A human homologue of the Drosophila sex determination factor

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 93, pp , August 1996 Developmental Biology A human homologue of the Drosophila sex determination factor transformer-2 has conserved splicing regulatory functions (sex determination/alternative splicing/rna recognition motif/ser/arg-rich domain/evolution) BRIGIrrE DAUWALDER, FELIPE AMAYA-MANZANARES, AND WILLIAM MATrOX* Department of Molecular Genetics, University of Texas, M. D. Anderson Cancer Center, Houston, TX Communicated by Bruce S. Baker, Stanford University, Stanford, CA, May 16, 1996 (received for review April 20, 1996) ABSTRACT Regulation of gene expression through alternative pre-mrna splicing appears to occur in all metazoans, but most of our knowledge about splicing regulators derives from studies on genetically identified factors from Drosophila. Among the best studied of these is the transformer-2 (TRA-2) protein which, in combination with the transformer (TRA) protein, directs sex-specific splicing of pre-mrna from the sex determination gene doublesex (dsx). Here we report the identification of htra-2a, a human homologue of tra-2. Two alternative types of htra-2a cdna clones were identified that encode different protein isoforms with striking organizational similarity to Drosophila tra-2 proteins. When expressed in flies, one htra-2a isoform partially replaces the function of Drosophila TRA-2, affecting both female sexual differentiation and alternative splicing of dsx pre-mrna. Like Drosophila TRA-2, the ability of htra-2a to regulate d&x is femalespecific and depends on the presence of the dsx splicing enhancer. These results demonstrate that htra-2a has conserved a striking degree of functional specificity during evolution and leads us to suggest that, although they are likely to serve different roles in development, the tra-2 products of flies and humans have similar molecular functions. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. Despite the fact that a large fraction of identified cellular pre-mrnas undergo alternative splicing, few vertebrate factors have been identified that affect splicing patterns. Of those factors so far studied, the evidence for a role in the regulation of splicing is best for the SR proteins, a family of RNA binding proteins that contain extensive regions rich in arginine and serine (RS domains) (for review, see ref. 1). Several lines of evidence suggest these domains may facilitate interactions with other RNA binding proteins. While SR proteins are known to play vital roles during constitutive pre-mrna splicing both in the initiation of spliceosome assembly and in interactions between small nuclear ribonucleoproteins they have also been shown to affect the selection of alternative 5' splice sites in a variety of artificial and natural substrates in a concentration dependent manner. In addition, several SR proteins are known to interact with the purine-rich splicing enhancer elements found in some vertebrate exons. While these observations strongly suggest a role for SR proteins in regulating splicing, there is still little direct evidence that vertebrate SR proteins normally direct the developmentally specific alternative splicing of any particular cellular pre-mrnas in vivo. Proteins with established roles in developmental regulation of splicing have been identified in Drosophila through genetic analysis (2-7). Many of these proteins form a cascade of splicing factors that directs sexual differentiation in the fly. Two SR-related proteins, encoded by the transformer (tra) and transformer-2 (tra-2) genes, play a central role in this pathway (8). However, unlike the vertebrate SR proteins described above, these SR-related splicing factors have sex-specific functions and therefore do not appear to play a role in constitutive RNA processing. TRA and TRA-2 are directly required for female-specific splicing of doublesex (dsx) pre-mrna (9-11). In the absence of functional TRA-2, the dsx male-specific mrna, encoding an alternative protein, is produced by default. The mechanism by which TRA and TRA-2 affect dsx splicing has now been studied in some detail (12-17). In vitro studies suggest that these proteins both bind directly to the dsx splicing enhancer, a 300-nt region of the dsx pre-mrna located in the femalespecific exon. Using a heterologous system, it was found that TRA and TRA-2 can interact with several human SR proteins to form a complex on the dsx enhancer that promotes recognition of the