1 MRC Human Genetics Unit, Western General Hospital, Crewe Rd, Edinburgh EH4
2XU, UK
2 School of Biology, Bute Medical Buildings, University of St Andrews, St
Andrews, Fife KY16 9TS, UK
* Present address: Centre for Research in Biomedicine, Faculty of Applied
Sciences, University of the West of England, Coldharbour Lane, Bristol BS16
1QY, UK
Department of Anatomy and Developmental Biology, University College London,
Gower Street, London WC1E 6BT, UK
Author for correspondence (e-mail:
nick.hastie{at}hgu.mrc.ac.uk)
Accepted 16 December 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Wilms' tumour suppressor, C2-H2 zinc fingers, ribonucleoprotein particles (RNP), Density gradients, Xenopus oocytes, Lampbrush chromosomes, B-snurposomes, Nucleoli
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Further structural complexity arises from alternative splicing. Of
particular interest is an evolutionarily conserved alternative splice that
inserts three amino acids (KTS) at the C-terminal end of the -helix of
zinc finger three. Structural studies have shown that the +KTS insertion
severely disrupts DNA binding (Laity et
al., 2000a
; Laity et al.,
2000b
; Laity et al.,
2000c
). The ratio of +KTS:KTS isoforms is tightly
controlled, and perturbations in this ratio are implicated in the aetiology of
Frasier syndrome, which is characterised by severe urogenital defects,
including sex reversal (Barbaux et al.,
1997
; Klamt et al.,
1998
). A recent mouse model has shown that the two isoforms have
distinct yet overlapping functions. By knocking out the ability of cells to
make either +KTS or KTS protein, Hammes and colleagues have shown that
mutants of both strains die after birth because of kidney defects. However,
mice specifically lacking the +KTS isoform (Frasier mice) show a complete XY
sex reversal as occurs in the human syndrome
(Hammes et al., 2001
).
WT1 is a multifunctional transcription factor and several candidate target
genes are known. Thus, WT1 represses the IGFIR gene
(Werner et al., 1994),
activates the amphiregulin (Lee
et al., 1999
), bcl-2
(Mayo et al., 1999
) and
SF1 genes (Wilhelm and Englert,
2002
), and co-activates the MIS gene
(Nachtigal et al., 1998
).
However, evidence suggests that WT1 is also involved in post-transcriptional
processes. WT1 (+KTS) colocalises and co-immunoprecipitates with splice
factors (Larsson et al.,
1995
), binds to the essential splice factor U2AF65 and associates
with active splice complexes (Davies et
al., 1998
). WT1 also binds to WTAP (Wilms tumour associated
protein) (Little et al.,
2000
), a nuclear protein with strong homology to the
Drosophila protein FL(2)D. FL(2)D is required for female-specific
splicing of Sxl and Tra pre-mRNAs mediated by alternative
3' splice site choice (Penalva et
al., 2000
). WT1 zinc fingers, both in the + and KTS
conformations, bind to RNA in vitro. In particular, zinc finger one is
required for binding to GC-rich RNA derived from exon 2 of the mouse
Igf2 gene (Caricasole et al.,
1996
). A SELEX (in vitro selection) assay using WT1 zinc fingers
resulted in the definition of three RNA aptamers
(Bardeesy and Pelletier, 1998
).
WT1 zinc fingers bind to the RNA aptamers with dissociation constants ranging
from 13.8 to 87.4 nM (Zhai et al.,
2001
). Consistent with its ability to bind to RNA, WT1 is present
in poly(A)+ nuclear RNP isolated from cell lines and fetal kidneys
(Ladomery et al., 1999
).
The aim of this study was to search for evidence that WT1 associates with transcripts in vivo. We focused on the role of zinc finger one and the ability of +/KTS isoforms to locate to intranuclear structures, and compared the properties of WT1 with the related transcription factor EGR1. Our approach was to express tagged constructs in mammalian cells and to exploit the many advantages of the Xenopus oocyte system.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nuclear and whole-cell extract preparation
Medium was removed and cells rinsed with 2x ice-cold
phosphate-buffered saline (PBS). Cells were scraped with a cell scraper and
collected in RNase-free tubes. Cells were centrifuged for 3 minutes at 4000
rpm at 4°C, the supernatant removed, and the pellet raised in 600 µl
(sufficient for each 14 mm confluent plate) of hypotonic swelling buffer (10
mM Tris-HCl, pH 7.4; 10 mM NaCl; 3 mM MgCl2; Boehringer Mannheim
protease inhibitor cocktail). Cells were left on ice for 10 minutes; 35 µl
of 10% Nonidet-P40 was added to the cells, which were then mixed by short
vortexing for 2 seconds and centrifuged for 1 minute at 13,000 rpm in a
microfuge. When required, the supernatant (cytoplasm) was kept aside to
combine with nuclear extract. The pellet (nuclei) was resuspended in an equal
volume of lysis buffer (20 mM Hepes, pH 7.9; 600 mM KCl; 0.2 mM EDTA; 20 U/ml
DNAse I (Boehringer Mannheim), 10 U/ml Superase RNase inhibitor cocktail
(Ambion) and protease inhibitor cocktail (Boehringer Mannheim). The
resuspension was left on ice for 30 minutes, with mixing every 10 minutes; it
was then centrifuged for 10 minutes at 13,000 rpm in a microfuge to remove
insoluble material.
