InteractiveFly: GeneBrief

transformer 2: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - transformer 2

Synonyms -

Cytological map position - 51B4--51B6

Function - mRNA splicing factor

Keyword(s) - Sex determination, Splicing factor

Symbol - tra2

FlyBase ID: FBgn0003742

Genetic map position - 2-70

Classification - Serine/Arginine motif, RNA recognition motif

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Li, J. and Handler, A. M. (2017). Temperature-dependent sex-reversal by a transformer-2 gene-edited mutation in the spotted wing Drosophila, Drosophila suzukii. Sci Rep 7(1): 12363. PubMed ID: 28959033
Summary:
Female to male sex reversal was achieved in an emerging agricultural insect pest, Drosophila suzukii, by creating a temperature-sensitive point mutation in the sex-determination gene, transformer-2 (tra-2), using CRISPR/Cas9-directed repair gene-editing. Ds-tra-2ts2 mutants developed as normal fertile XX and XY adults at permissive temperatures below 20 ° C, but at higher restrictive temperatures (26 to 29 ° C) chromosomal XX females developed as sterile intersexuals with a predominant male phenotype, while XY males developed with normal morphology, but were sterile. The temperature-dependent function of the Ds-Ds-tra-2ts2 protein was also evident by the up- and down-regulation of female-specific Ds-Yolk protein 1 (Ds-Yp1) gene expression by temperature shifts during adulthood. This study confirmed the temperature-dependent function of a gene-edited mutation and provides a new method for the more general creation of conditional mutations for functional genomic analysis in insects, and other organisms. Furthermore, it provides a temperature-dependent system for creating sterile male populations useful for enhancing the efficacy of biologically-based programs, such as the sterile insect technique (SIT), to control D. suzukii and other insect pest species of agricultural and medical importance.
Mikoluk, C., Nagengast, A. A. and DiAngelo, J. R. (2018). The splicing factor transformer2 (tra2) functions in the Drosophila fat body to regulate lipid storage. Biochem Biophys Res Commun 495(1): 1528-1533. PubMed ID: 29203241
Summary:
Excess nutrients are stored as triglycerides mainly in the adipose tissue of an animal and these triglycerides are located in structures called lipid droplets. Previous genome-wide RNAi screens in Drosophila cells identified splicing factors as playing a role in lipid droplet formation. This lab has recently identified the SR protein, 9G8, as an important factor in fat storage as decreasing its levels results in augmented triglyceride storage in the fat body. Previous in vitro studies have implicated 9G8 in the regulation of splicing of the sex determination gene doublesex (dsx) by binding to transformer (tra) and transformer2 (tra2); however, any function of these sex determination proteins in regulating metabolism is unknown. This study has uncovered a role of tra2 to regulate fat storage in vivo. Inducing tra2(dsRNA) in the adult fat body resulted in an increase in triglyceride levels but had no effect on glycogen storage. Consistent with the triglyceride phenotype, tra2 knockdown flies lived longer under starvation conditions. In addition, this increase in triglycerides is due to more fat storage per cell and not an increase in the number of fat cells. Interestingly, the splicing of CPT1, an enzyme involved in the breakdown of lipids, was altered in flies with decreased tra2. The less-catalytically active isoform of CPT1 accumulated in tra2(dsRNA) flies suggesting a decrease in lipid breakdown, which is consistent with the increased triglyceride levels observed in these flies. Together, these results suggest a link between mRNA splicing, sex determination and lipid metabolism and may provide insight into the mechanisms underlying tissue-specific splicing and nutrient storage.
BIOLOGICAL OVERVIEW

The splicing of Doublesex pre-messenger RNA has become the primary model for the process of alternative splicing of RNAs in all higher organisms. Essential to this splicing is Transformer 2, a protein containing arginine/serine rich domains involved in protein-protein interaction. This overview will outline the complex process of splicing, highlighting the specific importance of alternative splicing, with special attention paid to the role of Transformer 2 protein.

In the most general terms, splicing is the removal of an intron, a specified portion of the nucleotide chain, from between two flanking exons (portions of the pre-messenger RNA that code for proteins). The process is regulated by certain proteins called splice factors, and depends upon the successful construction of splice machinery, in particular a structure known as a spliceosome, a multiprotein complex that is assembled step by step during the splicing process. Alternative splicing generates alternative messenger RNA species, depending on the pattern of intron removal. For example, by alternative splicing in which exons are skipped, unwanted exons can be excluded altogether from mRNA species coding for a particular polypeptide chain, thus generating a functionally alternative protein.

Imagine the intron, a stretch of nucleotides to be removed, bracketed between two exons, one upstream and one downstream. In the case of Doublesex pre-mRNA, the intron to be removed lies between exons 3 and 4. When this particular intron is productively removed, a female specific Doublesex mRNA is produced. The female specific Doublesex mRNA, as its name implies, codes for a female specific Doublesex protein that will result in the development of female flies. When the intron is not productively removed, the exon 3 becomes spliced instead to a downstream exon 5, resulting in the production of a male specific Doublesex mRNA, and the consequent development of male flies.

This all important splice event is regulated by two proteins: Transformer and Transformer 2. Splicing of TRA and TRA2 pre-mRNA is regulated by the splice factor Sex lethal, the upstream master regulator of sex determination in Drosophila. Sex lethal assures that TRA and TRA2 proteins are present only in females. TRA and TRA2 proteins act subsequently on Doublesex pre-mRNA to regulate splicing that will result in a female specific Doublesex mRNA. In the absence of TRA and TRA2, a male specific Doublesex mRNA is attained, guaranteeing a male developmental fate.

How exactly do TRA and TRA2 accomplish the splicing of DSX pre-mRNA? To answer this question, it is necessary to look more closely at what happens biochemically during splicing. Critical to this process is the initial recognition of the two ends of the intron. The left hand, upstream end, known as the 5' splice site, is first recognized by U1 small nuclear ribonucleoprotein (snRNP), a multiprotein complex consisting of among others, an A subunit, a C subunit and a 70 kD ribonucleoprotein with an RNA recognition motif and two arginine-serine rich RS motifs (for more information on Drosophila U1 snRNP, see sans fille). The right hand downstream end, known as the 3' splice site is recognized by U2AF65, a protein with three RNA recognition motifs and an arginine-serine rich RS motif. Another protein (U2AF35) that interacts with U2AF65 must also assembling at the 3 ' splice site (Chabot, 1996).

At this point, after the proteins listed above are poised for action, another group of proteins (called SR proteins) become involved. The human SR protein family comprises at least six members, designated SRp75, Srp55, SRp40, SRp30a, SRP30b and SRp20; these are conserved from Drosophila to humans. In Drosophila, only the SR proteins RBP1 (homologous to SRp20) and B52 (homologous to SRp55) have been cloned (Heinrichs, 1995 and references). The SR proteins share a characteristic domain structure consisting of an N-terminal RRM type RNA binding domain known to be involved in RNA binding and a C-terminal serine-arginine SR domain. What happens is that the SR proteins act as a bridge between U1, associated with the 5' splice site and U2AF65/U2AF35, assembled at the 3' splice site. Essentially the SR proteins are involved in the assembly of the spliceosome. It is thought that SR proteins can regulate alternative splicing in many pre-mRNAs, but in DSX mRNA splicing the principle regulators are TRA and TRA2 (Chabot, 1996). Once the spliceosome is assembled, removal of the intron is achieved by a complex program involving additional steps (Lamond, 1994) that will not be described here.

So far this overview has focused on the intron stretched between upstream and downstream exons of DSX pre-mRNA. A closer look at the middle of exon 4 is now in order, the site from which TRA and TRA2 act. Here is found one of the wonders of nucleic acid: a sequence coding for a protein can have hidden in its triplet codes, a region with an altogether different function, in this case a sequence that binds splicing regulators and influences the intron splicing event taking place upstream from its hidden message. The message inside exon 4 is a highly structured region called the dsx repeat element (dsxRE); it contains six copies of a 13-nucleotide repeat sequence. Between repeat elements 5 and 6 of the dsxRE is another element, the purine-rich enhancer (PRE). Thus the dsxRE consists of two types of regulatory sequences, the repeat elements R1-6, and the PRE (Lynch, 1995 and references).

TRA2 binds to repeat elements R2-5 with significant specificity. In contrast, the SR proteins bind with very little, although measurable, specificity. TRA does not bind specifically to the dsxRE. The specificity of binding for the combination of TRA and an SR protein is 10-15 times greater than that observed with either protein alone. Moreover, the addition of TRA2 to TRA plus an SR protein further increases the specificity of the TRA + SR complex by two to threefold (Lynch, 1996). It is thought that the SR protein RBP1 binds the purine-rich enhancer (PRE) in carrying out its role as a catalyst for assembly of TRA and TRA2, which interact with the repeat elements in the dsxRE (Heinrichs, 1995). TRA, TRA2 and RBP1 serve to facilitate the assembly of a functional spliceosome associated with the intron between exons 3 and 4.

The 3' splice site of the DSX intron is actually thought to be suboptimal, that is, without help from the dsxRE, unaided splicing would favor the male outcome: exon 4 of the DSX gene would be passed over and splicing would occur between exons 3 and 5 of the DSX pre-mRNA. The splice enhancer is utilized in the females, and is functional because of the presence of TRA and TRA2 (Tian, 1994). TRA's role is special in that splicing of the TRA pre-mRNA of Drosophila is regulated by Sex-lethal-dependent 3' splice site blockage, generating a female specific TRA protein (Sosnowski, 1994). Thus presence of female specific TRA, but not TRA2 which does not differ between males and females, phenotypically distinguishes males and females, and the role of TRA2 is to lend specificity to joining of 3' and 5' splice junctions.

Sex determination genes control the development of the Drosophila genital disc, modulating the response to Hedgehog, Wingless and Decapentaplegic signals

Alternative mRNA splicing directed by SR proteins and the splicing regulators TRA and TRA2 is an essential feature of Drosophila sex determination. These factors are highly phosphorylated, but the role of their phosphorylation in vivo is unclear. Mutations in the Drosophila LAMMER kinase, Darkener of apricot (Doa), alter sexual differentiation and interact synergistically with tra and tra2 mutations. Doa mutations disrupt sex-specific splicing of doublesex pre-mRNA, a key regulator of sex determination, by affecting the phosphorylation of one or more proteins in the female-specific splicing enhancer complex. Examination of pre-mRNAs regulated in a similar manner as dsx shows that the requirement for Doa is substrate specific. These results demonstrate that a SR protein kinase plays a specific role in developmentally regulated alternative splicing (Du, 1998).

In both sexes, the Drosophila genital disc contains the female and male genital primordia. The sex determination gene doublesex controls which of these primordia will develop and which will be repressed. In females, the presence of DoublesexF product results in the development of the female genital primordium and repression of the male primordium. In males, the presence of DoublesexM product results in the development and repression of the male and female genital primordia, respectively. This report shows that DoublesexF prevents the induction of decapentaplegic by Hedgehog in the repressed male primordium of female genital discs, whereas DoublesexM blocks the Wingless pathway in the repressed female primordium of male genital discs. It is also shown that DoublesexF is continuously required during female larval development to prevent activation of decapentaplegic in the repressed male primordium, and during pupation for female genital cytodifferentiation. In males, however, it seems that DoublesexM is not continuously required during larval development for blocking the Wingless signaling pathway in the female genital primordium. Furthermore, DoublesexM does not appear to be needed during pupation for male genital cytodifferentiation. Using dachshund as a gene target for Decapentaplegic and Wingless signals, it was also found that DoublesexM and DoublesexF both positively and negatively control the response to these signals in male and female genitalia, respectively. A model is presented for the dimorphic sexual development of the genital primordium in which both DoublesexM and DoublesexF products play positive and negative roles (Sanchez, 2001).

dpp is expressed in the growing male genital primordium of male genital discs but not in the repressed male primordium (RMP) of female genital discs. This suggests that the developing or repressed status of the male genital primordium is determined by the regulation of dpp expression. As dsx controls the developmental status of the male genital primordium, and the expression of dpp depends on the Hh signal, the relationship between the Hh signal cascade and dsx in the control of RMP development was examined. To this end, a twin clonal analysis for the loss-of-function tra2 mutation was performed in tra2/+ female genital discs. In this way, the proliferation and the induction of dpp expression was examined in the clones homozygous for tra2 (male genetic constitution) and that of the twin wild-type clones, both in the repressed male and the growing female primordia. Recall that the effects of tra2 in the genital disc are entirely mediated by its role in the splicing of DSX RNA: the presence or absence of functional Tra2 product gives rise to the production of female DsxF or male DsxM product, respectively. Clones for tra2 (expressing DsxM) induced in the RMP of female genital discs show overgrowth and are always associated with dpp expression, indicating that the lower proliferation shown by the RMP is probably caused by the absence of dpp expression. This activation of dpp is restricted to only certain parts of the clone and never overlaps with Wg expression. Since wg is normally expressed in the RMP, the possibility exists that the cells that do not express dpp in the clone are expressing wg, owing to their antagonistic interaction. Double staining of Wg and Dpp in tra2 clones reveals an expansion of the normal domain of wg expression that abuts the dpp-expressing cells (Sanchez, 2001).

In the RMP, the two sister clones are different in size: the tra2 clone (male genetic constitution) is bigger than the wild-type twin clone (female genetic constitution). In contrast, when the clones are induced in the growing female genital primordium, both of them are of a similar size. Moreover, the pattern of dpp expression does not change in the tra2 cells induced in this primordium (Sanchez, 2001).

optomotor-blind, a target of the Dpp pathway, also responds to Dpp in the genital disc. Since dpp is de-repressed in tra2 clones induced in the RMP, the activation of omb was monitored in these clones. The activation of dpp in tra2 clones induces the expression of this target gene, whose function is required for the development of specific male genital structures. It is concluded that DsxF product prevents the induction of Dpp by Hh in the repressed male genital primordium of female genital discs (Sanchez, 2001).

In the male genital disc, which has DsxM product, the low proliferation rate of the repressed female primordium (RFP) cannot be attributed to a lack of dpp or wg, since both genes are expressed in this primordium. Failure to respond to the Dpp signal may also be ruled out because the RFP expresses the Dpp downstream gene, omb, indicating that the Dpp pathway is active in this primordium. However, Dll, a target gene for both Wg and Dpp, is not expressed in the RFP but is expressed in the developing female primordium of female genital discs. This suggests that the Wg pathway cannot activate some of its targets in the RFP. Thus, the analysis of dsx1 mutant genital discs, where both male and female genital primordia develop, becomes relevant. These mutant discs show neither DsxM nor DsxF products. The female genital primordium of these discs now expresses Dll. It is concluded that DsxM controls the response to the Wg pathway in the RFP of male genital discs (Sanchez, 2001).

The gene dachsund (dac) is also a target of the Hh pathway in the leg and antenna. In the present study, it was found that dac is differentially expressed in female and male genital discs. In the female genital discs, which have DsxF product, dac expression mostly coincides with that of wg in both the growing female primordium and the RMP. In contrast, in male genital discs, which have DsxM product, dac is not similarly expressed to wg but its expression partially overlaps that of dpp and no expression is observed in the RFP. In pkA minus clones, which autonomously activate Wg and Dpp signals in a complementary pattern, dac was ectopically expressed only in mutant pkA minus cells at or close to the normal dac expression domains in male and female genital discs. In pkA minus;dpp minus double clones, which express wg, dac is not ectopically induced in the male primordium of the male genital disc, but is still ectopically induced in both the growing female genital primordium and the RMP of female genital disc. Conversely, in pkA minus wg minus double clones, which express dpp, dac is not ectopically induced in the growing female or in the RMP of female genital discs, but is ectopically induced in the growing male primordium of the male genital disc. These results indicate that dac responds differently to Wg and Dpp signals in both sexes (Sanchez, 2001).

