From the
Inducible nitric oxide synthase, the critical enzyme responsible
for the enhanced synthesis of nitric oxide in inflammatory states, is
widely expressed in mammalian cells. To evaluate potential regulatory
roles of the 5`-untranslated region (UTR) in the human inducible nitric
oxide synthase gene, the transcription initiation sites and structure
of the 5`-UTR of human inducible nitric oxide synthase were examined.
Freshly isolated human alveolar macrophages, bronchial epithelial
cells, and several types of cultured cells were evaluated following
stimulation with cytokines ( i.e. interferon-
Nitric oxide (NO)
The human iNOS gene, located on chromosome 17 at position
17cen-q11.2, is a
There is increasing evidence that the
5`- and 3`-untranslated regions (UTRs) of many mRNAs play an important
role in the regulation of gene expression by influencing mRNA stability
and translational efficiency
(10) . We hypothesized that, due to
the diversity and multiplicity of transcription element consensus
sequences in the promoter region of the iNOS gene, previously
unidentified transcription initiation sites could exist in the 5`-UTR.
To evaluate this hypothesis, the 5`-UTR of human iNOS mRNA was
characterized by using reverse transcription-polymerase chain reaction
(RT-PCR), Northern analysis, and 5`-rapid amplification of cDNA ends
(RACE). The study demonstrates that, due to multiple transcription
initiation sites and alternative splicing, there exist multiple forms
of exon 1 in iNOS mRNA transcripts from human epithelial cells and
alveolar macrophages, with production of these iNOS mRNAs enhanced by
cytokines.
Oligonucleotides were
synthesized using an automated DNA synthesizer (Applied Biosystems) and
purified on Sephadex G-25M (PD-10) (Pharmacia Biotech Inc.). All PCR
amplifications were performed using the DNA thermal cycler
(Perkin-Elmer). The 5`-ends of human iNOS mRNA transcripts (after
conversion to cDNA) were amplified for 25 cycles (94 °C for 20 s,
60 °C for 20 s, and 72 °C for 40 s), except that 28 cycles were
used for alveolar macrophages. Taq DNA polymerase (Stratagene)
was used with sense primer NO7 (5`-AAACAACTCTCTGGATGGCATGG-3`,
-59 to -37 in the 5`-flanking region) and antisense primer
NO6 (5`-CTGGGTCCTCTGGTCAAACTTTTG-3`, 653 to 630 in exon 5) (see
Fig. 1
); the transcription initiation site described by Chartrain
et al.
(6) is defined as +1. The PCR products
were evaluated by Southern analysis
(16) . 20% of the PCR
product of each sample was size-fractionated on 1.5% agarose gel in 1
RNA from cytokine-stimulated A549 cells (2 µg
of poly(A)
Sequence analysis of the 5`-end of the 712-bp fragment (derived from
mRNA transcripts that included exon 1, as shown by Southern analysis of
RT-PCR products) demonstrated that, as expected, mRNA transcripts
corresponding to this amplification product contained sequences of the
5`-flanking region adjacent to exon 1, exons 1 and 2, and the 5`-end of
exon 3. Consistent with the Southern analysis, sequence analysis of the
486-bp fragment (derived from mRNA transcripts that did not include
exon 1) demonstrated that iNOS mRNA represented by this fragment indeed
did have a deletion of the entire sequence of the previously reported
exon 1 (1 to 191) plus the adjacent 35 bp of 5`-flanking region
(-1 to -35). Altogether, these data clearly demonstrated
that some iNOS mRNA transcripts were initiated upstream of the TATA
box, and more than half of these transcripts lacked exon 1 plus 35 bp
upstream (-35 to 191).
We describe here the complexity of the 5`-UTR of human iNOS
mRNA. Multiple forms of exon 1 were observed in human alveolar
macrophages and respiratory and intestinal epithelial cells, resulting
from multiple transcription initiation sites and alternative splicing
(Fig. 4). Of the 201 clones sequenced in the 5`-RACE study, only
12 (
Similar to the human iNOS gene, the mouse iNOS gene contains a TATA
box 30 bp upstream of the reported transcription initiation site and
multiple consensus sequences for the binding of transcription factors
in the 5`-flanking region
(23) . Using RT-PCR, TATA-independent
iNOS mRNA transcripts were found in RAW 264.7 mouse macrophages
stimulated by interferon-
It had been observed that a TATA-containing promoter can
yield transcripts with multiple downstream initiation sites
(24, 25, 26, 27) . For example,
transcription initiation occurs at multiple sites between 10 and 111 bp
downstream from the TATA box in the human L-plastin gene
(24) . Although heterogeneity in the upstream initiation sites
of TATA-containing promoters is not widely reported, the presence of
multiple consensus sequences for transcription factors might account
for the observed diversity.