Transient transfection into mammalian cells
Plasmids encoding T7-tagged proteins were generated as previously described
(Ladomery et al., 1999). Ten
micrograms of each plasmid was transfected into Cos7 cells by electroporation
(1.00 kV; 25 µF), and into AC29 and HeLa cells using Lipofectamine
(Gibco-BRL) as per manufacturer's specifications. Expression was tested by
western blotting and immunofluorescence was measured using a mouse monoclonal
antibody directed against the T7 epitope (Novagen). Two days after
transfection, cells were fixed for 10 minutes in 1:1 acetone:methanol, and
blocked in 2% BSA in PBS, 7% (v/v) glycerol and 0.02% (v/v) sodium azide.
Primary antibody dilutions used were 1:1000 (anti-T7 mouse monoclonal) and
secondary dilutions 1:100 (FITC-conjugated goat anti-mouse; Sigma
Immunochemicals). Immunofluorescence was observed and recorded using a Zeiss
Axioplan 2 microscope, 63x objective, with a Micro Imager 1400.
Nycodenz density gradients
Nuclear extracts containing 1.5 mg total protein in a volume of 300
µl were dialysed against Nycodenz (Sigma) gradient low-salt buffer (20 mM
Tris-HCl, pH 7.5; 2 mM MgCl2; 1 mM EDTA) and loaded onto a
pre-formed 5 ml gradient of 20-60% Nycodenz dissolved in the above buffer.
Samples were spun at 154,000 g for 18 hours at 0°C in a
Sorvall AH650 rotor. Gradients were manually fractionated into 18 samples of
250 µl. The density of the samples was determined by measuring the
refractive index and applying the formula, density (
) in
g/cm3=3.242
3.323, where
is the refractive index.
Proteins were analysed by adding SDS-PAGE buffer directly; the presence of
Nycodenz presented no hindrance to pipetting these samples onto SDS-PAGE gels.
Fractions were western blotted to detect the T7 tag (mouse monoclonal,
Novagen), p116 (rabbit polyclonal, gift of P. Fabrizio), U2AF65 (rabbit
polyclonal, gift of R. Davies) and EGR1 (rabbit polyclonal sc-189, Santa Cruz
Biotech.). For RNA extraction (see below), samples were precipitated by
diluting the Nycodenz fractions threefold with distilled water and adding
three volumes of ethanol. They were left overnight at 20°C and spun
for 20 minutes at maximum speed on a microfuge.
RNA extraction
RNA lysis buffer, 0.5 ml (4 M guanidine isothiocyanate; 25 mM sodium
citrate, pH 7; 1 mM ß-mercaptoethanol; 0.5% w/v sodium N-laurylsarcosine;
0.1% v/v Sigma Antifoam A), was added to precipitated samples. Then 50 µl 2
M sodium acetate pH 4 was added, and the sample vortexed. Water-saturated
phenol, 0.5 ml, was then added, not pH adjusted, containing 0.1% v/v
hydroxyquinoline. Chloroform, 0.2 ml, was added and the sample vortexed. After
spinning for 20 minutes at 13,000 rpm in a microfuge at 4°C, 2 volumes of
ethanol were added to the water phase, and the sample vortexed. Samples were
precipitated overnight at 20°C then spun for 30 minutes at 13,000
rpm in a microfuge. Pellets were washed in 70% ethanol and the pelleted RNA
was dissolved in 20-40 µl DEPC-treated water, to which 10 U Superase RNAse
inhibitor cocktail (Ambion) was added.
Analysis of RNA by RT-PCR and end-labelling
RNA samples (500 ng) were reverse transcribed as per cDNA synthesis kit
specifications (Boehringer-Mannheim). cDNA samples were quantified and diluted
such that 10 ng template was used in each PCR. Each 30 µl PCR reaction
contained 10 pmol of each primer, and was run for 30 cycles (45 seconds at
95°C, 45 seconds at 55°C and 2 minutes at 72°C). Product sizes
were compared against DNA markers on 2% agarose gels. The following primers
were obtained (Genosys) to amplify WT1 pre-mRNA: forward,
5'-CAGTAGTATCCAGGGTGGTGG-3', derived from the 3' end of
mouse intron 9; reverse, 5'-CCGACAGCTGAAGGG-CTTTTCAC-3', 5'
end of exon 10, yielding a 380 bp product. For end-labelling, 50-500 ng RNA
samples were labelled using T4 RNA ligase (Boehringer Mannheim) and pCp
(32P), using the method of England et al.
(England et al., 1980), and
separated on a 5% acrylamide/8M urea gel in 1 x TBE buffer
(Tris-borate-EDTA).
Oocyte isolation, injection and labelling
Oocytes were isolated from Xenopus laevis as described previously
(Ryan et al., 1999) and were
maintained in OR-2 medium (Evans and Kay,
1991
). For microinjection, sets of 20-40 mid-vitellogenic (stage
III/IV) oocytes were isolated and 10 nl aliquots containing
10 pg of
purified plasmid DNA were injected, through the animal pole, into the nucleus
of each oocyte. RNA was labelled in vivo by injecting oocytes with 0.1 µCi
of [3H]uridine (27 mCi/mmole, Amersham) or with 0.1 µg of BrUTP
(Sigma). Transcription could be inhibited by incubating the oocytes in OR-2
containing 5 µg/ml actinomycin D. Radioactivity incorporated into RNA was
measured after extraction at 60°C with phenol/chloroform buffered to pH
4.2 and precipitation with 2.5 vol ethanol.
