From the a Gene Expression Programme, European Molecular Biology Laboratory, Heidelberg, Germany, the c Medical Research Council Human Genetics Unit, Edinburgh, United Kingdom, the e Biochemical Instrumentation Programme, EMBL, Heidelberg, Germany, and the g Centro de Investigaciones Biológicas, Madrid, Spain
Received for publication, October 21, 2002
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ABSTRACT |
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Genetic and molecular data have implicated the
Drosophila gene female-lethal (2)d (fl
(2)d) in alternative splicing regulation of genes involved in
sexual determination. Sex-specific splicing is under the control of the
female-specific regulatory protein sex-lethal (SXL).
Co-immunoprecipitation and mass spectrometry results indicate that SXL
and FL (2)D form a complex and that the protein VIRILIZER and a
Ran-binding protein implicated in protein nuclear import are also
present in complexes containing FL (2)D. A human homolog of FL (2)D was
identified and cloned. Interestingly, this gene encodes a protein
(WTAP) that was previously found to interact with the Wilms' tumor
suppressor-1 (WT1), an isoform of which binds to and co-localizes with
splicing factors. Alternative splicing of transformer
pre-mRNA, a target of SXL regulation, was affected by
immunodepletion of hFL (2)D/WTAP from HeLa nuclear extracts, thus
arguing for a biochemical function of FL (2)D/WTAP proteins in splicing regulation.
Alternative splicing is a widespread mechanism of gene expression
regulation frequently used during cell differentiation and development
(1-3). Deficiencies in splice site selection have significant
implications for a variety of diseases, including tumor progression,
muscular dystrophy, and inflammatory responses (4, 5). The mechanisms
underlying the control of splice site usage, however, are still poorly understood.
Drosophila sex determination offers a system in which the
factors involved in cascades of RNA processing events have been identified genetically. The gene Sxl controls the processes
of sex determination, dosage
compensation, and sexual behavior (6). It encodes an RNA-binding
protein (SXL)1 that is present exclusively in female flies
and that induces female-specific patterns of alternative splicing of
target genes. Female somatic differentiation and sexual behavior, for
example, depend upon activation by SXL of a female-specific 3' splice
site in the gene transformer (7-12). Use of the
non-sex-specific 3' splice site results in mRNAs with little coding
capacity because of the presence of premature stop codons. The stop
codons are skipped when the female-specific 3' splice site is used,
thus generating mRNAs that encode full-length TRA protein. In
addition SXL controls splicing of its own pre-mRNA in an
autoregulatory loop essential for the maintenance of sexual identity
throughout the life of the fly (13).
Genetic analyses have revealed three additional genes involved in at
least some of the splicing events regulated by SXL: snf (sans-fille) (14), vir (virilizer)
(15), and fl (2)d (female-lethal (2)d)
(16). snf encodes the Drosophila homolog of two
human splicing factors, U1A and U2B", which are components of the U1 and U2 small nuclear ribonucleoprotein particles, respectively (14,
17). vir and fl (2)d encode nuclear proteins
without significant homologies to characterized proteins in data bases (18, 19).
fl (2)d is required throughout development and adult life
and is important for splicing regulation of Sxl and
tra (transformer) pre-mRNAs (16, 20), and
these activities can account for the sex-specific phenotype associated
with certain fl (2)d mutant alleles. The non-sex-specific
lethal phenotype of other fl (2)d alleles suggests an
additional function for the gene (21). The molecular mechanisms
underlying these genetic interactions, however, have remained elusive.
In this report we show that the FL (2)D protein forms complexes with
SXL and VIR and that depletion of a FL (2)D human homolog from nuclear
extracts affects tra splicing in vitro. These
results argue that FL (2)D has a biochemical role in splicing regulation.
Interestingly, hFL (2)D was independently identified as a protein that
interacts with the WT1 (Wilms' tumor
1) protein (22). The tumor suppressor gene WT1
is important for genitourinary development, and its mutation is
associated with Wilms' tumor, a common form of pediatric kidney cancer
(23, 24). The gene encodes various isoforms of a protein containing
four zinc fingers generated by alternative splicing, editing, and
differential use of translation start sites. Some isoforms differ by
the presence or absence of three amino acids (KTS) located between the
third and fourth C-terminal zinc fingers (23). Although the Drosophila Stocks--
Flies were cultured on standard food at
25 or 18 °C. For a description of chromosomes and mutants see Ref.