In dsxMas/+ intersexual genital discs, which have both DsxM and DsxF products, and in dsx1 intersexual genital discs, which have neither DsxM nor DsxF products, dac is expressed in Wg and Dpp domains although at lower levels than in normal male and female genital discs. These results suggest that DsxM plays opposing, positive and negative roles in dac expression in male and female genital discs, respectively; and that DsxF plays opposing, positive and negative roles in dac expression in female and male genital discs, respectively. To test this hypothesis, tra2 clones (which express only DsxM ) were induced in female genital discs. The expression of dac is repressed in tra2 clones located in Wg territory. Therefore, DsxF positively regulates dac expression in the Wg domain, and DsxM negatively regulates dac expression in this domain, otherwise dac would be expressed in tra2 clones at the low levels found in dsx intersexual genital discs. However, when the tra2 clones are induced in the RMP, in the territory competent to activate dpp, they show ectopic expression of dac (Sanchez, 2001).

Therefore, DsxM positively regulates dac expression in the Dpp domain, whereas DsxF negatively regulates dac expression in this domain, since in normal female genital discs with DsxF dac is not expressed in Dpp territory. This is further supported by the induction of dac in the Wg domain and repression of dac in the Dpp domain by ectopic expression of DsxF in the male genital primordium of male genital discs. It is concluded that in male genital discs, DsxM positively and negatively regulates dac expression in Dpp and Wg domains, respectively; and in female genital discs, DsxF positively and negatively regulates dac expression in Wg and Dpp domains, respectively (Sanchez, 2001).

Homozygous tra2ts larvae with two X-chromosomes develop into female or male adults if reared at 18°C or 29°C, respectively, because at 18°C they produce DsxF and at 29°C they produce DsxM. A shift in the temperature of the culture is accompanied by a change in the sexual pathway of tra2ts larvae. Analysis of the growth of genital primordia and their capacity to differentiate adult structures of tra2ts flies was performed using pulses between the male- and the female-determining temperatures in both directions during development (Sanchez, 2001).

Regardless of the stage in development at which the female-determining temperature pulse was given (transitory presence of functional Tra2ts product; i.e. transitory presence of DsxF product and absence of DsxM product), the male genital disc develops normal male adult genital structures and not female ones. This occurs even if the pulse is applied during pupation. Pulses of 24 hours at the male-determining temperature (temporal absence of functional Tra2 ts product; i.e. transitory absence of DsxF product and presence of DsxM product) before the end of first larval stage produces female and not male genital structures. However, later pulses always give rise to male genital structures, except when close to pupation. Further, the capacity of the female genital disc to differentiate adult genital structures is also reduced when the temperature pulse is applied during metamorphosis (Sanchez, 2001).

When the effect of the male-determining temperature pulses was analyzed in the genital disc, it was found that overgrowth of the RMP is always associated with the activation of dpp in this primordium. However, this activation and the associated overgrowth only occurs when the temperature pulse is given after the end of first larval instar. This suggests that there is a time requirement for induction of dpp (Sanchez, 2001).

The activation of this gene in the RMP and the cell proliferation resumed by this primordium, as well as its capacity to differentiate adult structures is irreversible, because they are maintained when the larvae are returned to the female-determining temperature, which is when functional Tra2ts product is again available (i.e. the presence of DsxF product and absence of DsxM product). This time requirement for induction of dpp is also supported by the fact that dsx11 clones (which lack DsxM) induce differentiated normal male adult genital structures in the developing male genital primordium of XY; dsx11/+ male genital discs (which express only DsxM ) after 24 hours of development. However, when the dsx11 clones are induced in the time period between 0 and 24 hours of development, they do not differentiate normally and give rise to incomplete adult male genital structures. This different developmental capacity shown by the dsx11 clones depending on their induction time is explained as follows. When the clones are induced after 24 hours of development, dpp is already activated. Indeed, these clones show no change in the expression pattern of dpp or their targets. Accordingly, these clones display normal proliferation and capacity to differentiate male adult genital structures. However, when the clones are induced early in development, dpp is not yet activated, since this gene is not expressed in the male genital primordium of male genital discs early in development. Therefore, when the male genital disc reaches the state in development when dpp is induced, the cells that form the clones activate this gene as in dsx mutant intersexual flies because the clones have neither DsxM nor DsxF products. Consequently, these clones do not achieve a normal proliferation rate, and then do not differentiate normal adult male genital structures (Sanchez, 2001).

As described above, it has been shown that dsx regulates the expression of gene dac. Recall that in male genital discs, DsxM positively and negatively regulates dac expression in Dpp and Wg domains, respectively; and in female genital discs, DsxF positively and negatively regulates dac expression in Wg and Dpp domains, respectively. The expression of the gene dac was analyzed in genital discs of tra2ts flies using pulses between the male- and the female-determining temperatures in both directions. It was found that the dac expression pattern switches from a 'female type' to a 'male type' when male-determining temperature pulses were applied to tra2ts larvae after first larval instar. Note that dac expression is reduced in the Wg domain of the RMP and is progressively activated in the Dpp domain. It should be remembered that these pulses lead to the transient presence of DsxM instead of DsxF product. Thus, these results are consistent with the previously proposed suggestion that DsxM activates dac in the Dpp domain and represses it in the Wg domain (again the converse is true for DsxF). When the pulse is given during first larval instar, dac is not activated in the Dpp domain of RMP, in spite of the fact that there is also a transient presence of DsxM instead of DsxF. This is explained by the lack of competence of cells to express Dpp, which is acquired after first larval instar. When the tra2ts larvae reach such a developmental stage, these cells now produce DsxF because they have returned to the female-determining temperature (Sanchez, 2001).

For information on the role of Sex Lethal in TRA pre-mRNA, see Sex lethal. For information on the biological role of Doublesex, see Doublesex.


REGULATION

Targets of Activity

In addition to its role in the sex-specific control of doublesex RNA splicing in somatic tissues, the transformer-2 gene also regulates the splicing of its own transcripts in the male germ line. The Drosophila transformer-2 gene uses alternative promoters and splicing patterns to generate four different mRNAs that together encode three putative RNA-binding polypeptides. The transformer-2 products expressed in somatic tissues function to regulate the RNA splicing of the sex determination gene doublesex, whereas products expressed in the male germ line play an unknown, but essential, role in spermatogenesis. Two alternatively spliced transformer-2 transcripts (C and E) are found only in the male germ line. These male germ line-specific mRNAs differ from each other by the presence or absence of a single intron called M1. M1-containing transcripts make up a majority of transformer-2 germ-line transcripts in wild-type males but fail to accumulate in males homozygous for transformer-2 null mutations. Germ-line transformation experiments using a variety of reporter gene constructs demonstrate that specific polypeptide products of the transformer-2 gene itself normally repress M1 splicing in the male germ line. It is proposed that this autoregulatory function may serve in negative feedback control of transformer-2 activity during spermatogenesis. The finding that transformer-2 controls multiple splicing decisions suggests that a variety of different alternative splicing choices could be regulated by a relatively limited number of trans-acting factors (Mattox, 1991).

In the male germline, where tra-2 has an essential role in spermatogenesis, a single isoform, C, is found to uniquely perform all necessary functions. This isoform appears to regulate its own synthesis during spermatogenesis through a negative feedback mechanism involving retention of intron 3. Two transcripts, A and B, code for two different isoforms that function redundantly to direct female differention and female specific doublesex pre-mRNA splicing (Mattox, 1996).

Somatic sex determination in Drosophila involves a hierarchy of regulated alternative pre-mRNA processing. Female-specific splicing and/or polyadenylation of Doublesex pre-mRNA, the final gene in this pathway, requires Transformer and Transformer-2 proteins. The mechanisms by which these proteins regulate RNA processing has not been characterized. TRA-2 produced in Escherichia coli binds specifically to a site within the female-specific exon of DSX pre-mRNA. This site, which contains six copies of a 13 nucleotide repeat, is required not only for female-specific splicing, but also for female-specific polyadenylation. These observations suggest that TRA-2 is a positive regulator of DSX pre-mRNA processing (Hedley, 1991).

Cotransfection analyses in which the dsx gene and the female-specific transformer and transformer-2 complementary DNAs were expressed in Drosophila Kc cells reveal that female-specific splicing of the DSX transcript is positively regulated by the products of the tra and tra-2 genes. Furthermore, analyses of mutant constructs of DSX mRNA show that a portion of the female-specific exon sequence is required for regulation of DSX pre-messenger RNA splicing (Hoshijima, 1991).

Sex-specific alternative processing of the Doublesex pre-mRNA controls somatic sexual differentiation in Drosophila melanogaster. Processing in the female-specific pattern results from the utilization of an upstream terminal exon and requires the activities of both the transformer and transformer-2 genes. Use of the more downstream male-specific terminal exons does not require the activities of these genes and is thus considered the default DSX-processing pattern. Transient expression of DSX pre-mRNAs in the presence or absence of tra and tra-2 gene products was carried out in Drosophila tissue culture cells to investigate the molecular mechanism controlling this alternative RNA-processing decision. These studies reveal that female-specific processing of DSX pre-mRNA is controlled by TRA and TRA-2 through the positive regulation of female-specific alternative 3'-terminal exon use. Delineation of cis-acting sequences necessary for regulation shows that a 540-nucleotide region from within the female exon is both necessary and sufficient for regulation. In addition, utilization of the female-specific 3'-splice site (3'SS) is regulated independently of female-specific polyadenylation. Regulated polyadenylation is obtained only in the presence of splicing, suggesting that activation of female-specific exon use occurs by 3'SS activation (Ryner, 1991).

Sex-specific alternative processing of doublesex precursor messenger RNA (pre-mRNA) is one of the key steps that regulates somatic sexual differentiation in Drosophila. By transfection analyses using dsx minigene constructs, six copies of the 13-nucleotide sequence TC(T/A)(T/A)C(A/G)ATCAACA have been identified in the female-specific fourth exon; these act as the cis elements for the female-specific splicing of DSX pre-mRNA. UV-crosslinking experiments reveal that both female-specific Transformer and Transformer-2 bind to the 13-nucleotide sequences of DSX pre-mRNA. These results strongly suggest that the female-specific splicing of DSX pre-mRNA is activated by the binding of these proteins to the 13-nucleotide sequences (Inoue, 1992).

TRA and TRA2 act by recruiting general splicing factors to a regulatory element located downstream of a female-specific 3' splice site. Remarkably, TRA, TRA2, and members of the serine/arginine-rich (SR) family of general splicing factors are sufficient to commit DSX pre-mRNA to female-specific splicing, and individual SR proteins differ significantly in their ability to participate in commitment complex formation. Characterization of the proteins associated with affinity-purified complex formed on DSX pre-mRNA reveals the presence of TRA, TRA2, SR proteins, and additional unidentified components. It is concluded that TRA, TRA2, and SR proteins are essential components of a splicing enhancer complex (Tian, 1993).

The Drosophila proteins Transformer (TRA) and Transformer2 (TRA2) regulate the sex-specific alternative splicing of Drosophila Doublesex pre-mRNA by specifically binding to a splicing enhancer (dsx repeat element, or dsxRE) located 300 nucleotides (nt) downstream from a female-specific 3' splice site. The dsxRE can function as a TRA and TRA2-independent splicing enhancer in vitro when located within 150 nucleotides of the 3' splice site. Based on the relative levels of SR proteins that bind stably to the dsxRE in the presence or absence of TRA and TRA2, it is proposed that the constitutive splicing activity of the dsxRE is mediated by its weak interactions with SR proteins and possibly other general splicing factors. In contrast, TRA and TRA2 allow the dsxRE to function at a distance from the intron by stabilizing the interactions between these proteins and the dsxRE (Tian, 1994).

In Drosophila melanogaster, the fruitless (fru) gene controls essentially all aspects of male courtship behavior. It does this through sex-specific alternative splicing of the FRU pre-mRNA, leading to the production of male-specific FRU mRNAs capable of expressing male-specific Fru proteins. Sex-specific FRU splicing involves the choice between alternative 5' splice sites, one used exclusively in males and the other used only in females. The Drosophila sex determination genes transformer (tra) and transformer-2 (tra-2) switch FRU splicing from the male-specific pattern to the female-specific pattern through activation of the female-specific FRU 5' splice site (SS). Activation of female-specific FRU splicing requires cis-acting tra and tra-2 repeat elements that are part of an exonic splicing enhancer located immediately upstream of the female-specific FRU 5' SS and are recognized by the Tra and Tra-2 proteins in vitro. This FRU splicing enhancer is sufficient to promote the activation by Tra and Tra-2 of both a 5' splice site and the female-specific doublesex (dsx) 3' splice site, suggesting that the mechanisms of 5' splice site activation and 3' splice site activation may be similar (Heinrichs, 1998).

To determine whether the tra/tra-2 repeat elements are essential for regulation of FRU splicing by Tra and Tra-2, a construct, fruM+FREmut, was tested in which the sequence of the conserved part of the tra/tra-2 repeat elements was changed from TCAATCAACA to GGCAGCTTAC. In construct fruM+FREmut, switching to female FRU splicing by Tra and Tra-2 is almost completely blocked, as indicated by the presence of significant amounts of male splicing product M in the presence of cotransfected tra and tra-2. In contrast, deletion of a 1-kb fragment between the repeat elements and the male-specific 5' SS, as in construct fruM+FB-M, does not affect regulation of FRU splicing by Tra and Tra-2. These findings show that the tra/tra-2 repeat elements are required for regulation of FRU splicing by Tra and Tra-2, suggesting that Tra and Tra-2 promote female-specific FRU splicing by acting through these elements (Heinrichs, 1998).

Is the tra/tra-2 repeat region in FRU sufficient to promote the activation of a 5' SS by tra/tra-2? Since the male-specific FRU 5'SS is normally unaffected by tra/tra-2, a 300-bp fragment of fru containing the tra/tra-2 repeats was inserted 4 nt upstream of the male-specific FRU 5' SS (construct fruM+REwt). Interestingly, spliced male product is detected upon cotransfection with tra/tra-2, suggesting activation of the male-specific Fru 5' SS by tra/tra-2 in this hybrid construct. Thus, the tra/tra-2 repeat elements are essential to promote the activation of a heterologous 5' SS by tra/tra-2. Lack of usage of the male-specific 5' SS in the constructs fruM+REwt and fruM+REmut in the absence of cotransfected tra/tra-2 was found to be due to the deletion of a stretch of sequence upstream of the male-specific 5' SS in these constructs. The insertion of the FRU repeat region in either orientation does not affect the usage of the male-specific FRU 5' SS in the absence of cotransfected tra/tra-2 (Heinrichs, 1998).

The Drosophila exuperantia (exu) gene encodes overlapping sex-specific, germline-dependent mRNAs. In this work, the structural differences between these sex-specific EXU mRNAs were determined by sequence analysis of 9 ovary and 10 testis cDNAs. The transformer 2 gene functions in sex determination of female somatic cells through its role in regulating female-specific splicing of Doublesex (dsx). tra-2 is required in male germ cells for efficient male-specific processing of EXU RNA; in the absence of tra-2, X/Y males produce a new mRNA that is processed at its 3' end so that it contains sequences normally specific to the female 3' untranslated region. Although the processing event that requires tra-2 occurs in an untranslated region of the EXU transcript, the isolation and characterization of a male-specific exu allele that deletes male 3' untranslated sequence indicates that this processing is biologically significant (Hazelrigg, 1994).

In male germline TRA-2 affects sex specific processing of Exuperantia and TRA2 itself, both required for spermatogenesis. Transformer 2 isoform B is necessary and sufficient for correct processing of Exuperantia pre-mRNA in the male germline. In DSX splicing, TRA and TRA2 bind directly to sequences dowstream of the female specific 3' splice site, enhancing its recognition by the general splicing machinery. In the case of TRA-2 male specific splicing, the TRA2 protein represses splicing of its own intron (Mattox, 1996).

