Biochemical Function of Female-Lethal (2)D/Wilms' Tumor Suppressor-1-associated Proteins in Alternative Pre-mRNA Splicing*

Angeles Ortegaab, Martina Niksiccd, Angela Bachief, Matthias Wilme, Lucas Sánchezgh, Nicholas Hastieci, and Juan Valcárcelaj

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha -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.

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 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 [alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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 beta  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.

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.


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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.

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.


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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.

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.


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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.

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.

                              
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Table I
In vitro alternative splicing of tra in mock depleted versus hFL (2)D/WTAP-depleted extracts
The ratios between non-sex-specific and female-specific 3' splice site are indicated as the averages and standard deviations for the number of experiments indicated in parentheses. The concentrations of SXL protein added to the assays are indicated.

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.

                              
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Table II
Effect of FL (2)D addition to hFL(2)D/WTAP-depleted extracts
The ratio between non-sex-specific and female-specific 3' splice site usage in vitro was analyzed in mock or depleted extracts in the absence or presence of the indicated concentrations of recombinant purified FL(2)D. The values represent the averages and standard deviations for the number of experiments indicated in parentheses.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta  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.

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.

    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.

    FOOTNOTES

* 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

    ABBREVIATIONS

The abbreviations used are: SXL, sex-lethal, AdML, adenovirus major late; GST, glutathione S-transferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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