34. Descriptions of the fl (2)d1 and fl
(2)d2 alleles can be found in Refs. 19 and 21.
Splicing Substrates--
AdML1 pre-mRNA (MINX)
was generated by in vitro transcription with SP6 RNA
polymerase using as template plasmid pAdML (pMINX) digested with
BamHI. M-tra pre-mRNA contains the 5' splice site of the
AdML pre-mRNA and the alternative 3' splice sites of the first
intron of the Drosophila gene transformer and was
generated by in vitro transcription with SP6 using as
template pGEM-M-tra digested with BamHI (12).
Recombinant Proteins--
GST-SXL was expressed in and purified
from Escherichia coli as described (12). The protein was
dialyzed against Dignam buffer D (20 mM HEPES, pH 8.0, 0.2 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 0.05% Nonidet P-40) supplemented with 0.1 M KCl. FL (2)D
protein was expressed in insect Sf9 cells infected with
recombinant baculovirus containing His-tagged FL (2)D cDNA under
the polyhedrin promoter using the pFASTBACTM vector (Invitrogen). The
recombinant protein was expressed and purified on nickel beads
according to the protocols provided by the manufacturer and dialyzed
against buffer D with 0.1 M KCl.
Antibodies--
Anti-FL (2)D and anti-hFL (2)D/WTAP antibodies
were described previously (19, 22). Anti-FL (2)D and hFL (2)D/WTAP antibodies were affinity-purified, eluted in 200 mM glycine
and 0.1% bovine serum albumin, pH 2.5, and neutralized immediately. Anti-SXL polyclonal antibodies were generated in rabbits using as
antigen GST-SXL and RIBI as adjuvant (ImmunoChem Research). The
antisera used in this study correspond to bleeds obtained after four
boosts with the same antigen.
Nuclear Extracts--
Nuclear extracts from
Drosophila embryos were prepared according to Ref. 35. HeLa
nuclear extracts were prepared according to Dignam et al.
(36) or purchased from 4C Biotech (Seneffe, Belgium).
Cloning of hFL (2)D--
Based in the homology between FL (2)D
and the product of a conceptual translation of the expressed sequence
tag human clone D14661, a fragment of hFL (2)D/WTAP cDNA was
obtained by reverse transcription-PCR. RNA ligase-mediated rapid
amplification of 5' and 3' cDNAs ends was carried out using the
Qiagen RNeasy kit for RNA isolation and the GeneRacer kit (Invitrogen)
for full-length cDNA amplification.
Immunoprecipitations and Mass Spectrometry--
Antibodies
against FL (2)D were bound to protein A-Sepharose by
dimethylpimelimidate-mediated cross-linking at room temperature in a
rotating wheel for 2 h. The beads were washed four times with 1 ml
of NEB buffer (25 mM HEPES, pH 7.6, 12.5 mM
MgCl, 40 mM KCl, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 1 mM dithiothreitol) and
then incubated with 2 ml of Drosophila embryo nuclear
extract for 2 h at 4 °C. After washing four times with buffer,
the pellets were resuspended in 100 µl of SDS sample buffer and
heated to 95 °C for 3 min to release immunoprecipitated proteins. After electrophoresis on SDS-polyacrylamide gels, the gels were fixed
for 30 min in 10% glacial acetic acid, 10% methanol. Silver staining
of polyacrylamide gels was carried out by washing with water twice for
2 min and then for an hour on a shaking platform. The gel was
sensitized with 0.02% sodium thiosulfate for 1-2 min, then
thiosulfate solution was discarded, and the gel quickly rinsed with two
30-s changes of water. The gel was developed with a solution of 0.04%
formaldehyde in 2% sodium carbonate. When a sufficient degree of
staining was obtained, development was quenched by discarding the
developing solution and washing the gel with 1% acetic acid. Bands of
interest were excised and in-gel digested with trypsin, and the
resulting peptides were extracted as described previously (37). For
matrix-assisted laser desorption ionization mass mapping, a thin film
technique was used for target preparation as described previously
(38).
