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INTRODUCTION |
GLI1 was originally isolated as a highly amplified gene
in a malignant glioma (1) and subsequently implicated in the
development of other tumor types, including liposarcoma,
rhabdomyosarcoma, osteosarcoma, and astrocytoma (2, 3). Later it was
shown that GLI1 encodes a transcription factor that is a
downstream nuclear component of the Sonic hedgehog-Patched signaling
pathway (4-6). This pathway is evolutionarily conserved and found to operate in a number of tissues during vertebrate development and especially in regions involving mesoderm-ectoderm interactions (7-10).
Intercellular signaling by this pathway is initiated when Sonic
hedgehog (a secreted protein) binds to Patched (a cell surface transmembrane protein), resulting in the activation of GLI1 in the
nucleus and subsequent expression of target genes. Overexpression of
Sonic hedgehog has been shown to up-regulate Gli1
in chick limb buds and in the epidermal ectoderm of frog embryos (11, 12), whereas Gli1 expression is undetectable in Sonic
hedgehog null mouse embryos (13, 14), confirming that Sonic
hedgehog signaling regulates Gli1 expression.
The discovery of Patched mutations in familial and sporadic
forms of basal cell carcinoma
(BCC),1 the most common skin
cancer, has associated aberrant signaling of the Sonic hedgehog-Patched
pathway with the formation of these tumors (15-17). The genetic data
are supported by experimental evidence showing that overexpression of
Sonic hedgehog and other components of this pathway results
in the induction of BCCs in transgenic mice and transgenic human skin
(18-20). Overexpression of GLI1 in the epidermis of
transgenic animal models produces BCC-like lesions (21, 22) and will
transform rodent epithelial cells in cooperation with adenovirus EIA
(23), indicating that unregulated expression of GLI1 is
oncogenic. Recent studies have shown that GLI1 expression is
greatly increased in BCCs but not in the surrounding normal tissue,
consistent with a central role in tumor formation (21, 24, 25).
In addition to GLI1, two other isoforms have been identified in
vertebrates (termed GLI2 and GLI3), each encoded by a separate gene (8,
10). The GLI genes are highly expressed during development, and their expression profiles correlate with organogenesis but show
only low-level expression in most adult tissues (7, 9, 10). In the
skin, GLI1 expression is readily observed in the epidermal
compartment of the developing hair follicle, whereas GLI2
and GLI3 transcripts are detected in the surrounding
mesenchyme (10, 21, 25). The role of each GLI in mediating the Sonic hedgehog signal is not yet clear, but recent gene ablation studies have
shown overlapping roles and indicated some functional redundancy (26,
27). A number of studies have indicated that GLI1 is a transcriptional
activator, whereas GLI2 and GLI3 can act as both activators and
repressors depending on specific post-translational modifications
(28-30). Interestingly, GLI2 and GLI3 are now thought to regulate
GLI1 transcription directly by binding to the
GLI1 promoter (28, 30).
The 5' untranslation region (UTR) has a large influence on translation
and plays a key role in post-transcriptional gene regulation (31-36).
The efficiency of translation initiation is largely governed by the
composition and structure of the 5' UTR of the mRNA, which is
determined by both its length and its sequence. Extensive secondary structures and one or two small upstream open reading frames (uORFs) within a 5' UTR can profoundly inhibit protein translation. Most highly
expressed mRNAs have relatively short (20-100 nucleotides) 5' UTRs
that lack uORFs and extensive secondary structures (for review, see
Ref. 31). In contrast, mRNAs encoding oncoproteins, growth factors,
transcription factors, and other regulatory proteins are poorly
translated and often have long, highly structured 5' UTRs with multiple
upstream ATGs (32, 35, 36). In this study, we have identified
alternative 5' UTRs of GLI1 transcripts in mouse and human
tissues, which are generated by exon skipping and have marked
differences in translation efficiency. Until now, post-transcriptional
regulation of GLI1 has been inferred (37), but a precise
mechanism has not been determined. Our results suggest that
post-transcriptional regulation of GLI1 is mediated by exon skipping and show an association of the most efficiently translated 5'
UTR transcript with BCC.