female-specific 3' splice site by the general splicing machinery (18, 19). Mutations affecting either of two ubiquitous Drosophila SR proteins (dsrp55 and rbpl) interact with tra and tra-2 mutations (20, 21), and one of these proteins has been shown to bind to sites in the dsx RNA that are necessary for female-specific splicing (22). Therefore, it is likely that in Drosophila cells TRA and TRA-2 normally act in combination with essential SR proteins to facilitate dsx splicing. Genetic experiments in Drosophila suggest that neither TRA nor TRA-2 alone is sufficient for female-specific dsx splicing (8, 23). However, Tian and Maniatis (18) found that the addition of either TRA or TRA-2 to human splicing extracts could activate the female-specific 3' splice site. This observation suggests that the HeLa splicing system used contains some factors with TRA and/or TRA-2-like functions. Here we report the identification of cdna sequences from htra-2a, a human gene encoding a protein homologous to TRA-2. We find that htra-2ac is able to control dsx RNA splicing when expressed in Drosophila, suggesting that its splicing regulatory function is conserved. Taken together with our current knowledge about the divergence of sex determination mechanisms in these organisms (24, 25), our findings suggest that TRA-2 proteins from humans and flies are likely to interact with similar RNA targets and cofactors while taking on different roles in development. MATERIALS AND METHODS Isolation and Characterization of cdna Clones. HT1 and HT2 cdnas were identified through the similarity of two expressed sequence tag sequences (GenBank accession nos. R09691 and R07280) to the tra-2 RNA recognition motif (RRM) linker region. These clone DNAs were provided by the IMAGE Consortium, Lawrence Livermore National Laboratories (26). To obtain pht3, pht4, and pht5 the entire insert Abbreviations: SR proteins, Ser/Arg-rich RNA binding proteins; RRM, RNA recognition motif; RS domain, Arg/Ser-rich domain; hsp70, heat shock protein 70; RNP-1 and -2, conserved ribonucleoprotein sequence 1 and 2, respectively; RT-PCR, reverse transcription-pcr; X-Gal, 5-bromo-4-chloro-3-indolyl 3-D-galactoside. Data deposition: The sequence reported in this paper has been deposited in the GenBank data base (accession no. U53209). *To whom reprint requests should be addressed. 9004

2 Developmental Biology: Dauwalder et al. from HT1 was used to screen 3 x 105 clones from a HeLa cdna library (27). Construction and Analysis of hs-htra-2a Transgenic Fly Strains. The NotI/HindIll fragment of pht4 containing all coding sequences for the predicted 282-aa protein (see Fig. 1) was inserted into the P transformation vector pcasper-hs (28). The resultant pcsphs-htra-2a was used for germ-line transformation of the stock wjjls/bsy;tra-2b/cyo (28). Heat shocks on transgenic flies and controls were done in plastic food bottles by air-incubation at 37 C for 1 h twice daily, starting 48 h after egg laying, until eclosion. To assess effects on male fertility and adult RNA levels, heat shocks were continued for 2-3 days of adult life for 20 min twice daily. RNA was prepared 6 h after the last heat shock. Isolation, Northern and Reverse Transciption-PCR (RT- PCR) Analysis of RNA. RNA was isolated as described by Mattox et al. (29), but starting with only 5-20 adult flies. For RT-PCR analysis, the DNase-treated RNA equivalent to five flies was reverse transcribed by using the Superscript II kit (GIBCO) with antisense primers (fl and ml in Fig. 4A). Half of the cdna product was amplified by using fl and ml in combination with a primer from exon 3 (16). Amplification was carried out in 50 mm potassium chloride, 10 mm Tris HCl (ph 9 at 25 C), 0.1% Triton X-100, 1.5 mm magnesium chloride, 1.5,uM primers (each), 0.1 mm dntps, and 2.5 units Taq polymerase (Fisher Scientific). Conditions were 5 min at 94 C, then 18 cycles of 1 min at 94 C, 1 min at 50 C, 1 min at 72 C, followed by 5 min at 72 C. Half of each reaction was analyzed by Southern blot hybridization using end-labeled oligonucleotides f2 and m2, spanning the female- and male-specific splice junctions. The sequences of these primers were as follows: f2, 5'-ACGTATTGGCCCTCTTCGA; and m2, 5'-TCCACTCG- AGCCTCTTCGA. Hybridizations were done at 42 C in 1 M sodium chloride, 5% dextran sulfate, 0.4% polyvinyl pyrollidone, 0.4% Ficoll, 0.4% BSA, 0.1 M Pipes (ph 7.0), 0.2% SDS, and 100,gg of salmon sperm DNA per ml. Blots were washed twice in 6x standard saline citrate (SSC) at 25 C for 10 min, then