Western Blot Analysis--
Western blots of protein preparations
from flies were obtained by freezing the animals and homogenizing them
in SDS loading buffer. Appropriate amounts of extract were fractionated
by electrophoresis on 10% polyacrylamide-SDS gels, transferred to
nitrocellulose membranes, and incubated with anti-FL (2)D rabbit
antiserum at 1:500 dilution and anti-mouse monoclonal anti- Immunodepletion--
1 ml of either of two different anti-hFL
(2)D/WTAP polyclonal antisera (or 300 µl of affinity-purified
antiserum) or the corresponding preimmune sera were coupled to 200 µl
of protein A-Sepharose 4 fast flow beads (Amersham Biosciences) by
dimethylpimelimidate-mediated cross-linking. After incubation for
2 h at room temperature in a rotating wheel, the unbound
antibodies were eliminated by serial washes with 0.2 M
ethanolamine, pH 8, and 0.1 M glycine, pH 3, and the beads
were extensively washed with phosphate-buffered saline and then
equilibrated with buffer D with 0.1 or 1 M KCl. The first
round of depletion was carried out using 200 µl of the beads and 300 µl of HeLa nuclear extracts at 0.1 M KCl. After incubation for 2 h at 4 °C on a rotating wheel, the beads were removed by centrifugation, and the supernatant was adjusted to 1 M KCl to carry out a second round of depletion at 1 M KCl. The depleted extract was separated from the beads by
centrifugation using Mobitec columns and dialyzed against buffer D with
0.1 M KCl.
In Vitro Splicing Assays--
RNAs transcribed in the
presence of CAP analog (m7G (5') ppp (5') G) (New England
Biolabs) and for some experiments [ Polypeptides Associated with FL (2)D in Embryo
Extracts--
Affinity-purified anti-FL (2)D antibodies were used for
immunoprecipitation assays in Drosophila embryo nuclear
extracts. Equivalent amounts of immunoglobulins from preimmune sera
were used as controls. First, the presence of SXL in the precipitates was analyzed by Western blot using specific antibodies. Fig.
1A shows that SXL could be
detected in the precipitates obtained using anti-FL (2)D antibodies but
not in control immunoprecipitates. This result indicates that FL (2)D
and SXL are part of a complex in embryo nuclear extracts. This
conclusion is also supported by the presence of both proteins in
similar fractions when nuclear extracts were fractionated on sucrose
gradients (data not shown). The results were not affected by treatment
of the extract with RNase A previous to immunoprecipitation, arguing
against the possibility that the two proteins are co-precipitated
because of their independent association to the same pre-mRNA. GST
pull-down and far Western blot assays, however, failed to demonstrate a
direct interaction between the two proteins (data not shown).
To identify additional components associated to FL (2)D, the
products of immunoprecipitation were fractionated on SDS-polyacrylamide gels, which were stained with Coomassie Blue or silver nitrate. Two
polypeptides of around 120 and 220 kDa could be identified that were
precipitated by anti FL (2)D antibodies but not by preimmune sera (Fig.
1B). The corresponding bands were excised from the gel and
sequenced by mass spectrometry (37, 38). Matrix-assisted laser
desorption ionization time-of-flight analyses identified these
polypeptides as the products of the genes virilizer (vir) (18) and dim-7 (40).
Previous genetic data have implicated the gene vir in SXL
function (15). The presence of VIR in FL (2)D immunoprecipitates opens
the possibility that the genetic interactions observed between Sxl, fl (2)d, and vir are based upon
physical association of the products of these genes. The gene
dim-7 encodes a protein homologous to the human RanBP7
(Ran-binding protein
7), which is a member of the importin FL (2)D Is Not Involved in SXL Accumulation--
One possible
explanation for the genetic interactions observed between
Sxl and fl (2)d is that FL (2)D influences SXL
synthesis or accumulation. To test this, we made use of a fl
(2)d mutation, fl (2)d1, which is a
recessive temperature-sensitive allele generated by substitution of
aspartic acid 179 to asparagine in FL (2)D protein (19). This allele is
homozygous lethal for females at 29 °C but not at 18 °C, and it
does not affect the viability of males at either temperature (16).
