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A poly(U) motif in the 5′ untranslated region enhances the translational efficiency of β-glucuronidase mRNA in transgenic tobacco
Plant Science 165 (2003) 621 /626 www.elsevier.com/locate/plantsci
A poly(U) motif in the 5? untranslated region enhances the translational efficiency of b-glucuronidase mRNA in transgenic tobacco Issei Nagao a,b, Junichi Obokata b,* a
Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060, Japan b Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan
Received 17 February 2003; received in revised form 7 May 2003; accepted 22 May 2003
Abstract We examined how the base composition of the 5? untranslated region (UTR) modifies the translational efficiency of plant mRNAs with the aid of chimeric reporter constructs, and transient and stable transformation of tobacco. We inserted 20 base homopolymers of G, A, T and C into the 5? UTR of a b-glucuronidase (GUS) reporter gene driven by the cauliflower mosaic virus 35S promoter, and the resultant constructs were introduced into tobacco seedlings by particle bombardment. The transient GUS activity of the seedlings became higher only when the (T)20 motif was inserted. To elucidate how the (T)20 motif altered gene expression, we introduced the (T)20-inserted GUS construct into tobacco plants by Agrobacterium -mediated stable transformation and examined its expression in comparison with the GUS construct with no insert. The apparent GUS activity of the (T)20-GUS transgenic plants was 4.5-fold higher than that of the control GUS transformants, although the GUS mRNA levels were similar between these transgenic lines. The GUS activity/GUS mRNA ratio was 5.6-fold higher in the (T)20-inserted GUS transformants than in the controls. This indicates that the (U)20 motif inserted into the 5? UTR enhances the translational efficiency of the GUS reporter mRNA in tobacco cells. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: b-Glucuronidase; Tobacco; Translation; Translational enhancer; 5? untranslated region
1. Introduction The translational efficiency of cellular mRNAs differs from gene to gene [1]. The untranslated regions (UTRs) of mRNAs are possible determinants of translational efficiency and there are many examples of translational enhancers and repressors located in 5? and 3? UTRs [2 / 8]. However, our knowledge about how the translational efficiencies of individual mRNAs are determined is still limited. According to the sequence information available from databases, the 5? UTRs of many mRNAs appear to have a biased base composition and G residues tend to be rare in comparison with others [9]. In higher plants, the omega element of tobacco mosaic virus [10]
* Corresponding author. Tel.: /81-52-789-3083; fax: /81-52-7893081. E-mail address: [email protected] (J. Obokata).
and the tobacco psaDb leader sequence [11] are known to act as translational enhancers and both lack G residues. This raises the question as to whether base composition of the 5? UTRs may have some influence on the translational efficiency of plant mRNAs. In this study, we attempted to explore how base composition of the 5? UTR modifies the translational efficiency of plant mRNAs by employing the widely used reporter gene 35S::GUS as a model. When introducing a biased base composition into the UTRs of mRNAs, we should bear in mind that mRNAs are single-stranded molecules, and hence substitution and deletion of the pre-existing bases could cause alteration of the secondary structure that may in turn result in unexpected effects on gene expression. Generally, secondary structure in the 5? UTR is known to diminish translational efficiency [12,13]. Therefore, in this study, we inserted unstructured homopolymers of G, A, U and C into the 5? UTR of a model mRNA, expecting that
0168-9452/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00232-2
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their effects on mRNA secondary structure would be minimal. Using this experimental strategy, we found that a poly(U) motif introduced into the 5? UTR of the GUS reporter mRNA greatly enhances translational efficiency.
2. Material and methods
activities of the powdered tissues were determined by GUS Light (PE Applied Biosystems) and the LUC Assay System (Promega), respectively. The GUS activity of the seedlings was normalized for the LUC activity, an internal control, to correct for the variable efficiency of gene delivery in each bombardment [16]. Enzyme activities were measured using a luminometer (Luminescencer-JNR; ATTO Co. Ltd., Tokyo).
