(Received for publication, November 1, 1995)
From the
The Wilms' tumor (WT) suppressor gene, WT1, is mutated in a small set of WTs and is essential for proper development of the urogenital system. The gene has three sites of transcriptional initiation and produces mRNA transcripts containing 5`-untranslated regions of more than 350 nucleotides. The mRNA, through two alternative splicing events, is predicted to direct the synthesis of four protein isoforms with molecular masses of 47-49 kDa. In this report, we identify and characterize novel WT1 protein isoforms having predicted molecular masses of 54-56 kDa. Mutational analysis of the murine wt1 mRNA demonstrates that the novel isoforms are the result of translation initiation at a CUG codon 204 bases upstream of and in frame with the initiator AUG. We show that these isoforms are present in both normal murine tissue and in WTs. Like WT1, the larger isoforms localize to the cell nucleus and are capable of mediating transcriptional repression. Our results indicate that regulation of WT1 gene expression is more complex than previously suspected and have important implications for normal and abnormal urogenital system development.
Wilms' tumor (WT) ()is a embryonal malignancy
of the kidney that afflicts one out of every 10,000 children
(Matsunaga, 1981). Approximately 7-15% of sporadic WTs contain
detectable mutations in the tumor suppressor gene WT1 (Coppes et al., 1993; Little et al., 1992; Varanasi et
al., 1994). The WT1 gene encodes a transcription factor
belonging to the early growth response family of
Cys
-His
zinc finger proteins. The pre-mRNA is
alternatively spliced at two exons to produce four WT1 isoforms (Haber et al., 1991). The first alternatively spliced exon inserts or
removes 17 residues upstream of the four zinc fingers and is capable of
mediating transcriptional repression when fused to a DNA binding domain
(Wang et al., 1993a). The second alternatively spliced exon
inserts or removes 3 amino acids, KTS, between zinc fingers III and IV
and alters the DNA binding specificity of the protein, as well as its
subnuclear localization (Drummond et al., 1994; Larrson et
al., 1995; Rauscher et al., 1990). The -KTS WT1
isoforms recognize a GC-rich motif (5`-GCGGGGGCG-3`), as well as a
(TCC)
repeat (Rauscher et al., 1990; Wang et
al., 1993b), and can affect expression of a number of genes
harboring these motifs in their regulatory regions (for a review see
Rauscher(1994)). These genes include insulin-like growth factor II
(Drummond et al., 1992), insulin-like growth factor 1 receptor
(Werner et al., 1993), platelet-derived growth factor A-chain
(Gashler et al., 1992; Wang et al., 1992),
colony-stimulating factor-1 gene (Harrington et al., 1993),
transforming growth factor-
1 (Dey et al., 1994), the
retinoic acid receptor-
(Goodyer et al., 1995), and the wt1 gene itself (Rupprecht et al., 1994). WT1 can
mediate both transcriptional repression and activation, depending on
the architecture of the promoter under study (Madden et al.,
1991; Drummond et al., 1992, 1994; Wang et al.,
1992). Activation and repression are mediated by distinct domains
within the WT1 protein (Wang et al., 1993a).
In addition to its role as a tumor suppressor gene, WT1 plays an essential role in the normal development of the urogenital system. The expression pattern of WT1 is not ubiquitous, being mainly restricted to components of the urogenital system: the gonads, developing glomeruli, and the uterus (Pelletier et al., 1991a). Many children with germline WT1 mutations suffer from malformations of the urogenital system, ranging in severity from minor genital anomalies to streak gonads and renal nephropathy (for a review see Bruening and Pelletier(1994)). Consistent with a role for WT1 in the development of the urogenital system is the observation that this system fails to differentiate in wt1-null mice (Kreidberg et al., 1993).
Similar to many other genes involved in growth regulation, the wt1 mRNA transcript contains an AUG-initiated open reading frame (ORF) preceded by a long, GC-rich, 5`-untranslated region (UTR) (Nagpal et al., 1992). There are three sites of transcription initiation within the murine wt1 promoter, producing mRNA species with 5`-UTRs of 375, 700, or 720 nucleotides. In the course of characterizing the protein isoforms produced from these transcripts, we noted the presence of isoforms having molecular masses greater than expected. In this report, we demonstrate that these isoforms arise from translation initiation at a CUG codon upstream of and in frame with the wt1 initiator AUG. These novel isoforms are present in normal and malignant tissue and are capable of repressing transcription.
