©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
5`-Leader of a Photosystem I Gene in Nicotiana sylvestris, psaDb, Contains a Translational Enhancer(*)

Yoshiharu Y. Yamamoto (1)(§), Hideo Tsuji (2), Junichi Obokata (1)(¶)

From the (1) Division of Biological Science, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060, Japan and the (2) Department of Biology, Suma, Kobe Women's University, Kobe 654, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Messenger RNA primary structures responsible for translational efficiency of a photosystem I gene, psaDb, of Nicotiana sylvestris were studied using a transgenic tobacco system. The entire 5`-leader (23 base pairs) with the first four amino acid codons of the protein coding region was fused in frame with the -glucuronidase (GUS) gene under the control of the 35 S promoter of cauliflower mosaic virus (CaMV). This construct (CaMV::psaDb-GUS`) was introduced into tobacco. GUS activity and GUS mRNA levels were determined for individual transformants, revealing that the insertion of the psaDb sequence greatly enhanced the GUS activity relative to GUS mRNA abundance. The GUS activity/GUS mRNA was 14 times higher in the CaMV::psaDb-GUS` transformants than in the control CaMV::GUS` transformants. The high GUS activity/GUS mRNA of the CaMV::psaDb-GUS` transformants was reduced 20-fold when 13 bases within the psaDb leader were altered. These 13 bases are common to the leaders of an Arabidopsis ferredoxin gene and the psaDb gene of N. sylvestris. Since GUS proteins encoded by these chimeric GUS genes have identical amino acid sequences, these results indicate that the 5`-leader of the psaDb mRNA contains a translational enhancer element.


INTRODUCTION

Translation is a potential step to regulate gene expression, but its significance is quite poorly understood compared with transcriptional regulation (1) . In eukaryotes, the most extensively studied cases involving translational regulation are the mammalian genes for ferritin and 5`-aminolevulinate synthase, whose products are involved in the storage and use of iron, respectively. Messenger RNAs of these genes have an iron responsible element within their untranslated leader (2) . When iron is depleted, this element is bound by iron responsible element-binding protein (3) , which depresses translation (2, 4, 5, 6, 7) .

Messenger RNAs of some plant viruses are translated with greater efficiency than host mRNAs. This phenomenon is ascribed to their leader sequences. This is true for tobacco mosaic virus, alfalfa mosaic virus RNA 4, brome mosaic virus RNA 3, potato virus X, and tobacco etch virus (8). In these cases the viral leaders contain translational enhancers; however, the underlying mechanisms remain to be elucidated. As for cellular mRNAs, there have been few reports on translational enhancer elements to date. In this study, we examined the 5`-portion of psaDb mRNA (see below) and found that its 5`-leader greatly enhances the translational efficiency of a chimeric -glucuronidase (GUS)() reporter gene in vivo.

psaDb is a nuclear gene for a photosystem I (PSI) subunit in Nicotiana sylvestris(9) , and its product, a PSI-D subunit, is known to associate with ferredoxin, which is a soluble electron carrier reduced by PSI. Recently, the untranslated leader of the Arabidopsis ferredoxin gene, fedA, was shown to enhance the expression of a reporter gene in a transient expression assay, but it is not known whether this enhancement occurs at translation or not (10) . In this study, we found that the fedA and psaDb leaders share common motifs and that mutations introduced into these motifs abolished the translational enhancer activity of the psaDb leader.


EXPERIMENTAL PROCEDURES

Materials

N. sylvestris is a diploid ancestor of Nicotiana tabacum, which is an amphidiploid (11) . The two species have highly homologous genomes. Constructs containing fragments from an N. sylvestris gene, psaDb(9) , were transformed into N. tabacum.

Construction of Chimeric GUS Genes

Four fusion genes (see Fig. 1and Fig. 6) were used in this study. 1) pBI121 contains the CaMV::GUS construct (12) . 2) For CaMV::psaDb-GUS` two oligonucleotides (5`-CTAGACTTCTCTCAATCCAACTTTTCTATGGCCATGGCAC-3` and 5`-GATCGTGCCATGGCCATAGAAAAGTTGGATTGAGAGAAGT-3`) were annealed and inserted into the XbaI-BamHI site of pBI221. After sequencing the introduced region, an EcoRI-HindIII fragment containing the resultant chimeric gene was introduced into a binary vector, pBI101, in place of the original promoterless GUS gene of pBI101 (12) . 3) For CaMV::GUS` two oligonucleotides (5`-CTAGATGGCTATGGCTC-3` and 5`-GATCGAGCCATAGCCAT-3`) were annealed and introduced into pBI101 as for CaMV::psaDb-GUS` described above. 4) For CaMV::psaDbLM-GUS` two oligonucleotides, (5`-CTAGACTTCGAGACCTCACCAGGGTCTATGGCCATGGCAC-3` and 5`-GATCGTGCCATGGCCATAGACCCTGGTGAGGTCTCGAAGT-3`) were annealed and introduced into pBI101 as described above.