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EXPERIMENTAL PROCEDURES |
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), 5'
Rapid Amplification of cDNA Ends (RACE), and PCR--
Skin, brain,
heart, kidney, liver, lung, muscle, stomach, spleen, testis, and tongue
were obtained from various strains of neonatal, juvenile, and adult
mice. Total RNA was isolated from these tissues using TRI Reagent
(Molecular Research Center). First-strand cDNA was synthesized from
5 µg of total RNA primed with oligo(dT)16 (PerkinElmer)
or random hexamers (CLONTECH) using Superscript II
reverse transcriptase (Life Technologies) in a total volume of 20 µl.
One-tenth by volume of the cDNA was used as the template for
subsequent PCR reactions. Primer pairs (listed in Table I) for mouse
GLI1 corresponding to sequences within exon 1 (mGliF1) and
exon 2 (mGliR2) and for human GLI1 corresponding to exon 1 (hGliF1) and 2 (hGliR2) sequences were used. Each PCR reaction was
repeated at least three times with different RNA preparations and
included negative controls for each set of reactions. For 5' RACE, we
used a cDNA template that was generated using the primer RACE1 and
C tailed using terminal transferase according to the manufacturer's
protocol (Life Technologies). PCR was performed using the RACE anchor
and adapter primers (Life Technologies) and the
GLI1-specific nested primer RACE2. Mouse genomic DNA was amplified using primers derived from exon 1a (mGliF1a) and exon 2 (mGliR2) sequences. All PCR products were separated on 0.8-2% agarose
gels and visualized with ethidium bromide. Separated fragments were
purified using QIAEX II (Qiagen) and sequenced directly using the Big
Dye termination kit and automated fluorescent sequencing on an
ABI-Prism 377 DNA sequencer (PerkinElmer).
12-O-Tetradecanoylphorbol 13-acetate (TPA) Treatment of Mouse
Skin--
Neonatal and 7-day-old mice (Swiss outbred) were treated
with 20-50 µl of 100 µg/ml TPA (Sigma) topically applied to back skin. Mice were killed at different time points (0, 3, 8, and 24 h) after application, and total skin RNA was prepared for RT-PCR as
described above.
5' UTR-Green Fluorescent Protein (GFP) Constructs and Functional
Analysis of 5' UTRs--
Each of the three alternative 5' UTRs of
mouse GLI1 was generated by RT-PCR using primers
mGliF1Nhe and mGliQR2Age (Table II) that contain
NheI and AgeI restriction sites, respectively. PCR products were gel purified and cloned into the pGEM-T Easy vector
(Promega). The four ATG codons of the
-UTR were mutated sequentially
using the QuikChange site-directed mutageneis kit (Stratagene) and
complimentary primers mGliMF1-4 and mGliMR1-4 (Table II). The
-UTR
sequence was multimerized using primers mGliF1Bam and
mGliR2Bgl that contain BamHI and BglII
sites, respectively. The amplified fragment was cloned into pGEM-T Easy
and sequenced, and the insert was released by digestion with
BamHI and BglII. The purified fragment was
ligated in the presence of both restriction enzymes to form similarly
oriented concatomers that were then used as templates for PCR
amplification using mGLiF1Nhe and mGliR2Age. The
products of this reaction were sized on an agarose gel and the band
corresponding to four copies of the
-UTR cloned into the pGEM-T Easy
vector. All inserts were verified by sequence analysis, released from
the pGEM-T Easy vector by restriction digestion with NheI
and AgeI, and subcloned into the corresponding sites of the
GFP expression vector pEGFP-N1 (CLONTECH).
HaCaT, a human keratinocyte cell line (38), Cos-1 (39), and BHK-21 (40)
cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, ampicillin, and streptomycin
(Life Technologies). Primary mouse skin fibroblasts were obtained from
newborn Swiss mice using an established protocol. Briefly, skin was
removed, washed in PBS, and incubated in 2.5% dispase (Life
Technologies) for 24 h at 4 °C. The dermis was separated from
the epidermis and incubated in 0.2% collagenase (Sigma) at 37 °C
for 1 h. Cells were then pelleted, washed in PBS, and cultured in
Dulbecco's modified Eagle's medium as described above. Primary fibroblasts were used within the first 2 weeks of culturing.