The gene alternative testis transcripts (att) is alternatively expressed at both the RNA and protein levels in testes and somatic tissues. The testis-specific RNA differs from somatic RNAs in both promoter usage and RNA processing and is dependent on the function of the transformer 2 gene. The differences between the somatic and testis RNAs have substantial consequences at the protein level. The somatic RNAs encode a protein with homology to the mammalian Graves' disease carrier proteins. The testis RNA lacks the initiation codons used in somatic tissue and encodes two different proteins. One of these begins in a testis-specific exon, uses a reading frame different from that for the somatic protein, and is completely novel. The other protein initiates translation in the frame of the somatic RNA at a Len CUG codon that is within the open reading frame for the somatic protein. This produces a novel truncated version of the Graves' disease carrier protein-like protein that lacks all sequences N terminal to the first transmembrane domain (Madigan, 1996).

The Drosophila fruitless gene encodes a transcription factor that essentially regulates all aspects of male courtship behavior. The use of alternative 5' splice sites generates fru isoforms that determine gender-appropriate sexual behaviors. Alternative splicing of fruitless is regulated by Tra and Tra2 and depends on an exonic splicing enhancer (fruRE) consisting of three 13 nucleotide repeat elements, nearly identical to those that regulate alternative sex-specific 3' splice site choice in the doublesex gene. Doublesex has provided a useful model system to investigate the mechanisms of enhancer-dependent 3' splice site choice. However, little is known about enhancer dependent regulation of alternative 5 splice sites. The mechanisms of this process were investigated using an in vitro system in which recombinant Tra/Tra2 could activate the female-specific 5' splice site of fruitless. Mutational analysis has demonstrated that at least one 13 nucleotide repeat element within the fruRE is required and sufficient to activate the regulated female-specific splice site. As was established for doublesex, the fruRE can be replaced by a short element encompassing tandem 13 nucleotide repeat elements, by heterologous splicing enhancers, and by artificially tethering a splicing activator to the pre-mRNA. Complementation experiments show that SR proteins facilitate enhancer-dependent 5' splice site activation. It is concluded that splicing enhancers function similarly in activating regulated 5' and 3' splice sites. These results suggest that exonic splicing enhancers recruit multiple spliceosomal components required for the initial recognition of 5' and 3' splice sites (Lam, 2003).

Transformer, a protein that interacts with Transformer 2: Splicing of Transformer mRNA

The protein Sex-lethal activates a female-specific 3' splice site in the first intron of Transformer pre-mRNA while repressing an alternative non-sex-specific site. Using an in vitro system, the molecular basis of the splice site switch has been determined. SXL inhibits splicing to the non-sex-specific (default) site by specifically binding to its polypyrimidine tract, blocking the binding of the essential splicing factor U2AF. This enables U2AF to activate the lower-affinity female-specific site. A splicing 'effector' domain present in U2AF but absent from SXL accounts for the different activities of these two polypyrimidine-tract-binding proteins: addition of the U2AF effector domain to SXL converts it from a splicing repressor to an activator and renders it unable to mediate splice-site switching (Valcarcel, 1993).

Sex determination and X chromosome dosage compensation in Drosophila melanogaster are directed by the Sex-lethal (Sxl) protein. In part, Sxl functions by regulating the splicing of the Transformer pre-mRNA by binding to a 3' splice site polypyrimidine tract. Polypyrimidine tracts are essential for splicing of metazoan pre-mRNAs. To unravel the mechanism of splicing regulation at polypyrimidine tracts the interaction of Sxl with RNA was studied. The RNA binding activity of Sxl maps to the two ribonucleoprotein consensus sequence domains of the protein. Quantitation of binding shows that both RNA binding domains (RBDs) are required in cis for site-specific RNA binding. Individual RBDs interacted with RNA more weakly and had lost the ability to discriminate between wild-type and mutant Transformer polypyrimidine tracts. Structural elements in one of the RBDs that are likely to interact with a polypyrimidine tract have been identified using nuclear magnetic resonance techniques. In addition, these data suggest that multiple imino protons of the Transformer polypyrimidine tract are involved in hydrogen bonding. Interestingly, in vitro Sxl binds with equal affinity to the polypyrimidine tracts of pre-mRNAs that it does not regulate in vivo (Kanaar, 1995).

Splicing of the transformer gene of Drosophila is regulated by Sex-lethal-dependent 3' splice site blockage. 40 nucleotides immediately upstream of the regulated splice site are sufficient to direct sex-specific regulated splicing in transgenic animals. This entire region appears to be necessary for regulation and for efficient Sex-lethal binding. Natural splice sites containing partial homology to Transformer do not show regulation. Mutations that replace the 16 nucleotides surrounding the branch point or that alter single nucleotides near the splice site eliminate or reduce regulation without eliminating splicing. Mutations that reduce or eliminate regulation in vivo reduce binding to Sex-lethal in vitro, consistent with the hypothesis that these mutations bring about their effects by altering Sex-lethal binding rather than by altering binding sites for additional non-Sex-lethal factors (Sosnowski, 1994).

The Sex-lethal (Sxl) protein regulates alternative splicing of the Transformer (TRA) messenger RNA precursor by binding to the tra polypyrimidine tract during the process of sex-determination. Sxl binds tightly to a characteristic uirdine-rich polypyrimidine tract at the non-sex-specific 3' splice site in one of the TRA introns, preventing the general splicing factor U2AF from binding to this site and forcing it to bind to the female specific 3' splice site. The crystal structure has now been determined at 2.6 A resolution of the complex formed between two tandemly arranged RNA-binding domains of the Sxl protein and a 12-nucleotide, single-stranded RNA derived from the tra polypyrimidine tract. The Sxl-binding polypyrimidine tract of TRA-mRNA does not form a tertiary base-paired structure. The two RNA-binding domains have their beta-sheet platforms facing each other to form a V-shaped cleft. The RNA is characteristically extended and bound in this cleft, where the UGUUUUUUU sequence is specifically recognized by the protein. This structure offers an insight into how a protein binds specifically to a cognate RNA without that RNA possessing any intramolecular base-pairing (Handa, 1999).

The Drosophila gene female-lethal(2)d [fl(2)d] interacts genetically with the master regulatory gene for sex determination, Sex-lethal. Both genes are required for the activation of female-specific patterns of alternative splicing on transformer and Sex-lethal pre-mRNAs. P-element-mediated mutagenesis has been used to identify the fl(2)d gene. The fl(2)d transcription unit generates two alternatively spliced mRNAs that can encode two protein isoforms differing at their amino terminus. The larger isoform contains a domain rich in histidine and glutamine but has no significant homology to proteins in databases. Several lines of evidence indicate that this protein is responsible for fl(2)d function. (1) The P-element insertion that inactivates fl(2)d interrupts this ORF. (2) Amino acid changes within this ORF have been identified in fl(2)d mutants, and the nature of the changes correlates with the severity of the mutations. (3) All of the phenotypes associated with fl(2)d mutations can be rescued by expression of this cDNA in transgenic flies. Fl(2)d protein can be detected in extracts from Drosophila cell lines, embryos, larvae, and adult animals, without apparent differences between sexes, as well as in adult ovaries. Consistent with a possible function in posttranscriptional regulation, Fl(2)d protein has nuclear localization and is enriched in nuclear extracts (Penalva, 2000).

How can Fl(2)d affect the function of Sxl? One possibility is that Fl(2)d plays an important role in post-translational modifications or proper subcellular localization of Sxl. A second possibility is that Fl(2)d has a direct role in the regulation of pre-mRNA splicing. It could act, for example, by facilitating Sxl binding to its target pre-mRNAs or by assisting its repressive activities. These putative functions could be based on direct interactions between Sxl and Fl(2)d. Alternatively, Fl(2)d could be part of a complex in which Sxl functions, which could also include the products of the genes snf and vir. Finally, a third possibility is that Fl(2)d facilitates the use of the distal splice sites in Sxl and tra that become activated when Sxl represses the use of the proximal ones. More indirect effects of Fl(2)d, as is likely to be the case for the recently reported effects on Sxl expression of mutations in an aspartyl-tRNA synthetase gene, cannot be ruled out (Penalva, 2000).

The non-sex-specific function of fl(2)d remains to be identified. Because Sxl activity is not required for male development, mutations that affect both male and female viability cannot be attributed to genetic interactions with Sxl. It is also very unlikely that Fl(2)d is a general splicing factor involved in an obligatory step in the splicing reaction for the following reasons: (1) no aberrant splicing pattern is detected in Sxl and tra RNAs in fl(2)d mutant males or females (the normal, default sites are used); (2) fl(2)d mutations are not cell lethal. Particularly interesting are the results of the clonal analysis of fl(2)d2, a mutation that produces a truncated, presumably nonfunctional protein. Clones homozygous for fl(2)d2 induced in fl(2)d2/+ males and females are viable, except that in females they develop male instead of female structures, due to the female-specific function of fl(2)d. Furthermore, transplanted male germ cells homozygous for fl(2)d2 can develop into functional spermatozoa, whereas transplanted female germ cells homozygous for fl(2)d2 follow an abortive spermatogenetic pathway, which is an indication of a sexual transformation of the mutant germ cells, due to the female-specific function of fl(2)d. If Fl(2)d was a component of the general splicing machinery, neither fl(2)d2 homozygous clones nor mutant germ cells would survive. One possible scenario is that Fl(2)d plays a role in splicing regulation of a gene(s) important for development in addition to those involved in sex determination. Recently, it has been reported that fl(2)d appears to be necessary for inclusion of mI and mII microexons in Ubx mRNAs. Other examples of splicing factors that are essential for viability but that are dispensable for processing of multiple pre-mRNAs have been described in yeast, Drosophila, and mammals. For some of these, mutations have been identified that disrupt the splicing of only particular substrates, similar to the effects of the fl(2)d1 mutation (Penalva, 2000).

The gene virilizer is needed for dosage compensation and sex determination in females and for an unknown vital function in both sexes. In genetic mosaics, XX somatic cells mutant for vir differentiate male structures. One allele, vir2f, is lethal for XX, but not for XY animals. This female-specific lethality can be rescued by constitutive expression of Sxl or by mutations in msl (male-specific lethal) genes. Rescued animals develop as strongly masculinized intersexes or pseudomales. They have male-specifically spliced mRNA of tra, and when rescued by msl, also of Sxl. These data indicate that vir is a positive regulator of female-specific splicing of Sxl and of tra pre-mRNA (Hilfiker, 1995).

All the tested alleles that cause lethality to males and females are also deficient for the female-specific functions of dosage compensation and sex determination. These pathways are controlled by Sxl, and vir could thus be a regulator of Sxl. The role of Sxl is well established in the pathway of somatic sex determination. Its protein, besides regulating the splicing of its own pre-mRNA (autoregulation), promotes the female-specific splicing of the tra pre-mRNA (transregulation) by blocking the 3' splice site that is used in males. The most obvious and best understood aspect of vir is its role in sex determination, as demonstrated by XX;vir mutant cells forming male structures in genetic mosaics. The temperature-sensitive intersexual phenotype of sturtevant/transformer;vir1ts/vir1ts animals is rescued by the construct hs-tra-female that constitutively expresses the female mode of tra. This indicates that vir acts upstream of tra. The new findings confirm the high hierarchical position of vir in the cascade of sex-determining genes and suggest that it plays a role in the regulation of Sxl and tra (Hilfiker, 1995).

These genetic arguments receive support from molecular analyses that show that surviving XX; vir2f mle pseudomales produce mRNAs of Sxl and tra that are male-specifically spliced. The male-specific products of tra could be the consequence of the male-specific expression of Sxl. The next experiments, however, indicate that vir is also directly engaged in the splicing of tra transcripts (Hilfiker, 1995).

Since SxlM/+;vir2f animals are efficiently rescued from the female lethal effect of vir2f, but remain sexually transformed pseudomales or strongly masculinized intersexes, it is concluded that the sex-transforming effect of vir mutations is not exerted via Sxl alone. When the female-specific function of Sxl is constitutively provided in XX;vir2f animals by SxlM or SxlcF1#19, the female-specific function of tra is still not guaranteed, but in addition appears to require an active product of vir. To test this conclusion the rescued animals were subjected to Western analysis. Substantial levels of Sxl protein are present in strong intersexes and pseudomales. Even animals with normal levels of Sxl, such as the intersexes, still splice the pre-mRNA of their tra gene largely in the male mode, as indicated by their strongly masculinized appearance. These intersexes are not mosaics of male and female cells, but display an intermediate sexual phenotype at the cellular level characteristic of flies expressing only low levels of female-specific products of tra. The results thus support the conclusion that vir is needed not only for female-specific expression of Sxl, but also of tra. This may also be the case for fl(2)d, although to a lesser extent, since XX animals mutant for fl(2)d are rescued by SxlM1 and are sexually transformed into intersexes at 29°C (Hilfiker, 1995).

Transformer, a protein that interacts with Transformer 2: Downstream action of Transformer protein

The isolation and analysis of Drosophila mutants with altered sexual orientation lead to the identification of novel branches in the sex-determination cascade that govern the sexually dimorphic development of the nervous system. One such example is the fruitless (fru) gene, the mutation of which induces male-to-male courtship and malformation of a male-specific muscle, the muscle of Lawrence (MOL). Since the MOL is formed in wild-type flies when the innervating nerve is male, regardless of the sex of the MOL itself, the primary site of Fru function is likely to be the motoneurons controlling the MOL. The fru gene produces multiple transcripts including sex-specific ones. A female-specific mRNA from the fru locus has a putative Transformer (Tra) binding site in its 5' untranslated region, suggesting that fru is a direct target of Tra. The fru transcripts encode a set of proteins similar to the BTB (Bric a brac, Tramtrack and Broad-complex)-Zn finger family of transcription factors. Mutations in the dissatisfaction (dsf) gene result in male-to-male courtship and reduced sexual receptivity of females. The dsf mutations also give rise to poor curling of the abdomen in males during copulation and in females, a failure of egg-laying. The latter phenotypes are ascribable to aberrant innervation of the relevant muscles. A genetic analysis reveals that expression of the dsf phenotypes depends on Tra but not on Doublesex (Dsx) or Fru, suggesting that dsf represents another target of Tra. The effect of dsf mutation was determined in chromosomal females after sexual transformation with tra- (Finley, 1997). In the dsf+;tra- XX flies, the ventral abdominal muscles are innervated by normal locking boutons, as is the case in wild-type males (XY dsf+). In contrast, the corresponding muscles in dsf-;tra- XX flies are associated with enlarged round boutons, just like those seen in mutant males (XY dsf-). The expression of the dsf mutant phenotype depends on tra due to the fact that dsf is located downstream of tra in the sex-determination cascade. In accordance with the idea that dsf is downstream of tra, a moderate reduction in tra+ activity in females eliminates motor innervation of the uterine muscle, inducing a dsf phenotype. Taken together, these findings suggest that the sex-determination protein Tra has at least three different targets: dsx, fru and dsf, each of which represents the first gene in a branch of the sex-determination hierarchy functioning in a mutually-exclusive set of neuronal cells in the Drosophila central nervous system (Yamamoto, 1998).

Few mutations link well defined behaviors with individual neurons and the activity of specific genes. In Drosophila, recent evidence indicates the presence of a doublesex-independent pathway controlling sexual behavior and neuronal differentiation. A gene, dissatisfaction (dsf), has been identified that affects sex-specific courtship behaviors and neural differentiation in both sexes without an associated general behavioral debilitation. Male and female mutant animals exhibit abnormalities in courtship behaviors, suggesting a requirement for dsf in the brain. Virgin dsf females resist males during courtship and copulation and fail to lay mature eggs. dsf males actively court and attempt copulation with both mature males and females but are slow to copulate because of maladroit abdominal curling. Structural abnormalities in specific neurons indicate a role for dsf in the differentiation of sex-specific abdominal neurons. The egg-laying defect in females correlates with the absence of motor neuronal innervation on uterine muscles, and the reduced abdominal curling in males correlates with alteration in motor neuronal innervation of male ventral abdominal muscles. Epistasis experiments show that dsf acts in a tra-dependent and dsx-independent manner, placing dsf in the dsx-independent portion of the sex determination cascade (Finley, 1997).

