©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Regulation of the Interferon--inducible Tryptophanyl-tRNA Synthetase Includes Alternative Splicing (*)

(Received for publication, August 30, 1994; and in revised form, October 17, 1994)

Anne B. Tolstrup Anette Bejder Jan Fleckner (§) Just Justesen (¶)

From the From Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have investigated the transcriptional control elements of the human interferon (IFN)--induced tryptophanyl-tRNA synthetase (hWRS) gene and characterized the transcripts. Transcription leads to a series of mRNAs with different combinations of the first exons. The full-length mRNA codes for a 55-kDa protein (hWRS), but a mRNA lacking exon II is present in almost as high amounts as the full-length transcript. This alternatively spliced mRNA is probably translated into a 48-kDa protein starting from Met in exon III. The predicted 48-kDa protein corresponds exactly to an IFN--inducible protein previously detected by two-dimensional gel electrophoresis. By isolation of genomic clones and construction of plasmids containing hWRS promoter fragments fused to the secreted alkaline phosphatase reporter gene we have mapped a promoter region essential for IFN-mediated gene activation. This region contains IFN-stimulated response elements (ISRE) as well as a Y-box and a -activated sequence (GAS) element. IFN- inducibility of hWRS depends on ongoing protein synthesis, suggesting that so far undescribed transcription factors apart from the latent GAS-binding protein p91 contribute to gene activation. This could be interferon-regulatory factor-1, which binds ISRE elements.


INTRODUCTION

Interferons (IFNs) (^1)are small, soluble proteins exerting pleiotropic effects on cells. Apart from the antiviral effect that led to the discovery of IFNs, these are now recognized as factors eliciting multiple effects in target cells, such as antitumor activity, antigrowth activity, immunoregulatory activity, as well as effects on the differentiation of various cells(1, 2, 3) . These diverse effects are believed to be mediated by a specific set of more than 30 proteins, regulated in response to IFN treatment. A few of these proteins, for example the 2-5A synthetases, p68 kinase, and indoleamine 2,3-dioxygenase (IDO), have been characterized with respect to enzymatic activity and implicated in the antiviral, antitumor, and antigrowth activities of IFNs(3, 4, 5, 6, 7, 8, 9) . However, most of the inducible proteins lack functional explanations with regard to their role in the IFN system.

IFN-inducible proteins are mainly regulated at the transcriptional level. Recently, remarkable progress has been achieved in the understanding of the IFN-alpha-induced activation of immediate early genes, i.e. genes transcribed within minutes after IFN-alpha stimulation. Following IFN-alpha binding to the receptor, a latent cytoplasmic transcription factor, the interferon stimulatory gene factor 3 (ISGF3), is activated by two protein tyrosine kinases (PTKs), Tyk2 and Jak1. ISGF3 then translocates to the nucleus and binds to the interferon-stimulated response element (ISRE), a consensus element found in the majority of IFN-alpha-inducible gene promoters. This leads to transcriptional activation(10, 11) .

The signal pathway(s) involved in IFN--inducible gene activation is less well described and appears to be more complicated. Ligand binding to the IFN- receptor leads to phosphorylation of two PTKs, Jak1 and Jak2(12, 13) , and one of the ISGF3 components, p91, is then activated by tyrosine phosphorylation(14, 15, 16) . However, Jak1 and Jak2 as well as p91 are involved in signal transduction of other cytokines as well, for example epidermal growth factor, colony-stimulating factor-1, platelet-derived growth factor, and interleukin-10(17, 18, 19, 20, 21, 22, 23) , suggesting that other factors are necessary to provide specificity.

Several of the genes activated by IFN-, for example the IDO and the MHC class II genes, are induced in a protein synthesis-dependent way, meaning that the factor(s) involved in signaling are not pre-existing in the cell(24) . It is likely that the transcription factor complexes activating these genes contain both general factors, such as p91 or related proteins, and as yet uncharacterized more specific proteins.

We and others have recently cloned and sequenced the IFN--inducible human tryptophanyl-tRNA synthetase (hWRS) (25, 26, 27, 28) . Although hWRS is strongly inducible by IFN- in many different cell types and to a lesser extent by IFN-alpha(29) , the role of this household protein in the IFN system is still not understood. Another IFN--inducible protein involved in tryptophan metabolism is the IDO, which catalyzes the first, rate-limiting step in tryptophan degradation. IDO is important for IFN--induced cytotoxity against the intracellular parasites Toxoplasma gondii and Chlamydia psittaci in macrophages (8) and it has also been implicated in cellular antigrowth effects on tumor cells(9) . As tryptophan is a common substrate for IDO and hWRS, a link between the IFN--induced functional effects of these proteins appears likely, also because the two genes are regulated in a very similar way by IFN-. The induction of both enzymes by IFN- is dependent on protein synthesis in most cell lines. However, a coupling of the two enzymatic activities with regard to IFN remains elusive(29) .

