(Received for publication, August 30, 1994; and in revised form, October 17, 1994)
From the
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.
Interferons (IFNs) ()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--induced activation of immediate early genes, i.e. genes transcribed within minutes after IFN-
stimulation.
Following IFN-
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-
-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-
(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 -
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.
Cytokines used were IFN-2b (IntronA) and IFN-
(Schering-Plough, Bloomfield, NJ). Cells were treated by addition to
the medium of 500 units/ml IFN-
and 100 units/ml IFN-
,
respectively.
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.
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- (500 units/ml) or IFN-
(100
units/ml) was used as template in PCR-reactions. U, untreated
cells;
, IFN-
-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.
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.
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.
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.
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-
.
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-
and IFN-
, demonstrating that the ISRE-dependent IFN-
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- (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-
(white bars) the day after transfection, or left untreated (black bars). Medium was assayed for phosphatase activity 24 h
after IFN-
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-
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-
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-
treatment. The 4xISRE construct confers high
inducibility to the reporter gene in these cells, compared to the hWRS promoter fragments.
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-- 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-
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-
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-
response observed previously(29) . Only constructs
containing the ISRE I responded to IFN-
, strongly suggesting this
element to be necessary for transcriptional activation by IFN-
.
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 Fc
RI, 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.