From the School of Biological Sciences, Seoul
National University, Seoul 151-742, Korea and the ¶ Hormone
Research Center, Chonnam National University,
Kwangju 500-757, Korea
Received for publication, September 25, 2002, and in revised form, March 13, 2003
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ABSTRACT |
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The mammalian
gonadotropin-releasing hormone (GnRH) gene consists of four short exons
(denoted as 1, 2, 3, and 4) and three intervening introns (A, B, and
C). Recently, we demonstrated that excision of the first intron (intron
A) from the GnRH transcript is regulated in a tissue- and developmental
stage-specific fashion and is severely attenuated in hypogonadal (hpg)
mouse because of its lack of exonic splicing enhancers (ESE) 3 and 4. In the present study, we examined the influence of intron A on
translational efficiency, thereby establishing a post-transcriptional
control over GnRH biosynthesis. First, we verified that an intron
A-retained GnRH transcript is a splicing variant but not a splicing
intermediate. Intron A-retained transcripts can be transported to the
cytoplasm in contrast to intron B-containing transcripts, which are
restricted to the nucleus. This result implicates the intron A-retained
GnRH transcript as a splicing variant; it has a long 5'-untranslated region, as the GnRH prohormone open reading frame (ORF) begins on exon
2. We investigated whether an intron A-retained GnRH transcript can
properly initiate translation at the appropriate start codon and found
that intron A completely blocks the translation initiation of its
downstream reporter ORF both in vivo and in
vitro. The inhibition of translation initiation appears to be due
to the presence of a tandem repeat of ATG sequences within intron A. Constructs bearing mutations of ATGs to AAGs restored translation initiation at the downstream start codon; the extent of this
restoration correlated with the number of mutated ATGs. Besides the
failure in the translation initiation of GnRH-coding region in the
intron A-containing variant, the present study also suggests that the interference between mature GnRH mRNA and intron A-retained
splicing variant could occur to lower the efficiency of GnRH
biosynthesis in the GT1-1-immortalized GnRH-producing cell line.
Therefore, our results indicate that the precise and efficient
excision of intron A and the joining of adjacent exons may be a
critical regulatory step for the post-transcriptional regulation of
GnRH biosynthesis.
Gonadotropin-releasing hormone
(GnRH)1 is a hypothalamic
neurohormone that plays a pivotal role in the neuroendocrine regulation of mammalian reproduction and sexual development. The majority of
GnRH-secreting neurons are located in the preoptic area (POA) of the
hypothalamus (1). The mammalian GnRH gene consists of four short exons
(denoted as 1, 2, 3, and 4) and three intervening introns (A, B and C).
The translation start site of GnRH gene resides on the exon 2. GnRH
exon 2 encodes a signal peptide, the GnRH decapeptide, and a part of
the GnRH-associated peptide. Exon 3 and 4 encode a remaining part of
the GnRH-associated peptide and the 3'-untranslated region (UTR) (2).
In GnRH-producing neurons, all three introns are efficiently excised
from the primary gene transcript, resulting in a mature GnRH mRNA
(3-5). Several extrahypothalamic tissues also express GnRH gene
transcripts with a relatively low abundance. It is of interest to note
that GnRH RNA species that retain intron A are expressed in human
reproductive tissues (6), and the primary transcript appears to be more prevalent than the mature mRNA in rat ovary (7). Our recent findings using an in vitro splicing system have shown that
the introns B and C are easily excised from the GnRH primary
transcript, but intron A is not (8, 9). The attenuation of intron A excision is most likely due to its suboptimal 3'-splice site. Exonic
splicing enhancers (ESEs) located in the exon 3 and 4 (denoted as ESE 3 and 4) and a subset of putative transacting factors specific for
GnRH-producing cells are thought to be required for the efficient removal of intron A in GnRH neurons (8-10).
In addition to cell type-specific regulation, the excision rate of
intron A from the GnRH gene transcript is regulated during sexual
maturation in the mouse POA (11). A functional significance of the
intron A excision in the regulation of GnRH synthesis is clearly
implicated in a nature's knockout hypogonadal (hpg) mouse, where exons 3 and 4 as well as ESE 3 and 4 are truncated. Even though
this mutant retains the intact sequence encoding the mature GnRH
decapeptide and expresses a detectable amount of GnRH transcript in the
hypothalamus (11, 12), no GnRH peptide can be produced resulting in
drastic reductions in serum gonadotropin levels and an undeveloped
gonad (12, 13). Recently, we found that, in the hpg mouse, the excision
rate of GnRH intron A is extremely low even in the POA (11), raising
the possibility that intron A can affect translation efficiency,
thereby establishing post-transcriptional control over GnRH prohormone synthesis.