The hWRS sequence was originally found to be almost identical to the mammalian peptide chain release factor (RF)(30) . However, clear-cut evidence has recently been provided showing that the putative RF cDNA was wrongly identified and actually encodes the rabbit WRS, and the WRS protein has no RF activity(31, 32) . Here we present analyses of the hWRS mRNA transcript and promoter region. We have examined the hWRS transcripts by reverse transcription-polymerase chain reaction (RT-PCR) and primer extension. This revealed the existence of two mRNAs alternatively spliced inside the coding region of the gene as well as several mRNAs differentially spliced in the 5` non-coding region. Furthermore, the major transcriptional initiation point has been mapped. Isolation of genomic clones covering the hWRS gene has allowed investigation of cis-elements conferring IFN- and -alpha inducibility to the gene. Homology search of the promoter region has revealed the presence of several elements formerly described to be involved in IFN-mediated gene regulation. By use of the secreted alkaline phosphatase (SAP) reporter gene, we have analyzed the promoter region to detect elements necessary for IFN inducibility. These analyses indicate that additional sequences other than a previously identified -activated sequence (GAS) element (33) are important for IFN inducibility of hWRS.


MATERIALS AND METHODS

Cells

Daudi (B-lymphoblastic) and HL60 (promyelocytic) cells were grown as suspension cultures in exponential phase in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 50 µg/ml streptomycin, at 37 °C, 6% CO(2). HT1080 (fibroblast), T98G (glioblastoma), AMA (amnion), HeLa (epithelial), and K14 (keratinocyte) cells were cultured in monolayers in Dulbecco's modified Eagles medium (Life Technologies, Inc.) supplied with 10% newborn calf serum and antibodies as described above, at 37 °C, 6% CO(2).

Cytokines used were IFN-alpha2b (IntronA) and IFN- (Schering-Plough, Bloomfield, NJ). Cells were treated by addition to the medium of 500 units/ml IFN-alpha and 100 units/ml IFN-, respectively.

RNA Preparation and 1-Strand cDNA Synthesis

RNA samples were prepared by the acid guanidinium thiocyanate/phenol/chloroform extraction method(34) . Oligo(dT) primed 1-strand cDNA synthesis was performed with 400 ng of total RNA in a reaction containing 50 units of AMV reverse transcriptase, 50 mM Tris-HCl, 140 mM KCl, 10 mM MgCl(2), 4 mM dithiothreitol, 200 µg oligo(dT) primer, 40 units of RNasin, and 0.5 mM dNTP for 2 h at 42 °C.

Polymerase Chain Reactions

PCR reactions were run according to Saiki et al.(35) using Taq polymerase. Cycling conditions were as follows: 35 cycles of denaturing (94 °C, 1.2 min), annealing (60 °C, 1.2 min), and extension (73 °C, 1.2 min) on a PCR apparatus from Microlab, Aarhus, DK.

Primers

Primers were synthesized on an Applied Biosystems 381B DNA synthesizer and purified on Oligo Purification Cartridges (DNA Technology ApS, Aarhus, DK). Oligonucleotides used were as follows (from 5` to 3`): AGCTCAACTGCCCAGCGTGACC (forward, exon Ia), GACCAGGTTGTGGAAGACACTGCAGAGGTGGC (reverse, exon Ia), GGAGTAGGCAGTTTTGCTCTTT (forward, exon Ib), GAGGCTGAGATGCCA AAAGGTT (reverse, exon Ib), CAGAGGATTTGTTCAGCAGACGCGT (reverse exon II), CAGTCAGCCTTGTAATCCTCCCCC (reverse, exon III), CAAGAAGGCACTCATAGAGGTTCTGCAGC (forward, exon XI) and TTGATAACATACACAGGCTTACAGAGGCC (reverse, exon XI), for PCR analysis of the hWRS mRNA transcript. CGATAAGCTTACAGTTTCCCTTTCCACCGC (forward, pTK-SAP-0.4), GCTCAAGCTTTTCTGAGAATCTGGGTGATT (forward, pTK-SAP-0.15), and GCTCTAGAACTAGTGGATCCAAGCCC (reverse, pTK-SAP-0.4 and pTK-SAP-0.15) were used for PCR cloning of the hWRS promoter. Underlined sequences correspond to restriction sites.