once in 6x SSC at 65 C for 20 min. Predicted female and male PCR products were 344 and 244 nt, respectively. The product from contaminating genomic DNA (460 nt) does not hybridize under the conditions used. RESULTS A Human Gene Encoding Proteins with Similarity to TRA-2. Comparison of the sequences of tra-2 genes from Drosophila melanogaster and Drosophila virilis revealed an unrecognized but highly conserved region of 19 aa immediately downstream of the RRM, which we refer to as the "linker" region (Fig. 1). Searches of sequence data bases with tra-2 sequences identified two human expressed sequence tags with strong similarity to this region. The corresponding cdna clones (HT1 and HT2), which derive from a 20-week fetal liver spleen library (26), were obtained and used to identify three additional cdnas (HT3, HT4, and HT5) from a HeLa cell library by hybridization. Sequence analysis of these cdnas revealed that both HT1 and HT2 encode a protein of 113 aa that contains both an RRM motif and linker region similar to Drosophila TRA-2 but lacks the RS domains at the amino and carboxy termini (Fig. 1A). HT3, HT4, and HT5 derive from alternatively processed mrnas that differ from HT1 and HT2 at both their 5' and 3' ends. Each of these cdnas encodes an identical 282-aa protein that contains amino and carboxy terminal RS domains (RS1 and RS2) in addition to the same RRM and linker sequences found in HT1 and HT2. The overall organization of this protein is highly similar to the Drosophila TRA-2264 isoform (Fig. 1A). When used to search current data bases the 282-aa protein was found to be similar to many RNA binding proteins but similarity was strongest to Drosophila tra-2 Proc. Natl. Acad. Sci. USA 93 (1996) 9005 (42% identical). The most conserved region between the fly and human proteins is the linker and last 11 aa of the RRM (80% identical). Although similarity extends throughout the entire protein, it should be noted that the RS domains are of low sequence complexity, diminishing the significance of the matches in these regions. RS domain sequences in the human and fly TRA-2 proteins align only in areas of alternating arginines and serines, suggesting that the Arg/Ser-rich composition of these domains rather than primary sequence is conserved (Fig. 1). Based on the organizational and sequence similarities of these cdnas to Drosophila tr-2, we tentatively designate the human gene encoding them htra-2a. Human tra-2-like cdnas Encode a Functional Homologue of Drosophila tra-2. To test whether the 282-aa protein is a functional homologue of Drosophila TRA-2, we fused the coding sequences from the HT4 cdna to the Drosophila heat shock protein (hsp70) promoter (Fig. 2) and introduced this construct (hs-htra-2a) into the fly genome. The ability of the transgene to functionally replace the endogenous tra-2 gene was tested in diplo-x (chromosomally female) individuals homozygous for the loss-of-function tra-2b mutation. Without the transgene these individuals are transformed into males due to the absence of functional TRA-2 protein. When each of five independent transgenic lines were subjected to periodic heat shocks during development (see Materials and Methods), they all produced diplo-x tra-2b/tra-2b individuals showing varying degrees of female sexual differentiation. As summarized in Table 1 and shown along with controls in Fig. 2 A-, such flies from two transgenic strains frequently developed with fully female abdominal pigmentation, while two other strains gave intersexual pigmentation. These lines also showed substantial, but not always complete, female differentiation in sex combs (Fig. 2H) and sixth sternite bristles (Table 1). Genitalia were often rotated in flies from all five lines (a characteristic of intersexuality), but these structures were never fully transformed to a female phenotype. Overall, the rescue observed was similar to that obtained when the hsp70 promoter was used to drive expression of a cdna encoding the Drosophila TRA-2179 isoform and somewhat less pronounced than when it was used to drive larger TRA-2 isoforms (32). The above results demonstrate that htra-2ac can partially replace tra-2 during sexual differentiation and strongly suggests that these genes are functional homologues. The htra-2av Gene Affects dsx pre-mrna Splicing in Chromosomal Females But Not Males. To test whether htra-2a expression affects dsx splicing, we used an RT-PCR strategy to examine dsx mrnas produced in diplo-x; tra-2b/tra-2b individuals with and without the