after the uAUG, and deleting a G nucleotide just before the termination codon of the uORF. Plasmids p-1Ter-cdc2 and p-2Ter-cdc2 were created by a point mutation (UGA to UGG) in the first (-1Ter) or in both (-2Ter) in-frame termination codons of the uORF. The deletion construct (plasmid pDel-cdc2) was created by digesting pWT-cdc2 with BsaBI and Acc65I and ligating into this plasmid the HincII-Acc65I fragment of pSKWTcdc2. Thus, RNA transcribed from all these constructs included the nucleotides GGGATCC (the underlined G is the first transcribed nucleotide and the other 6 nt are the BamHI site), the native or modified 5' UTR of AtMHX, and the cdc2 coding region. All amplification reactions were carried out with a proof-reading polymerase, and the fidelity of the amplified products was verified by sequencing.
Construction of plasmids for in vivo assays
Plasmid pWT-cdc2 was digested with NcoI and EcoRI to excise the coding sequence of cdc2. The vector was ligated with a fragment that was digested with the same enzymes from plasmid pJD330 (kindly provided by DR Gallie). This fragment included the coding sequence of ß-glucuronidase (GUS) and the nopaline synthase (NOS) terminator. The initiating AUG codon of GUS was included within the NcoI site. An NcoI-Ksp22I fragment from the GUS sequence was replaced with the corresponding fragment of pCAMBIA1301 (Cambia, Australia), to obtain the intron included in the GUS sequence of the latter vector. The BamHI-NcoI fragment of the resulting plasmid, including the WT 5' UTR, was then replaced with the corresponding fragments of p-AUG-cdc2. Two plasmids were thus obtained, carrying either the WT or the –AUG 5' UTRs. The cauliflower mosaic virus 35S promoter was amplified by PCR from pCAMBIA1301 using primers that introduced HindIII and BglII sites at its 5' and 3' ends, respectively, and ligated into the binary vector pGA492 (An, 1986) that was digested with the same enzymes. The resulting binary vector was digested with BglII and EcoRI, and ligated with the BamHI-EcoRI fragment of the two plasmids carrying the WT or -AUG 5' UTRs to create pWT-GUS and p-AUG-GUS, respectively. In the resulting binary vectors, the WT or mutated 5' UTRs were preceded by the AGATCC sequence. This sequence was created by the compatible BglII-BamHI ligation; transcription from the 35S promoter initiates at the underlined A nucleotide. The binary vectors were immobilized into the Agrobacterium strain EHA105 (Hood et al., 1993).
In vitro transcription and translation
The constructs used for in vitro translation were linearized with Acc65I, which leaves 54 nt of the vector sequence downstream to the cdc2 coding sequence. The restricted template DNA was purified using Qiagen PCR columns, and 5' capped RNA was transcribed using the Ambion Message Machine kit, according to the instructions of the manufacturers. Immediately before the translation reactions, RNA was incubated for 2 min at 50 °C, allowed to cool gradually to the temperature in which the translation reaction was carried out, quantified by optical spectrometry, and fractionated on agarose gels to verify its integrity. In vitro translation reactions were carried out with kits from Ambion, using 0.5 µg RNA and 30 °C for translation in reticulocyte lysate, and 4 µg RNA and 27 °C for translation in wheat germ extract, as recommended by the manufacturer. The amounts of RNA used for translation were within the linear range for each system, as determined by preliminary analyses. When an internal control was used, it consisted of 0.5 µg RNA of Xenopus laevis elongation factor 1-
(Krieg et al., 1989), which was supplied by the manufacturer of the kit. The 35S-methionine-labelled translation products were fractionated by SDS–PAGE. A PhosphorImager was used to detect and digitally quantify the products on dried gels.
Transient expression in Nicotiana benthamiana leaves
Agroinfiltration was carried out as described by Yang and coworkers (Yang et al., 2000) with slight modifications. Agrobacteria cells containing each construct were grown overnight in LB medium at 28 °C. Following centrifugation (5 min, 5000 g, 4 °C), cells were resuspended to OD600 of 1.5 in a medium including 10 mM MES (pH 5.6), 10 mM MgCl2, and 100 µM acetosyringone (Sigma-Aldrich), and kept at 4 °C until use. Bacterial suspensions were infiltrated into intracellular spaces of fully expanded Nicotiana benthamiana leaves using a 1 ml plastic syringe (without the needle). The margins of each infiltrated spot (typically 2 cm2) were marked to allow precise excising of the infiltrated areas 3 d later.