2.1. Chimeric constructs
2.3. Ti-mediated tobacco transformation
All constructs used in this study were derived from pBI121 [14], which contains a bacterial ?-glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S promoter. A modified GUS construct carrying the (G)20 motif in the 5’ UTR was made by inserting annealed oligonucleotide pairs of 5’ /CTAGAGGGGGGGGGGGGGGGGGGGG/3’ and 5’ /GATCCCCCCCCCCCCCCCCCCCCT /3’ into the Xba I/Bam HI site of pBI221 to give pBI-(G)20-221. Similarly, pBI-(A)20221, pBI-(T)20-221, and pBI-(C)20-221 were made by using the oligonucleotide pairs of 5’ /CTAGAAAAAAAAAAAAAAAAAAAAG /3’ and 5’ /GATCCTTTTTTTTTTTTTTTTTTTT /3’, 5’ /CTAGATTTTTTTTTTTTTTTTTTTTG /3’ and 5’ /GATCCAAAAAAAAAAAAAAAAAAAAT /3’, and 5’ /CTAGACCCCCCCCCCCCCCCCCCCCG /3’ and 5’ / GATCCGGGGGGGGGGGGGGGGGGGGT /3’, respectively. The pBIL221 construct, which harbors a firefly luciferase (LUC) gene and was used for the internal control in the transient expression analysis, was made by replacing the GUS coding region of pBI221 with a Bam HI /Sac I fragment containing the LUC coding region of pGEMluc (Promega). For Agrobacterium -mediated transformation, the Hin dIII /Eco RI fragment of pBI-(T)20-221 was inserted into the binary vector pBI101. The resultant construct carrying the 35S::(T)20::GUS was named pBI-(T)20-121.
The binary vectors pBI121 and pBI-(T)20-121, carrying chimeric GUS genes, were mobilized into Agrobacterium tumefaciens LBA4404, and leaf discs of N. tabacum cv Petit Havana SR1 were transformed as previously described [11,17]. From the regenerated transgenic plants, developed leaves of 3 /6 cm were harvested, frozen in liquid N2 and ground to fine powder. A portion of the powdered tissue was used for GUS assays [14] and the rest for RNA extraction. Protein concentration was determined according to Bradford [18] and used to normalize measured GUS activities. 2.4. RNA analysis Total RNA was extracted from the powdered tissue using an AGPC method [19] followed by three phenol/ chloroform extractions and LiCl precipitation. Preparation of riboprobes and subsequent RNase protection analysis were carried out as previously described [20]. Ten micrograms of tobacco leaf RNA was hybridized with 2 /103 cpm of [32P]-labeled riboprobes, digested with RNaseA/T1 and the protected fragments were electrophoresed on 8 M urea/6% polyacrylamide gels. The radioactivity of each band on the gels was analyzed using a Fujix BAS2000 Imaging Analyzer (Fuji Photo, Inc., Japan).
2.2. Transient expression analysis 3. Results Seeds of Nicotiana tabacum cv Petit Havana SR1 were surface sterilized, sown on agar plates containing halfstrength MS salts [15] and placed under a regime of 16-h light/8-h dark for 7 days at 25 8C. One microgram each of the test construct and the internal control, pBIL221, were mixed and precipitated onto 0.5 mg of gold particles (1.5 /3.0 mm in diameter; Aldrich Chemical Co., WI) with ethanol. DNA-coated gold particles were suspended in 100 ml of ethanol, and 4 ml aliquots were used for each bombardment. Each plate of tobacco seedlings was bombarded twice using a particle gun (IDERA GIE-III; TANAKA Co. Ltd., Sapporo), then incubated at 25 8C for 24 h. Subsequently, liquid N2 was poured onto the plates, and the frozen seedlings were harvested and ground into fine powder. GUS and LUC
3.1. The (T)20 motif inserted into the 5? UTR enhances the expression of the reporter gene in transient transformation experiments To explore how the base composition of the 5? UTR modifies plant gene expressions, we carried out an experiment with a widely used reporter gene, 35S::GUS (Fig. 1). This gene encodes a GUS reporter driven by the cauliflower mosaic virus 35S promoter and 3?-flanked by the nopalin synthase (NOS) gene terminator. We introduced 20 base homopolymers of G, A, T and C into the 5? UTR of this reporter gene and the resultant constructs were introduced into tobacco seedlings by particle bombardment.
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Fig. 1. The chimeric constructs used in this study and their entire 5? UTR sequences. Twenty base homopolymers of G, A, T and C were inserted into the 5? UTR of the GUS reporter gene of pBI221 by Xba I and Bam HI. Transcription initiation sites are denoted by /1.
The transient GUS activities of seedlings are shown in Fig. 2. Although the insertion of (G)20, (A)20, or (C)20 sequences gave little effect on the expression level of the GUS reporter gene, (T)20 insertion enhanced the apparent GUS activity. Because all the constructs used in this experiment had the same promoter sequence, it is likely that the (T)20 insertion altered GUS gene expression at the post-transcriptional level, i.e. it affected mRNA stability or translational efficiency.