The levels of FL (2)D were similar in homozygous fl
(2)d1 male flies maintained at 29 or 18 °C (Fig.
2A). This result indicates that the fl (2)d1 mutation does not cause a
reduction in the levels of the protein at the restrictive temperature.
The experiments were carried out after heat shock treatment for reasons
that will become obvious below.
Next we tested whether accumulation of SXL was affected. For this
purpose we used the very strong fl
(2)d1/fl (2)d2 mutant genotype,
which shows also temperature sensitivity. The fl
(2)d2 allele produces a nonfunctional truncated
protein (19), and therefore the presence of functional, full-length FL
(2)D in these flies results exclusively from expression of the fl
(2)d1 allele. Transgenic male flies transformed with a
Sxl cDNA (SxlcF#1) under a heat shock promoter (13) were subject to
heat shock, and accumulation of SXL in fl
(2)d1/fl (2)d2 flies was
measured at permissive and restrictive temperatures by Western blot
analysis. The levels of SXL 48 h after heat shock were similar at
both temperatures (Fig. 2B, compare lanes 3 and 4 or lanes 5 and 6). Although the
amounts of SXL detected were relatively low, previous work has proven
that efficient splicing regulation of transformer could be
achieved under these experimental conditions (13). We conclude that the
fl (2)d1 mutation affects the functional
properties of FL (2)D rather than the stability of the protein and that
FL (2)D is not involved in the synthesis or degradation of SXL.
Wilms' Tumor Suppressor-1-associated Protein (WTAP) Is a Human
Polypeptide with Homology to FL (2)D--
The product of conceptual
translation of human expressed sequence tags D14661 and DKFZp761K0722
show homology to a region comprising residues 112-216 of FL (2)D (19).
5' and 3' rapid amplification of cDNA ends was used to determine
the sequence of full-length hfl (2)d transcripts, and
alignment to RefSeq genomic sequences was used to establish the
exon/intron structure of the locus.
Interestingly, some of the exon/intron boundaries have been conserved
between human and Drosophila, further arguing that the two
genes are evolutionarily related (Fig.
3A). hFL (2)D/WTAP shows 40%
overall identity (50% similarity) to dFL (2)D and 63% identity and
76% similarity in the region between residues 136 and 298 (Fig.
3B). This region contains aspartate 179, which is mutated in
the fl (2)d1 allele mentioned in the previous
section. The motif with highest homology includes residues 204-238,
where the two proteins are 85% identical and 94% similar.
Effect of hFL (2)D/WTAP Depletion on in Vitro Splicing
Assays--
Antibodies raised against hFL (2)D/WTAP were used to
deplete the protein from HeLa nuclear extracts. After one round of depletion at 0.1 M and a second round at 1 M
KCl, the concentration of the protein was reduced by at least 90%
(Fig. 4A). In vitro splicing of a model adenovirus pre-mRNA was not reduced in depleted extracts compared with mock depleted extracts (Fig. 4B,
compare lanes 3 and 4). This result indicates
that depletion of hFL (2)D/WTAP does not compromise splicing of all
introns in vitro.
To address the possibility that hFL (2)D/WTAP has a role in splicing
regulation, in vitro alternative splicing assays were carried out in hFL (2)D/WTAP-depleted and mock depleted HeLa nuclear
extracts. Previous work has shown that the activation of a
female-specific 3' splice site in transformer pre-mRNA
can be reproduced in vitro by the addition of recombinant
purified SXL protein to human nuclear extracts (12, 42) (Fig.
4C, lanes 2-4). When the experiment was carried
out using hFL (2)D/WTAP-depleted extracts, the female-specific site was
activated more efficiently at low concentrations of SXL, with some
activation being observed even in the absence of regulator (Fig.