There is increasing evidence that the
5`- and 3`-UTRs of many mRNAs play an important role in the regulation
of gene expression
(10) . To assure efficient translation, most
eukaryotic mRNAs have a short 5`-UTR and no AUGs upstream of the
translation initiation site. However, in
It appears that NHBE cells and AMs,
as well as T84 and DLD-1 cells, without apparent cytokine stimulation,
contained significant amounts of iNOS mRNA (Fig. 1). In DLD-1
cells, the amount of iNOS mRNA was substantially increased by
cytokines. It would thus appear that a low level of iNOS mRNA is
present in some cells. In agreement, expression of human iNOS at the
protein level has been observed in the normal epithelium of large,
cartilaginous airways
(39) . It is possible that environmental
stimuli result in the induction of iNOS mRNA in NHBE cells and AMs,
secondary to an increase in cytokine levels above basal. In cells in
culture, such as DLD-1 and T84 colon carcinoma cells, autocrine
stimulation from endogenous production of cytokines or similar factors
might conceivably result in the increase in iNOS mRNA. In contrast to
these cells, no significant amount of iNOS mRNA was observed in
unstimulated A549 or primary culture of normal human bronchial
epithelial cells. These observations are consistent with the conclusion
that iNOS mRNA might be induced in some cells without stimuli from
other cells whereas other cells might be more dependent on outside
stimuli. Thus, the autocrine loop may be cell- or tissue-specific.
We thank Dr. Martha Vaughan, Dr. Vincent C.
Manganiello, and Dr. Bruce Trapnell for critical reviews of the
manuscript and Carol Kosh for expert secretarial assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
,
interleukin-1
, tumor necrosis factor-
, and interleukin-6).
The mRNA was analyzed by reverse transcription-polymerase chain
reaction, Northern analysis, and 5`-rapid amplification of cDNA ends.
Despite the presence of a TATA box in the promoter region, multiple
transcription initiation sites were observed, some extending several
hundred base pairs upstream from the main TATA-directed initiation
site. Alternative splicing in the 5`-UTR of human inducible nitric
oxide synthase mRNA resulted in further diversity. The TATA-independent
inducible nitric oxide synthase mRNA transcripts were up-regulated by
cytokines. The long and complex 5`-UTRs contain eight partially
overlapping open reading frames upstream of the putative inducible
nitric oxide synthase ATG, which may have an important role in
translational regulation of human inducible nitric oxide synthase mRNA.
(
)
appears to be critical
in the physiology or pathophysiology of every organ system
(1, 2, 3, 4) . The enzyme responsible
for NO synthesis, nitric oxide synthase (NOS), converts
L-arginine to L-citrulline and NO. The three human
NOS genes include an inducible type (iNOS), whose activity is
Ca
-independent, and two constitutive,
Ca
/calmodulin-dependent types termed neuronal and
endothelial constitutive NOS
(1, 2, 5) . The
genes for these three isoforms are located on different chromosomes.
37-kilobase pair, 26-exon gene with the
translational start codon in exon 2 and stop codon in exon 26
(6) . In 425 bp of its 5`-flanking region are found a TATA box
and consensus sequences for three interferon-
response elements,
an NF-
B site, an A activator-binding site, and a degenerate (but
palindromic) tumor necrosis factor response element. These findings are
consistent with the observation that the iNOS gene is inducible by
cytokines. Expression of the human iNOS gene has been documented in a
variety of cell types ( i.e. hepatocytes, respiratory
epithelial cells, and macrophages)
(2, 3) , and the
human iNOS cDNA has been cloned from hepatocytes, chondrocytes, and a
human colorectal adenocarcinoma cell line, DLD-1
(7, 8, 9) . Sequence analysis reveals only a few
nucleotide differences among these cDNAs, consistent with the existence
of a single human iNOS gene.
