The Sex Determination Genetic Cascade

The epistatic relationships between the sex determination genes revealed that a hierarchical interaction exists among these genes (Baker and Ridge, 1980). The characterization of these genes showed that their expression during development is controlled by sex-specific splicing of their primary transcripts: the product of a gene controls the sex-specific splicing of the pre-mRNA of the downstream gene in the genetic cascade.

Sxl is at the top of this cascade: its product controls the splicing of its own pre-mRNA and the splicing of the pre-mRNA from the downstream gene transformer (tra). The Tra product and the

Figure 3 Scheme showing the developmental meaning of the X/A signal. produced in females. See text for details.

For simplicity, it is assumed that the X/A signal is only

Figure 3 Scheme showing the developmental meaning of the X/A signal. produced in females. See text for details.

For simplicity, it is assumed that the X/A signal is only product of the constitutive gene transformer-2 (tra-2) control the sex-specific splicing of pre-mRNA from gene doublesex (dsx), which is transcribed in both sexes but gives rise to two different proteins, DsxF and DsxM, in females and males, respectively. The genes intersex (ix) and her are expressed in both sexes and in females, their products assist DsxF to activate female terminal differentiation and to repress male terminal differentiation, whereas Her helps DsxM to activate male terminal differentiation and to repress female terminal differentiation (Figure 4).

The gene dsx is the last gene in the genetic cascade that controls the development of sexual somatic characters, yet there are exceptions. These concern the sex-specific characteristics of the central nervous system and the neuronal induction of a male-specific abdominal muscle, which depend on the function of tra and not dsx through the genes fruitless and dissatisfaction (Figure 4). The gene takeout, which is expressed in the fat body tissue, is closely associated with the adult brain function and is involved in male courtship behavior (Dauwalder et al., 2002). The determination of sexual behavior will not be reviewed here. The interested reader is referred to other reviews on the subject (Yamamoto et al., 1998; O'Kane and Asztalos, 1999; Billeter et al., 2002).

1.1.1.3.1. Control of Sxl expression by sex-specific splicing of its primary transcript The mechanism by which Sxl precisely controls the skipping of its male-specific exon L3 is not totally understood. In contrast to most examples of exon-skipping events described in the literature, Sxl promotes a 100% switch between the two alternatively spliced forms of its own pre-mRNA, suggesting the existence of a complex mechanism of regulation. The role of the Sxl protein in the splicing of its own pre-mRNA is highlighted in brief. More extensive details have been reviewed in Penalva and Sanchez (2003).

The Sxl gene encodes one of the best-characterized members of the family of RNA-binding proteins. It has two segments, located in tandem in the central portion, with similarity to the RNA-binding domain sequences (RBD) or RNA recognition motifs (RRM). The amino terminus of Sxl is very rich in glycine. It has been shown that this domain is implicated in protein-protein interactions (Sxl multimerization) and absolutely required for proper control of Sxl RNA alternative splicing (Wang and Manley, 1997; Waterbury et al., 2000; Lallena et al., 2002). Deletions of the N- and C-termini does not interfere with the ability of the Sxl RBDs to properly bind in vitro to their target sequences, while both RNA-binding domains are required for site-specific RNA binding (Wang and Bell, 1994; Kanaar et al., 1995; Sakashita and Sakamoto, 1996; Samuels et al., 1998).

The Sxl-binding sequence consists of long stretches of poly(U) interrupted by two to four guanosines (Sosnowski et al., 1989; Inoue et al., 1990;

Figure 4 Sex determination gene network. SxlF and SxlM stand for functional female and nonfunctional truncated male Sxl protein, respectively. TraF and TraM stand for functional female and nonfunctional truncated male Tra protein, respectively. DsxF and DsxM stand for functional Dsx protein in females and males, respectively. FruF and FruM stand for functional Fru protein in females and males, respectively. Dsf is dissatisfaction. To is takeout. In the absence of the X/A signal in males, the default state corresponds to the production of SxlM, TraM, and DsxM proteins. Ix and Her proteins are produced in both sexes. CNS, central nervous system. For description of the genes and their regulation see text.

Figure 4 Sex determination gene network. SxlF and SxlM stand for functional female and nonfunctional truncated male Sxl protein, respectively. TraF and TraM stand for functional female and nonfunctional truncated male Tra protein, respectively. DsxF and DsxM stand for functional Dsx protein in females and males, respectively. FruF and FruM stand for functional Fru protein in females and males, respectively. Dsf is dissatisfaction. To is takeout. In the absence of the X/A signal in males, the default state corresponds to the production of SxlM, TraM, and DsxM proteins. Ix and Her proteins are produced in both sexes. CNS, central nervous system. For description of the genes and their regulation see text.