The transient expression system used here is a timesaving and convenient experimental system [16,21]. However, as is often the case for particle bombardment, the absolute expression level of the foreign gene was so low that direct quantitation of the mRNA level was difficult. Therefore, we next examined how poly(T) insertion modified GUS gene expression in stable transgenic plants. 3.2. The (U)20 motif functions as a translational enhancer in transgenic tobacco
Fig. 2. Transient expression analysis of the (N)20-inserted GUS constructs. Chimeric constructs were introduced into tobacco seedlings by particle bombardment and the GUS activity of the seedlings was determined after 24 h of incubation. The mean and S.D. of five experiments are shown.
35S::GUS chimeric constructs with or without (T)20 motif in their 5? UTRs were introduced into tobacco leaves by Agrobacterium -mediated stable transformation, and the regenerated transgenic plants were subjected to GUS assay and determination of GUS mRNA levels. The results from 14 and 13 transgenic lines, respectively, bearing the 35S::GUS and 35S::(T)20::GUS constructs are summarized in Fig. 3. As shown in Fig. 3A, (T)20 insertion greatly enhanced the GUS activity of the stable transgenic plants. The average GUS activity of the transgenic lines was 4.5 times higher in the (T)20 insertion lines than in the control GUS transformants (Fig. 3A). This enhancement was greater than that previously observed for the transient expression system (Fig. 2). We next determined the relative GUS mRNA levels of the individual transgenic lines by RNase protection
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method. The steady-state levels of the GUS mRNAs were similar between the 35S::GUS and 35S::(T)20::GUS transgenic lines (Fig. 3B). This indicates that (T)20 insertion in the 5? UTR scarcely altered the transcription
rate or the mRNA stability of the GUS reporter gene. In our GUS chimeric constructs, the 20 base insertion in the 5? UTR did not cause any change in the amino acid sequence of the encoded GUS protein (Fig. 1). Thus, it is very likely that the (T)20 insertion enhanced GUS gene expression by enhancing translation. As an indicator of the translational efficiency of the GUS reporter mRNAs, we calculated the ratio of GUS activity to GUS mRNA abundance (Fig. 3C) from the data presented in Figs. 3A and B. This value was 5.6fold higher for the (T)20-inserted GUS mRNA than for the control GUS mRNA (Fig. 3C). This result clearly shows that, in the case of a model GUS mRNA, insertion of the (U)20 sequence into the 5? UTR greatly enhanced translational efficiency.
4. Discussion
Fig. 3. Effects of (T)20 insertion into the 5? UTR on the expression of the GUS gene in stable transgenic tobacco plants. (A) GUS activities of transgenic plants carrying 35S::GUS (left) and 35S::(T)20::GUS (right). (B) GUS mRNA levels of transgenic plants determined by RNase protection analysis. (C) The GUS activity/mRNA ratio of transgenic plants carrying 35S::GUS (left) and 35S::(T)20::GUS (right). Black bars indicate mean9/S.D. of 14 35S::GUS transgenic lines (left) and of 13 35S::(T)20::GUS transgenic lines (right).