Primer Extension

This was performed according to standard procedures (36) as follows. End-labeling of primer: 20 pmol of primer was end-labeled at 37 °C for 30 min by incubation with 100 µCi of [-P]ATP using 5 units of T4 polynucleotide kinase (Amersham Corp.) and gel-purified on a 12% denaturing polyacrylamide gel. For annealing, 10 µg of RNA and 0.5 pmol of primer were mixed in a total volume of 6 µl of 10 mM Tris-HCl, pH 6.9, 40 mM KCl, 0.5 mM EDTA, heated to 95 °C for 30 s, and incubated at 50 °C for 20 min. For extension, 4 µl of extension mixture consisting of 1 µl of 10 times RT buffer (1.25 M Tris-HCl, pH 8.4, 250 mM MgCl(2), 50 mM dithiothreitol. 0.8 µl 2.5 mM dNTPs, 2.2 µl of H(2)O, and 1 unit of AMV reverse transcriptase (Boehringer Mannheim) was added to the annealing reaction and incubated at 42 °C for 30 min. The reaction was stopped by addition of 40 µl of 0.3 M sodium acetate, pH 6.0, and the extension product was EtOH-precipitated before analysis on a 6% sequencing gel.

Isolation of Genomic Clones

Four genomic clones each spanning approximately 15 kb of DNA were isolated from an MRC-5-V2 (SV40 transformed MRC-5 fibroblasts) genomic library using a 300-bp 5` hWRS cDNA fragment. This library, which was constructed by inserting Sau3A1-digested MRC-5-V2 genomic DNA into BamHI-digested Dash vector, was a gift from Dr. H. Leffers, Department of Medical Biochemistry, University of Aarhus. From one of these genomic clones a 4-kb NotI fragment was selected by hybridization to a reverse exon Ia primer (see Primers) and subcloned into pBS-KS+ (=clone B1).

DNA Sequencing

The Cyclist DNA sequencing kit (Stratagene, La Jolla, CA) was employed for sequencing of gel-purified PCR fragments according to instructions from the manufacturer.

Sequencing reactions of plasmids were performed using the Taq DyeDeoxy Terminator Cycle sequencing kit (Applied Biosystems) according to instructions from the manufacturer. 1 µg of plasmid DNA and 3.2 pmol of sequencing primer were used in each reaction. Cycling conditions were as follows: 25 cycles of denaturing (94 °C, 30 s), annealing (50 °C, 15 s), and extension (60 °C, 4 min). Sequencing was carried out on an automatic Applied Biosystems DNA sequencing machine. Sequencing primers were synthesized at DNA Technology ApS, Aarhus, DK.

Construction of Promoter Deletions in pTK-SAP

Two plasmids containing genomic clones with promoter sequences of hWRS, clone 493, which consists of a 3.5-kb sequence upstream of the transcriptional initiation point and 262 bases downstream, cloned into the BamHI site of pBS-SK+, and clone B1, which contains 1 kb upstream of the initiation point and 3 kb downstream, cloned into the NotI site of pBS-KS+, were used to subclone fragments of the promoter into the reporter plasmid pTK-SAP, kindly provided by Nigel Sharp, Welcome, London, UK. Constructs were made either by exploiting existing restriction sites or by PCR cloning. Briefly, pTK-SAP-3.4, which contains the entire genomic clone 493, was made by HindIII digest of clone 493 followed by partial digest with XbaI and ligation into HindIII/XbaI-digested pTK-SAP. pTK-SAP-2.5 was made by digesting clone 493 with XbaI and subcloning into XbaI-digested pTK-SAP. pTK-SAP-0.8 was made by SmaI digestion of clone 493 and religation of the fragment containing a 0.8-kb promoter and vector. The resulting plasmid, pBS-0.8 was HindIII/XbaI-digested and the fragment ligated into HindIII/XbaI digested pTK-SAP. All fragments were gel-purified before ligation. Plasmid preparations, restriction digests, gel purification, and ligations were made according to methods described in Sambrook et al.(36) . pTK-SAP-0.4 and pTK-SAP-0.15 were made by PCR clonings using clone B1 as template. The forward primers contained a HindIII site and the reverse primer an XbaI site used for cloning of the PCR products into HindIII/XbaI digested pTK-SAP. The resulting plasmids were sequenced to verify that the PCR products contained the correct sequence. pTK-SAP 4xISRE was constructed and kindly provided by Dirk Gewert, Welcome, London, UK.