Sakamoto et al., 1992; Horabin and Schedl, 1993a, 1993b; Sakashita and Sakamoto, 1994; Samuels et al., 1994; Singh et al., 1995; Wang and Manley, 1997). It has been observed that Sxl binds to RNAs containing two or more poly(U) sequences in a cooperative manner (Wang and Bell, 1994; Kanaar et al., 1995). Different models have been proposed for the molecular mechanism underlying the role of Sxl protein in controlling the female-specific splicing of its own pre-mRNA (Sakamoto et al., 1992; Horabin and Schedl, 1993a, 1993b; Wang and Bell, 1994; Wang and Manley, 1997; Waterbury et al., 2000; Penalva et al., 2001; Lallena et al., 2002; Nagengast et al., 2003). Nevertheless, there is a common aspect to all of them, namely, the fact that the Sxl protein participates in the skipping of the male-specific exon L3 (female-specific splicing of Sxl pre-mRNA) through binding to multiple U-rich sequences placed in introns 2 and 3, surrounding the male-specific exon L3 (Figure 5). Multiple poly(U) sequences are also present in the adjacent introns of the male-specific exon L3 of the Sxl genes of D. virilis (Bopp et al., 1996) and D. subobscura (Penalva et al., 1996).

The female-specific splicing of late Sxl pre-mRNA requires, in addition to the Sxl protein, the function of other genes, such as sans fille (snf) (Albrecht and Salz, 1993; Flickinger and Salz, 1994; Salz and

Flickinger, 1996; Samuels et al., 1998), female-le-thal-2-d (fl(2)d) (Granadino et al., 1990, 1992; Penalva et al., 2000; Ortega et al., 2003), and viri-lizer (vir) (Hilfiker and Nothiger, 1991; Hilfiker et al., 1995; Niessen et al., 2001). These genes, however, do not play any role in the splicing pattern of early Sxl transcripts (Horabin and Schedl, 1996).

1.1.1.3.2. Sxl protein as its own translation repressor Sxl gives rise to a variety of transcripts, some of them stage- and tissue-specific (Salz et al., 1989; Samuels et al., 1991; Keyes et al., 1992; Penalva et al., 1996). The Sxl mRNAs also contain several Sxl-binding sites at the 3' unstranslated region (UTR). It has been shown that the association of Sxl protein with the 3' UTR of Sxl mRNA significantly decreases Sxl protein expression (Yanowitz et al., 1999). Transcripts having a short 3' UTR are more abundant in the germline and during the early stages of embryogenesis (5-8 h postfertilization) (Salz et al., 1989). In later stages of development and during adult life, the most abundant transcripts have a long 3' UTR (up to 3600 nt) containing several Sxl binding sites (13 in the largest transcript) (Samuels et al., 1991).

It was speculated that the negative autoregulatory loop of Sxl might play an important homeostatic role and that the presence of short transcripts at

Figure 5 Sex-specific splicing of late Sxl pre-mRNA (autoregulatory Sxl function throughout development and adult life). Only the key exons (L2-L4) and introns (I2-I3) of the pre-mRNA are shown. The dot inside exon L3 represents translation stop codons. The hatched ellipsoids in introns I2 and I3 represent the sequences bound by the Sxl protein. Exons and introns are not drawn to scale and the numbers and distribution of Sxl-binding sequences are also not drawn to scale.

Figure 5 Sex-specific splicing of late Sxl pre-mRNA (autoregulatory Sxl function throughout development and adult life). Only the key exons (L2-L4) and introns (I2-I3) of the pre-mRNA are shown. The dot inside exon L3 represents translation stop codons. The hatched ellipsoids in introns I2 and I3 represent the sequences bound by the Sxl protein. Exons and introns are not drawn to scale and the numbers and distribution of Sxl-binding sequences are also not drawn to scale.

early stages of development allows a substantial amount of Sxl protein to be produced and a positive autoregulatory loop to be established via splicing. Once accumulation of the Sxl protein is no longer required, short mRNAs are substituted for larger ones whose expression can be repressed by Sxl. The balance between the negative and the positive posttranscriptional controls keeps the concentration of Sxl at levels that do not interfere with other cellular functions but are sufficient for regulation of Sxl splicing and splicing of its target genes (Yanowitz et al., 1999). Indeed, it has been shown that high levels of Sxl protein have toxic consequences for the cells (Meise et al., 1998; Saccone et al., 1998).