Figure 1: Constructs of the chimeric GUS genes. A, the 5`-terminal sequence of the psaDb mRNA. The transcription start site is shown as +1. Arrows indicate palindromic sequences. B, schematic representation of the chimeric GUS genes. Arrows indicate the transcription start sites. CaMV::GUS represents the original GUS reporter gene of pBI121 (12). The psaDb fragment shown in A was inserted into the untranslated leader of CaMV::GUS to form CaMV::psaDb-GUS`. CaMV::GUS` is a control gene identical to CaMV::psaDb-GUS` except for the lack of the 23-base pair psaDb leader sequence and the two base substitutions as shown in C. C, nucleotide sequences of the chimeric GUS genes downstream of the XbaI sites (TCTAGA), aligned with the N-terminal sequences of the encoded proteins. The underlined portion shows the inserted psaDb sequence. Arrowheads indicate the base substitutions to alter the palindromic sequences.




Figure 6: Substitution of LM1 and LM2 decreases translational efficiency. A, the indicated 5`-leader motifs LM1 and LM2 in the CaMV::psaDb-GUS` construct were altered (shadedboxes) to generate a new chimeric construct, CaMV::psaDbLM-GUS`. B, GUS activity/GUS mRNA values were determined for individual transformants of CaMV::psaDb-GUS` and CaMV::psaDbLM-GUS`, and the averages are shown.



Ti-Mediated Gene Transfer and GUS Assays

Binary vectors containing the chimeric GUS genes mentioned above were mobilized into Agrobacterium tumerfaciens LBA4404, and leaf discs of N. tabacum cv. Petit Havana SR1 were transformed (13) . Plants were grown at 25 °C under continuous light. From the regenerated transformants (M generation), fully developed leaves of 3-6 cm in Magenta culture boxes (Magenta GA-7, Sigma) were harvested and frozen in liquid N, ground to fine powder, and stored at -80 °C. A part of the powdered tissue was used for GUS assays (12) , and the rest was used for RNA extraction. Protein concentration was determined according to Bradford (14) and used to normalize measured GUS activities.

RNA Analysis

Total RNA was extracted from the powdered tissue by the acid guanidium thiocyanate-phenol-chloroform method (15) , followed by three phenol/chloroform extractions and LiCl precipitation. A GUS gene-specific primer (5`-GTCGAGTTTTTTGATTTCACGGGTTGGGGTTTCTA-3`) was labeled at the 5`-end with P and used for primer extension analysis as described previously (9) . Extension products were electrophoresed in 8 M urea, 6% polyacrylamide gel, and the radioactivity of each band was analyzed using a Fujix BAS2000 Imaging Analyzer (Fuji Photo Inc., Japan).


RESULTS

Experimental Strategy

psaDb mRNA has a 23-base untranslated leader (Fig. 1A). In this study, we examined the possible involvement of this leader in translational regulation of psaDb mRNA by use of a transgenic tobacco system. The entire 5`-leader with the initial four codons of psaDb (Fig. 1A) were introduced into the untranslated leader of the original CaMV::GUS construct of pBI121 to obtain a chimeric GUS gene named CaMV::psaDb-GUS` (Fig. 1, B and C). As a result of this insertion, the GUS protein gained 12 amino acids at its N terminus (Fig. 1C, CaMV::psaDb-GUS`), which might alter the specific activity or turnover rate of the protein. We refer to this modified GUS protein as GUS`. In order to assess the effect of the inserted psaDb sequence on GUS` gene expression, we prepared a control GUS` construct, CaMV::GUS`, whose protein coding region has the same nucleotide sequence as that of CaMV::psaDb-GUS` except for the sixth and the twelfth nucleotides from the initiator ATG (Fig. 1C, arrowheads). These nucleotides were substituted in CaMV::GUS` to alter the palindromic sequences at the translation initiation site but did not affect the amino acid sequence. These three types of chimeric genes, CaMV::GUS, CaMV::psaDb-GUS`, and CaMV::GUS`, were introduced into tobacco, and the resultant transformants were regenerated.