Cell Culture
A549 cells (American Type Culture
Collection (ATCC) CCL 185), a human alveolar type II epithelium-like
lung carcinoma cell line, were maintained in Ham's F-12K medium
supplemented with 10% heat-inactivated fetal bovine serum, 2
mM glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin (all from Biofluids). DLD-1 cells (ATCC, CCL 221), a human
colorectal adenocarcinoma cell line, were maintained in RPMI 1640
medium (BioWhittaker) with the same supplements used for the A549
cells. T84 cells (ATCC, CCL 248), a human colon carcinoma cell line,
were maintained in Dulbecco's modified Eagle's medium
(BioWhittaker) supplemented with 5% heat-inactivated fetal bovine
serum, 2 mM glutamine, 50 units/ml penicillin, and 50
µg/ml streptomycin. Normal human bronchial epithelial primary
culture (HBEC) cells (Clonetics), isolated from the large bronchi and
trachea of normal humans, were cultured in serum-free, modified LHC-9
medium (Clonetics). To induce iNOS mRNA, A549, DLD-1, and HBEC cells
were treated with a mixture of recombinant human cytokines containing
100 units/ml interferon-, 0.5 ng/ml interleukin (IL)-1
, 10
ng/ml tumor necrosis factor-
, and 200 units/ml IL-6
(9) .
All of these were from Boehringer Mannheim except IL-1
, which was
from Genzyme. All experiments were carried out with confluent cells.
Isolation of Normal Human Bronchial Epithelial Cells and
Alveolar Macrophages
Normal human bronchial epithelial (NHBE)
cells were obtained as described previously from the large bronchi and
trachea of six nonsmoking, normal volunteers (age range, 21-45
years) by fiberoptic bronchoscopy, using a standard cytology brush
(Microvasive)
(11, 12) . The cells were immediately
suspended in RPMI 1640 medium at 4 °C, pelleted by centrifugation,
and lysed in 4 M guanidinium thiocyanate, 25 mM
sodium acetate, pH 5.2, 0.5% Sarkosyl, and 0.1 M
-mercaptoethanol. Alveolar macrophages were obtained from one of
the six volunteers by bronchoalveolar lavage
(13) and purified
by adherence on plastic for 1 h at 37 °C in serum-free RPMI 1640
medium supplemented with 2 mM glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin
(14) . After washing
three times with ice-cold Hanks' balanced salt solution
(Mediatech), macrophages were lysed in 4 M guanidinium
thiocyanate, 25 mM sodium acetate, pH 5.2, 0.5% Sarkosyl, and
0.1 M
-mercaptoethanol.
Analysis by RT-PCR of the 5`-End of Human iNOS
mRNA
Total RNA was extracted from cells by the guanidinium
thiocyanate-CsCl gradient method
(15) . The 5`-ends of iNOS
mRNAs from all cell types were analyzed by RT-PCR. The cDNA was
synthesized using Moloney murine leukemia virus RNase H reverse transcriptase (Superscript II RNase H
RT; Life Technologies, Inc.), random hexanucleotide primer
(Pharmacia Biotech Inc.), and oligo(dT)
primer
(Pharmacia Biotech Inc.) in 20 µl of reverse transcriptase buffer
(50 mM Tris, pH 8.3, 75 mM KCl, 3 mM
MgCl
, 10 mM dithiothreitol, and 0.5 mM
each of dATP, dGTP, dCTP, and dTTP).
TBE buffer (89 mM Tris, pH 8.3, 89 mM boric
acid, 2 mM EDTA). DNA on duplicate gels was blotted onto
Nytran membranes (Schleicher & Schuell) and fixed to the membrane
by UV irradiation (UV Stratalinker; Stratagene). One membrane was
hybridized with an [
-
P]dATP-labeled (at the
3`-end) oligonucleotide probe (probe A,
5`-GATCCTCACATGCCGTGGGGAGGACAATGGGGTTGCATCCAGCTTGACCAG-3`, 525 to 475
in exon 4), washed, evaluated by autoradiography (see
Fig. 1A), and then scanned by a PhosphorImager
(Molecular Dynamics); relative amounts of exon 1
and
1
transcripts were calculated using the total units
of both transcripts as 100%. The other membrane was hybridized with a
191-bp, [
-
P]dATP-labeled iNOS cDNA probe
(probe Exon 1), prepared by random priming, encompassing the entire
sequence of exon 1 (see Fig. 1 B).