Relatively little is known about the neural circuitry underlying sex-specific behaviors. The feminizing gene transformer was expressed in genetically defined subregions of the brain of male Drosophila, and in particular within different domains of the mushroom bodies. Mushroom bodies are phylogenetically conserved insect brain centers implicated in associative learning and various other aspects of behavior. Expression of transformer in lines that mark certain subsets of mushroom body intrinsic neurons, and in a line that marks a component of the antennal lobe, causes males to exhibit nondiscriminatory sexual behavior: they court mature males in addition to females. Expression of transformer in other mushroom body domains, and in control lines, has no such effect. These data support the view that genetically defined subsets of mushroom body intrinsic neurons perform different functional roles (O'Dell, 1995).

Pheromones are intraspecific chemical signals important for mate attraction and discrimination. In Drosophila, hydrocarbons on the cuticular surface of the animal are sexually dimorphic in both their occurrence and their effects: Female-specific molecules stimulate male sexual excitation, whereas the predominant male-specific molecule tends to inhibit male excitation. Female flies produce dienes (two double bonds) with 27 and 29 carbons (cis,cis-7,11-heptacosadiene and cis,cis-7,11-nonacosadiene). A few tens of nanograms of both dienes together can elicit vigorous male precopulatory behavior. Male flies synthesize monoenes (one double bond) with 23 and 25 carbons (cis-7tricosene and cis-7-pentacosene). cis-7tricosene can inhibit dose-dependent male excitation, whereas cis-7pentacosene stimulates males of some strains. According to a previously proposed biosynthetic scheme, an elongase, controlled by ecdysone, perhaps coupled with a desaturase, would be sufficinet to replace 7-monoenes by 7,11-dienes. Complete feminization of the pheromone mixture produced by males is induced by targeted expression of the transformer gene in adult oenocytes (subcuticular abdominal cells) or by ubiquitous expression during early imaginal life. Early adult life is the critical period during which sexually dimorphic hydrocarbons replace immature hydrocarbons on the fly cuticle. The resulting flies, genetically male, generally exhibit male heterosexual orientation but elicit homosexual courtship from other males. Two out of seven transformed lines exhibit some bisexual behavior, possibly because they were feminized in the calyces of their mushroom bodies (strain A) and in a dorso-medial subset of their antennal lobes (strains A and B). However, these two brain structures, which function in mate recognition, are not feminized in the other tranformed strains, showing that these structures are not required for feminization of the pheromonal profile. This analysis shows that two aspects of individual sexual identity in Drosophila -- the perception of others and the presentation of self to others -- are under separate genetic and anatomical control (Ferveur, 1997).

Sexual differentiation in Drosophila is controlled by a short cascade of regulatory genes, the expression pattern of which determines all aspects of maleness and femaleness, including complex behaviors displayed by males and females. One sex-determining gene is transformer (tra): tra activity is needed for female development. Flies with a female karyotype (XX) but which are mutant for tra develop and behave as males (and are termed X2 flies). In such flies, a female phenotype can be restored by a transgene that carries the female-specific cDNA of tra under the control of a heat-shock promoter. This transgene, called hs[trafem], also transforms XY animals into sterile females. When these XX and XY 'females' are raised at 25 degrees C, however, they display vigorous male courtship while at the same time, as a result of their female pheromone pattern, they are attractive to males. Intriguingly, their male courtship behavior is indiscriminately directed toward both females and males. When expression of tra is forced by heat shock, applied during a limited period around puparium formation, male behavior is abolished and replaced by female behavior. The temperature-sensitive period extends from shortly before puparium formation into early metamorphosis. Cuticular hydrocarbons function as pheromonal cues between the sexual partners. Consistent with their attractiveness to males, the normal female pattern of pheromones is found in transformed flies, irrespective of the heat shock. Could the indiscriminate male behavior of these flies result from continuous self-stimulation by their own female pheromones? The female pheromone pattern was replaced with the male pattern without affecting the CNS. This was achieved by introducing the male-determining mutation dsxD, which acts downstream of tra. This mutation causes XX flies to produce a male pheromone pattern. They are no longer attracted to males. Similarly, X2 and XY 'females', now with male body and pheromonal status due to dsxD, are completely unattrative to males; but they still court both sexes. Such flies even manage to copulate with females, showing that X2 and XY 'females' are capable of performing the full repertoire of male courtship. These results rule out self-stimulation and leave a disturbed nervous system as the most probable cause for the indiscriminate mating behavior. It is concluded that sexual behavior is irreversibly programmed during a critical period as a result of the activity or inactivity of a single control gene. Such programming takes place during a period of accelerated growth in the brain and especially in the mushroom bodies, suggesting that sexual behavior is 'hard wired' in the CNS (Arthur, 1998).

In Drosophila, Sex-lethal (Sxl) controls autoregulation and sexual differentiation by alternative splicing but regulates dosage compensation by translational repression. To elucidate how Sxl functions in splicing and translational regulation, a full-length Sxl protein (Sx.FL) and a protein lacking the N-terminal 40 amino acids (Sx-N) were ectopically expressed. The Sx.FL protein recapitulates the activity of Sxl gain-of-function mutations, since it is both sex transforming and lethal in males. In contrast, the Sx-N protein unlinks the sex-transforming and male-lethal effects of Sxl. The Sx-N proteins are compromised in splicing functions required for sexual differentiation, displaying only partial autoregulatory activity and almost no sex-transforming activity. However, the Sx-N protein does retain substantial dosage compensation function and kills males almost as effectively as the Sx.FL protein. In the course of the analysis of the Sx.FL and Sx-N transgenes, a novel, negative autoregulatory activity, in which Sxl proteins bind to the 3' untranslated region of SXL mRNAs and thereby decrease Sxl protein expression, was also uncovered. This negative autoregulatory activity may be a homeostasis mechanism (Yanowitz, 1999).

While the Sx-N protein retains at least some autoregulatory and dosage compensation activities, the most striking finding is that the protein seems to lack the ability to regulate the tra sexual differentiation pathway. The feminization activities of the Sx.FL and Sx-N transgenes were compared in males that do not contain the endogenous Sxl locus. This background allows alterations in somatic sexual differentiation to the Sxl proteins expressed by the transgenes to be unambiguously ascribed. The extent of sex transformation that is seen in escaper Sx.FL males again appears to resemble that for the strong cF1 lines, in which the Sxl cDNA is expressed under the control of the inducible hsp70 promoter. The males are intersexual; they have lighter abdominal pigmentation, rotated genitalia, and fewer sex combs, and they are sterile. In sharp contrast, Sxl minus; Sx-N males are morphologically indistinguishable from wild-type males and are fertile. The absence of sex transformations in Sx-N males most likely reflects an inability to regulate the tra pathway. To determine if this is the case, a RT-PCR assay was used to examine the splicing pattern of tra mRNAs. Three amplification products can be detected in this assay, and these correspond to unspliced RNA, default (male) spliced RNA, and female spliced RNA. Of these, only unspliced and default spliced tra RNAs are observed in wild-type males, while all three species are found in females. Thus, while the Sx.FL transgene has tra regulatory activity, it is not as effective as the endogenous Sxl gene. These findings confirm the suggestion that the Sx-N protein is defective in its tra regulatory function (Yanowitz, 1999).

The integration of multiple developmental cues is crucial to the combinatorial strategies for cell specification that underlie metazoan development. In the Drosophila genital imaginal disc, which gives rise to the sexually dimorphic genitalia and analia, sexual identity must be integrated with positional cues, in order to direct the appropriate sexually dimorphic developmental program. Sex determination in Drosophila is controlled by a hierarchy of regulatory genes. The last known gene in the somatic branch of this hierarchy is the transcription factor doublesex (dsx); however, targets of the hierarchy that play a role in sexually dimorphic development have remained elusive. The gene dachshund (dac) is differentially expressed in the male and female genital discs, and plays sex-specific roles in the development of the genitalia. Furthermore, the sex determination hierarchy mediates this sex-specific deployment of dac by modulating the regulation of dac by the pattern formation genes wingless (wg) and decapentaplegic (dpp). The sex determination pathway acts cell-autonomously to determine whether dac is activated by wg signaling, as in females, or by dpp signaling, as in males (Keisman, 2001).

The behavior of tra + and tra2IR clones provides insight into the mechanism of repression in the undeveloped genital primordium. It was anticipated that such clones would be difficult to recover when they occurred in the male and female primordium, respectively, because they should adopt the repressed state. Instead, large tra + (female) clones were recovered in the male primordium of a male disc, and large tra2IR (male) clones were recovered in the female primordium of a female disc. Some of these clones constitute a substantial fraction of the primordium in question. Though tra + or tra2IR clones were not scored in adults, previous studies strongly suggest that such clones would fail to differentiate adult genital structures (Keisman, 2001).

It has been shown that tra - (male) clones cause large deletions in the female genitalia, indicating that genetically male cells like those in a tra2IR clone divide but cannot differentiate female genital structures. Further, it has also been shown that male structures are deleted when the mosaic border passes through the male genitalia, suggesting that female tissue cannot differentiate male structures. To reconcile these data, it is proposed that repression of the inappropriate genital primordium involves two separable processes: repression of growth and the prevention of differentiation. Thus, clones of cells of the inappropriate genetic sex cannot differentiate, but they can grow and contribute to a morphologically normal genital primordium. This poses yet another question. Cells in a tra + clone in the male primordium of a male genital disc are analogous to the cells in the repressed male primordium of a wild-type female genital disc: both are genetically female, and both have A9 segmental identity. Why do tra + clones in the male primordium grow, while the repressed male primordium in a female disc does not? One possibility is that the decision of the male primordium to grow in a male disc is made before tra + clones were induced and cannot be over-ridden by a later switch of genetic sex. However, temperature-shift experiments with tra-2 ts alleles suggest that the decision of a genital primordium to develop can be reversed later in development. Furthermore, occasional, large tra + clones can cause severe reductions in male genital discs. This observation leads to the suggestion of a model in which growth in the genital disc is regulated from within organizing zones, such as the domains of wg and dpp expression. According to this model, the sex of the cells in the organizing regions would determine how the disc grows, while cells in other regions would respond accordingly, regardless of their sex. The tra + clones that cause reduction could result when such a clone intersects with one of the postulated organizing centers within the disc. The implication is that the sex determination pathway acts in yet undiscovered ways to modulate the function of the genes that establish pattern in the genital disc. One such interaction was found in the regulation of dac; further study is needed to determine if others exist, and what role they play in producing the sexual dimorphism of the genital disc and its derivatives (Keisman, 2001).

Transformer, a protein that interacts with Transformer 2: Sex-specific apoptosis, functioning downstream of transformer, regulates sexual dimorphism in the Drosophila embryonic gonad

Sexually dimorphic development of the gonad is essential for germ cell development and sexual reproduction. The Drosophila embryonic gonad is already sexually dimorphic at the time of initial gonad formation. Male-specific somatic gonadal precursors (msSGPs) contribute only to the testis and express a Drosophila homolog of Sox9 (Sox100B: Loh, 2000), a gene essential for testis formation in humans. The msSGPs are specified in both males and females, but are recruited into only the developing testis. In females, these cells are eliminated via programmed cell death dependent on the sex determination regulatory gene doublesex. This work furthers the hypotheses that a conserved pathway controls gonad sexual dimorphism in diverse species and that sex-specific cell recruitment and programmed cell death are common mechanisms for creating sexual dimorphism (DeFalco, 2003).

To investigate when sexual dimorphism is first manifested in the somatic gonad, expression of SGP markers were examined in embryos whose sex could be unambiguously identified, at a developmental stage (stage 15) soon after gonad coalescence has occurred. Analysis of Eya expression reveals anti-Eya immunoreactivity throughout the female somatic gonad, though Eya expression is somewhat stronger in the posterior. In males, anti-Eya immunoreactivity is also found throughout the somatic gonad. However, the expression at the posterior of the gonad is much more intense than in females, as there appears to be a cluster of Eya-expressing cells at the posterior of the male gonad that is not present in females. In blind experiments, the sex of an embryo could be accurately identified by the Eya expression pattern in the gonad. Thus, sexual dimorphism is already apparent in the somatic gonad soon after initial gonad formation. A sex-specific expression pattern is also observed with Wnt-2 at this stage. As is observed with Eya, Wnt-2 is expressed in the SGPs of the female gonad, but its expression is greatly increased at the posterior of the male gonad. The SGP marker bluetail (see Galloni, 1993) exhibits a similar sex-specific pattern as Eya; however, the SGP marker 68-77 is expressed equally in both sexes (see below). Thus, the somatic gonad is sexually dimorphic by stage 15, but only a subset of SGP markers reveals this sexual dimorphism (DeFalco, 2003).

During Drosophila embryogenesis, Sox100B is expressed in a number of cell types, including the gonad (Loh, 2000). Since Sox100B is closely related to Sox9 (an important sex determination factor in humans and mice), whether Sox100B expression is sexually dimorphic in Drosophila was tested. Interestingly, it was found that after gonad coalescence (stage 15), Sox100B expression in the gonad is male-specific. Sox100B immunoreactivity is not observed in the coalesced female gonad, whereas it is detected in a posterior cluster of SGPs in the male gonad. While this expression pattern is seen in most wild-type backgrounds (including Canton-S and faf-lacZ), in certain 'wild-type' lines, such as w1118, a few Sox100B-positive cells are observed in the posterior of the coalesced female gonad (however, this is still clearly distinguishable from the number of Sox100B-positive cells in the male). Unlike Eya and Wnt-2, Sox100B is not expressed in all SGPs, since it is usually absent from female gonads and from the anterior region of the male gonad and does not colocalize with the SGP marker 68-77. Sox100B expression appears restricted to the posterior cluster of SGPs that is observed only in the male gonad. Thus, like Sox9 expression in vertebrates, Sox100B exhibits a male-specific pattern of expression in the Drosophila embryonic gonad, suggesting that it may indeed be an ortholog of Sox9 (DeFalco, 2003).

After having identified sexually dimorphic markers of the embryonic gonad, these markers were used to investigate how sexual dimorphism is established. It was asked whether proper gonad formation is necessary for the establishment of sexual dimorphism by examining Sox100B expression in fear-of-intimacy (foi) mutant embryos. In foi mutants, germ cells migrate and associate normally with the SGPs, but these two cell types fail to coalesce into a round and compact gonad. Despite the failure of gonad coalescence, a cluster of Sox100B-expressing cells was still observed at the posterior of the male gonad, while no Sox100B-expressing cells are observed in the female at this stage. Whether the presence of germ cells is necessary for the establishment of sexual dimorphism in the embryonic gonad was examined. Embryos that lack germ cells due to a hypomorphic mutation in oskar, a gene required for germ cell formation, were examined. Other aspects of embryonic development occur normally in these embryos, including the formation and coalescence of the SGPs. Agametic gonads show identical sexual dimorphism to wild-type embryos. Sox100B is coexpressed with Eya in the cluster of somatic cells in the posterior of the male gonad, but Sox100B expression is not observed in the female gonad. Thus, sexual dimorphism of the embryonic somatic gonad does not require proper gonad morphogenesis or the presence of germ cells (DeFalco, 2003).

The posterior cluster of Eya and Sox100B coexpressing cells could result from sex-specific differences in gene expression within the cells of the gonad. Alternatively, it could reflect a difference in gonad morphology, in which these cells are only present in males and not in females. To distinguish between these possibilities, the morphology of the male and female coalesced (stage 15) gonad was examined, using approaches that do not depend on cell-type-specific SGP markers. First, a CD8-GFP fusion protein was expressed broadly in the mesoderm. The fusion of the extracellular and transmembrane regions of mouse CD8 with GFP allows for visualization of cell and tissue morphology. A cluster of mesodermal cells is consistantly observed attached to the posterior of the male gonad that is not observed in the female. In blind experiments, the sex of the embryo can be predicted based on the presence of this posterior cluster of cells. Male and female gonads were also examined by transmission electron microscopy (TEM). Male and female embryos were first sorted using an X chromosome-linked GFP expression construct and then processed separately for TEM. In this analysis, a cluster of cells that is not present in the female gonad was consistently at the posterior of the male gonad. Both the size and morphology of these cells indicate that they are somatic cells rather than germ cells. Thus, the observed sexual dimorphism reflects a change in gonad morphology, not just a change in gene expression. Since the additional cells at the posterior of the male gonad express at least some markers in common with SGPs (e.g., Eya), these cells are referred to as male-specific SGPs (msSGPs) (DeFalco, 2003).