Determination of GUS activity
Infiltrated leaf sections were ground in liquid nitrogen, and approximately 50 mg plant powder were suspended in 50 µl of extraction buffer (50 mM NaPO4 pH 7.2, 10 mM ß-mercaptoethanol, 10% Triton X100, and 1 mM Na2EDTA). Following centrifugation (5 min, 14 000 g, 4 °C), the protein concentration of the supernatant was determined using the Bradford reagent (Bio-Rad). Protein samples (30 µg) were suspended in 250 µl extraction buffer including 1 mM (final concentration) of the fluorescent GUS substrate 4-methylumbelliferyl-ß-D-glucuronide (MUG) (Duchefa Biochemie BV). GUS activity was assayed on a 96-well fluorescent plate-reader (Fluoroscan II, Lab Systems) with the excitation wavelength set at 350 nm and the emission wavelength at 460 nm. GUS activity (milli-units mg–1 protein) was calculated from the slope of the line generated from measures taken at 3 min intervals for 2 h, with respect to the slope of commercial pure GUS enzyme (Roche Diagnostics GmbH).
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Results |
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The 5' UTR of AtMHX represses the translation of a reporter gene in vitro
An in vitro translation system was used to determine whether the 5' UTR of AtMHX can repress the expression of downstream coding sequences when included in a construct that is otherwise expressed efficiently. All chimeric genes used for in vitro analyses included the coding sequence of the Z. mays cdc2 kinase fused downstream to the viral T3 promoter (Fig. 2A). RNA transcribed from the control construct (Con) included 59 nt derived from the vector sequence between the site of transcription initiation and the initiating AUG codon of the reporter protein. RNA transcribed from the test construct (+5' UTR) included the 5' UTR of AtMHX downstream to the first 56 nt of this vector sequence. Following the in vitro production of 5' capped RNA, it was quantified by optical spectrometry, and its integrity was verified by agarose gel electrophoresis. Three different experiments, each using a new set of independently transcribed and quantified RNAs, were carried out in rabbit reticulocyte lysate and in wheat germ extract. Figure 2B shows the autoradiograms of typical experiments. The graph shows the average data obtained in the reticulocyte lysate. The control RNA (Con) was expressed well in rabbit reticulocyte lysate, and showed less intense expression in wheat germ extract. This variation probably resulted from a difference in the competence of the vector sequence to promote translation in the two systems. The presence of the 5' UTR of AtMHX inhibited reporter gene translation about 5-fold in the reticulocyte lysate, and abolished expression in the wheat germ extract (in which expression of the control construct was less efficient). These data show that the whole 5' UTR of AtMHX can repress the translation of a reporter gene in vitro.
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The uORF within the 5' UTR inhibits translation in a manner independent of the uORF peptide
The inhibitory effect of the 5' UTR of AtMHX on downstream translation may result from the presence of the uORF, or from a general inhibitory effect of long, structured leaders on the efficiency of ribosomal scanning (Kozak, 1989
; Morris and Geballe, 2000; Gaba et al., 2001; Kozak, 2002; Meijer and Thomas, 2002). The predicted internal stability of the control construct used before, that included the 5' UTR of AtMHX as well as 56 bp of the vector sequence, was higher than that of the native 5' UTR (
G= –65.6 and –46.3 kcal mol–1, respectively). To determine the impact of the stable structure and the uORF within the structural and sequence context of the native 5' UTR, the native 5' UTR was directly fused to the T3 promoter, leaving only an extra 7 nt at its 5' end (Fig. 3A, WT). A point mutation (AUG to AAG) that eliminated the uORF was then introduced into the 5' UTR (Fig. 3A, –AUG). To evaluate the general impact of the long, structured leader on the efficiency of translation, most of the central region of the 5' UTR (the region between the two open arrows in Fig. 1) was deleted. The resulting 5' UTR thus included only 50 nt of the original 5' UTR of AtMHX, did not include the uORF, and had a much lower internal stability compared to the WT 5' UTR (G= –8.1 and –46.3 kcal mol–1, respectively) (Fig. 3A, Del).