Thus, it is worthwhile examining the functional relevance of a retained
intron A in the regulation of GnRH biosynthesis. First, we investigated
whether intron A-retained GnRH transcripts could be transported into
the cytoplasm where translation occurs; then, we examined whether an
intron A-retained form of GnRH mRNA transcript can properly
initiate translation at the start codon downstream to intron A.
Plasmid Construction--
Mouse, human, and rat GnRH gene
fragments containing exon 1 and/or intron A were amplified from genomic
DNA by PCR. Various deletion constructs and point-mutated mouse
GnRH intron A fragments were produced from the intron A fragment by
PCR. All upper primers contained a HindIII restriction site
at their 5'-end, and all lower primers had an NcoI site. All
PCR products were cloned into the pGEM-T Easy vector (Promega, Madison,
WI), and sequence identities were confirmed by chain termination
sequencing methods. Luciferase reporter plasmids were prepared by
inserting each fragment into the pGL3-control vector (Promega) using
the HindIII/NcoI sites that reside between an
SV40 minimal promoter and the luciferase coding sequence. To avoid the
excision of the intron A region in the cells, the last G base of intron
A, was deleted. Primer sequences used for cloning are presented in
Table I.
Northern Blot Hybridization and Reverse Transcription (RT)
PCR--
Total RNAs from various tissues and cells were isolated as
described previously (8). Nuclear and cytoplasmic RNAs were isolated
separately in accordance with a previous report (14) with
modifications. Briefly, cytoplasmic RNAs were first fractionated by
homogenization of tissues or cells in lysis buffer (0.3 M
sucrose, 0.25% sodium deoxycholate, 10 mM Tris-HCl, pH
7.4, 1.5 mM MgCl2, and 0.5% Nonidet P-40) and
briefly centrifuged at 3000 × g to exclude the nuclear
fraction; nuclear RNAs were isolated from the precipitated nucleus
after washing out the cytoplasmic contaminant twice with lysis buffer.
RNAs were retrieved from each fraction by a single-step acid
guanidinium thiocyanate-phenol-chloroform method. For Northern blot
hybridization, 30 µg of each RNA were resolved on a 1.2%
formaldehyde agarose gel and transferred for 18 h by diffusion
blotting to a Nytran filter (pore size, 0.45 µm; Schleicher & Schuell). Complementary RNA probes to GnRH cDNA or intron A
were generated using a commercial in vitro transcription system (Promega) in the presence of [ Cell Culture and Transient Transfections--
All cell lines
were maintained in Dulbecco's modified Eagle's medium
supplemented with 4 mM glutamine, 1 mM sodium
pyruvate, 100 units/ml penicillin/streptomycin, and 10% fetal bovine
serum under a humidifying atmosphere containing 5% CO2 at
37 °C. For transfections, cells were plated in 60-mm dishes and
grown to 40-60% confluence for 1-2 days. Cells were washed twice
with Dulbecco's phosphate-buffered saline, and the medium was changed
to serum- and antibiotics-free Dulbecco's modified Eagle's medium
prior to transfection. One microgram of plasmid DNA was transfected using LipofectAMINE PLUS reagent (Invitrogen), and excess DNA complexes
were washed out with Dulbecco's phosphate-buffered saline on the
following day. After 24 more hours of incubation in regular medium, the
cells were harvested and subjected to luciferase assays or RT-PCR.
Luciferase Assay--
Each luciferase-reporter construct and a
CMV promoter-driven In Vitro Transcription/Translation-coupled
Reactions--
Mouse GnRH-luciferase fusion genes were subcloned into
pGEM-3Z vector (Promega) using HindIII/BamHI
restriction sites. In vitro
transcription/translation-coupled reactions were performed using a
coupled reticulocyte lysate system (Promega) in the presence of SP6 RNA
polymerase (Promega), according to manufacturer's instructions. To
detect transcribed RNA, [ Expression of the Intron A-retained GnRH Transcript in Various
Tissues--
It is well known that several extrahypothalamic tissues
express GnRH transcripts, though with relatively low abundance (6, 7);
we have recently demonstrated that the excision of GnRH intron A is
severely attenuated in these tissues (8). This finding suggests that
the intron A-containing GnRH transcript may be expressed as a splicing
variant. To explore this possibility, we examined the expression of
intron A-intact GnRH transcripts in several tissues (Fig.