Although AUG codons are
essentially exclusively used as initiation codons for eukaryotic mRNAs,
there are rare examples of cellular mRNAs where other codons (GUG, ACG,
and CUG) are also used for this purpose. These include proto-oncogenes (MYC, INT-2, PIM-1, and LYL-1)
(Acland et al., 1990; Hann et al., 1988; Saris et
al., 1991; Mellentin et al., 1989), as well as the basic
fibroblast growth factor gene (Prats et al., 1989), retinoic
acid receptor 4 (Nagpal et al., 1992), krox-24 (also a member of the early growth response family) (Lemaire et al., 1990), and the ltk receptor (Bernards and de
la Monte, 1990). The nature of the signals that dictate the use of a
CUG codon as an initiation codon within the 5`-UTR are not well
understood, but immediate downstream sequences can influence the
efficiency with which CUG codons are selected (Boeck and Kolakofsky,
1994; Grünert and Jackson, 1994). Our results show
that WT1 gene expression is more complex than previously
suspected and that the activity of these novel WT1 isoforms needs to be
considered in biological assays involving WT1.
For immunoprecipitations, cells were grown to 80%
confluency, washed in PBS, and preincubated in methionine-free medium
for 1 h. Cellular protein was labeled with 200 µCi/ml of
[S]methionine for 12 h. Following a wash with
PBS, the cells were lysed in RIPA buffer. For each immunoprecipitation,
approximately 10
cpm of lysate was precleared with protein
A-Sepharose (Pharmacia Biotech Inc.) and preimmune serum for 1 h at 4
°C. Incubation with specific antibody and protein A-Sepharose was
then performed for 4 h at 4 °C, after which time the
immunoprecipitate was washed three times with RIPA buffer and once with
PBS. Elution was performed in SDS-PAGE sample buffer. The eluted
proteins were fractionated on a 10% SDS-PAGE gel, treated with
EN
HANCE (New England Nuclear), and detected by exposure to
X-Omat film (Kodak).
Figure 2:
Detection of Wt1 in sporadic
WTs. A, schematic diagram of the genomic organization of the
human WT1 gene. The nucleotide sequence and predicted
polypeptide sequence of both the human and murine genes upstream of the
AUG codon is indicated. The white boxes represent
alternatively spliced exons, whereas hatched boxes represent
the zinc fingers. Each exon is numbered with a roman numeral. The
relative position of two CUG codons present upstream from and in frame
with the initiator AUG codon in the human and murine 5`-UTRs is shown.
The putative CUG initiation codons are underlined, whereas the
initiator AUG codon is underlined and in bold. The
peptide sequence used to generated the polyclonal serum S3 is boxed. Amino acid homology of the predicted amino-terminal
extension from the human and murine cDNAs is presented. Black
circles signify identity, whereas open circles signify
conservative changes. The methionine encoded by the initiator AUG is
denoted in bold. hWT1, human WT1 cDNA
sequence; mWT1, murine wt1 cDNA sequence; hwt1, human WT1 polypeptide sequence; mwt1, murine
Wt1 polypeptide sequence. B, analysis of WT1
in
WTs. A Western blot of nuclear extracts from murine TM3 cells (lane
1) or whole cell extracts from WTs (lanes 2-8) were
probed with anti-WT1
serum, S3. The WT1
isoforms are indicated by two arrows. Molecular mass
markers (in kDa) are indicated to the right of the
panel.
Figure 1:
Detection of a novel Wt1 isoform in
vivo. A, Northern blot analysis of RNA isolated from
3-day-old murine testis (lane 1) and TM3 cells (lane
2). The arrows indicate the positions of migration of the wt1 mRNA species. The position of migration of the 28 S and 18
S rRNA species is indicated to the right. The upper panel has been hybridized with a murine wt1 cDNA fragment. The bottom panel is the same blot reprobed with P-labeled
-actin. B, immunoprecipitation
from TM3 cells using the polyclonal anti-Wt1 605 antibody.