In this study, we explored how a biased base composition in the 5? UTR modifies plant gene expression using a model gene system of a GUS reporter construct and 20 base homopolymers in transient and stable tobacco transformants. The obtained results clearly show that the insertion of a (T)20 motif into the 5? UTR enhanced GUS gene expression (Fig. 2) and this effect was due to translational enhancement (Fig. 3). Since we still have limited knowledge about how translational efficiencies of individual plant mRNAs are determined [22,23], this finding could provide a unique clue to this subject. How does the (U)20 insertion enhance the translational efficiency? This insertion extended the 5? UTR from 40 to 60 nucleotides (Fig. 1), and this extension could be a possible cause for translational enhancement [24,25]. However, as shown in Fig. 2, insertion of the (G)20, (A)20 or (C)20 did not enhance GUS gene expression. When 5? leader sequence of tobacco psaEb gene [26] was inserted into the GUS construct instead of the above homopolymers and subjected to transient expression analysis, extension of the 5? UTR from 49 to 76 nucleotides did not enhance gene expression (Nagao et al., unpublished data), though the insertion of the psaDb leader resulted in a significant enhancement (Miyamoto et al., unpublished data). Taking these into account, it is unlikely that the translational enhancement by the (U)20 insertion was simply caused by the extension of the 5? UTR. If so, how could the (U)20 motif enhance the translational efficiency? Currently, there are two conceivable possibilities as follows. In eukaryotic cellular mRNAs, interaction of the poly(A)-binding protein with the initiation factors eIF4G and eIF4E can stimulate initiation of translation [27]. This protein /protein interaction between the 5? and 3? termini of the mRNA could circularize the mRNA molecules [28]. Currently, it is widely accepted that
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translationally active mRNAs could form a closed-loop structure [27,29]. In this view, it is tempting to think that the (U)20 sequence in the 5? UTR of the GUS reporter mRNA interacts with the 3? poly(A) tail to enhance the crosstalk between the 5? and 3? termini, which could in turn enhance translation initiation. In the case of the barley yellow dwarf virus, uncapped and non-polyadenylated viral mRNAs form a closed loop by direct basepairing between the stem loops of the 5? and 3? UTRs, and this structure brings about the efficient translation of this viral message in the host cytoplasm [8]. Polypyrimidine tract binding (PTB) protein is known to be involved in multiple steps of post-transcriptional gene regulation [30] including translation [31]. PTB binds to the polypyrimidine tract in the 5? UTR of the encephalomyocarditis virus and human rhinovirus-2 mRNAs; these steps promote the translation initiation by their internal ribosome entry sites [31,32]. Thus, it is another possibility that the (U)20 motif in the 5? UTR of the chimeric GUS mRNA may recruit cellular factors such as PTB that promote translation initiation. In this study, (U)20 insertion enhanced GUS gene expression about twofold in the transient expression system (Fig. 2) and more than fourfold in the stable transgenic plants. This difference is not surprising because these two experimental systems have many differences; particle bombardment was done on young seedlings, while mature leaves were used for the GUS assay in stable transgenic plants. In addition, particle bombardment physically damages plant cells, and injured plants are known to induce wounding signals to activate wound-inducible genes [33]. The translational enhancer activity of the 5? leader sequence of the tobacco psaDb gene [11] is also weak in transient expression systems when compared with stable transgenic plants (Miyamoto et al., unpublished data). The findings in this study not only provide an interesting example of a translational enhancer motif functional in plant cells, but also implies that there could be the plant mRNA population whose translational efficiencies are kept high because of the U-rich sequences in their 5? UTRs. This possibility as well as the underlying mechanism of translational enhancement awaits further investigation.
Acknowledgements The authors express their thanks to Drs. M. Sugiura, Y.Y. Yamamoto, M. Nakamura and T. Kubota for useful discussions and encouragement. Part of this study was carried out at the Faculty of Science, Department of Botany, Graduate School of Environmental Earth Sciences, Hokkaido University. This study was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture, Japan.
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[17] Y.Y. Yamamoto, Y. Kondo, A. Kato, H. Tsuji, J. Obokata, Light-responsive elements of the tobacco PSI-D gene are located both upstream and within the transcribed region, Plant J. 12 (1997) 255 /265. [18] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248 /254. [19] P. Chomczynski, N. Sacchi, Single step method of RNA extraction by acid guanidium thiocyanate /phenol /chloroform extraction, Anal. Biochem. 162 (1987) 156 /159. [20] Y.Y. Yamamoto, N. Nakamura, K. Kondo, H. Tsuji, J. Obokata, Early light-response of psaD, psaE and psaH gene families of photosystem I in Nicotiana sylvestris : PSI-D has an isoform of very quick response, Plant Cell Physiol. 36 (1995) 727 /732. [21] T. Miyamoto, T. Nakamura, I. Nagao, J. Obokata, Quantitative analysis of the transiently expressed mRNA level in particle bombarded tobacco seedlings, Plant Mol. Biol. Rep. 18 (2000) 101 /107. [22] W.A. Miller, P.M. Waterhouse, J.W. Brown, K.S. Browning, The RNA world in plants: post-transcriptional control III, Plant Cell 13 (2001) 1710 /1717. [23] J. Bailey-Serres, Selective translation of cytoplasmic mRNAs in plants, Trends Plant Sci. 4 (1999) 142 /148. [24] M. Kozak, Effects of long 5? leader sequences on initiation by eukaryotic ribosomes in vitro, Gene Expr. 1 (1991) 117 /125. [25] J.W.B. Hershey, W.C. Merrick, The pathway and mechanism of initiation of protein synthesis, in: J.W.B. Hershey, M.B. Mathews, N. Sonenberg (Eds.), Translational Control, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1996, pp. 33 /69.