DEAE-Dextran Transfection

Transfections were made as described in Seed and Aruffo(37) . Briefly, 50-75% confluent cells, plated in 10-cm Petri dishes the day before transfection, were transfected in complete medium containing 5 µg of DNA plus 400 µg/ml DEAE-dextran and 100 mM chloroquine (HeLa) or 200 µg/ml DEAE-dextran without chloroquine (HT1080). In selected experiments the DNA concentration was increased to 10 µg, resulting in increased, but relatively similar, responses, showing that the concentrations used were within the linear response range. Cells were incubated at 37 °C, 6% CO(2) and closely monitored during this time. When approximately 20% of the cells were dying (after 3-4 h for HeLa and 1 h for HT1080), cells were treated with 10% dimethyl sulfoxide in phosphate-buffered saline for 2 min, returned to complete medium, and left to incubate overnight. Cells were trypsinized, equally divided into three, and incubated for 24-48 h with IFN-alpha (500 units/ml, IFN- (100 units/ml), or left untreated.

Electroporation

Daudi cells, split the day before transfection, were electroporated according to an unpublished protocol from Nigel Sharp, Welcome, UK. First, cells were spun down and resuspended at 5 times 10^6/ml in complete medium. 0.8 ml of cell suspension was then supplied with 5 µg of DNA and 8 µg of DEAE-dextran. The addition of DEAE-dextran greatly reduces the required amount of DNA(38) . In selected experiments the DNA concentration was increased to 10 µg, resulting in increased, but relatively similar, responses as for the DEAE-dextran transfections. Electroporation was performed using a Bio-Rad Gene Pulser (Bio-Rad), at settings 270 V, 500 µF. 7.2 ml of medium were added to the cells, which were equally divided into two, replated, and treated with IFN-alpha (500 units/ml) or left untreated, respectively.

Secreted Alkaline Phosphatase Assay

A modified version of the assay described by Berger et al.(39) was used. Aliquots of 200 µl of medium were harvested from transfected cells 24 or 48 h after IFN stimulation. Medium was heated to 65 °C for 15 min to inactivate any endogenous phosphatases, cell debris was spun down, and 50 µl of the supernatant were added to 96-well plate wells. 150 µl of substrate (5 µMp-nitrophenyl phosphate in 50 mM Tris-HCl, pH 8.2) were supplied to each well, and the plate was left to incubate at 37 °C. Samples were assayed in duplicate, and phosphatase-mediated conversion of the substrate to p-nitrophenol was measured in an enzyme-linked immunosorbent assay reader at 405 nm.


RESULTS

Alternative Splicing within the Coding Region of hWRS

We have employed the RT-PCR technique to investigate the mRNA transcripts from the hWRS gene in untreated as well as IFN-alpha- or IFN--treated cells of different origin (HeLa, HL-60, and K14). Diagrams of the hWRS exon-intron structure (40) with indications of the positions of the different primers are shown in Fig. 1A. RT-PCR analysis of hWRS mRNA transcripts from all three cell types using pairs of primers situated in exons Ia and III, respectively, resulted in two major PCR products, one of about 300 bp as expected, as well as another, shorter product of about 150 bp. The two bands were detected both in IFN-alpha- or IFN--treated and in untreated cells (Fig. 1B). Sequencing of the two PCR products revealed the existence of two alternatively spliced mRNAs. One of these is encoded by exon Ia-II through XI and is identical to our published cDNA sequence(25) , whereas the other transcript lacks exon II (Fig. 1C). A similar PCR with primers located in exon Ia and XI resulted in two bands corresponding in size to the formerly detected bands, thus confirming the alternative splice pattern (Table 1). As the originally described translational start codon of hWRS is situated in the 5` part of exon II, the two alternatively spliced mRNAs will code for two different polypeptides. An additional weak band longer than the two others was also detected in the PCR reaction (Fig. 1B and Table 1). This band might correspond to a mRNA which includes exon Ib (see below).


Figure 1: Alternative splicing in the coding region of hWRS. A, diagram of the exon-intron structure of hWRS. Primers used in RT-PCR are shown. The enlargement shows the detailed organization of the first exons. B, gel analysis of RT-PCR with primers placed in exon Ia and III, respectively. Reverse transcribed RNA from K14 cells, either untreated or treated with IFN-alpha (500 units/ml) or IFN- (100 units/ml) was used as template in PCR-reactions. U, untreated cells; alpha, IFN-alpha-treated cells; , IFN--treated cells. Sizes of the two bands are indicated. C, schematic illustration of the two major splice events in hWRS mRNA processing.





Alternative Splicing Outside the Coding Region of hWRS

A cDNA clone encoding a 5` terminus differing from all other hWRS cDNA clones has been isolated(26) . This differentially spliced clone contains a 5` sequence encoded by a putative exon Ib (Fig. 1)(40) . To verify the existence of this splice variant, RT-PCR was performed with a pair of primers situated within exon Ib. Gel analysis of the resulting PCR product demonstrated a band of the expected size in reactions with template from HeLa or HL-60 cells either treated with IFN-alpha, IFN-, or left untreated (Table 1). A second PCR with the reverse primer placed in exon II was performed, again resulting in a band of the expected size. This confirms that the PCR product did not result from genomic DNA contamination of the template, as exons Ib and II are separated by a 4.5-kb intron. An additional larger band was also obtained, which presumably contains nucleotides from the intron between Ib and II (the putative exon Ic in Fig. 1).