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A construct having missense mutations in the uORF peptide was used to test whether the repression of translation results from ribosome stalling. In relatively rare cases, the peptide translated by uORFs stalls the ribosome in a sequence-specific manner (Geballe and Morris, 1994
; Cao and Geballe, 1996). In these cases, even uAUGs that are recognized at low efficiency are very inhibitory, because the stalled ribosomes block other ribosomes, that have skipped the uAUG, from reaching the main ORF. When this mechanism takes place, missense mutations, which alter the amino-acid sequence of the uORF peptide, are able to derepress the translation of the main ORF (Wang and Wessler, 1998). To create a missense construct, a frameshift was produced into the uORF by introducing an extra G nucleotide immediately after the uAUG, and deleting a G nucleotide just before the termination codon of the uORF. The sequence of the native and the mutated peptides that would be produced if the uORFs are translated is shown in Fig. 3B.
Figure 3C shows an autoradiogram of a typical in vitro translation experiment in wheat-germ extract. In contrast to the observation with the test construct used before, that included both the vector and the 5' UTR sequence, when the 5' UTR was directly fused to the T3 promoter expression in the wheat-germ system was above the level of detection. Thus, further experiments were carried out in this system, which is more suitable for studying plant genes. Independent RNA (supplied by the manufacturer of the translation kit) was included in the different reactions as an internal control, and showed no significant variation in its expression. The graph shows the average data of three different experiments, each using a new set of independently transcribed and quantified RNAs. The basal level of translation of RNA that had the native 5' UTR was rather low. The point mutation that eliminated the uAUG was sufficient to increase the rate of translation significantly. No further increase was observed in the deletion construct. The amount of RNA used for translation was within the linear range of the system (data not shown). This indicated that the main reason for inhibition of translation mediated by the 5' UTR of AtMHX was the presence of the uORF. The missense mutations did not relieve the inhibition of translation, indicating that the inhibitory effect is not dependent on the amino acid sequence of the uORF. When present, ribosome stalling by nascent peptides is preserved in in vitro translation systems (Wang et al., 1998).
The proportion of leaky-scanning is low
The efficiency of uAUG recognition can be further evaluated by determining the relative contribution of leaky-scanning and reinitiation to translation of the main ORF in RNA that has the native 5' UTR. When an uORF is present in a 5' UTR, translation of the main ORF can only occur by one of these alternative processes. Some ribosomes (the so-called leaky-scanners) scan past the uAUG. Alternatively, some of the ribosomes that have completed translation of the uORF may resume scanning and reinitiate translation at a downstream AUG. The approach used by Wang and Wessler (1998) to determine the proportion of leaky-scanning and reinitiation in the maize R gene was adopted to evaluate the relative contribution of these processes in the current case. Thus, the two in-frame termination codons located between the AUGs of the two ORFs were mutated (Fig. 4, -2Ter). If the uAUG of the resulting 5' UTR is recognized by the scanning ribosome, the first in-frame termination codon resides 
50 nt downstream to the AUG codon of the main ORF. Translation of the main ORF can thus occur only via leaky-scanning, and the contribution of reinitiating ribosomes to translation of the reporter gene is eliminated.
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For ribosomes that initiate at a uAUG, the efficiency of reinitiation depends on various factors, including the lengths of the uORF and of the intercistronic region. For reinitiation, the 40S subunit must reacquire Met-tRNAi as well as other initiation factors. If the elongation phase is brief, i.e. if the uORF is short and does not include inhibitory structures, the factors required for reinitiation would still be present when the 40S subunit resumes scanning. Reacquisition of Met-tRNAi is promoted by lengthening the intercistronic domain. However, no absolute limiting values can be assigned to the lengths of the uORF and of the intercistronic region (Kozak, 2002