Immunoblotting of oocyte extracts
At 18 or 30 hours after injection, nuclei and cytoplasms were isolated
under mineral oil (Sigma) from groups of 20-40 oocytes
(Ryan et al., 1999). Yolk and
lipid were extracted from cytoplasms with 1,1,2-trichlorotrifluoroethane
(Evans and Kay, 1991
) and,
after centrifugation at 13,000 rpm for 2 minutes in a microcentrifuge, the
clarified supernatant was carefully removed and mixed with an equal volume of
SDS-PAGE sample buffer (Sigma). The nuclei were disrupted by sucking up and
down through a fine pipette tip before solubilisation in sample buffer.
Proteins, equivalent to one cytoplasm or to four nuclei, were separated by
SDS-PAGE, transferred to nitrocellulose (Protran, Schleicher and Schull) and
immunoblotted as described previously
(Ryan et al., 1999
). A
monoclonal antibody against the T7 epitope (Novagen) and peroxidase-conjugated
(HRP) anti-mouse immunoglobulin G (IgG) (Sigma) were both used at a dilution
of 1:10,000 and bands were developed using the ECL (Amersham) procedure.
Immunostaining of germinal vesicle spreads
Germinal vesicle (GV) spread preparations from injected oocytes were
prepared at 18 or 30 hours post-injection as described
(Sommerville et al., 1993).
For salt stability studies the preparations were washed sequentially in PBS
(Sigma) adjusted to 200, 400 and 600 mM NaCl and stored in 70% ethanol. Before
immunostaining, nonspecific binding was blocked by incubating the preparations
for 30 minutes in PBS containing 0.05% Tween 20 (PBST) and 10% FCS. Monoclonal
anti-T7 epitope (Novagen) and monoclonal anti-bromodeoxyuridine (BrdU) (Sigma)
were each used at a dilution of 1:5000 in 10% FCS/PBST, and rabbit polyclonal
anti-p116 was used at a dilution of 1:1000. The secondary antibodies were
FITC-conjugated goat anti-mouse IgG and TRITC-conjugated goat anti-rabbit IgG
(Sigma), both at a dilution of 1:5000 in FCS/PBST. After extensive washing in
PBST the preparations were mounted in 50% glycerol, 1 mg/ml
p-phenylenediamine, pH 8.5, containing 20 ng/ml of
4,6-diamidino-2-phenylindole (DAPI) and viewed using a Leitz Ortholux
fluorescence microscope. Images were recorded on Kodak Ektachrome P1600
Professional film.
Immunoprecipitation and salt extraction
Anti-T7 IgG (2 µl) was linked to 20 µl of protein A-glass beads
(ProSep, Bioprocessing Ltd) as described previously
(Ryan et al., 1999). GVs
isolated from 50 injected oocytes were needle-sheared in 100 µl of a
solution containing: 100 mM NaCl; 1 mM MgCl2; 0.1% Nonidet P-40; 20
mM Tris-HCl, pH 7.5 (TBSMN). Extracts were mixed with 5 µl of the
antibody-beads for 60 minutes at 20°C. The beads were washed with TBSMN to
remove unbound material and then extracted sequentially with 100 µl of
TBSMN adjusted to 200, 400, 600 and 1000 mM NaCl. Protein from each of the
wash fractions was precipitated with four volumes of acetone, pelleted,
air-dried and immunoblotted.
Anion-exchange chromatography
Nuclear extract (1.5 mg) in 2 ml binding buffer (20 mM Tris-HCl, pH 7.5; 50
mM NaCl; 1.5 mM MgCl2) was applied to a 1 ml Q-Trap anion-exchange
column (Amersham Pharmacia Biotech). After reapplyling the extract to the
column twice, the flow through was collected, and a salt gradient applied, 100
mM up to 1 M NaCl in 100mM steps, 2x0.5 ml aliquots of buffer at each
salt concentration. Samples were precipitated by adding 2 volumes of ethanol,
left overnight at 20°C and spun at 13,000 rpm in a microfuge for 30
minutes. One third of each sample was set aside for RNA extraction.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Co-sedimentation of WT1 with RNP requires zinc finger one
Native WT1 co-sediments with hnRNP proteins and splice factors in
high-density fractions (1.27 g/cm3) in 20-60% Nycodenz
gradients (Ladomery et al.,
1999
). To assess the ability of tagged WT1 to become incorporated
into RNP, Cos7 cells were transiently transfected, and extracts prepared 48
hours after transfection were applied to Nycodenz density gradients
(Fig. 2). Like native WT1,
full-length tagged WT1 (+KTS) peaked in the pre-mRNP range, as did WT1
(KTS),
R
F34,
R
F234, the U5 snRNP-associated
splice factor p116 (Fabrizio et al.,
1997
) and WT1 pre-mRNA. By contrast, the closely related
transcription factor EGR1 peaked at a lower density (
1.15
g/cm3).