As the 5` limit of exon Ib has not been determined, we performed RT-PCR with primers located in exon Ia and Ib. This resulted in at least three bands (Table 1). Sequencing of one of these bands revealed splicing from exon Ia to the nucleotide located 130 bp upstream of the 3` limit of exon Ib (or 121 nucleotides downstream of the currently described 5` end of exon Ib). The 5` and 3` splice sites of the resulting intron contained the correct dinucleotides, GT and AG, respectively, necessary for RNA splicing(41) . Thus, a series of mRNAs varying within the 5`-untranslated region of hWRS exists.

Primer Extension Reveals One Major Transcriptional Start Site

To determine the transcriptional start point of the hWRS mRNAs, primer extension was performed with RNA from several cell lines. As shown in Fig. 2, one major start site was found. The start site was determined with both of two reverse primers, located in exon Ia and II, respectively, confirming that the band was not an artifact due to premature termination of the reverse transcriptase. Data with the reverse primer from exon II are not shown. Interestingly, primer extension with the exon II reverse primer revealed two additional bands in IFN--treated HT1080 cells, corresponding in size to the deduced mRNA transcripts of the two large PCR products obtained with the exon Ia-II primer set (Table 1). These bands were absent or weak in other cell lines tested (T98G, Daudi). The most likely explanation of these results is that one major transcriptional initiation site is used in all hWRS mRNA transcripts and that the level and/or presence of transcripts containing exon Ib varies between cell lines.


Figure 2: Primer extension analysis of the 5` termini of hWRS mRNA from IFN--treated cells. An oligonucleotide complementary to exon Ia, position 35-67, was used. 1, IFN--treated T98G cells; 2, IFN--treated HT1080 cells; 3, IFN--treated AMA cells. A sequence of the hWRS genomic clone (493), primed with the same oligonucleotide, is used as a size marker. The arrow indicates the exact position of the transcriptional initiation site.



Isolation of Genomic Clones Reveals Several IFN Response Elements in the hWRS Promoter

To analyze the IFN-mediated transcriptional activation of hWRS, we have isolated genomic clones covering the 5` end and promoter region of the gene. One 4-kb clone, B1, covers 1 kb of the proximal promoter upstream of the major transcriptional start site and ends 3 kb downstream of this, in intron 1b. Another clone, 493, kindly provided by Lyudmila Frolova, covers 3.4 kb of the promoter region plus the entire exon Ia and a small part of intron Ia. The promoter regions of these two clones were sequenced on an automatic DNA sequencing machine. The sequence of the 3.4-kb promoter has been deposited in the GenBank data base (accession no. X82107). Computer search for transcription factor binding sites revealed the presence of a putative TATA-box close to the transcriptional initiation site as well as several motifs previously found to be implicated in IFN-induced transcriptional regulation (Fig. 3). Two ISRE elements in opposite orientation are located at position -383 to -366 (ISRE I) and -55 to -44 (ISRE II), respectively. The ISREs also contain perfect IRF-1/IRF-2 binding sequences. Furthermore, several motifs sharing the palindromic motif of the GAS element are found, namely at positions -143 to -132, -2629 to -2620 and -2774 to -2765, respectively. Finally, two reversely oriented sequences highly homologous to the Y-box known to be important for IFN- induction in MHC class II genes are located at positions -187 to -181 and -1057 to -1051, respectively.


Figure 3: Structure of the hWRS promoter region. A, restriction map of the hWRS promoter region. Positions of restriction enzymes and IFN-related cis-elements are indicated, and the elements along with the recognized consensus sequences are outlined. The palindromic motif in the GAS elements is underlined. B, sequence of the 400 bases immediately upstream of the transcriptional initiation site. IFN-related elements are indicated as well as a putative TATA box.



Transient Transfections with the Reporter Gene Secreted Alkaline Phosphatase

To identify the cis-elements important for IFN inducibility, we have made a series of promoter constructs cloned into the reporter plasmid pTK-SAP (Fig. 4). Transient transfections of three different cell lines known to respond differentially toward IFN-alpha and IFN- treatment (29) have been performed. Results both from duplicate series of transfections and independent transfections were very reproducible (Table 2).