1). Mature GnRH mRNA was predominant in the POA of the hypothalamus, as expected. However, the GnRH transcript, which contains intron A but no other introns, was ubiquitously expressed in all examined tissues. Other GnRH transcripts were barely detectable in POA or extrahypothalamic tissues, indicating that the majority of intron-containing GnRH transcripts retains only
the first intron. Interestingly, the intron A-containing GnRH
transcript was expressed even in the POA; the expression level was not
significantly higher than that of other tissues. These results strongly
support the notion that the excision of intron A from the GnRH primary
transcript occurs specifically in GnRH-producing cells, and in other
cells the intron A-bearing GnRH transcript exists as a major splicing
variant.
Export of Intron A-containing GnRH Transcript from the
Nucleus--
To clarify whether the intron A-containing GnRH
transcript is not merely a splicing intermediate but a splicing
variant, we examined the translocation of intron A-containing
transcripts of the nucleus. Northern blot analysis showed that mature
GnRH mRNA exists exclusively in the cytoplasm and not in the
nuclear fraction of the rat POA; intron A-retained transcripts were,
however, detectable in both cytoplasmic and nuclear fractions of the
rat POA as well as the cerebral cortex (CTX) (Fig.
2A). We used RT-PCR to examine
the cellular localization of GnRH transcripts in various mouse tissues
and the GnRH-producing GT1-1 cell line. Again, mature GnRH mRNA was
abundant in the cytoplasmic fraction of the mouse POA and GT1-1 cells;
we also detected GnRH mRNA in other fractions, but at lower levels.
Intron A-retained transcripts were, however, detectable to a similar
extent in both cytoplasmic and nuclear fractions (Fig. 2B).
In contrast to the intron A-containing GnRH transcript, intron
B-containing transcripts were found only in the nuclear fraction (Fig.
2B). These results confirm the previous finding that intron
B is easily excised from the GnRH primary transcript (8) and strongly
suggest that the intron A-containing GnRH transcript is a splicing
variant and not simply a splicing intermediate of the gene
transcript.
Retained GnRH Intron A Blocks the Translation of Downstream Coding
Sequences--
The intron A-retained transcript may have a long 5'-UTR
as the coding region for the GnRH prohormone starts at the second exon
(2), and frame-shifted translation over the GnRH coding sequence cannot
occur. It is well known that long 5'-UTRs generally lower translation
efficiency (15), and our previous report suggests that intron A affects
the downstream open reading frame (ORF) translation in the Chinese
hamster ovarian cell line (CHO-K1) (11). In this study, we further
examined the effect of intron A on the translation of downstream ORF in
various cell lines, including both GnRH-producing and
non-GnRH-producing cell lines. Three GnRH-luciferase fusion constructs
(shown in Fig. 3A) were used
for transient transfection into several cell lines, such as GT1-1,
CHO-K1, mouse fibroblast (NIH-3T3), and human cervical cancer cell
(HeLa). When mouse GnRH exon 1 was fused to the 5'-end of the
luciferase coding sequence (mE1-Luc), luciferase activities varied from
42 to 108% as compared with those from the control luciferase reporter
plasmid (CTL-Luc), depending on cell type (Fig. 3B). Fusion
of the long GnRH 5'-UTR (mouse exon 1 and intron A) to the luciferase
ORF (mE1IA-Luc), however, showed a dramatic reduction in luciferase
activity; in fact, it was comparable to that of the promoterless
negative control vectors. The inhibitory effect of a long 5'-UTR
on the luciferase reporter was likely due to the presence of intron A,
as judged by the results from the mouse GnRH intron A-luciferase fusion
construct (mIA-Luc). To exclude the possibility that mE1IA-Luc and
mIA-Luc constructs have a certain defect in transcription, we examined
the expression of the GnRH-luciferase fusion mRNA by RT-PCR.
Because all constructs can produce luciferase mRNA at significant
levels in all cell lines tested (Fig. 3C), the intron
A-bearing transcripts must have a significant defect in
translation.
In addition to the mouse GnRH intron A, the equivalent human and rat
GnRH intron also strongly lowered the translation efficiency of
downstream ORF (Fig. 4). When the human
or rat GnRH exon 1 was fused to luciferase ORF, significant luciferase
activities were detected (human, 49-103% and rat, 27-118% of
CTL-Luc). Both human and rat GnRH intron A, however, strongly
suppressed luciferase activities to the levels of the promoterless
reporter. The results indicate that the translational regulation by
intron A is well conserved, at least among the human, rat, and mouse,
and that intron A-containing GnRH transcripts in these species could
scarcely contribute to the synthesis of GnRH prohormone.