Transient transfection of GFP constructs was performed using
LipofectAMINE Plus reagent (Life Technologies) according to the manufacture's instructions. Cells were seeded on round, glass coverslips in 24-well (fluorescence microscope study) or 6-well (flow
cytometry study) plates 24 h before transfection and incubated at
37 °C to a density of ~50-70% confluence. Cultures were washed twice in serum-free media and then incubated in DNA-LipofectAMINE Plus
complexes in OPTI-MEM (Life Technologies) for 3 h at 37 °C. Dulbecco's modified Eagle's medium containing serum was then added to
the culture. One day later, cells were fixed for microscopy or
harvested for flow cytometry analysis. Twenty thousand cells per sample
were analyzed on a FACSCalibur (Becton Dickinson) cell sorter, using
CELLQuest software (Becton Dickinson).
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RESULTS |
Identification of Alternative Mouse GLI1 5' UTRs--
To identify
the transcriptional start site of the mouse GLI1 gene, 5'
RACE was performed on total skin RNA from a BALB/c mouse, and the
resulting PCR product was sequenced directly. This analysis showed that
sequences around the translation start codon and at the beginning of
the transcript were identical to the published mouse GLI1
sequence obtained from F9 cells (41). However, the RACE product also
contained an additional 119 bases, not present in the published
sequence, located at the splice junction between exons II and III as
numbered by Liu et al. (41). We named this new 5' UTR
variant
-UTR and the published sequence
-UTR (Fig. 1A). To search for other
possible splicing variants, RT-PCR was conducted using a forward primer
located at exon 1 and a reverse primer that hybridizes immediately 5'
of the GLI1 ATG codon (Table I). This PCR
revealed a much smaller product, which was sequenced directly and
found to consist of only one 5' noncoding exon. This alternative mouse
GLI1 variant corresponds in both size and sequence to the
published human transcript obtained from a glioma cell line (7, 41) and
was denoted the
-UTR variant (Fig. 1A).

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Fig. 1.
Sequence, exon composition, and pre-mRNA
structure of the alternative 5' UTRs of mouse
Gli1. A, sequence alignment of the three
alternative GLI1 5' UTR variants (denoted -, -, and
-UTR) expressed in mouse. The novel 119-base pair sequence of exon
1a is shown in bold lowercase letters. The ATG codons
denoting the beginning of uORFs are underlined, and the main
ORF encoding Gli1 is shown in bold uppercase letters. The
intron-exon boundaries are indicated by arrows.
B, schematic showing the exon composition of the alternative
5' UTRs and the organization of the pre-mRNA from which they are
derived. Exons are denoted by open boxes, and introns are
denoted by solid lines with intron size shown. The
translation start site (ATG) of the main ORF is located in exon 2 and
indicated by a bent arrow.
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To determine how these alternative transcripts were generated, we
examined the genomic organization of this region. Mouse genomic DNA was
amplified using the primer pair mGliF1a and mGliR2 (Table I). Sequence
analysis of the 2.8-kilobase product identified the novel
119-nucleotide sequence within the
-UTR variant as an authentic exon
(which we denoted exon 1b) that is flanked by 2.5 kilobases (intron Ia)
and 115 bases (intron Ib) of intervening sequences (Fig.
1B). The identification of this additional noncoding exon
required a change in the nomenclature used in the earlier study
by Lui et al. (41), with exon II becoming exon 1a and exon
III (which encodes the translation start site) renumbered exon 2 (see
Fig. 1B).
Expression of Mouse GLI1 5' UTRs--
The expression of the
alternative 5' UTR variants was determined in neonatal, juvenile, and
adult tissues from various mouse strains by RT-PCR (Fig.