Immunoprecipitations performed on
[
S]methionine-labeled cell extracts were
resolved on a 10% SDS-PAGE gel. The 47-49-kDa Wt1 isoforms are
indicated by a dot and are not well resolved on this gel. The
57-kDa related protein is denoted by a
. Extracts were
prepared from untransfected COS-1 cells (lanes 1 and 4), COS-1 cells transfected with
CMV/wt1(-/-) (lanes 2 and 5), or
TM3 cells (lanes 3 and 6). Samples were
immunoprecipitated with the antibody 605 (lanes 1-3) or
with preimmune serum (lanes 4-6). Following
electrophoresis, the gel was treated with EN
Hance (New
England Nuclear), dried, and exposed to X-Omat (Kodak) film at
-70 °C for 2 days. C, Western blot analysis of
nuclear extracts prepared from COS-1 and TM3 cells. Blots were probed
with monoclonal antibodies 8A7 (lanes 1-3) and 13B5 (lanes 4-6), which recognize WT1 isoforms lacking or
containing the first alternatively spliced exon, respectively. Extracts
were prepared from untransfected COS-1 cells (lanes 1 and 4), COS-1 cells transfected with
CMV/wt1(-/-) (lane 2),
CMV/wt1(+/-) (lane 5), or TM3 cells (lanes 3 and 6). The dot indicates the
position of migration of the 47-49-kDa Wt1 isoforms; whereas the
delineates the position of the
57-kDa isoforms. Blots were
exposed to X-Omat film (Kodak) for 2 min. D, expression of
Wt1
in murine ovaries. Extracts were prepared from
untransfected COS-1 cells (lane 1), COS-1 cells transfected
with CMV/wt1(-/-) (lane 2), COS-1 cells
transfected with CMV/wt1(+/+) (lane 3), and
adult murine ovaries (lane 4). The arrows indicate
the positions of migration of the Wt1 isoforms. The blot was probed
with the antibody C-19 and exposed to X-Omat (Kodak) film for 5
min.
To detect Wt1 protein from TM3 cells, we made use of the polyclonal
anti-Wt1 antibody, 605. This antibody, directed against amino acids
123-299, is capable of immunoprecipitating
[S]methionine-labeled Wt1 from COS-1 cells
transfected with a CMV-wt1 expression vector (denoted by a dot in Fig. 1B, lane 2). This
polypeptide is not recognized by preimmune serum (Fig. 1B, lane 5), nor is it
immunoprecipitated from untransfected COS-1 cells (Fig. 1B, lane 1). In immunoprecipitates from
TM3 cells with the 605 antibody, we noted the presence of Wt1 protein (lane 3, denoted by a dot) as well as a polypeptide
of
57 kDa (indicated by a
in Fig. 1B, lane 3). Like Wt1, this polypeptide is not visible in
immunoprecipitates of TM3 cells with preimmune serum (Fig. 1B, lane 6).
To extend these results
and determine whether the 57-kDa polypeptide species was related
to Wt1, we made use of two anti-WT1 monoclonal antibodies (Mundlos et al., 1993). Antibody 8A7 specifically recognizes WT1
isoforms lacking the 17 amino acids introduced by alternative splicing
of exon 5 (-17 aa), whereas 13B5 specifically recognizes WT1
isoforms containing this alternatively spliced exon (+17 aa)
(Mundlos et al., 1993). On Western blots of COS-1 cells
transfected with CMV/wt1(-/-), 8A7 recognizes the
Wt1(-/-) protein isoform as expected (denoted by a dot, Fig. 1C, lane 2). This
polypeptide is not present in untransfected COS-1 cells (Fig. 1C, lane 1). In TM3 cells, a
57-kDa
polypeptide is also recognized by this antibody (indicated by a
in Fig. 1C, lane 3). Antibody 13B5 recognizes
the Wt1(+/-) isoform produced in COS-1 cells transfected
with CMV/wt1(+/-) (indicated by a dot in Fig. 1C, lane 5). In TM3 cells, a
57-kDa
polypeptide is also detected with this antibody (indicated by a
in Fig. 1C, lane 6). Upon prolonged exposure
of these blots, the 47-49-kDa wt1 isoforms can also be
detected in extracts of TM3 cells (data not shown). These results
strongly suggest the existence of novel Wt1 isoforms containing or
lacking the Wt1 exon V and having molecular masses
10 kDa higher
than Wt1. In this manuscript, we will refer to these larger isoforms as
Wt1
to distinguish them from the 47-49-kDa Wt1
isoforms.