4C, compare lanes 2 and 6 and
lanes 3 and 7). At sufficiently high
concentrations of SXL, however, use of the female-specific site was
induced with similar efficiency in both depleted and control extracts
(Fig. 4C, compare lanes 4 and 8).
Table I compiles quantitative analysis of
results from six independent experiments, which are compatible with the result shown in Fig. 4C. Two independently obtained anti-hFL
(2)D/WTAP antisera were used in these experiments, with similar
results.
To test whether the effects observed in immunodepleted extracts were
specific, recombinant purified FL (2)D protein was added to the
depleted extracts, and the relative use of the non-sex-specific and
female-specific 3' splice sites was quantified. The data presented in
Table II indicate that addition of FL
(2)D to depleted extracts can restore the ratio between the use of
non-sex-specific and female-specific sites observed in mock depleted
extracts. An excess of FL (2)D, however, caused a reduction of this
ratio both in mock-depleted and in hFL (2)D/WTAP-depleted extracts,
perhaps related to "squelching" effects of the recombinant purified
protein. Taken together, the results suggest that hFL (2)D/WTAP
influences the relative use of the alternative 3' splice sites in
tra and argue that the protein has a biochemical
function in splicing regulation.
female-lethal (2)d was identified through its genetic
interactions with Sex-lethal. The molecular basis for
genetic interactions can be various and indirect. For example, FL (2)D
products could be important for SXL expression or stability and could
influence SXL localization or the expression of splicing factors
interacting with SXL. Alternatively, FL (2)D could directly influence
the splicing regulation process. In this manuscript we report that FL
(2)D and SXL proteins are part of a complex that also contains the
product of the gene virilizer, also known to interact
genetically with Sxl. In addition we provide biochemical
evidence that regulation of an alternative 3' splice site choice of
transformer is affected by FL (2)D depletion.
Complexes containing SXL, FL (2)D, and VIR may play a direct role in
pre-mRNA splicing regulation, providing a potential explanation for
the genetic interactions between the three genes. We cannot rule out,
however, that FL (2)D and/or VIR do not have additional functions
related for example to SXL synthesis or localization. In this regard it
is intriguing that another component of FL (2)D complexes is DIM-7, a
protein homologous to human RanBP7 and RanBP8, which are members of the
importin SXL-dependent splicing regulation of
Sxl and tra is compromised in flies homozygous
for the temperature-sensitive allele fl (2)d1 at
restrictive temperatures (20, 43). Because the levels of FL (2)D and
SXL do not appear to be reduced under these conditions (Fig.
2B), the effect of the mutation could be explained by a
defect in the function of the FL (2)D1 protein as a
co-regulator of SXL function. It is possible that the amino acid
substitution responsible for the fl (2)d1
phenotype (aspartate to asparagine at position 179) (19) results in a
temperature-dependent change in the biochemical activities of the protein.
The mutant phenotype of fl (2)d1/fl
(2)d2 flies suggests that FL (2)D is involved in
promoting the activation of female-specific patterns of alternative
splicing. In the case of tra, for example, activation of the
female-specific 3' splice site is reduced at the nonpermissive
temperature in female mutant flies. This could be due to failure to
cooperate with SXL in the activation of the female-specific site,
either because FL (2)D helps to repress the non-sex-specific site or
because it facilitates activation of the female-specific site.
The results of biochemical depletion, however, indicate that the use of
the female-specific site is increased in the absence of hFL (2)D/WTAP,
even in the absence of SXL (Fig. 4C and Table I). Therefore,
these biochemical results argue for a role of hFL (2)D/WTAP in
repressing the female-specific site or in promoting the use of the
competing non-sex-specific site. The fact that SXL and FL (2)D form a
complex and that SXL binds to the non-sex-specific site may point to
the latter model as a more plausible scenario.