2). This analysis revealed that the UTR variants had no particular tissue-specific expression pattern but did
show marked strain-specific differences. In all tissues examined the
larger UTR forms (
- and
-UTR) predominate, whereas the
-UTR
appears as a minor amplified product (Fig. 2A). In some strains, such as BALB/c, DBA, and C57BL/6, the
-UTR variant was the
major form, whereas in CD1 and SV129 strains, the
-UTR form was the
predominant transcript (Fig. 2B). In Swiss outbred mice, expression of the
- and
-UTR was heterogeneous, with some animals expressing both forms and others expressing only one (data not shown).
In a given individual, the expression profile of the two larger
transcripts was identical in all tissues examined irrespective of the
strain used (Fig. 2A). We also followed the expression of
the UTR variants in postnatal skin development and found that the
apparent levels of all GLI1 transcripts were reduced with increasing age and that the
-UTR transcript was not detected at all
in adult skin (data not shown).

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Fig. 2.
The expression of alternative
GLI1 5' UTRs is not tissue specific but
does show strain variation. RT-PCR was performed on mRNA
isolated from brain (Br), liver (Li), Lung
(Lu), skin (Sk), stomach (St), and
tongue (To) tissues of a postnatal mouse (A). A
signal for the - and -UTR variants is also present in all tissues
examined but is barely discernible. B, in BALB/c, DBA, and
C57Bl/6 strains, the -UTR variant is the major transcript (BALB/c is
shown in lane 1), whereas the -UTR variant is the
dominant transcript in CD-1 and SV129 strains (CD-1 is shown in
lane 2). M, DNA marker.
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To evaluate whether the expression of these 5' UTR variants correlated
with proliferative status, newborn and 7-day-old animals were treated
with TPA topically applied to back skin. These experiments revealed
that expression of the larger UTR transcripts was maximally reduced at
3 h after application, whereas expression of the
form was
increased (Fig. 3). Reduced expression of
the
- and
-UTRs was still evident, albeit not as marked, 24 h after application (data not shown). Because acute TPA treatment
results in increased mitotic activity of basal layer keratinocytes
(42), these data indicate an association of the
-UTR transcript with
proliferation and the
- and
-UTRs with differentiation.

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Fig. 3.
Expression of the GLI1
5' UTR variants is altered by TPA treatment. The
expression of the -UTR transcript is increased, whereas expression
of the -UTR is reduced, in TPA-treated skin (+) relative to control
skin ( ). M, DNA size marker.
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Identification of Alternative Human GLI1 5' UTRs--
We next
searched for alternative human GLI1 transcripts in newborn
foreskin by RT-PCR with primers derived from exons 1 and 2 (Table I) of
the published sequence (7). Two PCR products were generated and
sequenced, with the smaller fragment corresponding to the published
sequence (7) and the larger containing an additional 144 bases located
at the splice junction of exons 1 and 2 (Fig.
4A). The larger transcript was
termed
-UTR, and the smaller sequence was termed the
-UTR variant
(Fig. 4, A and B). Notably, the novel
144-nucleotide sequence found within the human
-UTR has significant
homology with mouse exon 1a but is 30 bases larger and contains two
additional ATG codons (Fig. 4C).

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Fig. 4.
Sequence, exon composition, and pre-mRNA
structure of alternative human GLI1
5' UTRs. A, sequence alignment of the
alternative GLI1 5' UTR variants identified in human tissues
(denoted - and -UTR). The novel 144-base pair sequence of exon 1a
is shown in bold lowercase letters. The ATG codons denoting
the beginning of uORFs are underlined, and the main ORF
encoding GLI1 is shown in bold uppercase letters. The
intron-exon boundaries are indicated by arrows.
B, schematic showing the exon composition of the alternative
5' UTRs and the organization of the pre-mRNA from which they are
derived. Exons are denoted by open boxes, and introns are
denoted by solid lines with intron size shown. The
translation start site (ATG) of the main ORF is located in exon 2 and
indicated by a bent arrow. C, comparison of human
exon 1a sequences with mouse exon 1a. *, identical bases.
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The
- and
-UTRs were also present in a brain sample, and their
identity was confirmed by sequence analysis (Fig.