1.1.1.3.3. The role of transformer in sex determination The tra gene is transcribed in both sexes, but its RNA follows alternative splicing pathways (Figure 6). Intron 1 of tra has two alternative 3' splice sites. A non-sex-specific transcript is generated when the proximal 3' splice site is used. Use of this splice site introduces a stop codon in the open reading frame, leading to the production of a truncated, nonfunctional peptide. In females, approximately half of the tra pre-mRNA is spliced differently due to the intervention of the Sxl protein. In this case, the distal 3' splice site is used. As a result, the stretch containing the termination codon is not included in the mature transcript and synthesis of full length Tra protein occurs (Boggs et al., 1987; Belote et al., 1989) (Figure 6).

It has been determined that Sxl regulates female-specific tra pre-mRNA splicing by a blockage mechanism rather than by enhancing the use of the female specific 3' splice site (distal 3'ss). There is a Sxl binding site at the polypyrimidine tract of the non-sex-specific splice site (proximal 3'ss) (Sosnowski et al., 1989; Inoue et al., 1990). This region is also the binding site for the U2 auxiliary factor (U2AF), an essential splicing factor important for the recognition of the 3' splice site. U2AF but not Sxl binds also to the polypyrimidine tract associated with the female-specific 3' splice site, but with 100 times less affinity (Valcáírcel et al., 1993). Chimeric proteins containing the effector domain of U2AF fused to the complete RNA binding domain of Sxl inhibit rather than promote splicing to the non-sex-specific 3' splice site. This suggests that Sxl and U2AF compete for binding to the polypyrimidine tract associated with the non-sex-specific 3' splice site. Binding of Sxl to this sequence displaces U2AF, diverting it to the low affinity distal polypyrimidine tract and promoting the usage of the female-specific 3' splice site (Valcarcel et al., 1993; Granadino et al., 1997) (Figure 6).

The chimeric U2AF-Sxl protein, however, does not disrupt Sxl pre-mRNA splicing regulation, in

Figure 6 Sex-specific splicing of tra pre-mRNA. Boxes and thin lines represent exons (E1-E4) and introns, respectively. The dot inside exon E2 represents translation stop codons. The hatched ellipsoid in front of exon E2 represents the sequence to which both Sxl and U2AF proteins bind.

contrast to what occurs with tra splicing (Granadino et al., 1997). These data suggest that Sxl controls tra and Sxl pre-mRNA alternative splicing by different mechanisms. Notwithstanding, there is controversy concerning the function of the N-terminal region in tra RNA alternative splicing regulation. While it has been proposed that this region is not necessary for tra regulation (Granadino et al., 1997), others have proposed the opposite (Yanowitz et al., 1999). Differences in how the different groups have performed the experiments, which include different constructs (40aa deletion of the N-terminal region in one case and 94aa in the other), use of different promoters, and different real-time polymerase chain reaction (RT-PCR) methods to detect the splice forms, might explain the discrepancy.

The genes female-lethal-2-d (fl(2)d) (Granadino et al., 1996) and virilizer (vir) (Hilfiker et al., 1995) are also required for female-specific splicing of the tra pre-mRNA. Genetic analyses have ruled out a direct role of sans-fille (snf) in tra pre-mRNA splicing (Cline et al., 1999).

1.1.1.3.4. The role of doublesex, hermaphrodite, and intersex in sex determination At the bottom of the sex determination gene cascade are the genes dsx, her, and ix, which are expressed in both sexes.

The dsx pre-mRNA contains six exons: three common exons (E1-E3), a female-specific exon (E4), and two male-specific exons (E5-E6) (Figure 7). In females, the TraF protein, together with the constitutive Tra-2 protein (Goralski et al., 1989; Amrein et al., 1990; Mattox et al., 1990), directs the splicing of the dsx pre-mRNA to the female mode. The mRNA produced contains exons E1, E2, E3, and E4 and gives rise to the female DsxF protein, which promotes female sexual development. In males, where no functional Tra protein is available, the dsx pre-mRNA follows the male mode of splicing that gives rise to mRNA containing exons E1, E2, E3, E5, and E6, which produces male DsxM protein, which promotes male sexual development (Burtis and Baker, 1989; Hoshijima et al., 1991) (Figure 7). A further component that is crucial in the sex-specific splicing of dsx pre-mRNA, is the splicing enhancer dsxRE (doublesex repeat element) placed in the female-specific exon E4, 300 nt downstream of the 3' splice site (review: Maniatis, 1991). Six copies of a 13 nt repeat together with a purine-rich element (PRE) located between repeats 5 and 6, make up the dsxRE enhancer (Burtis and Baker, 1989; Lynch and Maniatis, 1996).