A variety of reasons could explain the apparent discrepancy between the
phenotype of fl (2)d1 mutant flies and the
results of biochemical depletion. First, human, not
Drosophila nuclear extracts were used to test the effects of
hFL (2)D/WTAP depletion. This was due to limitations in the activity of
Drosophila extracts, which, although capable of processing
constitutive introns in vitro, did not provide high enough levels of processing intermediates or products from
tra-derived pre-mRNAs to be used in assays of
SXL-mediated regulation. It is conceivable that the activities of the
FL (2)D homologs are different between vertebrates and invertebrates,
consistent with the limited similarity outside a region of high
conservation (Fig. 3B). The fact that Drosophila
FL (2)D restores the effects of hFL (2)D/WTAP depletion from human
extracts (Table II), however, argues against this possibility and in
addition suggests functional conservation across long evolutionary
periods. It is still possible that the activities of the two proteins
are quantitatively different. Difficulties in purifying recombinant hFL
(2)D/WTAP using a variety of expression systems has so far prevented us from carrying out a detailed quantitative comparison of their biochemical activities.
A more intriguing scenario could arise if the discrepancies between
in vivo and in vitro experiments were due to the
fact that the effects caused on FL (2)D function by the substitution of
aspartate 179 are not equivalent to the absence (or reduction in the
levels) of the protein. If FL (2)D is involved in repression of the
female-specific site or in activation of the non-sex-specific site,
reduction in the levels of FL (2)D should result in increased use of
the female-specific site, as observed in our in vitro
splicing assays. In contrast, substitution of aspartate 179 may lead,
for example, to a protein with increased activity as a repressor of the
female-specific site and therefore to failure of SXL to activate it.
Alternatively, the fl (2)d1 mutation could
affect aspects of the function of the protein relevant for its activity
as a co-factor of SXL (for example by disrupting the complex containing
both proteins), without affecting other activities of the protein (for
example to promote the use of the non-sex-specific site). This model
implies that FL (2)D could have antagonistic functions in splicing
regulation depending upon the presence or absence of SXL. Any of these
scenarios could explain why the fl (2)d1
mutation has no effect on tra splicing in males, where
activation of the female-specific site does not take place.
If FL (2)D plays a direct role in the splicing process, the protein may
represent a novel class of splicing co-regulators, because no
structural domain characteristic of proteins involved in splicing can
be found in the primary sequence features of FL (2)D/WTAP. The most
conspicuous sequence motifs of dFL (2)D are stretches rich in
histidines and glutamines. Glutamine-rich domains have been involved in
protein-protein interactions, and polyglutamine motifs associated to
CUG expansions have been linked to a variety of diseases, including
Hungtington disease (44). Neither histidine- nor glutamine-rich
stretches are present in hFL (2)D/WTAP, suggesting that if dFL (2)D and
hFL (2)D/WTAP share activities in the splicing process, they are not
related to these domains. The region of highest homology between human and Drosophila FL (2)D/WTAP proteins includes residue 179, which is mutated in the fl (2)d1 allele.
The human homolog of FL (2)D was identified independently in a
two-hybrid screen for proteins that interact with the WT1, an
interaction that was confirmed by co-immunoprecipitation experiments (22). The association of the WT1 +KTS isoform with U2AF65
and its co-localization with splicing factors suggest a function for
the isoform in splicing (30). Because WT1 expression is confined to
developing the kidney, gonads, spleen, and mesothelial lining of
abdominal organs (24), WT1 could play a role as a tissue-specific
splicing regulator. Given the interaction between hFL (2)D/WTAP and
WT1, it is tempting to speculate that hFL (2)D/WTAP can function as a
co-factor for alternative splicing events regulated by WT1, similar to
the association between dFL (2)D and SXL and to the involvement of dFL
(2)D in SXL-dependent alternative splicing decisions. In
this regard, it is intriguing that despite the mechanistic differences
in the molecular mechanisms of sex determination between
Drosophila and mammals, WTAP and FL (2)D may modulate the
activity of well characterized (SXL) or putative (WT1 +KTS) splicing
factors that play important roles as regulators of this process. The
recent identification of WT1 as a component of active spliceosomes (45)
underscores the significance of the biochemical results presented in
this manuscript and is consistent with the idea that FL (2)D/WTAP may
be the founding members of a novel class of splicing factors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
KTS
isoform binds nucleic acids and regulates transcription of genes
involved in cell proliferation/differentiation (25-28), the +KTS
isoform binds RNA rather than DNA and has been shown to be associated and localized with splicing factors (29-32), suggesting a role for
this isoform in RNA processing. Evidence for separable functions of the
/+ KTS isoforms was recently provided by studies in mice where each
of the isoforms were knocked out separately (33). This study
demonstrated that the +KTS isoform was essential for male sexual
determination, which is consistent with the finding that patients that
suffer from Frasier syndrome fail to produce this isoform, and males
frequently show sex reversal (23). We discuss in this manuscript the
involvement of FL (2)D-like proteins in
post-transcriptional regulation of gene expression during sex
determination in flies and mammals.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin
(Sigma) at 1:50.000 dilution for 1 h. Anti-rabbit or anti-mouse
horseradish peroxidase conjugates IgGs (Amersham Biosciences) were used
as secondary antibodies at a 1:5000 dilution for 1 h. The blots
were developed using an ECL detection kit (Amersham Biosciences) and exposed to film.