[26] A. Kubota, Y.Y. Yamamoto, J. Obokata, Cloning of a nuclearencoded photosystem I gene, psaEb , in Nicotiana sylvestris , Plant Physiol. 108 (1995) 1297 /1298. [27] A.B. Sachs, Physical and functional interactions between the mRNA cap structure and the poly(A) tails, in: N. Sonenberg, J.W.B. Hershey, M.B. Mathews (Eds.), Translational Control of Gene Expression, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2000, pp. 655 /670. [28] S.E. Wells, P.E. Hillner, R.D. Vale, A.B. Sachs, Circularization of mRNA by eukaryotic translation initiation factors, Mol. Cell 2 (1998) 135 /140. [29] A. Jacobson, Poly(A) metabolism and translation: the closed-loop model, in: J.W.B. Hershey, M.B. Mathews, N. Sonenberg (Eds.), Translational Control, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1996, pp. 451 /480. [30] E.J. Wagner, M.A. Garcia-Blanco, Polypyrimidine tract binding protein antagonizes exon definition, Mol. Cell. Biol. 21 (2001) 3281 /3288. [31] A. Kaminski, S.L. Hunt, J.G. Patton, R.J. Jackson, Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA, RNA 1 (1995) 924 /938. [32] S.L. Hunt, R.J. Jackson, Polypyrimidine-tract binding protein (PTB) is necessary, but not sufficient, for efficient internal initiation of translation of human rhinovirus-2 RNA, RNA 5 (1999) 344 /359. [33] J. Leon, E. Rojo, J.J. Sanchez-Serrano, Wound signaling in plants, J. Exp. Bot. 52 (2001) 1 /9.
A poly(U) motif in the 5? untranslated region enhances the translational efficiency of b-glucuronidase mRNA in transgenic tobacco Issei Nagao a,b, Junichi Obokata b,* a
Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060, Japan b Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan
Received 17 February 2003; received in revised form 7 May 2003; accepted 22 May 2003
Abstract We examined how the base composition of the 5? untranslated region (UTR) modifies the translational efficiency of plant mRNAs with the aid of chimeric reporter constructs, and transient and stable transformation of tobacco. We inserted 20 base homopolymers of G, A, T and C into the 5? UTR of a b-glucuronidase (GUS) reporter gene driven by the cauliflower mosaic virus 35S promoter, and the resultant constructs were introduced into tobacco seedlings by particle bombardment. The transient GUS activity of the seedlings became higher only when the (T)20 motif was inserted. To elucidate how the (T)20 motif altered gene expression, we introduced the (T)20-inserted GUS construct into tobacco plants by Agrobacterium -mediated stable transformation and examined its expression in comparison with the GUS construct with no insert. The apparent GUS activity of the (T)20-GUS transgenic plants was 4.5-fold higher than that of the control GUS transformants, although the GUS mRNA levels were similar between these transgenic lines. The GUS activity/GUS mRNA ratio was 5.6-fold higher in the (T)20-inserted GUS transformants than in the controls. This indicates that the (U)20 motif inserted into the 5? UTR enhances the translational efficiency of the GUS reporter mRNA in tobacco cells. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: b-Glucuronidase; Tobacco; Translation; Translational enhancer; 5? untranslated region
1. Introduction The translational efficiency of cellular mRNAs differs from gene to gene [1]. The untranslated regions (UTRs) of mRNAs are possible determinants of translational efficiency and there are many examples of translational enhancers and repressors located in 5? and 3? UTRs [2 / 8]. However, our knowledge about how the translational efficiencies of individual mRNAs are determined is still limited. According to the sequence information available from databases, the 5? UTRs of many mRNAs appear to have a biased base composition and G residues tend to be rare in comparison with others [9]. In higher plants, the omega element of tobacco mosaic virus [10]
* Corresponding author. Tel.: /81-52-789-3083; fax: /81-52-7893081. E-mail address: [email protected] (J. Obokata).