Figure 4: Constructions made for analysis of the hWRS promoter by transient transfection assays. Different lengths of the hWRS promoter were cloned into the pTK-SAP vector carrying the secreted alkaline phosphatase reporter gene 3` of a thymidine kinase minimal promoter. A diagram of the hWRS promoter with indications of 5` ends of each construct and positions of ISRE, GAS, and Y-box elements is shown above the constructs. The pTK-SAP-4xISRE contains four ISRE elements from the promoter of the small form of 2-5A synthetase in front of the reporter gene.





In HT1080 cells, the most prominent IFN- inducibility has been found using the 0.8- and 0.4-kb constructs, which both contain two ISRE elements, a Y-box, and a GAS element (Fig. 5A). On the other hand, the longer constructs pTK-SAP-3.4 and pTK-SAP-2.5 respond much less prominently, suggesting that some negative element is present in the promoter region upstream of the SmaI site (-0.8 kb). The construct pTK-SAP-0.15 responds only weakly to IFN- treatment and pTK-SAP-0.9, a construct containing a 0.9-kb HindIII/XbaI distal promoter fragment, including two putative GAS elements, does not respond at all. None of the constructs shows any response to IFN-alpha. In contrast, the pTK-SAP-4xISRE, which contains four ISRE elements from the 46/42-kDa 2-5A synthetase gene promoter, responds to both IFN-alpha and IFN-, demonstrating that the ISRE-dependent IFN-alpha signal pathway in these cells is intact.


Figure 5: Transient transfections of cell lines with hWRS promoter constructs using the secreted alkaline phosphatase reporter gene. All transfection series were performed in duplicate and the reproducibility was very good. Phosphatase activity assay was performed in duplicate with variations generally less than 5%. Mock represents transfection without the addition of DNA. Raw data from one representative transfection series are shown except that background activity from the medium was subtracted from the calculated absorbance. A, HT1080 cells transfected with promoter constructs of various length. The day after transfection cells were split equally into three and treated with IFN-alpha (white bars), IFN- (gray bars), or left untreated (black bars). Medium was assayed for phosphatase activity 48 h after IFN treatment. A representative transfection is shown. B, HeLa cells transfected with hWRS promoter constructs, treated with IFNs and assayed as in A. C, Daudi cells transfected with hWRS promoter constructs were split into two and treated with IFN-alpha (white bars) the day after transfection, or left untreated (black bars). Medium was assayed for phosphatase activity 24 h after IFN-alpha treatment.



In HeLa cells, the results from transient transfections show that the pTK-SAP-0.15, which contains a GAS element and an ISRE element, is sufficient to promote IFN- induction of the reporter, but that additional elements 5` of this fragment augment the response, as the pTK-SAP-0.4 and pTK-SAP-0.8 constructions also in these cells confer the most prominent transcriptional activation after IFN- treatment (Fig. 5B). Again, the longer constructs comprising 2.5 and 3.4 kb of the promoter respond more weakly. IFN-alpha does not induce the reporter gene significantly in any construct except the pTK-4xISRE.

Transient transfection of the promoter constructs into Daudi cells, which lack IFN- response, shows that the constructs containing the ISRE I respond to IFN-alpha compared to the untreated controls (Fig. 5C). In contrast to this, the shortest construct, pTK-SAP-0.15, does not contain sufficient sequences to respond to IFN-alpha treatment. The 4xISRE construct confers high inducibility to the reporter gene in these cells, compared to the hWRS promoter fragments.


DISCUSSION

In this study, we have detected alternative splicing of the hWRS transcript, both within and outside the coding region of the protein. All splicing variants found conform to the rules for splicing consensus sequences(41) . The existence of an exon Ib, so far only suggested by the isolation of an alternatively spliced cDNA-clone of the hWRS gene(26) , has now been verified by use of the sensitive RT-PCR technique. Exon Ib is expressed at low levels in untreated as well as IFN-alpha- or IFN--induced cells. Different lengths of this exon are present in various alternatively spliced mRNAs, and it is even likely that yet another exon (Ic), located in intron Ib, exists (Table 1). The significance of this extensive alternative splicing remains to be established.