To assure the translation inhibition by intron A of the downstream
reporter ORF, we performed in vitro
transcription/translation-coupled reactions. Both SP6 promoter-driven
mE1-Luc and mE1IA-Luc RNA transcripts were translated in
vitro using a reticulocyte lysate system. The mE1-Luc construct
yielded a single protein whose molecular mass was ~60 kDa as
expected. In contrast to mE1-Luc, mE1IA-Luc could not produce a
detectable amount of luciferase protein (Fig. 5). These results clearly indicate that
the translation of downstream coding sequence is completely blocked
when the mouse GnRH intron A resides upstream.
ATG Sequences in the Mouse GnRH Intron A Region Contribute to the
Inhibition of Translation--
To determine which regions are involved
in the inhibition of translation initiation at downstream start codon,
we generated several deletion constructs from the mIA-Luc reporter
plasmid and transfected these constructs into GT1-1 and CHO-K1 cells. Experiments using serial deletion constructs such as mIA-Luc
To verify this possibility, we introduced point mutations of the
mIA-Luc Interfering Influence of mE1IA-Luc on the Expression of mE1-Luc in
the GT1-1 Cell Line--
It can be postulated that mature mRNA and
its intron A-retained splicing variant can exist together in the cells,
although their relative amounts differ according to the cell- or
tissue-types. The notion was partly supported by the finding that
various tissues and even the immortalized GT1-1 cell line contained
both transcripts (Figs. 1 and 2). We further examined the possible
interactions between these two kinds of GnRH transcripts by
co-transfection experiment. When mE1-Luc and mE1IA-Luc constructs were
co-transfected into GT1-1 cells, increasing amounts of mE1IA-Luc
lowered luciferase activities from the same amount of an mE1-Luc
construct in a dose-dependent manner. When five times more
mE1IA-Luc plasmid was simultaneously introduced with mE1-Luc into GT1-1
cells, the luciferase activities from these cells were significantly
reduced to 63% of those from the cells transfected with the same
amounts of mE1-Luc and promoterless control luciferase plasmids.
mE1IA-Luc could not alter the luciferase activities from control
luciferase plasmid. In contrast to GT1-1 cells, mE1IA-Luc did not have
any influences on the expression of mE1-Luc fusion construct in NIH3T3
or CHO-K1 cells (Fig. 9). These results
suggest that the intron A-retained splicing variant may lower the
efficiency of GnRH biosynthesis, at least in GnRH-producing cells.
This study demonstrates that intron A-retained GnRH transcripts
are expressed ubiquitously in a variety of cell types and can be
exported from the nucleus to the cytoplasm. RNA splicing occurs
co-transcriptionally only in the nucleus by interactions between
splicing factors and the COOH-terminal domain (CTD) of RNA polymerase
II (16), and in situ hybridization studies using exon- and
intron-specific probes clearly demonstrate that intron-containing splicing intermediates from various genes are confined to the nucleus
(17-19). Indeed, intron B-containing GnRH transcripts are detectable
only in the nucleus, in contrast to intron A-retained transcripts. This
result is well in accordance with our previous finding that introns B
and C are "consensus" introns and are efficiently excised from the
primary transcript (8). This leads us to believe that an intron
A-retained GnRH transcript works as a splicing variant form of GnRH
transcripts that contains a long 5'-UTR and predominates in
non-GnRH-producing tissues.
It should be noted that the GnRH prohormone ORF begins at the second
exon; thus, we found it worthwhile to examine whether intron A-retained
GnRH transcripts can initiate translation properly. The 5'-leader
sequence size in this variant is larger than the average length of
eukaryotic 5'-UTRs, 50-100 nucleotides (15). In addition, GnRH intron
A contains multiple ATG sequences and putative stop codons, although
they cannot cause frame-shifted translation over the GnRH ORF. This
strongly suggested that intron A might affect translational efficiency
at a downstream start codon. Indeed, GnRH intron A strongly suppressed
the translation initiation of the downstream start codon both in
vivo and in vitro, and the elimination of ATG sequences
from intron A restored the translation efficiency of the downstream
reporter gene, suggesting that translation from AUG codons in intron A
is involved in the inhibitory effect on the downstream start codon.