To determine whether Wt1 could be detected in
normal tissues, nuclear extracts were prepared from murine ovaries and
probed with the anti-WT1 antibody, C-19, directed against the WT1
carboxyl terminus. Extracts prepared from COS-1 cells transfected with
CMV/wt1(-/-) (Fig. 1D, lane
2) or CMV/wt1(+/+) (Fig. 1D, lane 3) produced the appropriate wt1 isoforms and
acted as standards on this blot. No Wt1 protein was present in
untransfected COS-1 cells (Fig. 1D, lane 1).
Western blot analysis of nuclear extracts from murine ovaries revealed
the presence of the 47-49-kDa Wt1 isoforms, as well as the
Wt1
isoforms (Fig. 1D, lane 4). A
similar result was obtained with nuclear extracts from murine testis
(data not shown). These results indicate that Wt1
is
present in normal murine tissue.
Figure 3:
In
vitro synthesis of Wt1. A, diagram of in vitro
wt1 expression vectors. The black boxes represent the
coding regions, whereas the stippled and hatched boxes represent the murine and human 5`-UTRs, respectively. The dotted and checkered boxes represent the T7 and SP6
RNA polymerase promoters. The small black rectangle above the
murine 5`-UTR represents a small ORF present in this portion of the
cDNA. The two black dots represent the upstream CUG codons
highlighted in Fig. 2A. B, in vitro translation products obtained from RNA generated by 5`-UTR
deletion mutants. RNA was generated from constructs shown in A and translated in rabbit reticulocyte lysate at a mRNA
concentration of 16 µg/ml. RNA prepared from the following vectors
was used as input template: lane 1, no DNA; lane 2,
pKS/mwt1[+182]; lane 3,
pKS/mwt1[+475]; lane 4,
pSP/hWT1[+200]; lane 5,
pKS/hWT1[+378]. 3 µl of the translation
reaction were fractionated on a 10% SDS-PAGE gel. Following
electrophoresis, the gel was treated with EN
Hance, dried,
and exposed to Fuji x-ray film at -70 °C for 12
h.
To define
the codon responsible for directing translation initiation of
Wt1, a series of deletion and site-directed mutants were
generated within the murine 5`-UTR (Fig. 4A). In
addition to truncating various portions of the 5`-UTR, stop codons were
introduced that flanked the CUG codons identified above (Fig. 2A), and mutations affecting the nature of each
CUG codon were generated. Deletions within the first 507 nucleotides
from the 5`-UTR of the wt1 mRNA did not abolish production of
Wt1
(Fig. 4B, lanes 2-5),
whereas a deletion removing all but 182 nucleotides of 5`-UTR generated
a transcript unable to produce Wt1
(Fig. 4B, lane 11). Deletion of the Wt1
initiator AUG codon abolished production of Wt1, instead generating a
polypeptide having a molecular mass of
37 kDa (Fig. 4B, lane 12, indicated by a small
arrow). The molecular mass of this polypeptide is similar to that
expected from internal initiation of ribosomes at a downstream, in
frame AUG codon at position 378 (relative to the A of the initiator AUG
codon). Introduction of a UGA codon 229 nucleotides upstream from and
in frame with the AUG initiator codon did not affect production of
Wt1
(Fig. 4B, lane 6), indicating
that the Wt1
initiation codon must lie downstream of this
site. On the other hand, placing a UGA codon at position 183, in frame,
and upstream of the initiator codon generated an mRNA no longer capable
of synthesizing Wt1
(Fig. 4B, lane
7). These data suggest that the signals responsible for Wt1
translation initiation lie between nucleotides +183 and
+229. Site-directed mutagenesis was used to abolish the individual
CUG codons at position 204 and 192 (see Fig. 2A).
Converting CUG
to CUC
drastically affected
expression of Wt1
while having little effect on production
of Wt1 (Fig. 4B, lane 8). Expression of
Wt1
was reduced
10-fold but not completely abolished.
Mutagenesis of CUG
to CUC
had no effect on
production of Wt1
(Fig. 4B, lane
9), and mutagenesis of both CUG codons had the same effect as
altering only CUG
(Fig. 4B, lane
10). These results strongly indicate that translational initiation
at CUG
is responsible for production of Wt1
.
We interpret the residual production of Wt1
observed with wt1[+475](CTC@204) and wt1[+475](CTC@204/192) to indicate that
the CUG to CUC mutation is leaky and that nucleotide sequences flanking
the CUG codon are influencing translation initiation at this site
(Boeck and Kolakofsky, 1994; Grünert and Jackson,
1994).