Figure 1:
Alternative transcription initiation
sites and deletion of previously reported exon 1 in iNOS mRNA in human
cells. At the top is shown a portion of the 5`-flanking region
and the structure of the mature 26-exon human iNOS mRNA. The TATA box
in the 5`-flanking region, the start codon ( ATG) in exon 2,
and stop codon ( TGA) in exon 26 are indicated. Shown also are
the positions of primers ( NO7 in the 5`-flanking region and
NO6 in exon 5) used in RT-PCR, the antisense oligonucleotide
probe A ( A) in exon 4, and the PCR-generated Exon 1 probe
( Exon 1) used for hybridization with amplified mRNA
transcripts. Total RNA was extracted from cells with or without prior
stimulation by cytokine mixture ( CM) and converted to cDNA,
which was amplified by PCR for 25 cycles (28 cycles for AMs) using NO7
and NO6 primers and then evaluated by Southern analysis. A,
hybridization with [-
P]dATP-labeled
oligonucleotide probe A. PCR products of 712 and 486 bp, respectively,
corresponding to iNOS mRNA with (exon 1
) and without
previously reported exon 1 plus the adjacent 35 bp at its 5`-flanking
region (exon 1
) were detected. B,
hybridization with [
-
P]dATP-labeled iNOS
cDNA Exon 1 probe encompassing only the sequence of exon 1. The 486-bp
bands were not detected except for some nonspecific hybridization in
lanes 2 and 4. C,
-actin.
-Actin-specific primers and probe were used for RT-PCR and
Southern hybridization, respectively. Sources of RNA are indicated
above the lanes. A549 alveolar type II
epithelium-like lung carcinoma cells ( lanes 1 and 2),
DLD-1 colorectal adenocarcinoma cells ( lanes 3 and
4), HBEC cells ( lanes 5 and 6), T84 colon
adenocarcinoma cells ( lane 7), freshly isolated NHBE cells
( lane 8), and AMs ( lane 9) were evaluated.
H
O was used in the RT-PCR as a negative control ( lane
10).
As a control,
-actin mRNA was evaluated by a similar technique, except that the
primers were specific for
-actin transcripts (HAG3,
5`-ATGAAGATCAAGATCATCGCACCC-3` and HAG4,
5`-CACCAAGCCACCGACTTGTCTTCC-3`); amplification was performed for only
20 cycles, and a nested PCR-generated cDNA probe was used (see
Fig. 1C)
(17, 18) .
Nucleotide Sequence of iNOS mRNA Transcripts with and
without Previously Reported Exon 1
After preliminary
observations of the RT-PCR products suggested the presence of two
different iNOS transcripts (712-bp ``exon
1'' transcripts and 486-bp ``exon
1
'' transcripts), the exon 1
and exon 1
transcripts of RT-PCR products from
DLD-1 cells were excised from the agarose gel, purified (QIAEX gel
extraction kit; QIAGEN Inc.), and sequenced by the dideoxy chain
termination method
(19) using a double-stranded DNA cycle
sequencing system (Life Technologies, Inc.) with NO7 and NO26
(5`-TGATACTGAAGGTCATCCTGTGTC-3`, 404 to 381 in exon 3) as sequencing
primers.
Northern Analysis of the 5`-End Structure of Human iNOS
mRNA
The 5`-end of human iNOS mRNA transcripts was further
evaluated by Northern analysis
(20) . Total RNA (20 µg/lane)
from A549 and DLD-1 cells, with and without stimulation by cytokines,
was subjected to formaldehyde-agarose gel electrophoresis, transferred
to Nytran membranes, and hybridized with various oligonucleotide probes
(probe A, same as the probe A used for Southern analysis of RT-PCR
products; probe B,
5`-CCAAAGGGAGTGTCCCCAGCTTGTGTACAGTTAGCTAATTTATGACTGTGA-3`, -100
to -150; probe C,
5`-TCACAGTCATA-AATTAGCTAACTGTACACAAGCTGGGGACACTCCCTTTGG-3`, -150
to -100; probe D,
5`-GGCCAAGCCACATGGCCTCACTTTCAGCATCTGGGAGATTTTTTCCTCAGC-3`, -306
to -356) labeled with [-
P]dATP at the
3`-end (see Fig. 2, A-D). Probes A, B, and D were
antisense; probe C corresponded to the sense strand of probe B. As a
control, glyceraldehyde-3-phosphate dehydrogenase mRNA was evaluated
with a 1.0-kilobase pair human glyceraldehyde-3-phosphate dehydrogenase
cDNA probe (see Fig. 2 E)
(21) .