Evidence is accumulating that 5'-UTRs have a profound effect on the
translational efficiency because of their primary and secondary
structures. In addition, it is well known that an upstream start codon
can modulate translation efficiency. The presence of an upstream AUG
sequence (uAUG) and stop codon can generate a short or so-called
upstream ORF (uORF). These uORFs usually inhibit translation
from downstream start codons, although in some cases they have been
reported to be stimulative. As examples, genes such as mammalian
S-adenosylmethionine decarboxylase (20), HER-2 (neu, erbB-2)
protooncogene (21), suppressor of cytokine signaling 1 protein (22) and
CCAAT/enhancer binding protein (23) have been reported to contain
inhibitory uORFs, whereas the uORFs in the mouse glucocorticoid
receptor (24) and the human androgen receptor (25) are crucial for the
translation initiation of a major ORF located downstream.
It is of interest that uORFs are found in less than 10% of known
mammalian mRNAs; however, they are strongly biased toward certain
classes of genes such as growth factors and their receptors, tumor
suppressors, and regulated transcription factors (26), strongly
suggesting that translational control by uORFs serves as a fine
regulatory mechanism. Although the exact molecular mechanism underlying
the translational regulation by uORF is not fully understood, Morris
and Geballe (26) have proposed a possible explanation for the
inhibitory effect of uORFs on the translation of downstream major ORF
from the fact that the uORF itself must be translated to participate in
translational regulation and that inhibition is dependent on the
sequence and the length of the intercistronic region. In their proposed
model (26), a scanning ribosome encounters the initiator AUG of the
uORF and initiates translation. Upon reaching the stop codon of the
uORF, the carboxyl terminus of the nascent peptide sometimes interacts
with part of the translational machinery, depending on the surrounding
nucleotide sequences. This interaction is thought to reversibly inhibit
either translation termination or a release of the completed peptide,
which, in turn, arrests the translating ribosome over the termination
codon. The arrested ribosome fails to reinitiate translation and
creates a blockade to scanning by additional ribosomes entering at the cap, thus inhibiting translation of the downstream ORF (26). Our
results strongly suggest that the possible uORFs generated by a
retained intron A could act through this mechanism, inhibiting translation of a downstream ORF.
Along with extrahypothalamic tissues, intron A-retained GnRH
transcripts are implicated in the mutant hpg mouse, which has a
truncated GnRH gene. Sequence analysis showed that the GnRH gene of the
hpg mouse contains the intact promoter region and the first two exons
but lacks the remaining parts of the gene (12). The previous reports
have shown that GnRH transcripts could be detected in the POA of these
animals by RT-PCR and in situ hybridization even at a lower
level than that of normal mice (11, 12). They suggested that
transcription could be driven by the GnRH promoter sequence to produce
a certain kind of mRNA, which might be a fusion RNA species
produced by the joining of the remaining GnRH mRNA sequence with
another unidentified sequence. Indeed, this possibility can be
postulated from the fact that a part of GnRH promoter is known to be
sufficient for the transcription of downstream reporter genes in a
subset of hypothalamic neurons (27). It should be noted that, although
the GnRH gene of the hpg mouse retains the sequence encoding the GnRH
decapeptide and a part of the GnRH-associated peptide, no GnRH
decapeptide can be detected in the hypothalamic extract of the animals
even by sensitive methods such as high performance liquid
chromatography coupled with highly specific anti-GnRH antisera (13).
Interestingly, our recent study indicated that a majority of the GnRH
transcript in the hpg mouse retained intron A due to an extremely low
splicing efficiency for this intron (11). The decreased excision rate of GnRH intron A in the hpg mouse is presumably due to a lack of ESE 3 and 4, which were implicated in our previous report (8, 9). With these
previous results, the present study provides a possible explanation for
the hypogonadism of the hpg mouse. The failures in the translation
initiation of GnRH transcript caused by the retained intron A might be
a major cause of the complete lack of GnRH decapeptide in the
hypothalamus of the hpg mouse, even though a relatively low, but
significant amount of GnRH transcript can be detected in the POA of
these mice. As 5'-UTRs are also known to affect mRNA stability
(28), something the retained GnRH intron A might also do so. As
mentioned above, the hpg mouse has the intact GnRH promoter, but the
mRNA level of GnRH in the hpg mouse is much lower than that of the
normal mouse (11, 12). This raises the possibility that intron A may be contributing to GnRH gene expression via its mRNA stability. This possibility needs to be explored.