Figure 4:
Effect of wt1 5`-UTR deletion and point mutations on translation
initiation. A, diagram of deletion and point mutations
generated within the wt1 5`-UTR. The black boxes indicate the ORFs, the hatched boxes represent the
5`-UTRs, and the stippled boxes represent the SP6 RNA
polymerase promoters. The two upstream CUGs at nucleotides 204 and 192
are represented by filled circles, and the black
rectangular boxes above the 5`-UTRs represent short ORFs. The
position of the initiator AUG and stop codons are shown, as well as the
position of a downstream, in frame AUG codon. Stars represent
UGA stop codons, whereas small open boxes represent CUG to CUC
mutations. B, in vitro translation of RNA produced
from deletion and point mutations within the 5`-UTR. Indicated above
each lane is the SP6 construct from which the input RNA was derived. A dash represents no input RNA. The position of migration of Wt1
and Wt1 are indicated. The smaller arrow indicates
the position of migration of the truncated Wt1 polypeptide predicted to
arise from translation initiation at the downstream AUG codon.
Following electrophoresis in a 10% SDS/polyacrylamide gel, the gel was
treated with EN
Hance, dried, and exposed to X-Omat (Kodak)
film at -70 °C with an intensifying screen
overnight.
It is clear from the translation results obtained with wt1[+475](TGA@229) and wt1[+475](TGA@183) (Fig. 4B) that the Wt1 translation codon
must lie between nucleotides +183 and +229. To rule out the
possibility that the initiator codon for Wt1
was upstream
or downstream of CUG
and that the effects on translation
initiation of mutating CUG
were not the result of
altering a motif that indirectly influenced the efficiency of
initiation, four additional constructs were generated (Fig. 5A). One of these, wt1[+202], retains 202 base pairs of 5`-UTR and
terminates at the G residue of CUG
. Should translation of
Wt1
commence downstream of CUG
, then this
construct should generate RNA competent for Wt1
production.
Two other mutants alter the identity of CUG
, converting
it either to the more efficient AUG initiation codon, wt1[+475](ATG@204), or to a stop codon, wt1[+475](TGA@204). wt1[+475](
229-205) generates a
24-base pair in-frame deletion immediately upstream of the CUG
codon and will abolish production of Wt1
only if the
initiation codon lies upstream of CUG
. Following in
vitro transcription/translation of these plasmids and their
respective mRNAs, the [
S]methionine-labeled
protein products were analyzed by SDS-PAGE. Translation of wt1[+475] produced both Wt1
and Wt1
as expected (Fig. 5B, lane 2). Only Wt1 was
produced following translation of RNA synthesized from wt1[+202], consistent with the notion that the
Wt1
initiation codon does not lie downstream of
CUG
. Mutation of CUG
to an AUG codon
increased the production of Wt1
(Fig. 5B, lane 4), consistent with the assignment of CUG
as the initiation codon. Conversion of CUG
to a UGA
codon abolished production of Wt1
, whereas production of
Wt1 was still observed (Fig. 5B, lane 5).
Removal of nucleotides 229-205 affected the overall translational
efficiency of the mRNA produced (Fig. 5B, compare
intensity of products in lane 7 with those in lane
6); however, the production of both Wt1
and Wt1 was
still observed (Fig. 5B, lane 7), indicating
that the Wt1
initiator codon does not lie upstream of
CUG
. These results indicate that CUG
is the
Wt1
initiation codon.
Figure 5:
Translation analysis of mutations at or
upstream from CUG. A, schematic diagram of
mutations generated within the wt1 5`-UTR. The black boxes indicate the wt1 ORFs, the hatched boxes represent the 5`-UTRs, and the stippled boxes represent
the SP6 RNA polymerase promoter. The two upstream CUGs at nucleotides
204 and 192 are represented by filled circles, and the filled rectangular boxes above the 5`-UTRs represent short
ORFs. The nucleotide sequence flanking CUG
is shown for
each construct. The triplet at position 204 is underlined. The open triangle signifies a deletion. A star represents
a stop codon, and an open circle represents an AUG codon. B, in vitro translation of RNA with mutations at or
upstream from CUG
. Indicated above each lane is the SP6
construct from which the input RNA was derived. A dash represents no input RNA. The position of migration of Wt1 and
Wt1
are indicated by an arrow and an arrowhead, respectively. Translation products were analyzed as
described in the legend to Fig. 3and Fig. 4.