R
F1234 and CT
F1 were distributed
across the gradient in both lower and higher density fractions. In summary,
only constructs that included zinc finger one clearly co-sedimented with
pre-mRNP in mammalian cells.
|
Expression of WT1 (+/KTS) in Xenopus oocytes
To test the ability of T7-tagged mouse WT1 to associate with
Xenopus ooocyte nuclear structures, expression plasmids encoding
either full-length WT1 (+KTS) or WT1 (KTS) were injected into the GVs
of stage IV oocytes, and after 18 hours the GVs were isolated and nuclear
spreads prepared. The spread preparations were doubly immunostained for WT1
and the splice factor p116 (Fabrizio et
al., 1997), and counterstained with DAPI. Both isoforms located to
the lateral loops of chromosomes (Fig.
3). However, only the +KTS isoform was detected, along with native
p116, on Cajal bodies (Fig.
3A-D). The immunostaining on Cajal bodies was specifically on
B-snurposomes, some of which are located on the surfaces of the larger bodies
(Fig. 3E-F), with many others
scattered free throughout the preparation. The KTS isoform, although
extensively decorating the chromosomal loops, was not seen on Cajal bodies
(Fig. 3G-I).
|
Contribution of zinc finger one to lateral loop and snurposome
localizations
We expressed the C-terminus and CTF1 (both +KTS) in oocytes and
compared these with expressed EGR1. The deleted forms of WT1 were imported
into the nucleus as efficiently as full-length WT1, but the uptake of EGR1
appeared to be more complete after 18 hours
(Fig. 4A). On immunostained
spread preparations the C-terminus was seen to behave similarly to the
full-length protein and located on lateral loops and B-snurposomes
(Fig. 4B-D). However,
expression of CT
F1 caused chromosome compaction, loop retraction and
shedding of RNP matrix, with relatively more intense labelling of Cajal bodies
(Fig. 4E-G). Similarly, EGR1
expression also appeared to cause chromosome compaction and loop retraction
(Fig. 4H-J), although the
immunostaining differed in showing no RNP loops; rather, staining was
restricted to the chromosomal axes in the pattern similar to that seen with
DAPI. Furthermore, EGR1 did not localise to Cajal bodies or in free
B-snurposomes. Next, we tested a construct in which WT1 zinc finger one was
added to EGR1 upstream of the first zinc finger (EGR1+F1), resembling the zinc
finger domain of WT1 (KTS). Like WT1 (KTS), EGR1+F1 located to
the lateral loops, was not detected on snurposomes, and was not associated
with chromosome compaction and loop retraction
(Fig. 4K-M).
|
The N-terminus can influence localisation of co-expressed
isoforms
To check the distribution of full-length WT1 (+/KTS) WT1 isoforms on
co-expression, oocyte nuclei were co-injected with plasmids expressing
T7-tagged WT1 (+KTS) and Myc-tagged WT1 (KTS). At 30 hours
post-injection both isoforms had been efficiently imported into the nucleus
(Fig. 5A). On examining nuclear
spreads, the T7-tag was not seen in snurposome structures
(Fig. 5C-E), which were
immunostained on expressing the +KTS isoform alone
(Fig. 3). This apparent
dominance of the KTS isoform on localisation was ablated on
co-expression with CT (C-terminus +KTS) that is, in the absence of the
N-terminus. The CT (+KTS) protein was also efficiently imported into the
nucleus (Fig. 5B) and was
clearly seen to locate to B-snurposomes on Cajal bodies
(Fig. 5F-H).
|
Effect of expressed WT1 and EGR1 on endogenous transcription
rates
Chromosome compaction and loop retraction suggested that EGR1, but notably,
not WT1, severely affect transcription rates when overexpressed in oocytes.
The effects of WT1 and EGR1 on chromosomal RNA transcription could be compared
directly by labelling injected oocytes with BrUTP. In oocytes expressing WT1
(KTS), immunostaining of incorporated BrU was seen to be extensive over
the lateral loops (Fig. 6A-C).
By contrast, chromosomes isolated from oocytes expressing EGR1 were poorly
labelled with BrUTP, with immunostaining occurring at relatively few loci
(Fig. 6D-F). Transcription
rates were then checked by the incorporation of [3H]-labelled
uridine into transcripts. Rates of incorporation in oocytes expressing WT1
were found to be similar to rates in noninjected oocytes
(Fig. 6G). By contrast,
transcription in oocytes expressing EGR1 was severely reduced. After about 10
hours from EGR1 plasmid injection, the incorporation rate was little more than
the rate obtained with oocytes treated with actinomycin D, a potent inhibitor
of transcription (Fig. 6G).
|
Nucleolar localisation of WT1 and EGR1
We also observed that the expression of tagged proteins in oocytes for 30
hours resulted in an extension of the immunofluorescence signal to nucleoli.
Both full-length WT1 (+KTS) (not shown) and C-terminus (+KTS) were located
throughout the nucleolus, including the RNA-rich granular component, but
apparently not the DNA-rich fibrillar centres
(Fig. 7A-C). By contrast,
CTF1 (+KTS) had a more restricted location, primarily to the fibrillar
centres (Fig. 7D-F), and EGR1
was seen to be exclusively located to the fibrillar centres
(Fig. 7G-I). Strikingly, EGR+F1
again mimicked the properties of the intact WT1 zinc finger domain and located
to the RNA-rich granular component (Fig.