Since no sex-specific differences were observed in SGP proliferation in the gonad, it seems unlikely that the SGPs are dividing to produce the msSGPs. Therefore, Sox100B was used as a marker for the msSGPs to determine where and when these cells are first specified. At stages prior to gonad coalescence (stages 12 and 13), a cluster of Eya/Sox100B double-immunopositive cells is observed posterior and ventral to the developing clusters of SGPs, which express Eya alone. Interestingly, this cluster of Eya/Sox100B double-positive cells is initially observed in both males and females and appears identical, although Eya expression may be somewhat lower in the female cluster. During stage 13, as the SGPs and germ cells associate closely along PS 10-12, the Eya/Sox100B double-positive cells move toward the gonad in both sexes. In males, these cells join the posterior of the coalescing gonad. In contrast, these cells do not join the gonad in females, and only Eya-positive, Sox100B-negative cells are found in the coalesced gonad. It is concluded that the Eya/Sox100B double-positive cells are the msSGPs and that they form separately from the SGPs. These cells are initially specified in both males and females and move anteriorly to join the gonad in males. In females, these cells do not form part of the gonad, as judged by the above morphological analysis, and are no longer detected using available markers (DeFalco, 2003).

Since the msSGPs develop separately from the SGPs, it was of interest to address where the msSGPs arise and what controls their specification. By marking the anterior of each parasegment using an antibody against Engrailed, it was determined that the msSGPs are specified in PS13. This observation is consistent with these cells arising posterior to the SGPs, which form in PS 10, 11, and 12. Other Sox100B expression is observed in nongonadal tissues. Whether, like the SGPs, the msSGPs are specified in the dorsolateral domain of the mesoderm was also addressed. Mesodermal cell types that form in this region, such as the SGPs and the fat body, require the homeodomain proteins Tinman and Zfh-1 for their specification. However, in embryos double-mutant for tinman and zfh-1, the msSGPs are still specified, even though the SGPs fail to develop. Thus, msSGPs do not arise from the dorsolateral domain, consistent with the fact that the msSGPs are first observed in a position ventral to the SGPs. The msSGPs also differ from the SGPs in terms of their requirements for the homeotic gene abd-A. SGP specification absolutely requires abd-A, while msSGPs are still present in these mutants. Thus, despite the fact that the msSGPs and the SGPs share expression of some molecular markers such as Eya and Wnt-2, their specification is under independent control (DeFalco, 2003).

Since the msSGPs express both Eya and Sox100B, the requirements for each of these genes in msSGP specification was investigated. In eya mutants, Sox100B-positive cells are still observed posterior to the germ cells at early stages, in a position where the msSGPs normally develop. Since the SGPs are not maintained in these mutants, the germ cells disperse and the gonad does not coalesce. Therefore, it is impossible to tell if the msSGPs would join the posterior of the male gonad in eya mutants. However, initial msSGP specification does not require eya. Similarly, in a deletion that removes the Sox100B locus, a large cluster of Eya-positive cells was still observed at the posterior of the male gonad that does not appear in females. Thus, the initial development of the msSGPs does not require Sox100B. Expression of Eya and Sox100B are mutually independent and are likely to be downstream of factors controlling initial msSGP specification (DeFalco, 2003).

Since the msSGPs are initially specified in both males and females, a determination was made of how these cells receive information about their sexual identity that allows them to behave differently in the two sexes. tra plays a key role in the sex determination pathway in Drosophila and is required to promote female differentiation in somatic tissues. tra mutant gonads were examined to test if tra function is required for gonad sexual dimorphism (XX embryos are masculinized by mutations in tra). Sox100B-immunopositive cells are observed in the posterior somatic gonad of both XX and XY tra mutant embryos in a manner comparable to wild-type males. Analysis of the Sox100B expression pattern in the gonad reveals that there are no differences between XX and XY tra mutants, or between either of these genotypes and wild-type males. Conversely, when Transformer is expressed in XY embryos (UAS-traF, tubulin-GAL4), Sox100B-immunopositive cells are no longer observe in these gonads, and they now appear similar to wild-type females (DeFalco, 2003).

In most somatic tissues, the principle sex determination factor downstream of tra is dsx. Unlike tra, dsx is required for both the male and female differentiation pathway, since both XX and XY dsx mutant adults show an intersexual phenotype. However, in the somatic gonad, dsx mutant XY embryos are indistinguishable from wild-type males and show no change in Sox100B expression. Thus, unlike in most somatic tissues, this early characteristic of male development does not require dsx. In XX embryos that are mutant for dsx, a completely masculinized phenotype is observed, in which Sox100B expression in the gonad is similar to a wild-type male. When a dominant allele of dsx, dsxD, is used to express DsxM (dsxD/dsx) in XX embryos, these gonads are no more masculinized than dsx null XX gonads. Therefore, while DsxF is required for the proper female phenotype in XX gonads, it appears that the male Sox100B expression pattern is the 'default' state in the absence of dsx function (DeFalco, 2003).

Since the msSGPs join the posterior of the male gonad but are no longer detected in the female, the basis for the sexually dimorphic behavior of these cells was investigated. In the female, these cells could turn off Sox100B and Eya and contribute to some other tissue, or they might be eliminated altogether. To test this latter hypothesis, whether msSGPs are eliminated by sex-specific programmed cell death in the female was addressed. Since programmed cell death occurs in a caspase-dependent manner, the gonad phenotype was examined in embryos in which caspase activity was inhibited by expressing the baculovirus p35 protein in the mesoderm. In these embryos, XX gonads now appear masculinized; Sox100B-positive cells (msSGPs) persist and join the posterior of female gonads, and coexpress Eya, as in wild-type male embryos. There are not as many Sox100B-positive cells in females as in males, suggesting that p35 may not be completely suppressing cell death. The presence of such cells in the female gonad does not appear to drastically affect ovary formation or oogenesis, since embryos develop into fertile adult females (DeFalco, 2003).

To investigate how programmed cell death might be controlled in the msSGPs, the genes of the H99 region (head involution defective [hid], reaper [rpr], and grim), which are regulators of apoptosis in Drosophila, were examined. A small deletion (DfH99) removes all three of these genes and blocks most programmed cell death in the Drosophila embryo. In DfH99 mutants, an equivalent cluster of Sox100B-positive cells is observed in both males and females. Again, these posterior cells are also Eya positive. Furthermore, XX embryos mutant for hid alone also contain Sox100B-positive cells in the posterior of the gonad, although the posterior cluster of cells is slightly smaller than in the male. It is concluded that the msSGPs are normally eliminated from females through sex-specific programmed cell death, controlled by hid and possibly also other genes of the H99 region. However, if cell death is blocked in females, these cells can continue to exhibit the normal male behavior of the msSGPs, including proper marker expression and recruitment into the gonad. Therefore, the decision whether or not to undergo apoptosis is likely the crucial event leading to the sexually dimorphic development of these cells at this stage (DeFalco, 2003).

It is concluded that proper information from the sex determination pathway is required to control the sexually dimorphic behavior of the msSGPs. The female phenotype in the embryonic gonad is dependent on both tra and dsx. Interestingly, it seems that the male phenotype is the default state; in the absence of any tra or dsx function, msSGPs in both XX and XY embryos behave as in wild-type males. This is a different situation than in most other tissues, in which dsx is required in both sexes to promote proper sexual differentiation. In particular, while no role is found for DsxM in this process, DsxF is positively required either to establish the female fate in the posterior somatic gonad or to repress the male fate. This role for DsxF in msSGP development is analogous to its role in the genital disc, in which DsxF is required to block recruitment of btl-expressing cells into the disc; in both cases, dsx female function serves to repress incorporation of a male-specific cell type. Since the msSGPs are initially specified in a sex-independent manner, this may account for the fact that the persistence of these cells (the male phenotype) is the default state. It will be of interest in the future to address the role of the msSGPs in testis development, and how genes such as dsx, eya, and Sox100B act in this process (DeFalco, 2003).

Although sex determination schemes vary widely in the animal kingdom, there is evidence that the molecular and cellular pathways used to control sexual dimorphism may be conserved, even between vertebrates and invertebrates. One example is Sox9, which has been implicated as an ancestral sex-determining gene in vertebrates given its male-specific gonad expression in diverse species such as human, mouse, turtle, and chicken. A potential Drosophila ortholog of Sox9, Sox100B, is expressed in a male-specific manner in the embryonic somatic gonad. The manner of Sox100B expression is reminiscent of that in the mouse; Sox9 is initially expressed in both sexes, but is maintained and upregulated in the male gonad. It will be very interesting to compare the role that Sox100B plays in the development of the Drosophila testis to the one played by Sox9 in vertebrates (DeFalco, 2003).

Molecular conservation is also observed amongst the members of the Dsx/Mab-3 Related Transcription Factor (DMRT) family. DMRT family members have been shown to be essential for sex-specific development in Drosophila (Dsx), C. elegans (mab-3), medaka fish (DMY), and mice (DMRT1) and have been implicated in human sex reversal. This study demonstrates that dsx is essential for proper sex-specific development of the msSGPs. Thus, increasing evidence indicates that DMRT family members are also conserved regulators of sexual dimorphism (DeFalco, 2003).

Protein Interactions

Both subunits of U2AF, U2AF65 and U2AF65, associate with 3' splice sites during assembly of the E complex, the earliest functional complex formed at splice sites. When pre-mRNAs containing a strong 3' splice site are incubated in nuclear extracts, a mixture of a nonspecific H complex and a functional E complex is formed. The amount of E complex formed can be enhanced significantly by addition of SR proteins to the nuclear extract, indicating that SR proteins are limitiing in these extracts. The pyrimidine tract of the DSX 3' splice site is interrupted by purines and is therefore not a high-affinity binding site for U2AF65. Splicing enhancers promote the binding of U2AF to this weak 3' splice site during E complex formation. Enhancer complexes formed on the dsxRE contain significant amounts of U2AF35. An artificial DSX pre-mRNA lacking the dsxRE forms only an H complex under these conditions; the addition of SR proteins has no effect. Thus, the weak female-specific splice site is not recognized by the splicing machinery in the absence of an enhancer, and SR proteins alone are unable to drive complex formation (Zuo, 1996).

To examine the effect of a constitutive DSX splicing enhancer activity on E complex formation, an artificial dsxR2-5 mRNA was tested in which repeats were located within 100 nucleotides of the 3' splice site (in this case close by in contrast to the usual distal positioning). E complex formation is observed with this RNA, and the addition of SR proteins leads to a significant increase in the amount of complex formed Both the 65- and 35-kD subunits of U2AF are present in E complexes formed on pre-mRNAs containing such a strong 3' splice sites (Zuo, 1996).

Experiments were carried out determine whether the TRA- and TRA2-dependent splicing enhancer recruits U2AF to the weak dsx pre-mRNA enhancer containing the dsxRE at its normal position. DSX pre-mRNA containing the dsxRE at its normal position, distant from the 3' splice site, is not assembled into the E complex in the absence of TRA and TRA2. In the presence of TRA and TRA2, however, a significant amount of E complex is observed, and the addition of SR proteins substantially increases the amount of E complex formed. Both U2AF35 and U2AF65 are detected in the DSX RNA complexes. This association is TRA- and TRA2-dependent and is stimulated by SR proteins. In addition, U2AF35 is required for enhancer-dependent binding of U2AF65 to the weak female-specific 3' splice site. This work suggests that recognition of the correct 3' splice site is accomplished through the formation of a network of protein-protein interactions extending across the downstream exon (containing the enhancer site). A key element in this model is the ability of U2AF35 to form a bridge between U2AF65 and SR proteins (including TRA and TRA2) bound to the exon. Similarly, U2AF35 may play a crucial role in the interaction between the 5' and 3' splice sites by functioning as a bridge in the U1 70K-SR protein-U2AF network of protein interactions across the intron (Zuo, 1996 and references).

The Doublesex splice enhancer functions by assembling specific SR protein complexes. Transformer and Transformer 2 recruit different members of the SR family of splicing factors to the six 13-nucleotide repeats and the purine-rich element (PRE) of the splice enhancer. In mammalian cells, the complexes that form on the repeats consist of TRA, TRA2 and the SR protein 9G8. In Drosophila Kc cell extracts, TRA and TRA2 recruit the SR protein RBP1 to the repeats. These proteins are arranged in a specific order on the repeats, with the SR protein at the 5' end of each repeat, and the TRA2 at each 3' end. Although TRA does not cross-link strongly to the repeats, its presence is essential for the binding of TRA2 to the 3' end of the repeat. Individual SR proteins are also recruited to the PRE by TRA and TRA2; SF2/ASF in mammalian cells and dSRp30 in Drosophila cell extracts. The binding of TRA2, TRA, and the specific SR proteins to the repeats in the PRE is highly cooperative within each complex. Thus, TRA2, which contains a single RNA binding domain, can recognize distinct sequences in the repeats and the PRE in conjunction with specific SR proteins. The cross-linking results presented here strongly suggest that Drosophila RBP1 and human 9G8 are functional homologs. These observations show that the protein composition of each complex is determined by the RNA recognition sequence and specific interactions between SR proteins and TRA and TRA2 (Lynch, 1996).

The SR proteins represent a family of splicing factors, several of which have been implicated in the regulation of sex-specific alternative splicing of DSX pre-mRNA. Two RNA target sequence motifs recognized by the SR protein RBP1 have been identified. Several copies of these RBP1 target sequences are found within two regions of the DSX pre-mRNA that are important for the regulation of DSX alternative splicing: these areas are the repeat region and the purine-rich polypyrimidine tract of the regulated female-specific 3' splice site. RBP1 target sequences within the DSX repeat region are required for the efficient splicing of DSX pre-mRNA. RBP1 contributes to the activation of female-specific DSX splicing in vivo by recognizing the RBP1 target sequences within the purine-rich polypyrimidine tract of the female-specific 3' splice site (Heinrichs, 1995).

Within the doublesex repeat element (dsxRE) that contains six copies of a 13-nucleotide repeat sequence, there is, in addition, a purine-rich enhancer (PRE) sequence. This PRE element functionally synergizes with the repeat sequences. In vitro binding studies show that the PRE is required for specific binding of TRA2 to the dsxRE, and that TRA and SR proteins bind cooperatively to the dsxRE in the presence or absence of the PRE. Thus positive control of dsx pre-mRNA splicing requires the TRA- and TRA2-dependent assembly of a multiprotein complex composed on a site of at least two distinct elements (Lynch, 1995).

Specific recognition and pairing of the 5' and 3' splice sites are critical steps in pre-mRNA splicing. The splicing factors SC35 (the SR protein SRp30b) and SF2/ASF (the SR protein SRp30a) specifically interact with both the integral U1 small nuclear ribonucleoprotein (snRNP U1-70K) and with the 35 kd subunit of the splicing factor U2AF (U2AF35). Previous studies indicate that the U1 snRNP binds specifically to the 5' splice site, while U2AF35-U2AF65 heterodimer binds to the 3' splice site. Together, these observations suggest that SC35 and other members of the SR family of splicing factors may function in splice site selection by acting as a bridge between components bound to the 5' and 3' splice sites. Interestingly, SC35, SF2/ASF, and U2AF35 also interact with the Drosophila splicing regulators Transformer and Transformer-2, suggesting that protein-protein interactions mediated by SR proteins may also play an important role in regulating alternative splicing (Wu, 1993).