5). The expression of the
- and
-UTRs was further examined in HaCaT cells and seven BCC samples. We
found that the
transcript was present in proliferating cultures of
HaCaT cells and all BBC samples, but in contrast to foreskin
keratinocytes and brain tissue, we were unable to amplify the
transcript from these mRNAs (Fig. 5). Therefore, the
-UTR
transcript may represent the major variant expressed by proliferating
cells in human tissues as well.

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Fig. 5.
Detection of human GLI1
5' UTR variants by RT-PCR. RT-PCR results from
skin, brain, the HaCaT human keratinocyte cell line (HaC),
two BCC biopsies, and a no-RNA control
(dH2O) are shown. M, DNA size
marker.
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Functional Analysis of the GLI1 5' UTRs--
The 5' UTR is known
to regulate gene expression by influencing the efficiency of
translation. An examination of the GLI1 5' UTR variants
revealed three small uORFs in mouse
-UTR, two in human
-UTR, one
that overlaps the GLI1 ORF in the mouse
-UTR, and none in the
-UTRs (Table III). The secondary
structure for each 5' UTR was analyzed using the RNA folding prediction
program MFOLD (43). This program predicted extensive secondary
structures in the longer UTRs, with calculated free energy values of
72.7 to
94.5 kcal/mol for the mouse
-UTR,
65 kcal/mol for the
human
-UTR, and
55 kcal/mol for the mouse
-UTR. The human and
mouse
-UTRs were predicted not to form stable secondary structures and to have free energy values of
12 kcal/mol (Table III). The presence of uORFs and stable secondary structures in the larger UTRs
suggests that they will be less efficiently translated than the
variant, which lacks these features.
To test the above prediction in vivo, the three mouse
GLI1 5' UTR fragments were cloned upstream of a GFP reporter
gene (Fig. 6A), and the
constructs were transiently transfected into HaCaT, Cos-1, and BHK-21
mammalian cell lines and primary mouse skin fibroblasts. The difference
in GFP fluorescence produced by these constructs was striking (Fig.
6B), with the
-UTR construct producing the lowest
fluorescence levels, the
-UTR construct producing intermediate
levels, and the
variant producing the highest levels (even higher
than the GFP vector control). Importantly, there was no apparent
difference in the transfection efficiency of these constructs for a
given cell type, showing that the increase in the number of brightly
fluorescing cells transfected with the
form is caused by an
increase in GFP production.

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Fig. 6.
The Gli1 5'
UTR variants differentially express a GFP reporter construct in
transfected cells. A, diagram showing the GFP
constructs used in the transfection studies. The mouse Gli1
5' UTR sequences were cloned upstream of the GFP ORF as indicated.
pEGFP-N1, parent GFP construct. B, GFP
fluorescence observed in Cos-1 cells transfected with the GFP
expression vector alone (GFP) and -UTR-GFP ( ),
-UTR-GFP ( ), and -UTR-GFP ( ) constructs. Transfection
efficiency was determined by counting GFP-expressing cells, which
revealed that each construct was transfected with the same efficiency
(within experimental limits).
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To quantify these results, cells were also subjected to flow cytometry
analysis (Fig. 7A). The
advantages of this technique over enzymic reporter assays are that GFP
levels are determined in individual cells using a large number of cells
(20,000 cells/construct), and untransfected cells are discarded from
the calculations, thus removing any bias attributable to transfection
efficiencies. This analysis revealed that the
- and
-UTRs
expressed the reporter 60-90% lower than the GFP vector control,
whereas the
-UTR produced 2-3-fold enhancement of expression (Fig.
7B). Comparison of expression levels between the three
variants reveals a 14-23-fold increase in GFP production by the mouse
-UTR construct over the
-UTR (
/
) and a 5-13-fold increase
over the
-UTR (
/
; Fig. 7C). The greatest
differences in GFP intensities were seen in HaCaT cells. These data
show that the
-UTR facilitates expression of a heterologous protein,
whereas the
- and
-UTRs significantly suppress protein
production.

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Fig. 7.