7J-L).
|
WT1 is stably bound to transcripts
Binding of WT1 zinc fingers to RNA in vitro is salt stable
(Zhai et al., 2001). We tested
the stability of the interaction of full-length WT1, C-terminus and
CT
F1 (all +KTS) with transcripts in vivo. Nuclear spreads from oocytes
expressing WT1 were washed with increasing concentrations of salt buffer
(Fig. 8A-I). At 400 mM and 600
mM NaCl, T7-immunostaining was retained on the lateral loop matrix, albeit in
a more compact form (Fig.
8D-I). Chromosome structure was disrupted by washing in 1 M NaCl
buffer (not shown).
|
To obtain a more quantitative estimate of binding stability, we injected
the nuclei of sets of 50 oocytes with WT1, C-terminus, CTF1 and
co-injected BrUTP. After 18 hours the nuclear contents were needle-sheared and
centrifuged, and pellets were resuspended and incubated in the presence of
anti-BrU/protein A glass beads. The unbound material was retained and the
beads were washed with increasing salt concentrations. The eluted fractions
were then precipitated and immunoblotted using anti-T7 tag. Full-length WT1
was the most stably bound protein, eluting only at 1 M NaCl
(Fig. 8J). The C-terminus
eluted earlier, mainly at 600 mM (Fig.
8K), and CT
F1 was the least stably bound, eluting at
400 mM (Fig. 8L). These results
confirm the importance of zinc finger one for stable interaction between WT1
and RNP in oocytes, which is consistent with the density gradient data
(Fig. 2).
We examined whether native WT1 in mammalian cells is similarly stably
associated with transcripts. We applied nuclear extract from AC29 mouse
mesothelioma cells to anion exchange resin, which has high affinity for RNA
(Palfi et al., 1989). After
collecting the flow through, a series of salt washes was applied,
increasing from 100 mM to 1 M NaCl (Fig.
9). The majority of bound nuclear proteins eluted in the 300-500
mM range. By contrast, native WT1 eluted in the 1 M NaCl wash together with
the bulk of snRNA and pre-mRNA, consistent with results using Xenopus
oocyte (Fig. 8).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We previously reported that T7-tagged WT1 (+KTS) colocalises with the
essential splice factor p116 in nuclear speckles in both HeLa and Cos7 cells,
and that the C-terminus but not the N-terminus of WT1 associates with nuclear
poly(A)+ RNP (Ladomery et al.,
1999). We prepared additional constructs, which included a
truncation of the putative RRM and deletions of zinc fingers. We began by
testing the ability of the new constructs to target to the nucleus. In a
previous study of WT1 nuclear localisation signals, fusion of zinc fingers two
and three with ß-galactosidase resulted in nuclear targeting
(Bruening et al., 1996
). We
confirm the importance of zinc finger two for nuclear targeting. The reason
for its importance is the high proportion of basic amino acids in zinc finger
two, resembling a nuclear localisation signal. The ability of zinc fingers to
direct nuclear targeting is not unique to WT1. For example, zinc fingers two
and three are required for nuclear targeting of EGR1
(Gashler et al., 1993
). Next,
we transiently transfected Cos7 cells with the above constructs and ran
soluble extract on Nycodenz density gradients. Native WT1 present in mouse
cell lines and fetal kidneys co-sediments with RNP on Nycodenz
(Ladomery et al., 1999
). We
found that both (+ and KTS) isoforms of expressed WT1 co-sedimented
with splice factors and pre-mRNA, unlike EGR1. Constructs that retained zinc
finger one similarly peaked in the pre-mRNP range.
In Xenopus oocytes we observed an accumulation of both
+/KTS isoforms of WT1 on the lateral loops of lampbrush chromosomes,
indicating their ability to bind nascent transcripts. By stark contrast, the
transcription factor EGR1 located to the chromosomal axes and binding was
associated with loop retraction and transcriptional shut-down. Notably, WT1
(+KTS) accumulated in B-snurposomes. These are 20-30 nm particles
corresponding to the components of the interchromatin granules of somatic
nuclei described as `transcriptosomes'
(Gall et al., 1999).
Transcriptosomes are sites of storage for the machinery required for
transcription and RNA processing. These results agree with previous studies in
which WT1 (+KTS) was found to colocalise with splice factors in nuclear
speckles in mammalian cells (Larsson et
al., 1995
) and to interact with the splice factor U2AF65
(Davies et al., 1998
). More
recently, Hammes and colleagues (Hammes et
al., 2001
) reported that an isoform-specific mouse knockout in
which only the +KTS isoform is produced resulted in more prominent nuclear
speckle localisation. We also found that CT
F1 (+KTS) accumulated in
B-snurposomes even more prominently than full-length protein. This is
consistent with the apparent ability of zinc fingers 2-4 (+KTS) to interact
with U2AF65, independently of zinc finger one, in the yeast two-hybrid assay
(Davies et al., 1998
). It is
tempting to speculate that the localisation of WT1 (+KTS) on snurposomes may
depend on its interaction with endogenous Xenopus U2AF65. The absence
of zinc finger one could make more protein available for U2AF65, as opposed to
transcripts.