-32P]UTP (Amersham
Biosciences) were gel-purified. 20 fmol of RNA were used to set up 10 µl of in vitro splicing mixes containing 2.7 mM MgCl2, 1 mM ATP, 20 mM creatine phosphate, 4.8 units/µl RNasin, 3% polyvinyl
alcohol, 30% nuclear extracts, or 45% immunodepleted/mock depleted
nuclear extracts, complemented with either buffer D with 0.1 M KCl or with recombinant proteins (SXL or FL (2)D) in the
same buffer. After incubation, the RNAs were purified by proteinase K
treatment, phenol chloroform extraction, and precipitation. Spliced
products were analyzed directly by electrophoresis on 13% denaturating
polyacrylamide gels in Tris-borate-EDTA buffer when radioactively
labeled RNA was used in the assays. tra alternative splicing
was analyzed by primer extension using splice junction-specific primers
as described (12). The gels were exposed to PhosphorImager screens
(Fuji BAS-MP), and the intensity of the bands was quantified.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Proteins associated with FL (2)D in embryo
extracts. A, SXL is present in FL (2)D
immunoprecipitates. Products of immunoprecipitation using anti-FL (2)D
antibodies and preimmune serum were fractionated on SDS-polyacrylamide
gels, and the presence of SXL and FL (2)D in the immunoprecipitates was
analyzed by Western blot using anti-SXL and anti-FL (2)D antibodies.
The positions of the two proteins and of the 55-kDa immunoglobulin
subunits are indicated. A Western blot of nuclear extracts in the
absence of immunoprecipitation is also shown. B, additional
specific components of FL (2)D immunoprecipitates. Products of
immunoprecipitation using anti-FL (2)D antibodies and preimmune serum
were fractionated on SDS-polyacrylamide gels and analyzed by silver
staining. The positions of the 116- and 220-kDa polypeptides identified
by mass spectrometry as the products of the genes virilizer
and dim-7 are indicated. MW, molecular
mass.
family of nuclear
import receptors (40, 41). As was the case with SXL, association of VIR
and DIM-7 proteins with FL (2)D was not affected by RNase A digestion
previous to the precipitation assay, suggesting that the complex is not mediated by RNA.
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Fig. 2.
Effect of the fl
(2)d1 mutation on FL (2)D and SXL accumulation.
A, stability of FL (2)D1 protein. Wild type of
fl (2)d1 homozygous flies were subject to heat
shock, and the levels of FL (2)D and tubulin were analyzed by Western
blot after 24 or 48 h of recovery at 18 or 29 °C, as indicated.
B, accumulation of SXL in males transformed with a Sxl
cDNA (SxlcF1#1) in a fl (2)d1/fl
(2)d2 mutant background. The cross to generate these
flies was cm Sxlf1
ct6/FM6; cn fl (2)d1
SxlcF1#1/CyO x +/Y, cn fl (2)d1
bw/CyO, and the cross to produce fl
(2)d1 homozygous flies was cn
fl(2)d1 bw/CyO x +/Y; cn fl (2)d1
bw/CyO. Accumulation of SXL and tubulin was analyzed by
Western blot after induction of SXL expression by heat shock and
recovery at the restrictive (29 °C) or permissive (18 °C)
temperature for the times indicated. The signals corresponding to SXL
protein are indicated by asterisks. F and
M indicate females and males, respectively. The genotypes of
the flies used in the experiment are indicated at the top.