and the tobacco psaDb leader sequence [11] are known to act as translational enhancers and both lack G residues. This raises the question as to whether base composition of the 5? UTRs may have some influence on the translational efficiency of plant mRNAs. In this study, we attempted to explore how base composition of the 5? UTR modifies the translational efficiency of plant mRNAs by employing the widely used reporter gene 35S::GUS as a model. When introducing a biased base composition into the UTRs of mRNAs, we should bear in mind that mRNAs are single-stranded molecules, and hence substitution and deletion of the pre-existing bases could cause alteration of the secondary structure that may in turn result in unexpected effects on gene expression. Generally, secondary structure in the 5? UTR is known to diminish translational efficiency [12,13]. Therefore, in this study, we inserted unstructured homopolymers of G, A, U and C into the 5? UTR of a model mRNA, expecting that
0168-9452/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00232-2
622
I. Nagao, J. Obokata / Plant Science 165 (2003) 621 /626
their effects on mRNA secondary structure would be minimal. Using this experimental strategy, we found that a poly(U) motif introduced into the 5? UTR of the GUS reporter mRNA greatly enhances translational efficiency.
2. Material and methods
activities of the powdered tissues were determined by GUS Light (PE Applied Biosystems) and the LUC Assay System (Promega), respectively. The GUS activity of the seedlings was normalized for the LUC activity, an internal control, to correct for the variable efficiency of gene delivery in each bombardment [16]. Enzyme activities were measured using a luminometer (Luminescencer-JNR; ATTO Co. Ltd., Tokyo).
2.1. Chimeric constructs
2.3. Ti-mediated tobacco transformation
All constructs used in this study were derived from pBI121 [14], which contains a bacterial ?-glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S promoter. A modified GUS construct carrying the (G)20 motif in the 5’ UTR was made by inserting annealed oligonucleotide pairs of 5’ /CTAGAGGGGGGGGGGGGGGGGGGGG/3’ and 5’ /GATCCCCCCCCCCCCCCCCCCCCT /3’ into the Xba I/Bam HI site of pBI221 to give pBI-(G)20-221. Similarly, pBI-(A)20221, pBI-(T)20-221, and pBI-(C)20-221 were made by using the oligonucleotide pairs of 5’ /CTAGAAAAAAAAAAAAAAAAAAAAG /3’ and 5’ /GATCCTTTTTTTTTTTTTTTTTTTT /3’, 5’ /CTAGATTTTTTTTTTTTTTTTTTTTG /3’ and 5’ /GATCCAAAAAAAAAAAAAAAAAAAAT /3’, and 5’ /CTAGACCCCCCCCCCCCCCCCCCCCG /3’ and 5’ / GATCCGGGGGGGGGGGGGGGGGGGGT /3’, respectively. The pBIL221 construct, which harbors a firefly luciferase (LUC) gene and was used for the internal control in the transient expression analysis, was made by replacing the GUS coding region of pBI221 with a Bam HI /Sac I fragment containing the LUC coding region of pGEMluc (Promega). For Agrobacterium -mediated transformation, the Hin dIII /Eco RI fragment of pBI-(T)20-221 was inserted into the binary vector pBI101. The resultant construct carrying the 35S::(T)20::GUS was named pBI-(T)20-121.
The binary vectors pBI121 and pBI-(T)20-121, carrying chimeric GUS genes, were mobilized into Agrobacterium tumefaciens LBA4404, and leaf discs of N. tabacum cv Petit Havana SR1 were transformed as previously described [11,17]. From the regenerated transgenic plants, developed leaves of 3 /6 cm were harvested, frozen in liquid N2 and ground to fine powder. A portion of the powdered tissue was used for GUS assays [14] and the rest for RNA extraction. Protein concentration was determined according to Bradford [18] and used to normalize measured GUS activities. 2.4. RNA analysis Total RNA was extracted from the powdered tissue using an AGPC method [19] followed by three phenol/ chloroform extractions and LiCl precipitation. Preparation of riboprobes and subsequent RNase protection analysis were carried out as previously described [20]. Ten micrograms of tobacco leaf RNA was hybridized with 2 /103 cpm of [32P]-labeled riboprobes, digested with RNaseA/T1 and the protected fragments were electrophoresed on 8 M urea/6% polyacrylamide gels. The radioactivity of each band on the gels was analyzed using a Fujix BAS2000 Imaging Analyzer (Fuji Photo, Inc., Japan).