The sequent of the female-specific 3' splice site in intron 3 departs significantly from the consensus 3'

Figure 7 Sex-specific splicing of dsx pre-mRNA. Boxes and thin lines represent exons (E1-E6) and introns, respectively. The dots inside exon E4 represent the dsxRE enhancer to which the TraF-Tra2 complex binds. Poly(A) indicates the polyadenylation site of the mRNA arising from female-specific splicing.

Figure 7 Sex-specific splicing of dsx pre-mRNA. Boxes and thin lines represent exons (E1-E6) and introns, respectively. The dots inside exon E4 represent the dsxRE enhancer to which the TraF-Tra2 complex binds. Poly(A) indicates the polyadenylation site of the mRNA arising from female-specific splicing.

splice site and is weaker than the 3' splice site of the downstream intron 4. In males, the splicing machinery recognizes the 3' splice site of intron 4 instead of the 3' splice site of intron 3. Consequently, the male splicing follows and DsxM protein is produced. In females, however, because of the presence of TraF protein, TraF and Tra2, as well as the SR-protein RBP1, form a complex that binds to the dsxRE enhancer. This complex recruits U2AF and possibly other components of the general splicing machinery to the dsx pre-mRNA causing the splicing machinery to recognize the 3' splice site of intron 3 instead of the 3' splice site of intron 4. Consequently, female splicing follows and DsxF protein is produced (Hedley and Maniatis, 1991; Inoue et al., 1992; Tian and Maniatis, 1993, 1994; Zuo and Maniatis, 1996; Du et al., 1998; Nikolakaki et al., 2002). The activity and localization of the TraF, Tra-2, and RBD1 proteins depend on the activity of the LAMMER kinase encoded by the gene Darkener of apricot (Doa) (Du et al., 1998; Yun et al., 2000; Nikolakaki et al., 2002).

The two Dsx proteins, DsxF and DsxM, are transcription factors controlling the activity of the final target genes involved in sexual differentiation. The two proteins share the N-terminal domain, which contains a DNA-binding domain (DM domain), whereas they differ at their C-terminal domains, which endow these proteins with their specific function (Burtis and Baker, 1989; Hoshijima etal., 1991).

The her gene encodes a zinc finger protein, which endows to this protein the capacity to bind DNA, so that Her can function as a transcription factor (Li and Baker, 1998a). This agrees with the dual function shown by the her gene. Its maternal expression is necessary for early Sxl activation (see Section 1.1.1.2.5), while its zygotic expression is necessary for female terminal differentiation and some aspects of male terminal differentiation (Pultz and Baker, 1995; Li and Baker, 1998b).

The Dsx and Her products act either independently or interdependently to control different sexual differentiation processes. Thus, for example, the yolk protein (yp) genes ypl and yp2 are the best-characterized female terminal differentiation genes. The expression of the ypl and yp2 genes is under the control of gene dsx in the fat body (review: Bownes, 1994). DsxF and DsxM directly activate and inhibit the yp genes, respectively. They exert their function through the fat body specific enhancer (FBE). It has been found that in females, DsxF and Her independently activate the yp genes and that Her acts through sequences different than those of the FBE enhancer (Li and Baker, 1998b). In males, DsxM prevents the activation of yp genes by Her (Li and Baker, 1998b). However, the genes dsx and her also function interdependently to control the female differentiation of the sexual dimorphic foreleg bristles and the pigmentation of the 5th and 6th tergites (Li and Baker, 1998b).

The ix gene is transcribed in both sexes and its pre-mRNA does not follow a sex-specific splicing, indicating that the Ix protein is present in both sexes. Ix shows similarity to proteins thought to act as transcriptional activators, although a DNA-binding domain has yet to be identified (Garrett-Engele et al., 2002). By means of the yeast-2 hybrid and coimmunoprecipitation analyses, it has been possible to demonstrate that Ix interacts with DsxF but not DsxM. In addition, by gel shift analysis it has been shown that Ix and DsxF form a DNA-binding complex (Garrett-Engele et al., 2002).

Ix also participates in the control of yp gene expression through the FBE enhancer (Garrett-Engele et al., 2002). Ectopic expression of DsxF in ix mutant males is not sufficient to induce expression of the yp genes, indicating that DsxF requires ix function to activate the yp genes (Waterbury et al., 1999). Collectively, these results suggest that Ix and DsxF form a complex to control female terminal differentiation.

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