5`-Terminal 35 Bases of psaDb mRNA Enhance Translational Efficiency in Vivo

The steady-state GUS activity in leaves was determined for individual transgenic plants grown under continuous light (Fig. 2). In comparison with the control transformants (CaMV::GUS`), GUS activity was much higher in the transformants having the psaDb sequence (CaMV::psaDb-GUS`). This enhancement of GUS activity may have been caused by internal cis-elements for transcriptional activation, stabilization of the transcripts, and/or elevation of translational efficiency. For the purpose of comparing the translational efficiencies among these transgenic plants, we determined the steady-state mRNA levels of the chimeric GUS genes by primer extension analysis (Fig. 3). The lengths of the extension products were different according to the lengths of the inserted fragments (Fig. 3). GUS activities of the individual transformants were divided by their respective mRNA levels (Fig. 4, GUS activity/mRNA), to examine the translational efficiency of the given GUS mRNAs. This value was 14 times higher in CaMV::psaDb-GUS` than in CaMV::GUS`. Thus, the inserted psaDb leader of 23 bases and/or the palindromic sequences at the translational initiation site (Fig. 1, A and C) greatly enhances the translation of GUS` messages.


Figure 2: Insertion of the psaDb sequence elevates GUS activity in transgenic tobacco. Mature leaves of individual transgenic lines were assayed for GUS activity. Note the logarithmic scale.




Figure 3: mRNA level of the chimeric GUS genes in transgenic tobacco. Total RNAs prepared from an untransformed plant (SR1) and from the transformants indicated were subjected to primer extension analysis. Numbers indicate transgenic lines as in Fig. 2. Arrowheads indicate the bands of chimeric GUS mRNAs, which are absent from the untransformed plant (SR1).




Figure 4: The ratio of GUS activity to GUS mRNA. The average value of the CaMV::GUS transformants is presented as 1.0. A, the GUS activity/GUS mRNA was determined for each transformant. Numbers indicate transgenic lines as in Figs. 2 and 3. B, average values and standard deviations for each of the chimeric constructs are presented.



5`-Leader Motifs Are Shared between psaDb of N. sylvestris and fedA of Arabidopsis

Recently, Casper and Quail (10) reported that the 5`-untranslated region (UTR) of the Arabidopsis ferredoxin gene, fedA, enhances gene expression. They fused the 5`-UTR of fedA to a luciferase reporter gene under the control of the fedA promoter, and the expression of this fusion gene was examined by a transient expression assay using Arabidopsis seedlings. When the 5`-UTR was deleted, luciferase activity was reduced 25-fold. The apparent similarity of the 5`-UTRs of psaDb and fedA in enhancing the reporter activity of chimeric genes led us to speculate that the translation of both genes may be enhanced by similar mechanisms. Therefore, the 5`-leader sequences of psaDb and fedA were compared (Fig. 5). These leaders share two common motifs, and these motifs cover more than half of the psaDb leader. We designate these 5`-leader motifs LM1 (TCTCAA) and LM2 (CAACTTT).


Figure 5: Comparison of the entire 5`-leader sequences of the psaDb gene of N. sylvestris and the fedA gene of Arabidopsis. Two conserved motifs designated as LM-1 and LM-2 are underlined.



Substitution of LM1 and LM2 within CaMV::psaDb-GUS` Greatly Decreases Translational Efficiency

In order to clarify whether LM1 and LM2 really operate as translational enhancers, these two motifs within the CaMV::psaDb-GUS` construct were altered to form CaMV::psaDbLM-GUS` (Fig. 6A). This construct with leader mutations was introduced into tobacco, and its GUS activity/GUS mRNA was determined for each transformant. As shown in Fig. 6B, mutations in LM1 and LM2 reduced the translational efficiency of the psaDb-GUS` message 20-fold. This indicates that LM1 and/or LM2 are integral parts of the translational enhancer.