Most interestingly, alternative splicing within the coding region of hWRS has also been revealed. The alternative splicing takes place irrespective of IFN-treatment and gives rise to two different mRNA transcripts, one of these lacking exon II (Fig. 1). Two in-frame ATG codons are located in the 5` part of exon III. A putative protein product resulting from either of these translational start sites lacks 41 or 47 amino acids, respectively, compared to the full-length hWRS protein of 471 amino acids. During our cloning of the hWRS gene, difficulties in performing N-terminal peptide microsequencing at first suggested that the N-terminal of this protein was blocked. However, thorough analysis of the obtained signals after isolation of hWRS cDNA clones clearly demonstrated that translation starts at the first ATG-codon in exon II(25) . Following the discovery of alternatively spliced mRNAs, we have reanalyzed the N-terminal microsequence data in order to detect the translational start codon of the mRNA lacking exon II. Although signals are weak and of course mixed up with the exon II N-terminal sequence, we did find signals from 8 amino acids out of the first 13 fitting with a start at the second Met codon (Met) in exon III. In contrast, signals resembling the sequence following Met were absent, making us conclude that Met is most likely used as the start codon, thus resulting in a 424-amino acid protein (calculated mass: 48.2 kDa). Indeed, this molecular mass and the calculated pI of 6.16 corresponds very well to an IFN--inducible protein termed 3 (and later renamed IGUP I-3421) previously detected by two-dimensional gel electrophoresis(42, 43) .

The genomic organization of the hWRS gene suggests that the protein contains a core, encoded by exons V-XI, which is responsible and sufficient for the tryptophanyl-tRNA charging activity(40, 44) . As it has been difficult to relate this housekeeping activity to IFN, the existence of an alternative IFN-related property, located in the N-terminal 141 amino acids of the protein encoded by exons II-IV, has been proposed(44) . However, the data presented here, demonstrating alternative splicing in the N-terminal region irrespective of IFN-treatment, do not support the idea of an IFN-induced activity in this part of the protein. On the other hand, the available data do not allow for exclusion of this hypothesis per se. At present, the significance of the differentially spliced mRNA transcripts is not clear. Comparison with the murine WRS protein, which was recently released from the GenBank data base, accession no. X69656, reveals a homology of 89% in the coding region on the protein level. Also, the rabbit and the bovine WRS proteins are very homologous to hWRS in the entire coding region(25) . In contrast, homology to the shorter bacterial WRS is found only within the core(44) . Whether the other mammalian WRS proteins are differentially spliced remains to be established. Heterologous expression of the various hWRS mRNAs will be important to assess the enzymatic activity of the different forms. Further characterization of the purified WRS is also necessary to confirm the existence of several WRSs.

The transcriptional start site found in this study matches the site detected as the major initiation site by RNase protection(33) . In contrast, it does not correspond to the 5` site of the cDNA cloned by Frolova et al.(26) , which extends 44 nucleotides into the promoter. Although multiple minor start sites are likely to exist, our primer extension experiments as well as the RNase protection studies clearly demonstrate the presence of a major transcriptional start site of the hWRS mRNA. The primer extension studies of different cell lines with a reverse primer located in exon II suggest that transcripts comprising exon Ib sequences also initiate at the same major start site as the mRNAs lacking this exon, and that the presence and/or level of transcripts containing exon Ib varies between cell lines.

Sequencing of genomic clones encoding up to 3.4 kb of the promoter region of the hWRS gene has revealed the presence of several cis-elements formerly demonstrated to be involved in IFN-induced transcriptional activation (Fig. 4). These are two inversely oriented ISRE elements, present in all known IFN-alpha inducible genes, two inversely oriented Y-boxes, formerly found to be necessary for the IFN- induction of MHC class II genes, as well as several GAS elements, implicated in binding of p91 or related transcription factors.

Transient transfection experiments with different hWRS promoter fragments placed in front of the reporter gene SAP resulted in maximal IFN- inducibility in both cell lines tested using pTK-SAP-0.4. This construct contains the two ISRE consensus elements, a Y-box and a GAS element. Deletion of 250 nucleotides, resulting in pTK-SAP-0.15, comprising only one ISRE as well as a GAS element, nearly abolished the IFN- inducibility of the reporter gene in HT1080 cells. In contrast to this, IFN- induction with this construct in HeLa cells was clear, but turned out to be much lower than the inducibility observed with pTK-SAP-0.4 (Table 2, Fig. 5B). The IFN-alpha response was only seen in the Daudi cells and it was clearly much weaker than the IFN- response, correlating with the transient nature of the IFN-alpha response observed previously(29) . Only constructs containing the ISRE I responded to IFN-alpha, strongly suggesting this element to be necessary for transcriptional activation by IFN-alpha.

Two putative GAS elements were found in the distal part of the 493 genomic clone (Fig. 3). However, pTK-SAP-0.9, a reporter construct containing these elements, could not be induced by IFN- in transient transfection experiments, suggesting that these GAS consensus elements are non-functional. Alternatively, negative element(s) could be located in the same region. Previously, it has been reported that a fragment including the 0.9 kb promoter region of pTK-SAP-0.9 was able to compete for -activated factor binding to the IFN--inducible Ly6E/A gene promoter GAS(33) . The two pseudo-GAS elements described here might explain this competition, even though these sequences cannot confer IFN- inducibility upon a heterologous promoter.