It is also of interest to note that the intron A-containing reporter
can lower the expression efficiency of the GnRH E1-Luc constructs in a
GT1 cell-specific manner. The result suggests a possible interfering
influence of an intron A-retained GnRH variant on the efficient
biosynthesis of GnRH. In this regard, it should be noted that the
excision rate of GnRH intron A is significantly low in prepubertal
mouse POA (11). It showed a gradual increase during postnatal
development. This result strongly suggested that the maturation of
splicing machinery responsible for GnRH neuron-specific excision of the
intron A could occur with sexual maturation. With the previous
findings, our present result indicates that the efficient excision of
the intron A may contribute to the efficient production of GnRH. An
exact molecular mechanism underlying the interference is presently
unknown. However, competition between mature GnRH mRNA and its intron
A-retained variant to the translation machinery in GnRH-producing cells
would be most probable, because these two transcripts contain
the same ribosomal entry sequence. As shown in Figs. 3 and 4, the
translation efficiency for E1-Luc constructs varied according to cell
type when compared with that of CTL-Luc containing no GnRH UTR
sequences. The translation efficiencies of E1-Luc were generally
impaired in non-GnRH-producing cell lines, although significant amounts of luciferase can be still produced in these cells. It appears that the
translation machinery in GT1 cell is specified to translate GnRH
mRNA more efficiently. Similar results were also observed in other
genes. For instance, translation efficiencies of heterogeneous neuronal
nitric oxide synthase (nNOS) transcripts were significantly affected by
cell types. Even in the same cell line, cell differentiation significantly altered the translation efficiency of that transcript. These results strongly suggest that there must be cell type-specific translation machinery that is specified for a subset of gene
transcripts (29). Thus, GT1 cell-specific inhibitory influence of
E1IA-Luc on the expression of E1-Luc may occur by competitively
reducing the translation efficiency of that E1-Luc transcript.
In conclusion, we clearly demonstrate that the GnRH transcript bearing
its intron A actually acts as a splicing variant form of the GnRH
transcript that is predominant in non-GnRH-producing cells. In contrast
to the exon 2-skipped splicing variant of GnRH mRNA, which lacks
the coding sequence for the GnRH decapeptide region and its surrounding
parts (30), this third variant retains an intact ORF. It cannot,
however, be appropriately translated because of the presence of
multiple uAUGs on intron A, and it inhibited the expression of the
reporter construct mimicking a mature GnRH mRNA in a GT1
cell-specific manner. These results indicate that the precise and
efficient excision of intron A and the joining of exons serves as key
regulatory steps for the synthesis of GnRH prohormone, thereby
contributing to the tissue- and developmental stage-specific expression
of this gene product, and also provides a possible molecular mechanism
for the absence of functional GnRH biosynthesis in the hypothalamus of
the mutant hpg mouse.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Primer sequences for gene cloning and RT-PCR
-32P]UTP.
Hybridization procedures were performed as described previously (8).
For RT-PCR analysis, 1 µg (for tissues and non-GnRH-producing cell
lines) or 100 ng (for the GT1-1 cell line) of the RNA templates were
reverse-transcribed with 200 units of Moloney murine leukemia virus
(MMLV) reverse transcriptase (Promega) using the manufacturer's instructions. Subsequently, a 3-µl aliquot of each RT sample was subjected to PCR in a 40 µl reaction mixture containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 1 mM dNTP, 20 pmol of
upstream and downstream primers, and 2.5 units of Taq
polymerase (PerkinElmer Life Sciences). Ten-microliter aliquots of PCR
products were electrophoresed on 2% agarose gels in Tris acetate-EDTA
buffer and visualized by ethidium bromide staining. The primer
sequences used for RT-PCR experiments are presented in Table I.
-galactosidase (
-gal) expression construct
were cotransfected. Cell extracts were prepared by incubating cells in
0.3 ml of reporter lysis buffer (Promega) for 15 min at room
temperature. After a brief centrifugation, supernatants were stored at
70 °C until assay.
-gal and luciferase assays were performed
using commercial enzyme assay kits (Promega) and the
-gal activity
was used to normalize for transfection efficiency.
-32P]UTP was added to the
reaction mixture, and labeled RNAs were resolved on a 6%
urea-polyacrylamide gel. [35S]methionine was used to
label translated peptide, and the reaction products were
electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel;
dried gel was exposed to x-ray film (Fuji, Japan) for 1 day.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Ubiquitous expression of intron A-retained
GnRH transcript in rat tissues. Total RNAs from various rat
tissues were resolved on a 1.2% formaldehyde gel and transferred to a
Nytran membrane. Hybridizations were performed using
32P-labeled RNA probes complementary to rat GnRH cDNA
(top panel) or intron A (middle
panel). Electrophoresed RNA was stained with ethidium
bromide and is shown in the bottom panel.