The ribonucleoprotein consensus sequence (RNP-CS) of TRA2 is required for male fertility and positive and negative control of alternative splicing in transgenic flies, as well as for in vitro binding of recombinant TRA2 to Doublesex and TRA2 pre-mRNAs. Thus, all of the known functions of TRA2 require specific protein-RNA interactions. One of the two arginine-serine (RS)-rich domains of TRA2 is dispensable, while the other is essential for all of the in vivo functions. Part of this domain is also required for RNA binding in vitro. Significantly, the essential RS domain is also required for specific protein-protein interactions. TRA2 interacts with itself, with the splicing regulator Transformer, and with the general splicing factor SF2 in vitro and in the yeast two-hybrid system. These results demonstrate that both protein-RNA and protein-protein interactions are involved in tra2-dependent activation and repression of alternative splicing (Amrein, 1994).

Transformer, a protein that interacts with Transformer 2: Downstream action of Transformer protein - Effects on germ line cells

In Drosophila, compatibility between the sexually differentiated state of the soma and the constitution of the sex chromosome in the germline is required for normal gametogenesis. In this study, important aspects of the soma-germline interactions controlling early oogenesis are defined. In particular, the sex-specific germline activity of the ovarian tumor (otu) promoter has been demonstrated to be dependent on somatic factors controlled by the somatic sex differentiation gene transformer. This regulation defines whether there is sufficient ovarian tumor expression in adult XX germ cells to support oogenesis. In addition, the ovarian tumor function required for female germline differentiation is dependent on the activity of another germline gene, ovo, whose regulation is transformer-independent. These and other data indicate that ovarian tumor plays a central role in coordinating regulatory inputs from the soma (as regulated by transformer) with those from the germline (involving ovo). transformer-dependent interactions influence whether XX germ cells require ovarian tumor or ovo functions to undergo early gametogenic differentiation. These results are incorporated into a model that hypothesizes that the functions of ovarian tumor and ovo are dependent on an early sex determination decision in the XX germline -- a decision that is at least partially controlled by somatic transformer activity (Hinson, 1999 and references).

With respect to interactions with the germline, transformer (tra) is the most extensively studied of the somatic sex regulatory genes. The masculinization of XX soma due to loss-of-function tra mutations causes germ cell aberrations during first instar larval stages and misregulates sex-specific germline gene expression in the embryo. Furthermore, when XY soma is feminized by ectopic tra expression (to form 'pseudofemales', the somatic components of the ovaries are sufficiently 'female' so that they can support the maturation of transplanted XX germ cells. The pseudofemale soma also appears to partially feminize the XY germline, since these cells now require the normally female-specific otu function for optimal proliferation. These observations indicate that tra controls a substantial portion of the somatic-germline interactions affecting early gametogenic differentiation (Hinson, 1999 and references).

In Drosophila, the sexual differentiation of the germline requires a complex interplay between cell autonomous factors controlled by the X:A ratio of the germ cells and sex-specific somatic functions. For example, certain allele combinations of transformer, transformer-2 and doublesex can cause chromosomally female (XX) flies to develop with most of their somatic tissues having a male identity, i.e., ‘XX pseudomales’. In these flies, oogenesis is aborted and there is even occasionally what appears to be early spermatogenic development. Since the germline expressions of these sex regulatory genes are not required for early stages of gametogenesis, the aberrant germline phenotypes must result from the male transformation of the soma (Hinson, 1999 and references).

It is not clear which germline genes are influenced by the proposed somatic interactions. Three possible candidates based on their early and sex-specific roles in female germline differentiation are ovarian tumor, ovo and Sex-lethal (Sxl). During oogenesis, the expression of otu is required in the germline at several stages, if not continually. The null mutant phenotype is characterized by the absence of egg chambers in an otherwise normal ovary, denoted as the quiescent phenotype, although substantial numbers of germ cells are still present in the germarium. Null and severe loss-of-function mutations can also produce 'ovarian tumors', a phenotype characterized by egg chambers containing hundreds of seemingly undifferentiated germ cells. Both the quiescent and tumorous cells are aborted at early oogenic stages, during the cystocyte divisions prior to cyst formation. Mutations in otu have no significant effect on spermatogenesis, although some aberrations in male courtship behavior have been reported. The ovo gene has been implicated in regulating sex determination and dosage compensation in the germline. This is based primarily on observations that ovo null XX germ cells are typically not found in the adult ovary, presumably because of reduced cell viability. In addition, certain ovo allele combinations produce tumorous germ cells that morphologically resemble primary spermatocytes. These phenotypes make ovo a candidate target for a somatic signal regulating early oogenesis, although the expression of ovo in adult germ cells does not appear to be responsive to somatic influences. ovo might directly regulate otu. The Ovo protein can bind to sites in the otu promoter, which displays sensitivity to changes in the dosage of ovo + function. It is not known when this putative regulation of otu occurs nor what role it plays in oogenesis (Hinson, 1999 and references).

The effects of an ovo null mutation on XX germ cells developing in pseudomale testes and female ovaries were examined. In females, ovo mutant XX germ cells typically arrest beginning at larval gonial stages. Occasionally, mutant germ cells survived to the adult stage. However, these cells generally failed to undergo gametogenic differentiation as seen by the absence of spectrosomes, fusomes or ring canals. It was reasoned that, if the requirement for ovo is solely dependent on the X:A ratio, then the phenotype of ovo mutant germ cells in pseudomales should be at least as severe. In this case, the ovo mutant XX pseudomale gonads should be either atrophic or contain a few clusters of mostly undifferentiated germ cells. There is an increase in the frequency of atrophic gonads (82%) compared to normal pseudomales (48%), many of the non-oogenic type. The non-oogenic gonads contained VASA-positive germ cells. This indicates that not only are a substantial fraction of the mutant germ cells viable in adults, but gametogenic differentiation occurs as well. The frequency of the non-oogenic gonads in ovo mutant pseudomales is essentially unchanged from that observed in normal pseudomales. This suggests that the observed increase in the atrophic category is due primarily to the loss of the oogenic class. Mutations in otu gave results similar to those described for ovo. This suggests that otu and ovo mutations specifically disrupt only those germ cells attempting female differentiation, rather than the indiscriminate elimination of the entire XX germline (Hinson, 1999).

Heat shock-otu can alter the XX pseudomale gonadal phenotype; to examine whether and to what degree otu expression could induce oogenic development in pseudomales, immunohistochemical studies were performed. When continually cultured at 20-25°C, hs-otu pseudomale gonads are as much as two to three times longer than normal. In addition, 88% of the hs-otu gonads examined show extensive Hu-li tai shao-labeling of ring canals (Hts is an adducin-like protein). These feminized gonads display a developmental progression of gametogenic stages. In section III of the gonad, the pseudomale germ cells have differentiated to postgermarial stages as defined by the expression of kelch. Kelch, an actin binding protein, is localized to female ring canals after the ring canal deposition of Hts and f-actin . Kelch is first detected in female ring canals in stage 1 egg chambers, but is not seen in all ring canals until stage 4. In hs-otu XX pseudomales, the germ cell clusters in section III contain thick ring canals, with virtually all of them showing Kelch deposition along the inner surface of the f-actin layer. In comparison, no Kelch-labeled ring canals are observed in XX pseudomales without hs-otu, indicating that oogenesis is not only less frequent, but also more limited. Taken together, these results indicate the masculinizing effect of male soma (or the absence of female soma) on XX germ cells can be partially, but consistently, overridden by the expression of otu from a heterologous promoter. The resulting fusome and ring canal development follows the same sequence of events as occurs in normal oogenesis. Therefore, pseudomale germ cells are competent to both initiate and undergo substantial oogenesis if provided with adequate levels of otu. Both ovo and Sxl were shown to be required for otu induced oogenic differentiation in XX pseudomales. However, an additional role for otu in some process affecting germline viability and/or proliferation can be identified that is separable from oogenic differentiation and independent of ovo and, possibly, Sxl functions (Hinson, 1999).

The finding that hs-otu can feminize XX pseudomale germ cells suggests oogenesis is blocked because of insufficient otu levels. Therefore, an examination was carried out to see whether tra-induced sexual transformation affects the level of otu gene expression. otu-lacZ is expressed in most, if not all, larval and pupal germ cells in both female and male gonads. Sex-specific regulation only becomes apparent in the adult testis where male germline expression become restricted to a few cells at the apical tip. As with otu, the ovo promoter is initially active in both male and female larval gonads. However, ovo-lacZ becomes sex-specific at an earlier stage, showing restricted expression in male gonads during the third instar larval and pupal periods. These results demonstrate that the otu and ovo promoters are under different regulatory control in the pre-adult germline. However, otu, but not ovo, promoter activity is influenced by tra-induced sexual transformation. These data demonstrate that the tra-induced sexual transformation specifically inhibits otu promoter activity. Also carried out was the reciprocal experiment, in which otu-lacZ activity was examined in XY germ cells developing in a female somatic background. XY pseudofemales produced by the ectopic expression of tra result in ovaries containing tumorous egg chambers. Because XY pseudofemale germ cells become sufficiently 'feminized' so that they acquire a need for otu function for optimal proliferation, it was anticipated they would also be permissive for otu promoter activity. This is in fact the case. Even in the absence of ovo function, XY pseudofemale germ cells consistently express otu-lacZ. This indicates that the feminizing effects of tra, but not ovo, are necessary for otu transcription. In comparison, the ovo promoter is not detectably active in XY pseudofemales, again illustrating differential regulation of ovo and otu (Hinson, 1999).

It is thought that during the pupal and adult stages, two critical events occur in the female germarium: (1) ovo activity allows XX germ cells to become receptive to the otu function controlling oogenic differentiation, and (2) tra-dependent somatic signals allow continued expression of otu in the female germline by maintaining otu promoter activity. The combination of these events constitutes a mechanism by which the otu gene serves to link the somatic sex differentiation pathway controlled by tra with a female germline developmental pathway controlled by ovo (Hinson, 1999).

In Drosophila, the sex of germ cells is determined by cell-autonomous and inductive signals. XY germ cells autonomously enter spermatogenesis when developing in a female host. In contrast, XX germ cells non-autonomously become spermatogenic when developing in a male host. In first instar larvae with two X chromosomes, XX germ cells enter either the female or the male pathway depending on the presence or absence of transformer activity in the surrounding soma. In somatic cells, the product of tra regulates the expression of the gene doublesex which can form a male-specific or a female-specific product. In dsx mutant larvae, XX and XY germ cells develop abnormally, with a seemingly intersexual phenotype. This indicates that female-specific somatic dsx products feminize XX germ cells, and male-specific somatic dsx products masculinize XX and XY germ cells. The results show that tra and dsx control early inductive signals that determine the sex of XX germ cells and that somatic signals also affect the development of XY germ cells. XX germ cells that develop in pseudomales lacking the sex-determining function of Sxl are spermatogenic. If, however, female-specific tra functions are expressed in these animals, XX germ cells become oogenic. Furthermore, transplanted XX germ cells can become oogenic and form eggs in XY animals that express the female-specific function of tra. Therefore, TRA product present in somatic cells of XY animals or in animals lacking the sex-determining function of Sxl, is sufficient to support developing XX germ cells through oogenesis (Steinmann-Zwicky, 1994).

What are the genetic requirements for high ovo expression in gonads? In autosomes, the X to autosome ratio is sufficient for Sex lethal expression. Does a similar mechanism hold in the gonads? To study this question, expression of ovo was examined in gonads of mutant flies where the chromosomal sex does not match the somatic sexual identity. If a 2X karyotype is necessary and sufficient for high levels of ovo expression, then female somatic sex should have no effect on ovo expression. XY females can be produced by using a strain of flies bearing a transformer gene driver by a heat-shock promoter (trahs). In XY genetic males bearing the trahs transgene, TRA activity is sufficient to direct female somatic development in flies that would otherwise develop as males, but the germ line remains male. If a female somatic sexual identity is sufficient for a high level of ovo expression, then both XX females and XY trahs females should be expected to show similar ovo expression. In fact ovo expression is not activated to a high level by a female somatic identity, suggesting that either an XX karyotype or female germ-line identity is required for high ovo expression (Oliver, 1994).

Sex determination in Drosophila depends on the post-transcriptional regulatory activities of the gene Sex-lethal (Sxl). Sxl maintains the female determined state and activates female differentiation pathways by directing the female-specific splicing of Sxl and tra pre-mRNAs. While there is compelling evidence that Sxl proteins regulate splicing by directly binding to target RNAs, previous studies indicate that the two Sxl RNA-binding domains are not in themselves sufficient for biological activity and that an intact N-terminal domain is also critical for splicing function. To further investigate the functions of the Sxl N terminus, a chimeric protein consisting of the N-terminal 99 amino acids fused to beta-galactosidase (hsp83:N beta-gal) was ectopically expressed. The Nbeta-gal fusion protein behaves like a dominant negative, interfering with the Sxl autoregulatory feedback loop and killing females. This dominant negative activity can be attributed to the recruitment of the fusion protein into the large Sxl:Snf splicing complexes that are found in vivo and the consequent disruption of these complexes (Deshpande, 1999).

Since the Sxl gene can be completely deleted in males without any detectable effects on viability, morphology or behavior, the N beta-gal transgene would not, in principle, be expected to have any effect on male-specific developmental pathways. However, contrary to this expectation, approximately 10% of the males in all of the lines exhibit morphological alterations characteristic of sex transformations. These transformations include reduced or patchy abdominal pigmentation, extra sternite hairs, rotated genitalia and alterations in the number or shape of the sex combs. In females, the phenotypic effects of the hsp83:N beta-gal transgene can be enhanced by raising the temperature and suppressed by lowering the temperature. To test whether this is also true for male feminization, wild-type females were crossed to males heterozygous for the N-41 transgenic line, and examined the viability and morphology of the resulting transgenic animals at either 25°C or 18°C. The lethal effects of a single copy of the transgene on female progeny from this cross decreases from about 16% at the higher temperature to about 6% at the lower temperature. On the contrary, decreasing the temperature does not reduce the frequency of feminization; the number of partially feminized males increases from 7% to about 21% as the temperature is dropped from 25°C to 18°C. A low but readily detectable level of yp1 mRNA is present in transgenic N-172 and N-21 males, but not in the control wild-type males. Expression of yp1 mRNA in transgenic males does not require the Sxl gene but does require tra (Deshpande, 1999).

The gain-of-function activity of the Nbeta-gal fusion protein is difficult to reconcile with the prevailing blockage model for the regulation of tra splicing. In this model, Sxl protein prevents the generic splicing factor U2AF from binding to the polypyrimidine tract of the default 3' splice site, forcing it instead to interact with the weaker downstream female-specific 3' splice site. A strong prediction of this model is that Sxl must be able to bind to the default polypyrimdine tract in order to block the binding of U2AF. Since specific binding to target RNAs requires the two Sxl RRM domains, the Nbeta-gal fusion protein should have absolutely no tra regulatory activity. This prediction is not fulfilled. A second, weaker, prediction is that the two Sxl RRM RNA-binding domains should be sufficient to regulate tra splicing. Taken together these findings call into question the simple blockage model and suggest that regulation of tra splicing may be different from that previously envisioned. The most likely mechanism is through interactions with generic RNA-binding proteins (or other components of the splicing machinery) that associate with TRA pre-mRNAs (Deshpande, 1999).

It has been suggested that sexual identity in the germline depends upon the combination of a nonautonomous somatic signaling pathway and an autonomous X chromosome counting system. The roles of the sexual differentiation genes transformer (tra) and doublesex (dsx) in regulating the activity of the somatic signaling pathway have been examined. It was asked whether ectopic somatic expression of the female products of the tra and dsx genes could feminize the germline of XY animals. TraF, the female form of transformer, is sufficient to feminize XY germ cells, shutting off the expression of male-specific markers and activating the expression of female-specific markers. Feminization of the germline depends upon the constitutively expressed transformer-2 (tra-2) gene, but does not seem to require a functional dsx gene. However, feminization of XY germ cells by TraF can be blocked by the male form of the Dsx protein (DsxM). Expression of the female form of dsx, DsxF, in XY animals also induces germline expression of female markers. Taken together with a previous analysis of the effects of mutations in tra, tra-2, and dsx on the feminization of XX germ cells in XX animals, these findings indicate that the somatic signaling pathway is redundant at the level of tra and dsx. Finally, these studies call into question the idea that a cell-autonomous X chromosome counting system plays a central role in germline sex determination (Waterbury, 2000).