Figure 2:
Effect of cytokines on human iNOS mRNA
with transcription initiation sites upstream of the TATA box. At the
top is shown a portion of the 5`-flanking region and the
structure of the mature 26-exon human iNOS mRNA, as well as positions
of oligonucleotide probes used for Northern analysis: antisense probe
A in exon 4, antisense probe B, sense probe
C, and antisense probe D upstream of TATA box. A549
and DLD-1 cells were incubated with or without a cytokine mixture
( CM) for 8 h before extraction of RNA. Northern blots were
hybridized with [-
P]dATP-labeled iNOS
oligonucleotide probes A, B, C, or D ( panels A-D,
respectively) or with [
-
P]dATP-labeled
1.0-kilobase pair human glyceraldehyde-3-phosphate dehydrogenase
( GAPDH) cDNA clone ( panel
E).
Analysis by 5`-RACE of Human iNOS mRNA
The results
of RT-PCR and Northern analysis indicated that only a small fraction of
iNOS mRNA transcripts was transcribed from upstream of the reported
transcription initiation site, but more than half of these lacked the
sequence -35 to 191 (see ``Results''). 5`-RACE (instead
of primer extension) using gene-specific primers and a 5`-AmpliFINDER
RACE kit (Clontech) were used to further characterize the 5`-end of
human iNOS mRNA.
RNA, which was purified from total RNA on
oligo(dT)-cellulose (5 Prime
3 Prime, Inc.)), and from freshly
isolated NHBE cells (20 µg of total RNA) was reverse transcribed
with a gene-specific antisense primer (NO32,
5`-CTGCCCCAGTTTTTGATCCTCAC-3`, 539 to 517 in exon 4) by avian
myeloblastosis virus reverse transcriptase. The first strand of cDNA
was ligated to an anchor (ANCH,
5`-P-CACGAATTCACTATCGATTCTGGAACCTTCAGAGG-NH
-3`) containing
a phosphate group at the 5`-end to facilitate ligation and an amine
group at the 3`-end to prevent self-ligation and amplified by two
rounds of PCR. For the first round PCR, the sense primer was ANCH-2
(5`-CTGGTTCGGCCCACCTCTGAAGGTTCCAGAATCGATAG-3`) and the antisense primer
was NO60 (5`-GGGGGCTTTCTCCACATTGTTG-3`, 361 to 339 in exon 2). For the
second round PCR, the sense primer was ANCH-2 and antisense primer was
NO30AP (5`-CTCGCTCGCCCACTCTATGGCTTTACAAAGCAGGTC-3`). PCR products were
subcloned into pDIRECT vector (Clontech) and subjected to sequence
analysis using a double-stranded DNA cycle sequencing system (Life
Technologies, Inc.).
Human iNOS mRNA Transcripts with Deletion of Previously
Reported Exon 1
Evaluation of iNOS mRNA transcripts in the
region encompassing the 5`-flanking region and exons 1-5 by
RT-PCR (Fig. 1, top panel) demonstrated two different
transcripts in cytokine-stimulated A549 cells, DLD-1 cells, and HBEC
cells (Fig. 1 A, lanes 2, 4, and
6), resting DLD-1 and T84 cells (Fig. 1 A,
lanes 3 and 7), and freshly isolated NHBE cells and
alveolar macrophages (Fig. 1 A, lanes 8 and
9). iNOS transcripts were not observed in unstimulated A549 or
HBEC cells (Fig. 1 A, lanes 1 and 5).
Results from NHBE samples from five other normal volunteers were
similar to lane 8 of Fig. 1(data not shown). The size
difference (226 bp) between these two amplified fragments (712 and 486
bp) was a little larger than the size of exon 1 (191 bp). Southern
analysis with an exon 4-specific probe (probe A) demonstrated that both
fragments contained the exon 4 sequence. In contrast, Southern analysis
with an exon 1-specific probe (probe Exon 1) demonstrated that the
486-bp fragment did not contain the exon 1 sequence
(Fig. 1 B), although some hybridization with the PCR
products from cytokine-stimulated A549 and DLD-1 cells was observed.