The interaction between the +KTS and KTS isoforms in vivo is poorly
understood. Strikingly, co-expression of both tagged WT1 isoforms in oocytes
impaired the accumulation of +KTS protein in snurposomes, as long as the
N-terminus was present. Significantly, WT1 is reported to dimerise via its
N-terminus (Moffett et al.,
1995). We speculate that WT1 (KTS) titrates WT1 (+KTS) away
from snurposomes. These observations provide evidence that +/KTS
isoforms, whose intracellular ratio is crucial in development, dimerise in
vivo.
To our surprise, expressed EGR1 caused loop retraction and chromosome
compaction. However, this may be explained by a generic interaction with DNA
and/or chromatin. We also found that CTF1 (+KTS) caused loop
retraction, although not as clearly as EGR1. This was not the case for
full-length WT1 (KTS) which, similarly overexpressed, did not interfere
with normal transcription rates. We speculate that by associating with nascent
transcripts, expressed WT1 is unable to interfere with the transcriptional
machinery. Interestingly, the inclusion of WT1 zinc finger one in EGR1
impaired loop retraction. This suggests that EGR1+F1 has acquired the ability
to interact with RNA. We also observed full-length WT1 (+KTS) and C-terminus
(+KTS) in the RNA-rich granular component of nucleoli. By contrast,
CT
F1 (+KTS) and EGR1 accumulated in central foci that stain with DAPI
and correspond to the DNA-rich fibrillar centres. As before, the inclusion of
WT1 zinc finger one in EGR1 gave rise to a protein with properties similar to
WT1 (KTS). Future work will address whether or not native WT1 passes
through nucleoli.
All of the above was consistent with the ability of WT1 to bind to RNA in
vitro, in a salt-stable manner (Zhai et
al., 2001). To test the stability of the association between WT1
and nascent transcripts, we washed lampbrush chromosome spreads in high salt
buffer. This treatment did not remove WT1 from the chromosomes. Full-length
WT1 in immunoprecipitated RNP was also stably bound, in contrast to
CT
F1, which again points to a central role for zinc finger one.
However, WT1 was more stably bound than the C-terminus alone, suggesting that
additional domains in the N-terminus may contribute to RNA binding for
example, the putative RRM or dimerisation domains in the N-terminus. It is
also likely that other zinc fingers contribute to RNA binding in vivo as they
do in vitro. Zinc finger proteins that bind to RNA generally use multiple zinc
fingers. To relate these findings to native WT1, we fractionated nuclear
extract obtained from mouse mesothelioma cells on anion exchange resin. WT1
clearly co-eluted with snRNAs and pre-mRNA in anion exchange in a high-salt
wash, consistent with results obtained with Xenopus oocyte.
In summary, we present evidence that both isoforms (+/KTS) of WT1
bind to transcripts in vivo and that zinc finger one has a crucial role to
play in RNA binding. Only WT1 (+KTS) was detected in Xenopus oocyte
B-snurposomes, both free and on Cajal bodies, which is consistent with an
interaction of WT1 (+KTS) with endogenous splice factors. That WT1
(KTS) also interacts with transcripts is corroborated by its
sedimentation properties on density gradients, accumulation on lateral loops
(this study) and ability to bind to RNA in vitro
(Caricasole et al., 1996;
Bardeesy and Pelletier, 1998
;
Zhai et al., 2001
). By
contrast, the related transcription factor EGR1, which lacks the equivalent of
WT1 zinc finger one, behaved differently. We suggest that during evolution,
WT1 has acquired the ability to influence gene expression at the
post-transcriptional level, thanks to the properties of zinc finger one and
the +KTS alternative splice. The +KTS alternative splice is unique to WT1 in
the EGR family of transcription factors. However, the question arises as to
what is particular about WT1 zinc finger one. Unlike EGR1, other related
transcription factors possess the equivalent of zinc finger one. An alignment
between zinc finger domains of WT1, EGR1 and other related proteins suggests
that WT1 zinc finger one has at least seven distinct amino acids, and it is
tempting to speculate that these differences relate to the ability of zinc
finger one to bind RNA (Fig.
10).
|
WT1 is not unique in its ability to interact both with DNA and RNA. There
are now many examples of such multifunctional proteins, and many of these are
zinc-finger proteins (Ladomery,
1997; Wilkinson and Shyu,
2001
; Ladomery and Dellaire,
2002
). The priority now is to search for in vivo RNA targets and
the role of WT1 in post-transcriptional gene regulation. Future studies will
also focus on the molecular basis of WT1: RNA interactions, a possible link
between DNA and RNA targets, and their connection to pathways involved in
development and disease.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barbaux, S., Niaudet, P., Gubler, M. C., Grunfeld, J. P., Jaubert, F., Kuttenn, F., Fekete, C. N., Souleyreau-Therville, N., Thibaud, E., Fellous, M. et al. (1997). Donor splice-site mutations in WT1 are responsible for Frasier syndrome. Nat. Genet. 17,467 -470.[Medline]
Bardeesy, N. and Pelletier, J. (1998).