DISCUSSION

This study clearly showed that the 5`-terminal 35 bases of the psaDb mRNA contain a translational enhancer element (Fig. 4), and that base substitution within the 5`-leader abolished this enhancer activity (Fig. 6). These results lead us to conclude that the 5`-leader of psaDb contains a translational enhancer element. This element enhances translation as much as 20-fold (Fig. 6). Since translational enhancer elements have rarely been documented in eukaryotic cellular messages, characterization of this element deserves much attention. The primary question is how this element activates translation. In recent years, cap-independent translation initiation mediated by the 5`-leader has been reported in poliovirus (16) , encephalomyocarditis virus (17) , the human immunoglobulin heavy-chain binding protein (BiP) gene (18) , and Antennapedia of Drosophila(19) . In these cases, an internal ribosomal entry site within the leader is a determinant for the rate of translation initiation. However, in contrast to the short psaDb leader of 23 bases, these leaders are generally very long (several hundred nucleotides or more). Whether the translational enhancer of the psaDb leader exerts its function in a cap-dependent or independent manner should be examined. Computer analysis of only the 35-base psaDb sequence using an energy-minimizing algorithm indicates that it does not form a stable secondary structure (data not shown). It is possible, though unlikely, that the psaDb leader sequence in the chimeric CaMV::psaDb-GUS` construct interacts with the GUS coding sequence to form a secondary structure of functional significance. To study this, a chimeric construct using a reporter gene other than GUS is currently being examined.

This study revealed that the psaDb leader shares common motifs, LM1 and LM2 (Fig. 5), with the leader of the Arabidopsis ferredoxin gene, fedA. The fedA leader was reported to enhance the expression of a chimeric reporter gene 25-fold in a transient expression assay, but it is not known if this enhancement was exerted transcriptionally or post-transcriptionally (10) . Judging from the result that substitution of LM1 and LM2 in the psaDb leader abolished the translational enhancer activity (Fig. 6), LM1 and/or LM2 are integral parts of the translational enhancer. Therefore, it is likely that LM1 and/or LM2 in the leaders of both psaDb and fedA enhance translation. Since the psaDb product (a PSI-D subunit) is the binding site of ferredoxin on PSI's surface (20) , it is reasonable that psaD and fedA are co-regulated, in this case at translation. Further dissection of the LM1 and LM2 motifs in respect to their enhancer activity and to their possible interactions with cellular proteins may provide insight into translational regulation in plant cells.

The GUS activity/GUS mRNA value was 5-fold higher in the CaMV::GUS` construct than in the original CaMV::GUS construct (Fig. 4). This difference is probably, at least in part, due to the two ATG codons in the 12 amino acids introduced at the N terminus of the protein, since the number of N-terminal methionines is known to affect translational efficiency (21, 22) . In plant genes, AACAATGGC is highly conserved at the translation initiation site, and a G residue at the +4-position (relative to the A of the ATG) is especially conserved (23, 24) . In this respect, CaMV::GUS` has a better context (ATGG) than the original CaMV::GUS (ATGT), and this may be another contributing factor for the noted difference in translational efficiency. In addition, the 12 amino acids introduced at the GUS` N terminus may slightly alter the protein stability and/or enzyme activity. However, though worthy of study, this difference in translational efficiency between CaMV::GUS` and CaMV::GUS is not the main concern of this paper; the enhancer activity of the psaDb leader is.

Last, we mention the applicability of the psaDb enhancer to plant gene technology. In transgenic tobacco, apparent GUS activity of the CaMV::psaDb-GUS` construct was about 2 orders of magnitude higher than that of the CaMV::GUS construct (Fig. 2). For what we have examined, the expression of the CaMV::psaDb-GUS` construct showed no apparent organ specificity.() Thus, the CaMV::psaDb-GUS` construct could be used instead of CaMV::GUS for a 100-fold increase of expression. Furthermore, the psaDb leader itself could be used to increase expression levels for a wide variety of plant vectors.


FOOTNOTES

*
This study was carried out at the Research Center for Molecular Genetics of Hokkaido University and supported in part by grants-in-aid from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
A recipient of the Research Fellowship for Young Scientists from the Japanese Society for Promotion of Science.

To whom correspondence should be addressed: Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060, Japan. Tel.: 81-11-706-5291; Fax: 81-11-757-5994; E-mail: jo@bio.hokudai.ac.jp

The abbreviations used are: GUS, -glucuronidase; PSI, photosystem I; CaMV, cauliflower mosaic virus; UTR, untranslated region.

Y. Kondo, Y. Y. Yamamoto, and J. Obokata, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. A. Kato of the Hokkaido Agricultural Experimental Station for advice in tobacco transformation studies, Dr. J. Maune for critical reading of this manuscript and useful discussions, and M. Ohtaki for help with some experiments.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.