Figure 6:
Nuclear localization of Wt1. A, schematic representation of expression vectors used in this
study. A black box signifies the wt1 ORF, a hatched box represents the 5`-UTR, and a stippled box represents the HA epitope tag (Pelletier et al., 1991a),
whereas a checkered box signifies the CMV promoter.
CUG
is represented by a filled circle. B, Western blot analysis of COS-1 cells transfected with wt1 expression vectors. Cell extracts were prepared and
processed for immunoblotting with the anti-HA antibody 12CA5 as
described previously (Goodyer et al., 1995). C and D, immunofluroescent detection of wt1 and wt1
. COS-1 cells were electroporated in PBS +
20 mM Hepes, pH 7.5, at 260 V/960 microfarads with 10 µg
of plasmid DNA. The cells were seeded into chamber slides and allowed
to recover for 48 h. After washing with PBS, the cells were fixed for
20 min in 3.7% formaldehyde/PBS followed by 5 min in methanol at
-20 °C. Slides were blocked with 10% goat serum/0.3% Triton
X-100 and probed with the anti-HA antibody 12CA5. Protein was
visualized with an fluorescein isothiocyanate-conjugated goat
anti-mouse IgG (Jackson Labs). After washing, the cells were mounted in
Immunomount (Fisher) and photographed using T-Max 100 black and white
film (Kodak). Cells were transfected with
CMV/wt1[+42] (C) or with
CMV/wt1[HA/wt1
] (D).
Figure 7:
Trans-repression by Wt1 and
Wt1. A, schematic representation of vectors used
in this study. See legend to Fig. 6for details. The cross-hatched box represents the cat gene, whereas
the TK promoter is shown by an open box. The two dark
rectangles within the TK promoter illustrate GAL4 binding sites. B, CAT assays of transient transfections performed with the
expression and reporter vectors in A. NIH 3T3 cells were
transfected by calcium phosphate and allowed to recover for 48 h. The
cells were lysed by freezing and thawing 3 times in 0.25 M Tris, pH 8.0. Following removal of the cellular debris by
centrifugation (12,000
g for 5 min), CAT assays were
performed on the supernatant as described previously (Gorman, 1985). A
vector containing the CMV promoter driving expression of the
-gal gene was included in all transfections and used to
standardize efficiencies. CMV/
is the backbone vector used to
generate the expression vectors in A but lacking a gene
downstream of the CMV promoter (Goodyer et al.,
1995).
The WT1 gene products are necessary for regulating
normal differentiation of the urogenital system. Mutations in the WT1 gene result in malformations of the urogenital system as
well as predispose to WTs. The structure of the WT1 gene
indicates that it should produce four alternatively spliced mRNAs that
direct the synthesis of isoforms having molecular masses of 47-49
kDa. In this report, we demonstrate that WT1 gene expression
is more complex than anticipated, with a non-AUG translational
initiation event producing additional protein isoforms of 54-56
kDa (WT1). We have found WT1
to be expressed in
a number of tissues and cells including murine TM3 cells (Fig. 1), murine ovaries (Fig. 1), murine testis (data
not shown), immortalized rat granulosa cells (data not shown), and
human K562 cells (data not shown). We have been unable to accurately
establish the relative ratio of WT1 to WT1
in these cells
and tissues, because the antibodies we are employing recognize the
isoforms with different efficiencies (e.g.Fig. 1,
compare C with B), perhaps due to differences in
protein conformation or isoform-specific post-translational
modifications. However, our data directly demonstrates that WT1
isoforms containing or lacking the first alternatively spliced
exon are generated. Using isoform-specific antibodies, we were able to
detect both isoforms in TM3 cells (Fig. 1C). In
addition, two WT1
isoforms are clearly detectable on
Western blots of whole cell extracts of WTs (Fig. 2B).
Although we have not analyzed the presence or the absence of the second
alternative splice site within WT1
, there is no reason not
to suspect its presence. Our data, taken together with the documented
alternative splicing of the WT1 gene (Haber et al.,
1991) and RNA editing (Sharma et al., 1994), indicate that
this gene may produce as many as 16 different protein isoforms. To
date, WT1 functional studies have generally been performed using
expression vectors capable of producing only the 47-49-kDa
isoforms. Given that the WT1 gene products can oligomerize
(Reddy et al., 1995; Moffett et al., 1995) and that
the isoform ratio is important for proper urogenital system development
(Bruening et al., 1992), functional studies need to take into
account the contribution of WT1
isoforms to the overall
biological function of the WT1 gene, including the possible
interplay of the various isoforms.