transgene (Fig. 3). Predominantly male-specific dsx mrna is expressed in such individuals when no transgene is present (Fig. 3B). In contrast, tra-2b individuals carrying hs-htra-2ac produced substantial amounts of both male and female dsx mrnas without heat shock and only female dsx mrna when subjected to periodic heat shocks throughout development. These results indicate that hs-htra-2a can function in place of the endogenous tra-2 gene to facilitate dsx female-specific splicing. RNAs encoding the Drosophila tra-2 proteins are present in the somatic tissues of both chromosomally male and female individuals but these proteins affect dsx splicing only in females due to the additional requirement for interactions with the female-specific TRA protein (23, 29, 32). In vitro studies suggest that these factors interact with one another directly and that TRA-2 enhances specific binding of TRA/SR protein complexes to dsx RNA (16, 17). To determine whether htra-2a also depends on TRA, we examined the sexual differentiation phenotypes and dsx mrnas of transgenic XY (chromosomally male) individuals. Transgenic chromosomal males subjected to periodic heat shocks through development had no overt signs of intersexuality, suggesting that these individuals are not competent to

3 9006 Developmental Biology: Dauwalder et alp A Drosophila LI human r. / _i / lxrril92.'s' -' S A' 33% 49% 79% 42% Proc. Natl. Acad. Sci. USA 93 (1996) TRA TRA-2226 TRA-2264 HT3, HT4, HT5 HT1, HT2 B D R E L s s- G L H C S Al-G-- -Y-K R- AUSIS A TTES G H K D. mel TRA-2 (264) SDV E E N N F E G RE S RSQS KS T T PA V KS E S SG SRS P SRV SE H R RSK SR RS RR Human TRA-2 alpha (28Z) DRRSD DYCGSR HQ RSfS H S H R R T R SUS H S HH S S E S P PMPEHIS GHS RDEM H K E H AD. mel TRA-2 (264) YT IYIRIR SIH S P Human TRA-Z alpha (282) human TRA-2 alpha (113) S REIN T NES H K VELEN K_II E R I O MMIA QEMQIECEIEEK L SEA R AK D S.~~~., I... I... ii3 II RNP-2 SIS RNP-1 linker PYR NGREREN D R Y D R N LM- -S P S RR-TNMYY_ N P Q L R R T S SR - A S Y D R G R E Y -_ R S P S P Y S Y R - _-y S P R_ * _ R -K A P R S F S P RIGJRVHES D. mel TRA-2 (264) Human TRA-2 alpha (282) human TRA-2 alpha (113) D. mel TR-2 (64) H S G G G G G GEG G G G G G G G GERD SYG Human TRA-2 alpha (Z8Z) Q human TRA-2 alpha (113) D. mel TRA-2 (264) Human TRA-Z alpha (28Z) FIG. 1. Sequence similarity between human cdnas and Drosophila TRA-2. (A) Comparison of the organization of the predicted Drosophila and human tra-2 isoforms. The gray areas labeled RS1 and RS2 indicate RS domains. Filled areas labeled L indicate the linker region. Cross-hatching indicates the RRM. The horizontal striped box indicates the glycine-rich hinge region, and open boxes are regions containing sequences of unknown function. Percentages of identity are indicated for each of the major sequence domains. (B) Alignment is shown comparing sequences from the Drosophila TRA-2264 protein to those encoded by human cdnas. Identical residues are shaded. Consensus RRM positions (30) matched by htra-2ac proteins are underlined, whereas RRM consensus positions where htra-2ac does not match are indicated with an asterisk. The highly conserved ribonucleoprotein sequence 1 (RNP-1) and RNP-2 regions found in most RRMs are indicated. The linker region, where human and Drosophila tra-2 proteins share extended similarity not found in other RRM proteins, is indicated by a heavy underline. undergo female differentiation in response to htra-2a expression (not shown). RT-PCR analysis of dvx transcripts produced in both control and transgenic males are shown in Fig. 3C. Only male dsx mrnas were observed both with or without periodic heat shocks. These results show that the effect of htra-2a on dsx splicing is highly specific to chromosomally female individuals and suggests that, like Drosophila TRA-2, htra-2a is likely to interact directly with the TRA protein. The Regulation ofdsx Splicing by htra-2a Is Dependent on the dsx Splicing Enhancer. It is unclear from the above experiments whether htra-2a affects dsx splicing through specific interactions with the dsx enhancer element or through more general effects on the preference of the general splicing machinery for sites based on their relative positions and strength as has been observed for other SR proteins (33-35). To test whether or not regulation of splicing by htra-2a is dependent on the dxx splicing enhancer, we took advantage of the dsxs mutation, which results from a 448-nt deletion of sequences in the dsx female-specific exon that closely encompasses