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Fig. 2.
Transport of intron A-containing GnRH gene
transcripts to the cytoplasm. A, cytoplasmic
(C) and nuclear (N) RNAs separately isolated from
the rat POA and cerebral cortex (CTX) were resolved on a
1.2% formaldehyde gel and transferred to a Nytran membrane.
Hybridization was performed using 32P-labeled RNA probes
complementary to rat GnRH cDNA (left panel)
or intron A (middle panel). B,
cytoplasmic and nuclear RNAs from mouse POA, cerebral cortex
(CTX), testis (TES), ovary (OV), and
GT1-1 cells (GT1) were reverse-transcribed using Moloney
murine leukemia virus reverse-transcriptase, and each kind of cDNA
was amplified by PCR. PCR products were resolved on 2% agarose gels
and visualized by staining with ethidium bromide.
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Fig. 3.
Influence of 5'-UTR sequences from mouse GnRH
on downstream luciferase activity. A, schematic diagram
for GnRH-luciferase fusion gene constructs is shown. B,
these constructs and the CTL-Luc vectors were transfected into GT1-1,
CHO-K1, NIH-3T3, or HeLa cells. Luciferase activities were measured
24 h after transfection and normalized by cotransfected
CMV- -gal activity. Data are shown as mean ± S.E.
(n = 6-12). C, total RNAs were isolated
from transfected cells, and DNA contaminants were removed by incubation
with DNase I at 37 °C for 30 min. One microgram of each RNA sample
was subjected to RT-PCR analysis (RT (+) lanes).
To assure the removal of plasmid contaminants, the PCR reaction without
RT was also performed for each RNA sample (RT (
)
lanes). PCR products were resolved on 2% agarose gels and
visualized by staining with ethidium bromide.
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Fig. 4.
Influence of human and rat GnRH intron A on
translation efficiency of a downstream luciferase gene. Control
luciferase reporter and the constructs containing human (A)
or rat (B) GnRH exon 1 and/or intron A upstream to
luciferase ORF were transfected into GT1-1, CHO-K1, NIH-3T3, or HeLa
cells. Luciferase activity was measured 24 h after transfection
and normalized by cotransfected CMV- -gal activity. Data are shown as
mean ± S.E. (n = 6).
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Fig. 5.
In vitro
transcription/translation-coupled reactions using mouse GnRH exon
1- or exon 1 and intron A-luciferase fusion constructs. The
mE1-Luc or mE1IA-Luc constructs were fused to the SP6 promoter.
The schematic diagram for these constructs are shown in panel
A. RNAs and peptides were synthesized from the plasmids using SP6
RNA polymerase and reticulocyte lysate in the presence of
[32P]UTP or [35S]methionine in
vitro. Synthesized RNAs were resolved on a 6% urea-polyacrylamide
gel, and peptides were electrophoresed on a 10% SDS-polyacrylamide
gel. Dried gels were exposed to x-ray films for 1 day
(B).
1,
2,
3, and mIA-Luc
7 revealed that a short proximal fragment of intron
A was sufficient to inhibit luciferase expression in both cell lines
(Fig. 6); the region just prior to this
short fragment (designated as mIA-Luc
4) also strongly interfered
with luciferase translation. However, luciferase activity was almost normal when the mIA-Luc
5 construct was used. In CHO-K1 cells, IA-Luc
6 produced a luciferase activity slightly higher than that of
other constructs (Fig. 6). Interestingly, sequence analysis revealed
that the intron A of mouse GnRH contains 14 ATG sequences, and at least
five of these are putative translation initiation sites; only
mIA-Luc
5 contains no ATG sequence in the fragment. This strongly
suggests that ATG sequences in the mouse GnRH intron A play an
inhibitory role at the downstream start codon.
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Fig. 6.
Effect of various partial intron A fragments
on the downstream luciferase gene translation. The fusion
constructs, consisting of partial GnRH intron A fragments and the
luciferase coding region, were cloned as described under
"Experimental Procedures." Schematic diagrams for the constructs
are shown on the left. Short bars on
intron A represent locations of ATG sequences. Each construct was
transiently transfected into GT1-1 or CHO-K1 cells. Luciferase
activities were determined 24 h after transfection and normalized
by cotransfected CMV- -gal activity. Data are shown as mean ± S.E. (n = 4).