; Poyry et al., 2004), and the impact of these parameters on the efficiency of reinitiation has to be determined experimentally in each case. To determine whether the efficiency of reinitiation is limited by these factors in the current case, only the first termination codon was mutated, while the second in-frame termination codon remained intact (Fig. 4A, -1Ter). Thus, the uORF was lengthened from 13 to 19 amino-acid codons, whereas the intercistronic length was shortened from 50 to 32 nt. The autoradiogram of a typical in vitro translation experiment in wheat-germ extract, as well as the average data of three independent experiments, are shown in Fig. 4A. Eliminating the first termination codon (-1Ter) resulted in only a slight reduction in the rate of translation. This suggests that in the context of the current sequence, the lengths of the uORF and of the intercistronic region are not close to the value that limits reinitiation. Eliminating the two termination codons (-2Ter) inhibited translation by about 80% relative to the native 5' UTR. This indicates that the proportion of leaky-scanning is low, and that most of the translation of the main ORF in the native 5' UTR occurs via reinitiation. Figure 4B illustrates a model that estimates what fraction of the ribosomes that started scanning the WT 5' UTR initiated translation at the uORF, and, among them, what fraction reinitiated. In this illustration, the letter L represents leaky-scanner ribosomes, which skipped the uORF. The letters R and F represent ribosomes that initiated translation at the uORF and either reinitiated at the main ORF (R), or failed to reinitiate (F). The level of expression seen with the deletion construct and the –AUG mutation (which is equal) is assumed to represent the full potential of ribosome binding and initiation of scanning of the supplied RNA. This quantity is defined as 100% of the ribosomes. As shown in Fig. 3C, RNA that had the native 5' UTR was translated by 38% of the ribosomes (compare the WT to the deletion and –AUG constructs). RNA that has the native 5' UTR can be translated by either leaky-scanning or reinitiation. As determined by the expression seen when all termination codons between the two ORFs were eliminated (the -2Ter construct, Fig. 4A), leaky-scanner ribosomes were 19% of those that translated RNA with the native 5' UTR. Thus, only 7% (19% of 38%) of the total ribosomes that started scanning RNA with the native 5' UTR became leaky-scanners, whereas 93% initiated translation at the uORF. Reinitiating ribosomes constituted 31% of the total ribosomes in the current experiments (this value is obtained by subtracting the leaky-scanner ribosomes (7%) from the ribosomes that translated the main ORF of the WT 5' UTR (38%). These data indicate that a high proportion (two-thirds) of the ribosomes that initiated at the uORF failed to reinitiate. Thus, efficient initiation at the uORF and a low rate of reinitiation were the major causes of the observed repression.
In vivo studies in transiently transformed tobacco plants
The in vitro analyses indicated that the presence of the uORF within the 5' UTR of AtMHX significantly represses downstream translation. An in vivo expression system was employed to validate this effect. The WT and the –AUG 5' UTRs were cloned upstream to the coding sequence of ß-glucuronidase (GUS) and downstream to the 35S promoter (Fig. 5A). The chimeric genes were then cloned into a binary vector and tested by agroinfiltration (Yang et al., 2000). In short, Agrobacteria cells were infiltrated into leaves of hydroponically grown Nicotiana benthamiana (tobacco) plants, and transient GUS expression was determined 3 d later. The GUS coding sequence included an intron, to prevent expression in the Agrobacteria cells present in the infiltrated leaves. In contrast to Arabidopsis, Nicotiana benthamiana plants can be easily transformed by this technique, and are widely used in agroinfiltration studies (Tampakaki and Panopoulos, 2000; Goodin et al., 2002). Transient transformation systems avoid the variability that results from the ‘position effect’ in stable transformations (Yang et al., 2000).
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Divalent cations, and Mg2+ in particular, can affect the secondary structure of mRNAs (Anwander et al., 1990
; Misra and Draper, 1998; Wu and Tinoco, 1998; Serra et al., 2002; Misra et al., 2003; Schaak et al., 2003). As mentioned, AtMHX sequesters Mg2+ and Zn2+ ions into the plant vacuole. Increased expression of AtMHX may be desired under conditions of high Mg2+ or Zn2+ levels to facilitate sequestration of the excess ions. It was, thus, interesting to test whether variation in the Mg2+ or Zn2+ levels can affect the rate of reporter gene translation. Alternating the Mg2+ levels in the in vitro reactions resulted in a similar effect on the translation of the tested constructs and of the internal control (data not shown), indicating that the observed effects were non-specific. The in vivo expression system was therefore employed to address this question as well.
Agroinfiltration experiments were carried out in hydroponically-grown tobacco plants to allow precise control of the growth medium. Different (but similar) regions of each leaf were injected with the two tested constructs. The margins of each infiltrated spot were marked to allow precise excising of the infiltrated areas. Following infiltration, plants were either maintained in the basic mineral solution, or supplied with increased levels of Mg2+, Zn2+, or Na+ (the latter was used as a control, irrelevant ion, and also to test the effect of a mild salt stress). These minerals were added in amounts which, according to previous experience, were sufficient to increase their content significantly in leaves of tobacco plants, but were not associated with visible stress symptoms (Shaul et al., 1999; data not shown). Three days following infiltration, the infiltrated leaf sections were collected and GUS activity was determined.