all known elements of the dsx splicing enhancer but does not affect dsx protein coding sequences or splice sites (8, 36). Due to the absence of the enhancer, the mrna produced by the dsxfs allele is processed exclusively in the male-specific pattern regardless of chromosomal sex. Diplo-X dsxs/dsx+ individuals develop as intersexes expressing roughly equal amounts of both male (from the dsxs allele) and female mrna (from the wild-type allele). Results from a comparison of the sexual phenotypes of diplo-x tra-2b/tra-2+; dsxs/dsxx, P[hs-htra-2a] individuals and diplo-x tra-2b/tra-2+; dsxs/dsx+ controls raised with periodic heat shocks are presented in Table 2. If htra-2a promotes female dsx splicing independently of the enhancer, we would expect to observe a shift toward female characteristics in the phenotype of such individuals expressing this protein. However, sex combs from individuals of both genotypes were similarly intersexual in both morphology and number. Similar observations were made for other sexually differentiated characteristics of these flies including genitalia and abdominal pigmentation. This strongly indicates that htra-2a cannot induce female splicing from the dsxs allele. We conclude that the effect of htra-2ca on sexual differentiation is dependent on the presence of the dsx splicing enhancer. htra-2a is therefore likely to recognize the same RNA sequences that are normally recognized by Drosophila TRA-2. The htra-2a Gene Does Not Affect Male Germ-Line Functions in Drosophila. In addition to its role in somatic sexual differentiation, TRA-2 also affects processing of pre-mrnas in the male germ line and is required for normal spermatogenesis and fertility (37-39). We therefore examined the fertility of transgenic chromosomal male tra-2b/tra-2b individ-

4 Developmental Biology: Dauwalder et al. A Proc. Natl. Acad. Sci. USA 93 (1996) 9007 H A :g ATG TGA hsp7o htra-2ax hso7 promoter FIG. 2. Sexual differentiation phenotypes of transgenic XX; tra-2b flies expressing htra-2a. Sexually differentiated tissues of flies from transgenic lines produced by microinjection of the wjjj8/bsy;tra-2b/ CyO strain with hs-htra-2a are shown. Unless otherwise noted, all individuals were subjected to the same heat shock regimen. In control tra-2+ males, tergites in the posterior abdomen are uniformly dark in pigment (A) and sex combs are thick and darkly pigmented (B). In tra-2+ females only the posterior regions of the same tergites are pigmented (I) and bristles analogous to sex combs are thinner and fewer in number (J). Chromosomal females homozygous for the tra-2b mutation have male-like abdominal pigmentation and sex combs (C and D) as do transgenic (line 30.2) individuals when heat shock is omitted (E and F). Heat shock treatment of the same transgenic line causes such individuals to develop with female-like abdominal pigmentation and sex comb morphology (G and H). The structure of the hs-htra-2a fusion gene used to produce these phenotypes is shown (K). uals after periodic heat shocks. Both heat shocked and untreated transgenic tra-2b/tra-2b male individuals bearing two copies of the hs-htra-2a gene were completely sterile (not shown) indicating that the htra-2a is unable to affect splicing of at least some targets in the male germ line. Further evidence for this idea came from examination of an autoregulated splicing choice in the Drosophila tra-2 premrna itself. The splicing of the third intron of tra-2 premrna is normally repressed by the presence of the Drosophila TRA-2226 isoform as part of a negative feedback mechanism (38, 40). We have previously shown that the reporter transgene pczp-orf3 expresses,3-galactosidase only from mrnas retaining this intron (38). When this reporter was introduced into XY individuals that carry a copy of the hs-htra-2a trarasgene and are homozygous for the tra-2b allele, no 5-bromo-4-chloro-3-indolyl P3-D-galactoside (X-Gal) staining above Table 1. Rescue of female sexual differentiation in chromosomally female w'118/w1118; tra-2b/tra-2b individuals carrying two copies of the hs-htra-2a transgene Female Average no. Transgenic abdominal of sixth Sex line pigmentation* sternite bristlest Genitalia* combs* No transgene *Sexual differentiation for each tissue indicated as fully male (-), male-like intersex (+), intermediate intersex (+ +), female-like intersex (+ + +), and fully female ( ). tnormal w"' 8/w"' 8; tra-2b/tra-2+ females have an average of 18.3 sixth sternite bristles. B = =- II,.1 it female inrna =AAA mizle mrna