Transplantation experiments and clonal analysis have suggested that germline sexual identity in XX animals depends upon a combination of cell-autonomous factors that somehow assess the X/A ratio and nonautonomous factors that signal sexual identity from the soma to the germline. A plausible pathway for linking somatic sexual identity to the mechanism that generates the nonautonomous signal is the well-characterized Sxl -> tra/tra-2 -> dsx cascade. In previous studies, the effects were tested of mutations in tra, tra-2, and dsx on the sexual identity of germ cells in XX animals. Unexpectedly, only in the case of the sex-nonspecific gene, tra-2, does loss-of-function mutation lead to a switch in sexual identity of the XX germ cells from female to male. To account for these findings, it has been proposed that the somatic signal must be generated by a novel tra-2-dependent regulatory cascade. Since dsx is dispensable for this process in XX animals, it has been postulated that an unidentified tra-2 regulatory target, z, directly or indirectly generates the signal. To explain the fact that XX germ cells retain partial female identity in tra mutants, it has been suggested that there must be another gene q whose activity overlaps or is redundant with tra. In this view, both tra and q would be able to function with the cofactor tra-2 to promote the female-specific expression of z (Waterbury, 2000).

This model was tested by introducing transgenes that ectopically express the female forms of tra and dsx into XY animals and by assaying their effects on germline sexual identity. The findings are generally consistent with predictions of the original model: there were some unexpected results that altered an understanding of the nature of the germline sex determination process and the role of dsx. Experiments with the tra transgene are considered below (Waterbury, 2000).

According to the model, ectopically expressed tra is predicted to activate the regulatory cascade that signals female identity from the soma to the germline. Activation of this signaling pathway should require tra-2 and the target gene z, while dsx would be dispensable. The results are generally consistent with these predictions. Ectopically expressed Tra switches the sexual identity of germ cells in XY animals from male to female, turning off male-specific germline markers and inducing female-specific markers. This switch in sexual identity is blocked by mutations in tra-2, but is not prevented by loss-of-function mutations in dsx (Waterbury, 2000).

In XX animals, the available evidence indicates that the tra/q -> tra-2 -> z feminization pathway functions in the soma. Hence, an expectation of the model is that ectopically expressed Tra would also feminize XY germ cells through its action in the soma, not in the germline. However, since the hsp83 promoter is known to be active in both soma and germline, it is possible that Tra protein ectopically expressed in XY germ cells feminizes these cells by a novel mechanism that is independent of the somatic signaling pathway that normally operates in XX animals. Two lines of evidence argue against this: (1) since several of the hsp83-traF lines were recovered from males, it would appear that expression of Tra in XY germ cells is not in itself sufficient to feminize these cells; (2) the available evidence suggests that ectopically expressed Tra feminizes the germline in XY animals by a pathway resembling that used in XX animals; it requires tra-2 but is independent of a functional dsx (Waterbury, 2000).

Although the hsp83-traF transgene does not require dsx to feminize the germline of XY animals, feminization can be prevented if the only source of Dsx protein is provided by an allele that constitutively expresses DsxM. This result was unexpected since DsxM has no effect on the sexual identity of the germline in XX animals. There are several possible explanations for this discrepancy. It has been proposed that there is another gene, q, which occupies the same position in the regulatory cascade as tra. If this gene is downstream of Sxl, as expected, it would be expressed in the male mode in XY; P[hsp83-traF] animals and hence would not contribute to the production of the feminizing signal. Because DsxM alters the development of the soma surrounding the germline and consequently the cell-cell contacts between soma and germline, the signal produced by tra alone might not be sufficient to feminize. A second possibility is that XY germ cells are intrinsically less responsive to the feminizing signal than XX germ cells. For example, given the lack of strong evidence for germline dosage compensation, the signal could be sensitive to a twofold difference in X-linked genes. A third possibility is that DsxM produces a masculinizing signal that is able to counteract the effects of the feminizing signal produced by TraF. At the present, these explanations cannot be distinguished (Waterbury, 2000).

Since the dsx gene can be removed or expressed exclusively in the male mode without affecting germline sexual identity in XX animals, it has been suggested that dsx has no role in germline sex determination. However, contrary to this suggestion, ectopic expression of DsxF in XY; dsx- animals can feminize the germline and this feminizing activity can be blocked if DsxM is also present in the soma (Waterbury, 2000).

Why is DsxF capable of feminizing XY germ cells, yet dispensable in XX animals? One way to reconcile these two observations is to postulate that z regulates the synthesis of the feminizing signal ('fes') instead of encoding the signal itself. If this were the case, both Z and DsxF could independently promote the production of fes. In females, since q and tra would be active, Z would be able to induce sufficient levels of fes to feminize the germline in the absence of DsxF or in the presence of DsxM. Furthermore, since the female and male Dsx proteins recognize the same target sequences, ectopically expressed DsxF would be able to activate fes synthesis in XY animals only when DsxM is absent (Waterbury, 2000).

In the revised model for the somatic signaling pathway, Sxl has been placed at the top of the regulatory cascade where it is responsible for activating the female-specific expression of both tra and q. While Sxl is known to be required for sex-specific regulation of tra, it should be noted that there is no evidence that it is responsible for controlling the activity of q. However, unless q is itself a target for the X/A counting system, there are no other known mechanisms that could promote female expression. If q is downstream of Sxl, results with SxlM1,fm3 and SxlM1,fm7 (revertant alleles of SxlM1) suggest that q is regulated by a different mechanism than tra. q and/or tra, together with tra-2, would then activate the female-specific expression of z and dsx. The female products of z and dsx would in turn direct the synthesis of the feminizing signal. By this model, the germline would assume male identity whenever Sxl is off in the soma. However, it is not clear whether the male pathway requires production of a male somatic signal by the male form of Dsx (or Z) or occurs in XY germ cells by default in the absence of a female signal. In favor of the former possibility is the finding that constitutively expressed DsxM prevents Tra from feminizing XY germ cells. However, functional dsx is not required in XY animals to select male identity (Waterbury, 2000).

One question raised by these studies is the role of the postulated autonomous X chromosome counting system in germline sex determination. In particular, it has been argued from pole cell transplantation experiments that this autonomous system overrides input from the soma in XY germ cells, forcing them to assume male identity. However, data has been presented indicating that the sexual identity of XY germ cells can be switched from male to female by ectopic expression of TraF and DsxF. If TraF and DsxF activate the signaling pathway(s) that normally functions in XX animals, this result would imply that there may be no cell-autonomous system that selects sexual identity by measuring the germ cell X/A ratio. In this view, the autonomous components of the germline sex determination system would play an entirely different role. They would be subordinate to the somatic signaling pathway, being responsible only for responding correctly to the somatic signal and having no role in making the actual choice. Of course, if the default pathway within the germline is male, then this pathway will be followed 'autonomously' in the absence of a feminizing signal from the soma (Waterbury, 2000).

From a phylogenetic perspective, the simplest solution for germline sex determination is that germ cells strictly follow the same sexual fate as that of the soma in securing the development of a fully functional organism. In fact, this appears to be the mechanism for germline sex determination in other dipteran species such as Musca domestica and Chrysomya rufifacies. In these organisms, somatic sex alone is necessary and sufficient to dictate sexual fate to the germline. Irrespective of their sexual karyotype, when germ cells are surrounded by ovarian tissue, eggs are produced, and when surrounded by testicular tissue, sperm are produced. Studies in the nematode C. elegans and in the mouse further support the idea that somatic sex is widely used to dictate the sexual fate of gametes. In Drosophila, it is believed that somatic sex is the primary determinant. Why then is this soma-to-germline signaling mechanism insufficient to direct complete female or male germline differentiation independent of the chromosome composition of the germ cells in Drosophila? A likely explanation is that XY and XX germ cells in Drosophila have lost the ability to respond equally well to somatic cues. For example, in XY; P[hsp83-traF] pseudofemales, most of the ovarioles have a tumorous ovary phenotype and ovarioles that have normal-looking egg chambers are observed very infrequently. Given that there is no strong evidence for germline dosage compensation, one plausible explanation for the abnormal development of these sex-transformed XY germ cells is that the dose of X-linked gene products is insufficient to properly execute an oogenic developmental program. The Sxl gene would be a good example of a gene that is required for oogenesis and, because of its autoregulatory activity, is highly sensitive not only to its own dose but also to the dose of other X-linked genes such as the splicing factor snf. It is reasonable to suppose that there may be a variety of steps in oogenesis (or spermatogenesis) that are sensitive to the dose of X-linked genes (Waterbury, 2000).

Within the germline, otu, ovo, and Sxl have been identified as candidate genes that respond to the feminizing signal from the soma and determine the sex of the germ cells. Mutations in all three genes have sex-specific effects on germline development. Perhaps the most striking result is the fact that loss-of-function ovo and otu mutations markedly reduce the viability of XX but not XY germ cells. Thus one important question is whether these mutations have similar effects on the viability of XY germ cells feminized by the hsp83-traF transgene. Somewhat surprisingly, it was found that otu and ovo mutations behave differently. Strong loss-of-function otu mutations reduce the viability of XY germ cells feminized by the traF transgene. This finding suggests that the lethal effects of strong otu mutations arise because the germ cells assume a female identity, and not because of their number of X chromosomes. In contrast, ovo mutations have no apparent effect on the viability of feminized XY germ cells. One explanation for this difference is that lethal effects are not observed in ovo mutants because the feminizing signal produced by the traF transgene in XY animals is weaker than the feminizing signal found in wild-type XX animals. Alternatively, it is possible that XX germ cell death in ovo mutants does not depend upon the choice of sexual identity, but rather is a function of the X chromosome dose (Waterbury, 2000).

If otu, ovo, or Sxl functions as a master sex determination switch within the germline, one would expect to find that mutations in any of these genes would completely block the feminization of germ cells much like mutations in Sxl prevent feminization in the soma. While the results indicate that none of these genes fits this criterion for a master regulatory switch, effects are observed in the expression of sex-specific markers. Mutations in all three genes prevent the traF transgene from inducing the expression of female bruno (and Sxl) gene products. However, in all three cases the transgene still induces the expression of female orb gene products. One interpretation of these findings is that the sex determination pathway in the germline is split into at least two branches -- one branch that contains bruno and Sxl and another branch that contains orb. For both bruno and orb, sex-specific expression depends upon the activation of distinct sex-specific promoters. If these two genes are in independent branches of the germline sex determination pathway, this would imply that there must be distinct 'male' and 'female' transcription factors for the four promoters. Moreover, it seems likely that one important function of the somatic signaling system would be to control the expression of these transcription factors. Clearly, it will be important to identify these transcription factors and to learn how they are regulated (Waterbury, 2000).

Transformer, a protein that interacts with Transformer 2:Population and species variation of Transformer sequence

This paper reports the results from an evolutionary approach to investigate tra regulation and function, by isolating the tra-homologous genes from selected Drosophila species, and then using the interspecific DNA sequence comparisons to help identify regions of functional significance. The tra-homologous genes from two Sophophoran subgenus species, Drosophila simulans and Drosophila erecta, and two Drosophila subgenus species, Drosophila hydei and Drosophila virilis, were cloned, sequenced and compared to the D. melanogaster tra gene. This comparison reveals an unusually high degree of evolutionary divergence among the tra coding sequences. These studies also highlight a highly conserved sequence within intron one that probably defines a cis-acting regulator of the sex-specific alternative splicing event (O'Neil, 1992).

The transformer locus produces an RNA processing protein that alternatively splices the doublesex pre-mRNA in the sex determination hierarchy of Drosophila melanogaster. Comparisons of the tra coding region among Drosophila species have revealed an unusually high degree of divergence in both synonymous and nonsynonymous sites. In this study, the hypothesis that the tra gene will be polymorphic in synonymous and nonsynonymous sites within species is tested by investigating nucleotide sequence variation in eleven tra alleles within D. melanogaster. Of the 1063 nucleotides examined, two synonymous sites were polymorphic and no amino acid variation was detected. Three statistical tests were used to detect departures from an equilibrium neutral model. Two tests fail to reject a neutral model of molecular evolution because of low statistical power associated with low levels of genetic variation (Tajima/Fu and Li). The Hudson, Kreitman, and Aguade test rejects a neutral model when the tra region is compared to the 5'-flanking region of alcohol dehydrogenase (Adh). The lack of variability in the tra gene is consistent with a recent selective sweep of a beneficial allele in or near the tra locus (Walthour, 1994).


DEVELOPMENTAL BIOLOGY

Embryonic

The gene transformer-2 of Drosophila is necessary not only for female sexual differentiation but also for normal spermatogenesis in males. The transcript is five to eight times more abundant in females than in males or pseudomales. In both sexes, a low level of transcript is present in the soma and a high level in the germ line. Transcripts are also present in the male soma and in the ovaries where they are not required (but see below). This indicates that tra-2 is regulated in a way different from other sex-determining genes (Amrein, 1988).

Two transcripts, A and B, code for two different isoforms that function redundantly to direct female differention and female specific doublesex pre-mRNA splicing (Mattox, 1996).

Adult

Two alternatively spliced transformer-2 transcripts, each encoding a different putative RNA-binding protein, are found only in the male germ line. These male germ line-specific mRNAs differ from each other by the presence or absence of a single intron called M1. M1-containing transcripts make up a majority of transformer-2 germ-line transcripts in wild-type males but fails to accumulate in males homozygous for transformer-2 null mutations (Mattox, 1991).

In the male germline, where tra-2 has an essential role in spermatogenesis, a single isoform (Type B) is found to uniquely perform all necessary functions. This isoform appears to regulate its own synthesis during spermatogenesis through a negative feedback mechanism involving retention of intron 3 (Mattox, 1996).


EFFECTS OF MUTATION

Differentiation of a male-specific muscle in Drosophila melanogaster does not require the sex-determining genes doublesex or intersex

In Drosophila melanogaster adults, a pair of muscles span the fifth abdominal segment of males but not females. To establish whether genes involved in the development of other sexually dimorphic tissues controlled the differentiation of sex-specific muscles, flies mutant for five known sex-determining genes were examined for the occurrence of male-specific abdominal muscles. Female flies mutant for alleles of Sex-lethal, defective in sex determination, or null alleles of transformer or transformer-2 are converted into phenotypic males that form male-specific abdominal muscles. Both male and female flies, when mutant for null alleles of doublesex, develop as nearly identical intersexes in other somatic characteristics. Male doublesex flies produced the male-specific muscles, whereas female doublesex flies lacked them. Female flies, even when they inappropriately express the male-specific form of Doublesex mRNA, fail to produce the male-specific muscles. Therefore, the wild-type products of the genes Sex-lethal, transformer and transformer-2 act to prevent the differentiation of male-specific muscles in female flies. However, there is no role for the genes doublesex or intersex in either the generation of the male-specific muscles in males or their suppression in females (Taylor, 1992).

Differential control of yolk protein gene expression in fat bodies and gonads by the sex-determining gene tra-2 of Drosophila. EMBO J 9: 3975-80

The regulation of the yolk protein (YP) genes in the somatic cells of the gonads has been studied, using temperature sensitive mutations (tra-2ts) of transformer-2, a gene required for female sexual differentiation. XX;tra-2ts mutant animals were raised at the permissive temperature so that they developed as females and were then shifted to the restrictive male-determining temperature either 1-2 days before or 0-2 h after eclosion. These animals form vitellogenic ovaries. Likewise, mutant gonads transplanted into either normal female hosts or normal male hosts, kept at the restrictive temperature, undergo vitellogenesis. Thus, the ovarian follicle cells can mature and express their YP genes in the absence of a functional product of the tra-2 gene. Although the gonadal somatic cells of ovary and testis may derive from the same progenitor cells, the testicular cells of XX;tra-2ts pseudomales do not express their YP genes nor take up YP from the hemolymph at the permissive female-determining temperature. It is concluded that in the somatic cells of the gonad, the YP genes are no longer under direct control of the sex-determining genes, but instead are regulated by tissue specific factors present in the follicle cells. It is the formation of follicle cells which requires the activity of tra-2 (Bownes, 1990).