Thus, some iNOS mRNA transcripts included previously reported exon 1
sequences whereas others did not. The exon 1 transcripts were more abundant than exon 1
transcripts in all samples with visible bands on autoradiography.
Regulation by Cytokines of Human iNOS mRNA Initiated
Upstream of the TATA Box
Northern analysis with an exon
4-specific antisense probe (probe A) demonstrated that iNOS mRNA
transcripts of A549 and DLD-1 cells were increased by a cytokine
mixture of interferon-, IL-1
, tumor necrosis factor-
,
and IL-6 (Fig. 2 A). Using antisense probe B in the
5`-flanking region (-100 to -150), Northern analysis
demonstrated a similar pattern (Fig. 2 B) to that with
probe A, although the exposure time during autoradiography required to
obtain bands of similar density was much longer. The data suggested
that only a small proportion of iNOS mRNA was transcribed from upstream
of the TATA box. In contrast, with the more upstream antisense probe D
(-306 to -356) or the sense probe C (-150 to
-100, sense strand of probe B), no iNOS mRNA could be detected by
Northern analysis except for some nonspecific hybridization on 28 S
rRNA (Fig. 2, C and D). The data clearly
demonstrated that cytokines enhanced the production of iNOS mRNA
transcribed from upstream, as well as downstream, of the TATA box.
5`-RACE of Human iNOS mRNA
Shown in
Fig. 3
are the results of 5`-RACE with mRNA from
cytokine-stimulated A549 cells (Fig. 3 B) and freshly
isolated NHBE cells (Fig. 3 C). A total of 128
independent A549 clones and 73 NHBE clones were sequenced to determine
the 5`-end of the first strand cDNA. Consistent with a previous report
(6) , a predominant transcription initiation site (55 of 128
A549 clones and 26 of 73 NHBE clones) was located 30 bp downstream of
the first nucleotide of the TATA box. Consistent with the results of
RT-PCR and Northern analysis, eight A549 clones and five NHBE clones
had transcription initiation sites upstream of the major site. Of
these, four A549 clones and four NHBE clones lacked exon 1 and 35 bp
upstream (-35 to 191), three A549 clones and one NHBE clone did
not have this deletion, and one A549 clone lacked the sequence
-221 to 191. Many clones had apparent transcription initiation
sites downstream of the major initiation site. Some of these likely
resulted from incomplete reverse transcription by the avian
myeloblastosis virus reverse transcriptase ( i.e. some
transcription initiation sites might actually be located upstream of
those defined by 5`-RACE). The structural diversity in the 5`-UTR of
human iNOS mRNA, demonstrated by the 5`-RACE study, is summarized in
Fig. 4
.
Figure 3:
Analysis by 5`-RACE of human iNOS mRNA
from A549 and freshly isolated NHBE cells. A, the 5`-flanking
region, exon 1, and part of exon 2 of human iNOS mRNA are shown.
Positions of consensus sequences for transcription factors, the TATA
box ( TATA), the major transcription initiation site
( +1), and the start codon ( ATG) are indicated.
Shown also are sequences of 5`-splice sites of exon 1B (5`-GTGGGT-3`;
GTGGGT) and exon 1A (5`-GTGAGT-3`; GTGAGT; see Fig. 4
and ``Results'' for details). B, 5`-RACE of human
iNOS mRNA from cytokine-stimulated A549 cells. 2 µg of
poly(A) RNA was reverse transcribed with a
gene-specific antisense primer (NO32 in exon 4). First strand cDNA was
ligated to an anchor primer. First round PCR (sense primer ANCH-2;
antisense primer NO60 in exon 2) and second round PCR (sense primer
ANCH-2; antisense primer NO30AP) followed. PCR products were subcloned
into pDIRECT vector and subjected to DNA sequence analysis. Each clone
is represented by a separate symbol.
, clone transcribed from 1 or
downstream;
, clone transcribed from upstream of 1 that includes
previously reported exon 1;
, clone transcribed from upstream of
1 with deletion of sequence -35 to 191;
, clone transcribed
from upstream of 1 with deletion of sequence -221 to 191. Of 128
clones sequenced, 55 had transcription initiation sites at 1.