none none line XX XX XX XX XX XX XX XX XY XX sex chromni heat shock tral-2"/trka-2" C [lo traldl"g.llc 3( A 2(1. xx xx XY XY XY XY XY *U -emalle _4 male n tra-.ii /+ genotype line chroinosomal sex - 1'emale minatle -/_ +/_ +/- +/_ +/ ± ID)mtrnra-2 genotype FIG. 3. Alternative splicing of dsx pre-mrna in flies carrying the hs-htra-2a transposon. (A) Schematic showing the structure of the dsx gene as well as the female-and male-specific transcripts generated by alternative splicing (9). Exons that are common to male and female transcripts are shown in gray, the female-specific exon in black, and the male-specific exons in white. The locations of the primers used for RT-PCR and for identification of PCR products are indicated. PCR products were visualized by Southern blot hybridization with primers f2 and m2, which span the female- and male-specific splice junctions, respectively. (B) Southern blots of products generated by RT-PCR from diplo-x tra-2b/tra-2b flies carrying the hs-htra-2a transposon and controls are shown. Splicing of dsx RNA was assayed in each transgenic line both with and without heat shock treatment. All control flies were subjected to parallel heat shocks. The transgenic lines assayed (20.1, 30.2, and 50.1) are indicated and compared with flies of the parental strain without the transgene ("none"). (C) Southern blot of dsx RT-PCR products from heat-treated XY;tra-2B/tra-2+ males carrying the hs-htra-2a transposon is shown. Products from chromosomal males of several independent transformed lines are compared with controls. The genotypes with respect to the endogenous tra-2 gene are indicated below each lane as either -/- (for tra-2b/tra-2b) or +/- (for tra-2b/tra-2+). background was observed in testes after periodic heat shocks (Fig. 4 C and D). In contrast, control flies without the transgene but carrying one copy of the endogenous tra-2+ gene showed significant staining after heat shock (Fig. 4B). These results suggest that htra-2a is not able to repress RNA splicing of the third intron of the tra-2 gene. DISCUSSION Here we have presented evidence that tra-2, a key splicing regulator in Drosophila sex determination, has a conserved function in human cells. The tra-2 protein is one of two factors known from genetic analysis to be directly required for processing of dsx pre-mrna along the female-specific pathway (11). There are good reasons to suspect that the homologue of tra-2 described here may facilitate alternative splicing of specific human pre-mrnas. When expressed in Drosophila, the htra-2a protein is able to recognize and affect the splicing of the dsx pre-mrna in a manner that is dependent

5 9008 Developmental Biology: Dauwalder et al. Table 2. Effect of a dsx splicing enhancer mutation on regulation of sexual differentiation by htra-2a Chromosomal Sex comb Genotype* sex Morphology Numbert +/+ Male Male 10.7 ± 0.8 (n = 12) +/+ Female Female 4.3 ± 0.5 (n = 12) dsxs/+ Female Intersexual 6.6 ± 0.7 (n = 20) dsxs/p[hs-htra-2a] Female Intersexual 6.6 ± 0.6 (n = 20) *Genotype shown for doublesex and transgene only. Progeny were obtained from the cross of w/bsy; tra-2+/tra-2+; dsxs/tm3, Serdsx+ males with w1118/w"'8 tra-2b/tra-2+; P[hs-htra-2a4 w+], dsx+/dsx+ virgins from line Progeny from all crosses were heat shocked periodically throughout development. tdata on sex comb number from tra-2b/tra-2+ and tra-2+/tra-2+ individuals were pooled. There were no significant differences observed between these two classes for any dsx genotype. on both the presence of the dsx splicing enhancer region and of at least one sex-specific cofactor that is almost certainly TRA. This strongly suggests that htra-2a participates appropriately in the formation of the multicomponent regulatory complex that directs female specific dsx splicing (19). The tra-2 proteins expressed in flies and humans are therefore likely to have a conserved ability both to recognize particular sequence elements in pre-mrna and to interact directly with other splicing factors that assemble on dsx pre-mrna. Although no natural human targets for htra-2a are yet known, it is worth noting that TRA-2 has been shown to interact with a purinerich sequence that is similar to those identified in several mammalian enhancer elements (17). Such elements are thought to play key roles in both constitutive and alternative RNA splicing and in some cases have been shown to associate with SR proteins and other unidentified protein factors (41-43). We propose that htra-2a interacts with specific human