2.2. Transient expression analysis 3. Results Seeds of Nicotiana tabacum cv Petit Havana SR1 were surface sterilized, sown on agar plates containing halfstrength MS salts [15] and placed under a regime of 16-h light/8-h dark for 7 days at 25 8C. One microgram each of the test construct and the internal control, pBIL221, were mixed and precipitated onto 0.5 mg of gold particles (1.5 /3.0 mm in diameter; Aldrich Chemical Co., WI) with ethanol. DNA-coated gold particles were suspended in 100 ml of ethanol, and 4 ml aliquots were used for each bombardment. Each plate of tobacco seedlings was bombarded twice using a particle gun (IDERA GIE-III; TANAKA Co. Ltd., Sapporo), then incubated at 25 8C for 24 h. Subsequently, liquid N2 was poured onto the plates, and the frozen seedlings were harvested and ground into fine powder. GUS and LUC
3.1. The (T)20 motif inserted into the 5? UTR enhances the expression of the reporter gene in transient transformation experiments To explore how the base composition of the 5? UTR modifies plant gene expressions, we carried out an experiment with a widely used reporter gene, 35S::GUS (Fig. 1). This gene encodes a GUS reporter driven by the cauliflower mosaic virus 35S promoter and 3?-flanked by the nopalin synthase (NOS) gene terminator. We introduced 20 base homopolymers of G, A, T and C into the 5? UTR of this reporter gene and the resultant constructs were introduced into tobacco seedlings by particle bombardment.
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Fig. 1. The chimeric constructs used in this study and their entire 5? UTR sequences. Twenty base homopolymers of G, A, T and C were inserted into the 5? UTR of the GUS reporter gene of pBI221 by Xba I and Bam HI. Transcription initiation sites are denoted by /1.
The transient GUS activities of seedlings are shown in Fig. 2. Although the insertion of (G)20, (A)20, or (C)20 sequences gave little effect on the expression level of the GUS reporter gene, (T)20 insertion enhanced the apparent GUS activity. Because all the constructs used in this experiment had the same promoter sequence, it is likely that the (T)20 insertion altered GUS gene expression at the post-transcriptional level, i.e. it affected mRNA stability or translational efficiency.
The transient expression system used here is a timesaving and convenient experimental system [16,21]. However, as is often the case for particle bombardment, the absolute expression level of the foreign gene was so low that direct quantitation of the mRNA level was difficult. Therefore, we next examined how poly(T) insertion modified GUS gene expression in stable transgenic plants. 3.2. The (U)20 motif functions as a translational enhancer in transgenic tobacco
Fig. 2. Transient expression analysis of the (N)20-inserted GUS constructs. Chimeric constructs were introduced into tobacco seedlings by particle bombardment and the GUS activity of the seedlings was determined after 24 h of incubation. The mean and S.D. of five experiments are shown.
35S::GUS chimeric constructs with or without (T)20 motif in their 5? UTRs were introduced into tobacco leaves by Agrobacterium -mediated stable transformation, and the regenerated transgenic plants were subjected to GUS assay and determination of GUS mRNA levels. The results from 14 and 13 transgenic lines, respectively, bearing the 35S::GUS and 35S::(T)20::GUS constructs are summarized in Fig. 3. As shown in Fig. 3A, (T)20 insertion greatly enhanced the GUS activity of the stable transgenic plants. The average GUS activity of the transgenic lines was 4.5 times higher in the (T)20 insertion lines than in the control GUS transformants (Fig. 3A). This enhancement was greater than that previously observed for the transient expression system (Fig. 2). We next determined the relative GUS mRNA levels of the individual transgenic lines by RNase protection
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method. The steady-state levels of the GUS mRNAs were similar between the 35S::GUS and 35S::(T)20::GUS transgenic lines (Fig. 3B). This indicates that (T)20 insertion in the 5? UTR scarcely altered the transcription
rate or the mRNA stability of the GUS reporter gene. In our GUS chimeric constructs, the 20 base insertion in the 5? UTR did not cause any change in the amino acid sequence of the encoded GUS protein (Fig. 1). Thus, it is very likely that the (T)20 insertion enhanced GUS gene expression by enhancing translation. As an indicator of the translational efficiency of the GUS reporter mRNAs, we calculated the ratio of GUS activity to GUS mRNA abundance (Fig. 3C) from the data presented in Figs. 3A and B. This value was 5.6fold higher for the (T)20-inserted GUS mRNA than for the control GUS mRNA (Fig. 3C). This result clearly shows that, in the case of a model GUS mRNA, insertion of the (U)20 sequence into the 5? UTR greatly enhanced translational efficiency.