The proximal GAS element in the hWRS promoter has previously been located by others, and the presence of p91 in an IFN--inducible GAS-binding complex has been demonstrated(33) . However, our results demonstrate that additional elements, located in the 250 nucleotides upstream of the GAS, are required to confer maximal transcriptional activation of a reporter gene by IFN-. Furthermore, transcription factors other than p91, which is present in a latent form in the cytoplasm, are definitely involved, since the protein synthesis inhibitor cycloheximide inhibits IFN- induction of hWRS(29) . This means that de novo synthesized protein(s) are required for gene activation. Cis-elements in promoters of other IFN--inducible proteins, for example FcRI, IRF-1, and interferon consensus sequence binding protein, bind several transcription factor complexes in response to IFN- treatment. Some of these complexes contain p91 as well as other as yet unidentified proteins(45, 46, 47, 48, 49, 50) . The promoter elements necessary for IFN- induction of these genes contain GAS motifs, but additional nucleotides are also required. A similar situation might exist for hWRS induction. The presence of a Y-box, which is one of several elements important for IFN- induction of MHC class II genes, upstream of the GAS element supports this hypothesis.

On the other hand, the effect of the ISRE has not been ruled out. Our transient transfections with the pTK-SAP-4xISRE, where the ISRE is derived from the 2-5A synthetase gene, clearly show that IFN- has an inducing effect through this element. The ISRE has formerly been shown to be implicated in IFN--mediated gene activation of MHC class I, 2-5A synthetase, 9-27, as well as the IDO gene(51, 52, 53, 54) . The IFN- inducible transcription factor IRF-1 (and not ISGF-3) has been reported to be involved in this activation, at least in some cases(55, 56) . Very recently, it was reported that induction of the guanylate-binding protein by IFN- was greatly reduced (40-fold) in IRF-1-deficient transgenic mice(57) . Earlier reports have demonstrated a role of p91 in the induction of this gene(14) . As both guanylate-binding protein and hWRS contain ISRE and GAS elements in the promoter region, it could be hypothetized that IRF-1 is involved in transcriptional activation of hWRS also. Further studies will show whether the ISRE-elements of the hWRS promoter are important for maximal IFN- induction as well.

Recent studies of signal transduction and transcriptional activation mediated by IFNs and other cytokines have demonstrated the use of general factors on many levels of signaling (e.g. receptors, tyrosine kinases, transcription factors, cis-elements)(11, 17) . Although cytokines are redundant, exerting overlapping effects on cells, the range of activities for each cytokine is specific and each cytokine regulates a specific set of proteins. This implicates the existence of as yet undetected factors providing specificity. Characterization of such factors, e.g. transcription factors and cis-elements, must be the next step in the investigation of the regulatory mechanisms in the IFN-system.


FOOTNOTES

*
This research was funded by the Danish National Research Council and by the Danish Cancer Society. 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.

§
Present address: University of Massachusetts Medical Center, Worchester, MA 01605.

To whom correspondence should be addressed: Dept. of Molecular Biology, University of Aarhus, C:F: Møllers Allé, Bldg. 130, 8000 Aarhus C, DK. Tel.: 45-8942-3188; Fax: 45-8619-6500.

(^1)
The abbreviations use are: IFN, interferon; IDO, idoleamine 2,3-dioxygenase; ISRE, interferon-stimulated response element; WRS, tryptophanyl-tRNA synthetase; hWRS, human tryptophanyl-tRNA synthetase; RF, release factor; RT, reverse transcription; PCR, polymerase chain reaction; SAP, secreted alkaline phosphatase; GAS, -activated sequence; ISRE, IFN-stimulated response elements; AMV, avian myeloblastosis virus; PTK, protein tyrosine kinase; MHC, major histocompatibility complex; bp, base pair(s); kb, kilobase pair(s); IRF-1, interferon-regulatory factor-1.


ACKNOWLEDGEMENTS

We are very grateful to Lyudmila Frolova, Engelhardt Institute of Molecular Biology, Moscow, for the genomic hWRS clone 493, to Nigel Sharp and Dirk Gewert, Welcome, London, for the pTK-SAP and pTK-SAP-4xISRE vectors, to Henrik Leffers, Department of Medical Biochemistry, Aarhus, for the MRC-5-V2 genomic library, and to Jørgen Kjems, Department of Molecular Biology, Aarhus, for help with primer extension. We thank Bente J. Christensen and Joan Hansen for expert technical assistance. We also wish to thank Niels Ole Kjeldgaard and Pia Møller Martensen for critical reading of the manuscript.


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