MW, molecular mass; HS, heat shock.
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[in a new window]
Fig. 3.
Genomic structure and predicted protein
product of WTAP, a putative human homolog of fl
(2)d. A, comparison between the exon/intron
structure of dfl (2)d and hfl (2)d/WTAP genes.
Exons are represented by rectangles, and introns are
represented by thin lines. Translation start and stop codons
for the main open reading frames identified are indicated.
B, alignment between the primary amino acid sequence of dFL
(2)D and hFL (2)D/WTAP. The position of aspartic acid 180, which is
mutated to asparagine in the fl (2)d1 allele, is
boxed.
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[in a new window]
Fig. 4.
Effect of hFL (2)D/WTAP immunodepletion on
pre-mRNA splicing in vitro. A,
extent of hFL (2)D/WTAP depletion. hFL (2)D/WTAP was detected in HeLa
nuclear extracts or extracts immunodepleted with anti-hFL (2)D/WTAP
antibodies or preimmune serum by Western blot. B, effect of
hFL (2)D/WTAP depletion on splicing of a pre-mRNA derived from the
AdML promoter. After incubation of AdML pre-mRNA with HeLa nuclear
extracts or mock depleted or hFL (2)D/WTAP-depleted extracts, in the
absence or presence of ATP, RNAs were purified and fractionated on
denaturing polyacrylamide gels. The positions of the pre-mRNA,
splicing products, and intermediates are indicated. C,
effect of hFL (2)D/WTAP depletion on alternative splicing of a
pre-mRNA containing tra alternative 3' splice sites.
M-tra pre-mRNA was incubated with mock depleted or hFL
(2)D/WTAP-depleted extracts and the accumulation of mRNAs
corresponding to use of the non-sex-specific or female-specific 3'
splice sites analyzed by primer extension using splice-junction
oligonucleotides as described (12). The products of primer extension
are shown. Concentrations of SXL present in the assays and presence or
absence of ATP are indicated. MW, molecular mass.
In vitro alternative splicing of tra in mock depleted versus hFL
(2)D/WTAP-depleted extracts
Effect of FL (2)D addition to hFL(2)D/WTAP-depleted extracts
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
family of nuclear import receptors (41). Consistent with
this putative function, DIM-7 has been shown to be important for
nuclear import of activated D-ERK kinase (40). It is
therefore possible that DIM-7 is involved in nuclear localization of FL
(2)D, SXL, and/or VIR and that FL (2)D and/or VIR could influence this process.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Markus Niessen and Rolf Nöthiger for providing the sequence of the gene virilizer before publication. We are also very grateful to Daniel Bopp for technical advice and discussions, to Luiz Penalva for discussions and help with data base searches, and to colleagues in our departments for comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by a Human Frontiers Science Program Organization grant (to J. V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
b Supported by fellowships from University of Granada, Ministerio de Educación y Ciencia (Spain) and Marie Curie Research Fellowships Program. Present address: Dept. of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Granada, Spain.
d Recipient of an EMBO short-term fellowship.
f Present address: Dibit, San Raffaelle Scientific Institute, 20132 Milano, Italy.
h Supported by Dirección General de Investigación Científica y Técnica Grant PB98-0466.
i Supported by the Medical Research Council and the European Union.
j To whom correspondence should be addressed. Present address: Centre de Regulacio Genomica, Passeig Maritim 37-49, 08003 Barcelona, Spain. Tel.: 34-93-2240956; Fax: 34-93-2240899; E-mail: juan.valcarcel@crg.es.
Published, JBC Papers in Press, November 19, 2002, DOI 10.1074/jbc.M210737200
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ABBREVIATIONS |
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The abbreviations used are: SXL, sex-lethal, AdML, adenovirus major late; GST, glutathione S-transferase.
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REFERENCES |
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