pre-mrnas to affect their splicing patterns in a manner analogous to the way TRA-2 affects dsx splicing. Given that htra-2a can specifically affect dsx pre-mrna splicing, it is surprising that the RRM, which is thought to constitute the major RNA binding domain for this protein, is only 49% identical to the Drosophila protein. Of the 36 identical residues in the RRM, 17 are at positions that make up the RRM consensus (see Fig. 1) and thus are very similar to sequences found in many proteins that do not interact specifically with dsx pre-mrna (30). Many of the remaining 19 identities are concentrated in two regions of RRM. One is immediately upstream of the RNP-1 subdomain and is analogous to the loop 3 segment of the RRM in the UlA and U2B" proteins that contains residues shown to be essential for RNA binding specificity (44, 45). The other region where nonconsensus identities are concentrated is at the carboxy terminus of the RRM directly adjacent to the conserved linker region. The RRM-linker junction region is not similar to any other known protein and thus is likely to perform conserved functions that are specific to TRA-2. The importance of the linker region is also suggested by the fact that two of the three mapped, loss-of-function missense mutations affecting both sex determination and spermatogenesis occur within it (16). Thus integrity of the linker region appears to play an important role in tra-2 function in both the soma and germ line. Interestingly, we have recently identified human cdnas encoding a second TRA- 2-like protein (htra-2p3) that also shares strong identity at the RRM-linker junction (F.A.-M. and W.M., unpublished data). The similarity of htra-2a to a Drosophila sex factor and its ability to affect sexual differentiation in flies raises the issue of whether this factor can affect human sex determination. Although sexual differentiation is a conserved feature of nearly all metazoans, sex determination mechanisms in different organisms show a remarkable degree of divergence that suggests that the molecular strategies used for regulating sex Proc. Natl. Acad. Sci. USA 93 (1996) C e D FIG. 4. Failure of the htra-2a gene to repress tra-2 splicing in the male germ line. X-Gal staining of testes from individuals that simultaneously carry both hs-htra-2a and the splicing reporter construct pczp-orf3 (38). The reporter expresses f3-galactosidase only when splicing of intron 3 is repressed by functional TRA-2 as is shown by comparing testes from tra-2b/tra-2b; P[pCZP-ORF3]) (A) and tra-2b/ tra-2+; P[pCZP-ORF3] (B) chromosomal males. Testes from heat shock treated tra-2b/tra-2b; P[pCZP-ORF3]/P[hs-htra-2a] individuals derived from lines 20.1 and 30.2 are shown in C and D, respectively. change extremely rapidly or have arisen independently during evolution (24). Although a number of genes involved in sex determination in nematodes, flies, and mammals have now been identified, these various types of organisms are known to share no homologous sex factors (25). Mammalian homologues have been identified for both daughterless (the E12 HLH protein) and sans fille (UlA and U2B" small nuclear ribonucleoprotein proteins), two genes that play roles in Drosophila sexual differentiation but also perform non-sexspecific general functions that are essential for viability in flies (7, 46-49). In contrast to these factors, Drosophila TRA-2 affects only spermatogenesis and female somatic sexual identity. The absence of this protein has no effect on viability in either sex (8, 37). Given that TRA-2 does not have a known role outside sexual differentiation, it seems surprising that this factor has conserved its biochemical specificity in two organisms that use entirely different pathways to regulate sex. While we view it as unlikely that htra-2ac or homologues of other Drosophila sex determination genes play critical roles in human sex determination, it is possible that a "regulatory module" consisting of several components of the Drosophila sex determination pathway has been conserved and put to some other purpose in human development. Indeed, a number of such conserved regulatory modules from Drosophila have served as useful paradigms for studying aspects of embryonic patterning in mammals (31, 50, 51). We thank Mitzi Kuroda, Ken Burtis, Tom Cooper, Elaine McGuffin, Dawn Chandler, Steve Marcus, and Richard Behringer for their comments and helpful discussions. Darshna Somaiya provided excellent technical assistance. 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