Overlapping RNA and DNA binding domains of the WT1 tumor suppressor
gene product. Nucleic Acids Res.
26,1784
-1792.
Bellini, M., Lacroix, J. C. and Gall, J. G. (1995). A zinc-binding domain is required for targeting the maternal nuclear protein PwA33 to lampbrush chromosome loops. J. Cell Biol. 131,563 -570.[Abstract]
Bruening, W., Moffett, P., Chia, S., Heinrich, G. and Pelletier, J. (1996). Identification of nuclear localization signals within the zinc fingers of the WT1 tumor suppressor gene product. FEBS Lett. 393,41 -47.[CrossRef][Medline]
Call, K., Glaser, T., Ito, C. Y., Buckler, A. J., Pelletier, J., Haber, D. A., Rose, E. A., Kral, A., Yeger, H. and Lewis, W. H. (1990). Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms' tumor locus. Cell 60,509 -520.[Medline]
Caricasole, A., Duarte, A., Larsson, S. H., Hastie, N. D.,
Little, M., Holmes, G., Todorov, I. and Ward, A. (1996). RNA
binding by the Wilm's tumor suppressor zinc finger proteins. Proc.
Natl. Acad. Sci. USA 93,7562
-7566.
Davies, R. C., Calvio, C., Bratt, E., Larsson, S. H., Lamond, A.
I. and Hastie, N. D. (1998). WT1 interacts with the splicing
factor U2AF65 in an isoform-dependent manner and can be incorporated into
spliceosomes. Genes Dev.
12,3217
-3225.
Davis, M. R., Manning, L. S., Whitaker, M. J., Garlepp, M. J. and Robinson, B. W. S. (1992). Establishment of a murine model of malignant mesothelioma. Int. J. Cancer 52,881 -886.[Medline]
England, T. E., Bruce, A. G. and Uhlenbeck, O. C. (1980). Specific labeling of 3' termini of RNA with T4 RNA ligase. Methods Enzymol. 65, 65-74.[Medline]
Evans, J. P. and Kay, B. K. (1991). Biochemical fractionation of oocytes. Methods Cell Biol. 36,133 -148.[Medline]
Fabrizio, P., Laggerbauer, B., Lauber, J., Lane, W. S. and
Luhrmann, R. (1997). An evolutionarily conserved U5
snRNP-specific protein is a GTP-binding factor closely related to the
ribosomal translocase EF-2. EMBO J.
16,4092
-4106.
Gall, J. G., Bellini, M., Wu, Z. and Murphy, C.
(1999). Assembly of the nuclear transcription and processing
machinery: Cajal bodies (coiled bodies) and transcriptosomes. Mol.
Biol. Cell 10,4385
-4402.
Gashler, A. L., Swaminathan, S. and Sukhatme, V. P. (1993). A novel repression module, an extensive activation domain, and a bipartite nuclear localisation signal defined in the immediate-early transcription factor Egr-1. Mol. Cell. Biol. 13,4556 -4571.[Abstract]
Gessler, M., Konig, A., Arden, K., Grundy, P., Orkin, S., Sallan, S., Peters, C., Ruyle, S., Mandell, J. and Li, F. (1994). Infrequent mutation of the WT1 gene in 77 Wilms' Tumors. Hum. Mutat. 3,212 -222.[Medline]
Hammes, A., Guo, J. K., Lutsch, G., Leheste, J. R., Landrock, D., Ziegler, U., Gubler, M. C. and Schedl, A. (2001). Two splice variants of the Wilms' tumour 1 gene have distinct functions during sex determination and nephron formation. Cell 106,319 -329.[Medline]
Hastie, N. D. (2001). Life, sex, and wt1 isoforms-three amino acids can make all the difference. Cell 106,391 -394.[Medline]
Kennedy, D., Ramsdale, T., Mattick, J. and Little, M. (1996). An RNA recognition motif in Wilms' tumour protein (WT1) revealed by structural modelling. Nat. Genet. 12,329 -331.[Medline]
King-Underwood, L. and Pritchard-Jones, K.
(1998). Wilms' tumor (WT1) gene mutations occur mainly in acute
myeloid leukemia and may confer drug resistance. Blood
91,2961
-2968.
Klamt, B., Koziell, A., Poulat, F., Wieacker, P., Scambler, P.,
Berta, P. and Gessler, M. (1998). Frasier syndrome is caused
by defective alternative splicing of WT1 leading to an altered ratio of WT1
+/KTS splice isoforms. Hum. Mol. Genet.
7, 709-701.
Ladomery, M. (1997). Multifunctional proteins suggest connections between transcriptional and post-transcriptional processes. BioEssays 19,903 -909.[Medline]
Ladomery, M. and Dellaire, G. (2002). Multifunctional zinc finger proteins in development and disease. Ann. Hum. Genet. 66,331 -342.[CrossRef][Medline]
Ladomery, M., Slight, J., McGhee, S. and Hastie, N. D.
(1999). Presence of WT1, the Wilm's tumour suppressor gene
product, in nuclear poly(A)+ RNP. J. Biol.
Chem. 274,36520
-36526.