Flow cytometry analysis of 5' UTR-GFP
constructs in transfected cells. A, fluorescence
intensity histogram compiled from the analysis of 20,000 cells per
sample for each construct (see Fig. 6) in transfected Cos-1 cells. The
peak closest to the vertical axis is attributable to
untransfected cells (negative cells), which show low fluorescent
intensities because of autofluorescence. The second peak represents
fluorescence from transfected cells that express GFP. The four
histograms representing each GFP construct were merged to allow direct
comparison of fluorescence intensities. This analysis revealed that the
-UTR construct produced the highest GFP intensities (mean value,
1.045 × 103) and the -UTR produced the lowest
(mean value, 6.2 × 101). B, graphical
representation of GFP intensities of the 5' UTR variants relative to
the empty GFP vector (defined as 100) in transfected BHK-21
(BHK), Cos-1, and HaCaT cells. C, graphical
representation of the ratios of GFP intensities among 5' UTR variants
in the cell lines indicated. The -UTR/ -UTR ( / ) and
-UTR/ -UTR ( / ) ratios are shown for constructs
transfected into BHK-21 cells (BHK), Cos-1 cells, primary
mouse skin fibroblasts (MSF), and HaCaT cells. The largest
differences in GFP fluorescence between the -UTR and the - and
-UTR variants were observed in HaCaT cells. The values shown are the
averages of three independent transfection and cell sorting
experiments.
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To determine whether this suppression is caused by the presence of
uORFs or some other property of the longer UTRs, such as increased
secondary structure, we made two additional constructs. The uORFs of
the
-UTR variant were removed by mutating all four upstream ATG
codons to TTG. Significantly, the mutant
-UTR construct expressed
the reporter at levels that approached that of the
-UTR construct
(Fig. 8). To ascertain whether the length
of the UTR sequence could influence expression levels, we produced a
-UTR multimer containing four copies of this sequence in the same
orientation. The
-UTR multimer, which contained 314 base pairs
compared with the 307 base pairs of the
- variant, produced GFP
levels that were only marginally lower than a single copy of the
-UTR (Fig. 8). Taken together, these data show that the uORFs
of the Gli1 UTRs play a major role in the suppression of protein
production.

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Fig. 8.
Flow cytometry analysis of mutant 5' UTR-GFP
constructs in transfected cells. A graphical representation of GFP
intensities of wild-type -UTR ( ), mutant -UTR
( mut), wild-type -UTR ( ), and the 4-mer -UTR
( x4) constructs relative to the empty GFP vector in Cos-1
cells is shown. The values shown are the averages of three independent
transfection and cell-sorting experiments.
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DISCUSSION |
The expression of alternative mRNAs from a single gene is an
important mechanism for gene regulation and for the generation of
functionally different proteins (44, 45). In this study, we have
identified alternative GLI1 transcripts that differ in their
5' UTRs and are generated by exon skipping. The longer transcripts (
and
in mouse and
in human) were present in all normal tissues examined, whereas the shortest transcript (
-UTR) was present in
neonatal tissues but rarely detected in adult tissues. Significantly, the
form is the only variant found in BCCs and in a proliferating human keratinocyte cell line. Moreover, expression of the
form could be induced by TPA treatment of mouse skin. We have found that the
GLI1 5' UTRs determine the expression levels of a
heterologous coding sequence in transfected cells, with the
form
associated with the highest levels of expression and the
- and
-UTRs associated with significantly lower levels. These findings
suggest that GLI1 levels may be regulated through the use of
alternative 5' UTRs and predict that an unregulated increase in the
transcript over the
and
forms may be tumorigenic.
We initially examined several tissues from various strains of mice,
both neonatal and adult, for evidence of tissue-specific expression of
the GLI1 alternative transcripts. We found that expression
of these transcripts did not show tissue specificity, but the levels of
the
and
transcripts (relative to each other) did vary between
strains. Because BALB/c mice predominantly express the
form and CD1
mice predominantly express the
form, we sequenced the intron-exon
boundaries of this region in these strains and found them to be
identical (data not shown), suggesting that the expression differences
between strains was not caused by polymorphic variation at these sites.