Our data indicate that
CUG is the initiation codon responsible for directing
synthesis of Wt1
. A number of mRNAs use non-AUG initiation
events to generate amino-terminally extended isoforms (see
Introduction). The Wt1 initiator CUG is in a favorable consensus
sequence (5`-XXAXXCUGG-3`, where the important
nucleotide residues are underlined) (Kozak, 1987), and these likely
play a role in directing translational initiation. The identity of the
codon at this position is important in determining the efficiency with
which this site is recognized, because mutagenesis of the initiation
codon CUG
to CUC
significantly reduced
(
15-fold) the amount of Wt1
produced (Fig. 4).
However, it is clear that additional signals influence initiation at
this site since the CUG to CUC mutation reduced but did not completely
abolish translation initiation (Fig. 4). Surprisingly, deleting
24 nucleotides immediately upstream of CUG
significantly
reduced the overall translational efficiency of the template mRNA (Fig. 5B, compare lanes 7 and 6).
Sequences downstream of non-AUG initiation codons have been shown to be
important for determining efficiency of recognition of the start codons
(Boeck and Kolakofsky, 1994; Grünert and Jackson,
1994). However, in the case of the wt1 template, the deletion
of sequences upstream of CUG
has a global effect on
translation initiation, including at the downstream AUG codon. In the
case of fibroblast growth factor-2 mRNA, a ribosome landing pad is
postulated to directly recruit 43S preinitiation complexes and guide
them to a non-AUG codon (Vagner et al., 1995). An internal
ribosome binding site near CUG
on the wt1 mRNA
template could account for the effects seen on global initiation by wt1[+475](
229-205) (Fig. 5B). Alternatively, the overall stability of the
template may be affected by this 24-nucleotide deletion.
The degree
of conservation between the amino-terminal extension of the murine and
human WT1 isoforms is not as high as between the WT1
proteins. At the amino acid level, the murine and human WT1 isoforms
are 96% identical (Buckler et al., 1991), but the
amino-terminal extensions of the human and murine WT1
isoforms are only 65% identical. However, the first 35 amino
acids of WT1
are quite conserved (88% identical) and may
encode a functional domain. Alternatively, overall charge or
conformation may be more important to the function of this domain
rather than primary sequence per se.
We have analyzed the
genomic region encoding the amino-terminal extension of WT1 for possible mutations in WTs using polymerase chain reaction
single stranded conformational polymorphisms (Varanasi et al.,
1994).
Examination of this region in over 100 WTs failed to
detect any mutations. This is not particularly surprising because the
majority of WT1 mutations occur within the DNA binding domain
(Bruening et al., 1992; Pelletier et al., 1991b;
Varanasi et al., 1994) with very few mutations occurring
outside of this region.
Several investigators have noted the
presence of a 54-56-kDa contaminating polypeptide in studies
involving immunoprecipitation of WT1. Given our results, these
polypeptides likely represent WT1
isoforms generated by
translation initiation at CUG
. The presence of such
isoforms is apparent in Fig. 3F of Larrson et
al.(1995), displaying an immunoprecipitation on nuclear extracts
with anti-WT1 antibodies from the mesonephric cell line, M15. A protein
with similar electrophoretic properties as WT1
was also
detected by Rackley et al.(1993) on an immunoblot analysis of
nuclear extracts prepared from the WT1-expressing kidney cell
line, 293 (see Fig. 3a; Rackley et al.(1993)).
The functional significance of producing WT1 isoforms with an
amino-terminal extension is not obvious. The amino-terminal domain on
its own does not have a strong trans effect on transcription
because fusion constructs containing this domain fused to the GAL4 DNA binding domain did not significantly affect transcription of
an appropriate reporter in transient transfection experiments.
This domain may be involved in a number of other activities such
as heterodimer formation, subnuclear localization, or regulation of WT1
intrinsic activity. Our description of a non-AUG translation event at
the WT1 locus indicates that the overall complexity of WT1 regulation is greater than previously suspected. Biological assays
involving all WT1 isoforms should present a better picture of the role
of WT1
in normal and abnormal urogenital system
development.