Ladomery, M., Marshall, R., Arif, L. and Sommerville, J. (2000). C4SR, a novel zinc finger protein with SR-repeats, is expressed during early development of Xenopus. Gene 256,293 -302.[CrossRef][Medline]
Laity, J. H., Chung, J., Dyson, H. J. and Wright, P. E. (2000a). Alternative splicing of Wilms' tumor suppressor protein modulates DNA binding activity through isoform-specific DNA-induced conformational changes. Biochemistry 39,5341 -5348.[CrossRef][Medline]
Laity, J. H., Dyson, H. J. and Wright, P. E. (2000b). DNA-induced alpha-helix capping in conserved linker sequences is a determinant of binding affinity in Cys(2)-His(2) zinc fingers. J. Mol. Biol. 295,719 -727.[CrossRef][Medline]
Laity, J. H., Dyson, H. J. and Wright, P. E.
(2000c). Molecular basis for modulation of biological function by
alternate splicing of the Wilms' tumor suppressor protein. Proc.
Natl. Acad. Sci. USA 97,11932
-11935.
Larsson, S. H., Charlieu, J. P., Miyagawa, K., Engelkamp, D., Rassoulzadegan, M., Ross, A., Cuzin, F., van Heyningen, V. and Hastie, N. D. (1995). Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing. Cell 81,391 -401[Medline]
Lee, S. B., Huang, K., Palmer, R., Truong, V. B., Herzlinger, D., Kolquist, K. A., Wong, J., Paulding, C., Yoon, S. K., Gerald, W. et al. (1999). The Wilms tumor suppressor WT1 encodes a transcriptional activator of amphiregulin. Cell 98,663 -673.[Medline]
Little, M., Holmes, G. and Walsh, P. (1999). WT1: what has the last decade told us? BioEssays 21,191 -202.[CrossRef][Medline]
Little, N. A., Hastie, N. D. and Davies, R. C.
(2000). Identification of WTAP, a novel Wilms' tumour
1-associating protein. Hum. Mol. Genet.
9,2231
-2239.
Mayo, M. W., Wang, C. Y., Drouin, S. S., Madrid, L. V.,
Marshall, A. F., Reed, J. C., Weissman, B. E. and Baldwin, A. S.
(1999). WT1 modulates apoptosis by transcriptionally upregulating
the bcl-2 proto-oncogene. EMBO J.
18,3990
-4003.
Moffett, P., Bruening, W., Nakagama, H., Bardeesy, N., Housman, D., Housman, D. E. and Pelletier, J. (1995). Antagonism of WT1 activity by protein self-association. Proc. Natl. Acad. Sci. USA 92,11105 -11109.[Abstract]
Nachtigal, M. W., Hirokawa, Y., Enyeart-VanHouten, D. L., Flanagan, J. N., Hammer, G. D. and Ingraham, H. A. (1998). Wilms' tumor 1 and Dax-1 modulate the orphan nuclear receptor SF-1 in sex-specific gene expression. Cell 93,445 -454.[Medline]
Palfi, Z., Bach, M., Solymosy, F. and Lührmann, R. (1989). Purification of the major UsnRNPs from broad bean nuclear extracts and characterization of their protein constituents. Nucleic Acids Res. 17,1445 -1458.[Abstract]
Penalva, L. O., Ruiz, M. F., Ortega, A., Granadino, B., Vicente,
L., Segarra, C., Valcarcel, J. and Sanchez, L. (2000). The
Drosophila fl(2)d gene, required for female-specific splicing of Sxl
and tra pre-mRNAs, encodes a novel nuclear protein with a HQ-rich domain.
Genetics 155,129
-139.
Ryan, J., Llinas, A. J., White, D. A., Turner, B. M. and
Sommerville, J. (1999). Maternal histone deacetylase is
accumulated in the nuclei of Xenopus oocytes as protein complexes with
potential enzyme activity. J. Cell Sci.
112,2441
-2452.
Smillie, D. A. and Sommerville, J. (2002). RNA
helicase p54 (DDX6) is a shuttling protein involved in nuclear assembly of
stored mRNP particles. J. Cell Sci.
115,395
-407.
Sommerville, J., Baird, J. and Turner, B. M. (1993). Histone H4 acetylation and transcription in amphibian oocytes. J. Cell Biol. 120,277 -290.[Abstract]
Werner, H., Rauscher, F. J., III, Sukhatme, V. P., Drummond, I.
A., Roberts, C. T., Jr and LeRoith, D. (1994).
Transcriptional repression of the insulin-like growth factor I receptor
(IGF-I-R) gene by the tumor suppressor WT1 involves binding to sequences both
upstream and downstream of the IGF-I-R gene transcription start site.
J. Biol. Chem. 269,12577
-12582.
Wilhelm, D. and Englert, C. (2002). The Wilms
tumor suppressor WT1 regulates early gonad development by activation of Sf1.
Genes Dev. 16,1839
-1851.
Wilkinson, M. F. and Shyu, A. B. (2001). Multifunctional regulatory proteins that control gene expression in both the nucleus and the cytoplasm. BioEssays 23,775 -787.[CrossRef][Medline]
Zhai, G., Iskandar, M., Barilla, K. and Romaniuk, P. J. (2001). Characterization of RNA aptamer binding by the Wilms' tumor suppressor protein WT1. Biochemistry 40,2032 -2040.[CrossRef][Medline]