Whether these strain differences were caused by polymorphic differences
in intronic cis-acting elements or by differences in
transacting factors remains to be determined. We did not observe any
transcripts equivalent to the mouse
-UTR in the human tissues
examined, and we were unable to find sequences equivalent to mouse exon
1b in the human gene, suggesting that humans do not have the capacity
to express an
-UTR variant, although the presence of three upstream
ATG codons in exon 1a of the human message compared with just one in
mouse, together with the increased stability of the human
-UTR
(Table III), appears to compensate for
the lack of exon 1b sequences in the human sequence. However, we cannot
exclude the possibility that expression of the
-UTR variant may be
idiosyncratic in human as it is in mice because of the small sample
size that was available in this study.
The expression of the 5' UTR variants did show an apparent correlation
with proliferative status. The longest transcripts are expressed in
normal tissues but not in BCCs or in a human keratinocyte cell line,
whereas the shortest transcript is the only variant present in BCCs and
in HaCaT cells. In addition, expression of the
form was induced by
TPA treatment of mouse skin, whereas the levels of the longer
transcripts were concomitantly reduced. These observations suggest an
association of the shortest 5' UTR with actively proliferating
keratinocytes and the
and
transcripts with quiescent cells. The
association of the most actively translated transcript with
proliferation fits neatly with the observation of increased GLI1
expression in BCCs and with experimental in vivo data
showing overexpression of GLI1 resulting in BCC-like lesions
(21, 22, 24, 25). The concomitant reduction in
and
transcripts
with increased levels of the
form after TPA treatment is consistent
with a post-transcriptional modification of a single pre-mRNA
species. Although the precise mechanism is unknown, the activity of
transacting factors that control alternative splicing decisions have
been shown to be modulated by mitogens such as TPA (46-49). It can
also be reasoned that alternative splicing of the GLI1
message may be regulated by Sonic hedgehog signaling. We are currently
testing the possibility that unregulated signaling of the Sonic
hedgehog-Patched pathway (as occurs in BCCs) results in the generation
of increased levels of the
form through changes in the composition
of splicing factors bound to the nascent GLI1 pre-mRNA.
It is also conceivable that tumor-causing mutations could occur in the
splice sites of upstream noncoding exons that would enhance the
formation of the
form. Alternatively, mutations that alter the
regulation of factors involved in splice site selection may also
contribute to tumor formation. In addition, GLI1 has been shown to bind
RNA directly at a conserved motif that is distinct from the GLI DNA
binding cis-element and to regulate translation of the bound
mRNA (37, 50). Because these putative GLI binding motifs are
present in the pre-mRNA sequence of GLI1, an intriguing
possibility is that GLI1 itself or a related protein may determine exon
selection. Two recent reports have indicated that GLI2 and GLI3
transcriptionally regulate GLI1 by binding directly to
promoter sequences, but the same data do not preclude up-regulation of
GLI1 via a post-transcriptional mechanism by these factors
as well (28, 30).
Our results suggest that the
- and
-UTR variants contain potent
translation inhibitors and are in agreement with other studies showing
that 5' UTRs with uORFs and stable secondary structures are features of
poorly translated mRNAs (31-36). Furthermore, we have shown that
the uORFs present in the
- and
-UTRs play an important role in
mediating this inhibition. The increased GFP levels observed for the
form may be wholly attributable to the lack of uORFs, because
increasing the length of the
-UTR variant 4-fold only marginally
decreased GFP levels. However, it is also possible that the
-UTR
transcript may be more stable than the longer variants and consequently
is able to produce more product per transcript than the
- and
-UTR variants. Several studies have shown that impaired translation
can initiate mRNA decay and that both stem-loop structures and
uORFs within 5' UTRs can modulate mRNA stability (51). Regardless
of whether these sequences modulate translation efficiency or mRNA
stability (or both), the retention or skipping of 5' noncoding exons
generates alternative UTRs that differentially express a heterologous
reporter, and it is assumed of GLI1 as well. In conclusion, this study
suggests that alternative splicing is an important mechanism in the
regulation of this gene, which may in part contribute to the
up-regulation of GLI1 observed in BCCs.