In all treatments, GUS activity obtained in leaf sections infiltrated with plasmids having the WT 5' UTR was close to, and often below, the level of detection (Fig. 5B). The –AUG mutation significantly increased GUS expression. This indicates that the uAUG codon is recognized at high efficiency in vivo also. Exposing the plants to increased levels of Mg2+ or Zn2+ did not derepress the translation, at least under the experimental conditions used here.
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Discussion |
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Northern and western blot hybridization experiments suggested that AtMHX expression is regulated or repressed at the translational level. To study the role of the 5' UTR of AtMHX in the expression of this gene, it was fused to reporter genes, and expression of the chimeric genes was studied in vitro and in vivo. The 5' UTR of AtMHX inhibited expression when inserted downstream to a short leader sequence that could otherwise mediate sufficient expression in rabbit reticulocyte lysate and in wheat-germ extract. This shows that the whole 5' UTR of AtMHX can repress the translation of downstream coding sequences in vitro. A point mutation that eliminated the uAUG codon was sufficient to increase the rate of translation in wheat-germ extract significantly. No further increase was observed in the deletion construct, in which most of the long leader, including the uORF, was deleted. This deletion resulted in a significant reduction in the predicted internal stability of the 5' UTR. Thus, the main cause of the observed repression was the presence of the uORF. As shown here, the efficient inhibition did not result from ribosome stalling. In the wheat-germ system, only 7% of the total ribosomes that started scanning RNA that had the native 5' UTR became leaky-scanners, whereas 93% initiated translation at the uORF. The uORF also mediated strong repression in the in vivo expression system, indicating that the uAUG codon was recognized at even higher efficiency in vivo. Differences in the efficiency of uAUG recognition between in vitro and in vivo experimental systems are a well-documented phenomenon (reviewed by Kozak, 2002
). Still, the data presented here show that, in both experimental systems, the uAUG codon was recognized by over 90% of the scanning ribosomes. The context of this uAUG seems highly unfavourable for both monocotyledonous (wheat-germ extract) and dicotyledonous (Arabidopsis and tobacco) translation systems (Table 1). Its efficient recognition is thus exceptional, since upstream AUG codons having such a weak context are usually not recognized (Kozak, 2002).
In the wheat-germ system, a high proportion (two-thirds) of the ribosomes that initiated translation at the uORF failed to reinitiate. The data presented here show that in the context of the current sequence, the lengths of the uORF and of the intercistronic region are not close to the value that limits reinitiation. Study of the maize Lc gene showed that the intercistronic sequence may have an inhibitory effect beyond the mere impact of its length (Wang and Wessler, 1998), but whether this is also the case for the AtMHX gene is currently unknown.
Elements that inhibit translation, such as uORFs, are often observed in transcripts of genes involved in cellular regulation, such as proto-oncogenes, growth factors, and transcription factors (Kozak, 1991). AtMHX exchanges vacuolar protons with Mg2+ and Zn2+ ions, and can thus participate in balancing the levels of these ions between the cytosol and the vacuole (Shaul et al., 1999). The vacuole plays a major role in the regulation of ion homeostasis in the cell and in detoxification of the cytosol (Marschner, 1995). Both an excess and a deficiency of Mg2+ and Zn2+ ions in the cytosol can seriously impair cellular function. Still, the rate of translation mediated by the native 5' UTR of AtMHX was not affected by increasing the levels of these ions in the growth medium of agroinfiltrated tobacco plants. However, the transient transformation system used here does not mimic the endogenous expression of AtMHX in terms of spatial and temporal regulation. Further research will be necessary to determine whether the 5' UTR of AtMHX merely plays an inhibitory role or might function, under certain developmental and physiological conditions, to regulate the expression of this transporter.
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Acknowledgements |
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The authors thank G Grafi for a kind gift of plasmid pbsZmCDC2AF, and DR Gallie for a kind gift of plasmid pJD330. This research was supported by the Israel Science Foundation (grant no. 437/99-1).
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Footnotes |
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* These authors contributed equally to this work.
Abbreviations: GUS, ß-glucuronidase; Mg2+, magnesium ion; MUG, 4-methylumbelliferyl-ß-D-glucuronide; NOS, nopaline synthase; nt, nucleotides; uAUG, upstream AUG; uORF, upstream open-reading frame; 5' UTR, 5' untranslated region; Zn2+, zinc ion.
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