4. Discussion
Fig. 3. Effects of (T)20 insertion into the 5? UTR on the expression of the GUS gene in stable transgenic tobacco plants. (A) GUS activities of transgenic plants carrying 35S::GUS (left) and 35S::(T)20::GUS (right). (B) GUS mRNA levels of transgenic plants determined by RNase protection analysis. (C) The GUS activity/mRNA ratio of transgenic plants carrying 35S::GUS (left) and 35S::(T)20::GUS (right). Black bars indicate mean9/S.D. of 14 35S::GUS transgenic lines (left) and of 13 35S::(T)20::GUS transgenic lines (right).
In this study, we explored how a biased base composition in the 5? UTR modifies plant gene expression using a model gene system of a GUS reporter construct and 20 base homopolymers in transient and stable tobacco transformants. The obtained results clearly show that the insertion of a (T)20 motif into the 5? UTR enhanced GUS gene expression (Fig. 2) and this effect was due to translational enhancement (Fig. 3). Since we still have limited knowledge about how translational efficiencies of individual plant mRNAs are determined [22,23], this finding could provide a unique clue to this subject. How does the (U)20 insertion enhance the translational efficiency? This insertion extended the 5? UTR from 40 to 60 nucleotides (Fig. 1), and this extension could be a possible cause for translational enhancement [24,25]. However, as shown in Fig. 2, insertion of the (G)20, (A)20 or (C)20 did not enhance GUS gene expression. When 5? leader sequence of tobacco psaEb gene [26] was inserted into the GUS construct instead of the above homopolymers and subjected to transient expression analysis, extension of the 5? UTR from 49 to 76 nucleotides did not enhance gene expression (Nagao et al., unpublished data), though the insertion of the psaDb leader resulted in a significant enhancement (Miyamoto et al., unpublished data). Taking these into account, it is unlikely that the translational enhancement by the (U)20 insertion was simply caused by the extension of the 5? UTR. If so, how could the (U)20 motif enhance the translational efficiency? Currently, there are two conceivable possibilities as follows. In eukaryotic cellular mRNAs, interaction of the poly(A)-binding protein with the initiation factors eIF4G and eIF4E can stimulate initiation of translation [27]. This protein /protein interaction between the 5? and 3? termini of the mRNA could circularize the mRNA molecules [28]. Currently, it is widely accepted that
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translationally active mRNAs could form a closed-loop structure [27,29]. In this view, it is tempting to think that the (U)20 sequence in the 5? UTR of the GUS reporter mRNA interacts with the 3? poly(A) tail to enhance the crosstalk between the 5? and 3? termini, which could in turn enhance translation initiation. In the case of the barley yellow dwarf virus, uncapped and non-polyadenylated viral mRNAs form a closed loop by direct basepairing between the stem loops of the 5? and 3? UTRs, and this structure brings about the efficient translation of this viral message in the host cytoplasm [8]. Polypyrimidine tract binding (PTB) protein is known to be involved in multiple steps of post-transcriptional gene regulation [30] including translation [31]. PTB binds to the polypyrimidine tract in the 5? UTR of the encephalomyocarditis virus and human rhinovirus-2 mRNAs; these steps promote the translation initiation by their internal ribosome entry sites [31,32]. Thus, it is another possibility that the (U)20 motif in the 5? UTR of the chimeric GUS mRNA may recruit cellular factors such as PTB that promote translation initiation. In this study, (U)20 insertion enhanced GUS gene expression about twofold in the transient expression system (Fig. 2) and more than fourfold in the stable transgenic plants. This difference is not surprising because these two experimental systems have many differences; particle bombardment was done on young seedlings, while mature leaves were used for the GUS assay in stable transgenic plants. In addition, particle bombardment physically damages plant cells, and injured plants are known to induce wounding signals to activate wound-inducible genes [33]. The translational enhancer activity of the 5? leader sequence of the tobacco psaDb gene [11] is also weak in transient expression systems when compared with stable transgenic plants (Miyamoto et al., unpublished data). The findings in this study not only provide an interesting example of a translational enhancer motif functional in plant cells, but also implies that there could be the plant mRNA population whose translational efficiencies are kept high because of the U-rich sequences in their 5? UTRs. This possibility as well as the underlying mechanism of translational enhancement awaits further investigation.
Acknowledgements The authors express their thanks to Drs. M. Sugiura, Y.Y. Yamamoto, M. Nakamura and T. Kubota for useful discussions and encouragement. Part of this study was carried out at the Faculty of Science, Department of Botany, Graduate School of Environmental Earth Sciences, Hokkaido University. This study was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture, Japan.
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