3 construct, which contains five ATG sequences. We changed
these ATGs to AAG sequences designated as mIA-Luc
3mut1 and found
that it produces luciferase activity comparable with that of mE1-Luc,
which lacks intron A. Interestingly, the restoration of luciferase
activity correlated well with the number of remaining ATG sequences
rather than any particular ATG sequence location. This result strongly
suggests that the proximal five ATGs, at least, act on the downstream
ORF cooperatively (Fig. 7). In addition to these experiments, we also performed in vitro
transcription/translation-coupled reactions to verify this prominent
role of ATG sequences of intron A and their inhibition of the
downstream start codon. In agreement with the transient transfection
experiments, the presence of an intact or proximal fragment of intron A
strongly inhibited the synthesis of luciferase. The translation of
luciferase ORF in the mIA-Luc
3mut1, however, did produce a
significant amount of luciferase protein (Fig.
8). Together, these results strongly indicate that multiple ATG sequences of the GnRH intron A block the
appropriate translation initiation at the downstream start codon and
may cause the failure in GnRH prohormone synthesis.
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Fig. 7.
Effect of mutating upstream ATGs on
translation of the downstream luciferase gene. Five ATGs of mouse
IA-luciferase 3 (mIA-Luc
3) were mutated to
AAGs by PCR-based mutagenesis, and the mutations were confirmed by
chain termination sequencing. Schematic diagrams for the gene
constructs are shown on the left. Short
bars on intron A region represent ATG sequences. CTL-Luc
vector, mE1-Luc, mIA-Luc, deletion construct mIA-luciferase
3
(mIA-Luc
3), and mutated IA-luciferase
3s
(mIA-Luc
3mut1 to
IA-Luc
3mut7) were transiently transfected into
GT1-1 or CHO-K1 cell lines. Luciferase activity was measured 24 h
after transfections, normalized by cotransfected CMV-
-gal activity,
and presented as a percentage of the CTL-Luc value. Data are shown as
mean ± S.E. (n = 6).
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[in a new window]
Fig. 8.
In vitro
transcription/translation-coupled reactions using intact or
mutated mouse GnRH-luciferase fusion constructs. Mouse
GnRH-luciferase fusion constructs (mE1-Luc, mE1IA-Luc, mIA-Luc,
deletion construct mIA-Luc 3, and mutated deletion construct
mIA-Luc
3mut1) were fused to the SP6 promoter. A schematic diagram
for these constructs are shown in the top panel.
Luciferase from each construct was synthesized using SP6 RNA polymerase
and reticulocyte lysate in the presence of
[35S]methionine in vitro. Synthesized peptides
were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide
gel. Dried gels were exposed to x-ray films for 1 day.
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[in a new window]
Fig. 9.
Effect of co-transfected mE1IA-Luc on
luciferase activity from mE1-Luc. Various amounts of SV40
promoter-driven CTL-Luc, promoterless-Luc, mE1-Luc, and/or mE1IA-Luc
constructs were transfected into GT1-1, CHO-K1 or NIH-3T3 as indicated.
Luciferase activities were determined 24 h after transfection and
normalized by cotransfected CMV- -gal activity. Data are shown as
mean ± S.E. (n = 8, *, p < 0.05).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We express our appreciation to the English-editing efforts of Biomedical English Editing Service, Portland, OR.
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FOOTNOTES |
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* This work was supported by the Korea Ministry of Science and Technology through Korean Brain Science and National Research Laboratory Grant 2000-N-NL-01-C-149 and a grant from Basic Research Program of the Korea Science and Engineering Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These two authors contributed equally to this work and were supported by a Brain Korea 21 Research Fellowship from the Korea Ministry of Education.
To whom correspondence should be addressed. Tel.:
82-2-880-6694; Fax: 82-2-884-6560; E-mail: kyungjin@snu.ac.kr.
Published, JBC Papers in Press, March 13, 2003, DOI 10.1074/jbc.M209850200
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ABBREVIATIONS |
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The abbreviations used are:
GnRH, Gonadotropin-releasing hormone;
POA, preoptic area;
UTR, untranslated
region;
ESE, exonic splicing enhancer;
hpg, hypogonadal;
RT, reverse
transcription;
CMV, cytomegalovirus;
-gal,
-galactosidase;
ORF, open reading frame;
uORF, upstream ORF;
CHO, Chinese hamster ovary;
CTL, control;
Luc, luciferase;
mIA, mouse GnRH intron A;
mE1, mouse
GnRH exon 1;
mE1IA, mouse GnRH exon 1 and intron A (long 5'-UTR).
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REFERENCES |
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