C, 5`-RACE of iNOS mRNA from NHBE cells. Experimental
procedures were the same as those in B except that 20 µg
of total RNA from freshly isolated NHBE were used for reverse
transcription. Of the 73 clones sequenced, 26 had transcription
initiation sites at 1.
Figure 4:
Structural diversity in the
5`-untranslated region of human iNOS mRNA. Shown are 5`-end structures
of human iNOS mRNA transcripts with exon numbers in boxes. The
start codon ( ATG) in exon 2 is indicated. Exon 1a contains the
sequence -35 to -1. Exon 1A contains a sequence with the
5`-end located between -221 and -37 and the 3`-end at
-36. Exon 1B contains a sequence with 5`-ends either at or
upstream of -223 and the 3`-end at -222. A, iNOS
mRNA transcribed from or downstream of the major transcription
initiation site ( in Fig. 3). Sequences of the 5` splice site and
part of the 3` splice site of intron 1 are shown. B, iNOS mRNA
transcribed from upstream of the major transcription initiation site
containing exon 1 (
in Fig. 3). C, iNOS mRNA transcribed
from upstream of the major transcription initiation site lacking
sequence -35 to 191 (
in Fig. 3). The 5` splice site
sequence of exon 1A (5`-GTGAGT-3`) is identical to that of exon 1.
D, iNOS mRNA transcribed from upstream of the major
transcription initiation site lacking sequence -221 to 191
(
in Fig. 3). The 5` splice site sequence of exon 1B
(5`-GTGGGT-3`) differs from that of exons 1 and 1A by one
nucleotide.
Five AUGs were noted in the 5`-UTR (-270 to
264), located at -256, -65, -45, -40, and 87,
which possibly serve as start codons for upstream open reading frames
(uORFs). Combined with deletion of the sequence -35 to 191
(Fig. 4 C) or -221 to 191 (Fig. 4 D),
eight small uORFs were found: 1) -256 to -212; 2)
(-256 to -222)-(192 to 210); 3) -65 to 1; 4)
(-65 to -36)-(192 to 230); 5) -45 to 9; 6) (-45
to -36)-(192 to 199); 7) (-40 to -36)-(192 to 210);
and 8) 87 to 149.
6%) had transcription initiation sites located upstream of the
TATA box. In contrast, no structural diversity was found in the 3`-UTR
by using RT-PCR (data not shown). The major transcription initiation
site defined by 5`-RACE in our study is the same as the only
transcription initiation site reported previously
(6) . No other
TATA-directed transcription initiation site was found in the upstream
395 bp. Although the majority of iNOS mRNA transcripts were generated
as expected in a TATA-containing gene, a small proportion of iNOS mRNA
transcripts were apparently TATA-independent. The TATA-independent mRNA
transcripts, including the alternatively spliced forms, were also
up-regulated by cytokines (Fig. 2), which is consistent with the
findings in many TATA-less genes
(22) and with the presence of
multiple transcription element consensus sequences involved in
cytokine-stimulated gene expression. The occurrence of alternative
splicing in the 5`-UTR may be explained in part by the presence of a
sequence in this region either identical (Fig. 4 C) or
similar (Fig. 4 D) to the reported 5` splice site
sequence for intron 1 (Fig. 4, A and B).
and bacterial lipopolysaccharide,
although no alternative splicing in the 5`-UTR was observed (data not
shown).
5-10% of vertebrate
mRNAs, the first AUG is not the translational initiation site of the
major open reading frame (ORF)
(28, 29) . Some 5`-UTRs
of these mRNAs dramatically inhibit translation at major ORFs
(30, 31, 32, 33) . For example, the
translational inhibition by uORFs of the GCN4 gene, which encodes a
transcriptional activator of amino acid biosynthetic genes, has been
well studied in Saccharomyces cerevisiae(34, 35, 36) . Similar to human iNOS mRNA,
the retinoic acid receptor
2 mRNA has a long (461 nucleotides)
5`-UTR, which contains five partially overlapping uORFs
(37) .
Recently, Zimmer et al.(38) demonstrated that uORFs
in the 5`-UTR of the retinoic acid receptor
2 mRNA inhibited its
translation in a tissue-specific manner. In human iNOS mRNA, the long
and complex 5`-UTR contains eight partially overlapping uORFs prior to
the expected iNOS AUG, which could have an important role in the
regulation of protein synthesis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.