Determination of male-specific gene expression in Drosophila accessory glands

The developmental regulation of three male-specific somatic transcripts is investigated. These RNAs are synthesized exclusively in the adult male accessory gland, an internal tissue derived from the genital disk of Drosophila melanogaster. The expression of these male-specific transcripts (msts) is under the control of the sex determination regulatory hierarchy, as demonstrated by the expression of all three msts in chromosomal females carrying mutant alleles at the doublesex (dsx), intersex (ix), or transformer-2 (tra2) loci. Although transcription of all three male RNAs is initiated late in pupation, temperature shifts of sturtevant/transformer; tra2ts2 homozygotes during development indicate that this expression is irreversibly determined earlier, during the third larval instar. A shift of sturtevant/transformer; tra2ts2 homozygotes to the male-determining temperature, for the duration of the late larval period only, is sufficient to elicit the expression of the msts during the adult stage. This critical period for the determination of these transcripts appears to correlate with the time of morphological determination of the accessory gland in these animals. Thus, the expression of these genes could be specified by the morphological determination of the male-specific tissue in which they are active (Chapman, 1988).

Autoregulation of transformer-2 alternative splicing is necessary for normal male fertility in Drosophila

In the male germline of Drosophila the Transformer-2 protein is required for differential splicing of pre-mRNAs from the exuperantia and alternative-testes-transcript (att) genes: Transformer-2 also autoregulates alternative splicing of its own pre-mRNA. The role of alternative splicing in these targets is largely unknown, however in the case of EXU mRNA it has been shown that mutations affecting the alternatively spliced 3' UTR lead to a significant reduction in the level of the EXU mRNA that accumulates in male germ cells. Null mutations in exu result in male sterility and, like tra-2 mutations, lead to formation of spermatids with defects in nuclear elongation. Autoregulation of TRA-2 splicing results in production of two mRNAs that differ by the splicing/retention of the M1 intron and encode functionally distinct protein isoforms. Splicing of the intron produces an mRNA encoding Tra-2226, which is necessary and sufficient for both male fertility and regulation of downstream target RNAs. When the intron is retained, an mRNA is produced encoding Tra-2179, a protein with no known function. Repression of M1 splicing is dependent on Tra-2226, suggesting that this protein quantitatively limits its own expression through a negative feedback mechanism at the level of splicing. This idea is examined by testing the effect that variations in the level of tra-2 expression have on the splicing of M1 and on male fertility. Consistent with the hypothesis, as tra-2 gene dosage is increased, smaller proportions of TRA-2226 mRNA are produced, limiting expression of this isoform. Feedback regulation is critical for male fertility, since it is significantly decreased by a transgene in which repression of M1 splicing cannot occur and TRA-2226 mRNA is constitutively produced. The effect of this transgene becomes more severe as its dosage is increased, indicating that fertility is sensitive to an excess of Tra-2226. These results suggest that autoregulation of Tra-2226 expression in male germ cells is necessary for normal spermatogenesis (McGuffin, 1998).

A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship

Reproduction in higher animals requires the efficient and accurate display of innate mating behaviors. In Drosophila, male courtship consists of a stereotypic sequence of behaviors involving multiple sensory modalities, such as vision, audition, and chemosensation. For example, taste bristles located in the male forelegs and the labial palps are thought to recognize nonvolatile pheromones secreted by the female. A putative pheromone receptor, GR68a, is expressed in chemosensory neurons of about 20 male-specific gustatory bristles in the forelegs. Gr68a expression is dependent on the sex determination gene doublesex, which controls many aspects of sexual differentiation and is necessary for normal courtship behavior. Tetanus toxin-mediated inactivation of Gr68a-expressing neurons or transgene-mediated RNA interference of Gr68a RNA leads to a significant reduction in male courtship performance, suggesting that GR68a protein is an essential component of pheromone-driven courtship behavior in Drosophila (Bray, 2003).

If Gr68a encodes a male-specific pheromone receptor, it would be predicted that the sex determination genes, which control all aspects of sexual differentiation, would regulate its expression. Thus, Gr68a expression was investigated in chromosomally female (XX) flies that were sexually transformed into Ψ males by mutations in tra2 or dsx. Both types of Ψ males show the normal male expression pattern of the p[Gr68a]-Gal4 driver. Since sex-specific fru expression is directly controlled by Tra and Tra2, and hence, independent of dsx (i.e., XX; dsx Ψ males express no Frum), male-specific expression of Gr68a is fru independent. Thus, Gr68a is a dsx-dependent effector gene expressed in chemosensory neurons of taste bristles in the foreleg, which is consistent with a function for this gene in pheromone recognition (Bray, 2003).


EVOLUTIONARY HOMOLOGS

SR proteins are RNA binding proteins with a conserved serine/arginine domain involved in protein-protein interactions. Included among SR proteins are a group of highly conserved proteins, numbering six or more, involved in the alternative splicing of many pre-messenger RNAs. In addition to what appears to be a highly conserved repertoire of SR proteins, a number or SR proteins with very specialized functions have been identified. These include TRA and TRA2 of Drosophila, involved in the sex determination hierarchy. The literature below represents a sample of the SR protein literature, and includes some information on the highly conserved proteins, information on proteins that interact with SR proteins, and several examples of SR proteins with specialized functions.

Tra2 in other Drosophila species and other SR proteins in insects

The splicing factor TRA-2 affects sex-specific splicing of multiple pre-mRNAs involved in sexual differentiation. The tra-2 gene itself expresses a complex set of mRNAs generated through alternative processing that collectively encode three distinct protein isoforms. The expression of these isoforms differs in the soma and germ line. In the male germ line the ratio of the two isoforms present is governed by autoregulation of splicing. However, the functional significance of multiple TRA-2 isoforms has remained uncertain. The structure, function, and regulation of tra-2 were examined inDrosophila virilis (DV), a species diverged from D. melanogaster by over 60 million years. The DV homolog of tra-2 produces alternatively spliced RNAs encoding a set of protein isoforms analogous to those found in DM. When introduced into the genome of DM, this homolog can functionally replace the endogenous tra-2 gene for both normal female sexual differentiation and spermatogenesis. Examination of alternative mRNAs produced in DV testes suggests that germ line-specific autoregulation of tra-2 function is accomplished by a strategy similar to that used in DM. The similarity in structure and function of the tra-2 genes in these divergent Drosophila species supports the idea that sexual differentiation in DM and DV is accomplished under the control of similar regulatory pathways (Chandler, 1997).

SR proteins are essential for pre-mRNA splicing in vitro, act early in the splicing pathway, and can influence alternative splice site choice. Dominant and loss-of-function alleles were isolated for B52, the gene for a Drosophila SR protein. The allele B52ED was identified as a dominant second-site enhancer of white-apricot (wa), a retrotransposon insertion in the second intron of the eye pigmentation gene white with a complex RNA-processing defect. B52ED also exaggerates the mutant phenotype of a distinct white allele carrying a 5' splice site mutation (wDR18), and alters the pattern of sex-specific splicing at Doublesex mRNA under sensitized conditions, so that the male-specific splice is favored. In addition to being a dominant enhancer of these RNA-processing defects, B52ED is a recessive lethal allele that fails to complement other lethal alleles of B52. A comparison of B52ED with the B52+ allele from which it was derived reveals a single change in a conserved amino acid in the beta 4 strand of the first RNA-binding domain of B52, which suggests that altered RNA binding is responsible for the dominant phenotype. Reversion of the B52ED dominant allele using X rays leads to the isolation of a B52 null allele. Together, these results indicate a critical role for the SR protein B52 in pre-mRNA splicing in vivo (Peng, 1995).

Balbiani rings, the most active genes in the polytene chromosomes of the midge Chironomus (Diptera) code for secretory giant peptides. A heterogeneous nuclear ribonucleoprotein (hnRNP), Ct-hrp45, is one of the major components of pre-mRNP particles in Chironomus tentans. hrp45 belongs to the SR family of splicing factors and exhibits high sequence similarity to Drosophila SRp55/B52 and human SF2/ASF. The distribution of hrp45 within the C. tentans salivary gland cells has been found to be abundant in the nucleus, whereas it is undetectable in the cytoplasm. The fate of hrp45 in specific pre-mRNP particles (the Balbiani ring (BR) granules), has been revealed by immunoelectron microscopy. hrp45 is associated with the growing BR pre-mRNP particles and is added continuously concomitant with the growth of the transcript, indicating that hrp45 is bound extensively to exon 4, which comprises 80-90% of the primary transcript. Furthermore, hrp45 remains bound to the BR RNP particles in the nucleoplasm and is not released until the particles translocate through the nuclear pore. Thus, hrp45 behaves as an hnRNP protein linked to exon RNA (and perhaps also to the introns) rather than as a spliceosome component connected to the assembly and disassembly of spliceosomes. It seems that hrp45, and possibly also other SR family proteins, is playing an important role in the structural organization of pre-mRNP particles and is perhaps participating not only in splicing but also in other intranuclear events (Alzhanova-Ericsson, 1996).

The medfly Ceratitis capitata contains a gene (Cctra) with structural and functional homology to the Drosophila melanogaster sex-determining gene transformer (tra). The Ceratitis homolog of Sxl does not appear to have a switch function: the gene is expressed in both sexes, irrespective of whether the male-determining Y is present or absent, which is inconsistent with a main sex-determining function. However, preliminary data suggest that the bottom-most component of the pathway, dsx, is not only present in Ceratitis (Ccdsx), but has conserved a role in sexual differentiation. The pre-mRNA of this gene is also alternatively spliced giving rise to sex-specific products that show a remarkable structural conservation when compared with the corresponding male and female products in Drosophila. Sequence analysis of Ccdsx revealed the presence of putative TRA/TRA-2-binding sites close to the regulated splice site, suggesting that the underlying mechanism of sex-specific splicing is conserved and under the control of proteins homologous to TRA and TRA-2. Similar to tra in Drosophila, Cctra is regulated by alternative splicing such that only females can encode a full-length protein. In contrast to Drosophila, however, where tra is a subordinate target of Sex-lethal (Sxl), Cctra seems to initiate an autoregulatory mechanism in XX embryos that provides continuous tra female-specific function and acts as a cellular memory maintaining the female pathway. Indeed, a transient interference with Cctra expression in XX embryos by RNAi treatment can cause complete sexual transformation of both germline and soma in adult flies, resulting in a fertile male XX phenotype. The male pathway seems to result when Cctra autoregulation is prevented and instead splice variants with truncated open reading frames are produced. It is proposed that this repression is achieved by M, the Y-linked male-determining factor (Pane, 2002).

The results show that Ceratitis and Drosophila sex-determining cascades share a conserved tra-->dsx genetic module to control sex determination and sexual differentiation as well as that tra sex-specific splicing regulation differs in the two species. In Drosophila, TRA protein, together with TRA-2, binds to the TRA/TRA-2 recognition sequences on the Drosophila dsx pre-mRNA and promotes the use of a nearby female-specific acceptor site. Cctra is needed to impose the female-specific splicing of Ccdsx, most probably by a similar mechanism as in Drosophila, invoking the existence of a Cctra2 homolog. This hypothesis is also supported by the finding of TRA/TRA-2 recognition sequences located in close vicinity to the female-specific acceptor site in Ccdsx pre-mRNA (Pane, 2002).

In Drosophila, tra female-specific splicing is promoted by SXL, which blocks the use of the non-sex-specific splice site present in the tra pre-mRNA. In Ceratitis, the presence of multiple TRA/TRA-2-binding elements within the Cctra male-specific exonic sequences strongly suggests that CcTRA and a hypothetical CcTRA-2 protein could bind to these sequences, thus mediating a direct autoregulation. The unusually strong phenotypic effects of the RNAi against this gene also support this model of Cctra regulation. The localization of the putative regulatory elements within the Cctra gene indicates a repression mode by which CcTRA in females prevents the recognition of male-specific splice sites. The mechanism by which Cctra seems to promote the female mode of processing of its own pre-mRNA by TRA/TRA-2-binding elements appears to be different also from the female-specific splicing of dsx. Rather than activating a splice site nearby the regulated exon, as in the case of dsx, inclusion of male-specific Cctra sequences is suppressed when CcTRA is present. Although this would be a novelty with respect to known Drosophila TRA/TRA-2 activities, it has been previously shown that the 'behavior' of these cis elements is context dependent and that changing the location of splicing enhancers can transform them into negative regulatory elements (Pane, 2002).

In Drosophila, the presence of the Y chromosome is necessary for male fertility but not for male development. By contrast, RNAi-treated Ceratitis embryos with a female XX karyotype can develop into fertile males, which indicates that transient repression of Cctra by RNAi is sufficient to implement fully normal male development. The cases of complete sexual transformation of genetic Ceratitis females (XX) into fertile males by RNAi demonstrate that the Y chromosome, except for the dominant male determiner M, does not supply any other contribution to both somatic and germline male development, as suggested by previous Y-chromosome deletion analysis. Other dipteran species, such as Musca domestica and Chrysomya rufifacies show a female and male germline sex determination that is completely dependent on the sexual fate of the soma. However, in Drosophila, the XX and XY germ cells seem to respond differently to sex determining somatic cues. Indeed the XY germ cells have also an autonomous stage-specific sex determination mechanism that probably integrates the somatic signal. In Ceratitis, Cctra could be required in XX somatic cells to let them induce the XX germ cells to differentiate as oogenic cells. Alternatively, Cctra could be required in XX germ cells to 'feminize' them. This case would be a novelty with respect to the known Drosophila transformer gene functions (Pane, 2002).

Since zygotes that carry a Y chromosome do not activate Cctra female-specific splicing and autoregulation, it is proposed that the Y-linked male-determining M factor prevents this activation. It is conceivable that Cctra is a direct target of the M factor. Presence of this M factor in the zygote may prevent the production of CcTRA protein. The Cctra positive feedback loop is a probable target for regulation, because of its sensitivity (already shown by RNAi). An important question to be addressed is how autoregulation of Cctra is initiated in XX embryos of C. capitata and how this is prevented in XY embryos. A possible explanation is suggested by the Cctra female-specific mRNAs encoding the full-length protein, which have been detected in unfertilized eggs. Depositing these Cctra transcripts in eggs may provide a source of activity that can be used later for 'female-specific' processing when Cctra is zygotically transcribed. Once zygotically activated in XX embryos, Cctra promotes its own female-specific splicing, maintaining the female sex determination and the female-specific splicing of the downstream Ccdsx gene. Taken together, these events induce the female differentiation. In the current model for sex determination of medfly, the M factor is directly involved in the Cctra sex-specific regulation. Thus, in the presence of M Cctra, autoregulation is blocked and the gene produces male-specific transcripts encoding short and possibly non-functional CcTRA peptides. The absence of CcTRA leads Ccdsx to produce male-specific transcripts by default, promoting male differentiation. The control of the M factor upon Cctra expression could be exerted at different levels. The male determiner M could, for example, act at the pre-translational level blocking the production of CcTRA protein from the maternal transcripts. M could act at the post-translational level antagonizing the formation of protein complexes necessary for the female splicing mode. Or M could act as a transient transcriptional repressor of Cctra to reduce the amount of active CcTRA below a threshold needed to maintain the feedback loop. The proposed autoregulatory model of Cctra may also explain the remarkable efficiency of sex reversal by Cctra RNAi: a transient silencing of Cctra by injecting dsRNA is sufficient to let the loop collapse. Furthermore, the sensitivity of this positive autoregulation could be an evolutionary widely conserved pre-requisite to permit a 'faster' recruitment/replacement of different upstream regulators and to easily evolve different sex determining primary signals, as observed in dipteran species (Pane, 2002).

Sex can even be determined by a maternal effect in dipteran species such as Sciara coprophila and Chrysomya rufifacies. The hypothesis of a Cctra maternal contribution to the activation of the zygotic Cctra gene has similarities to the model of sex determination proposed for Musca domestica. In the common housefly, the maternal product of the key switch gene F is needed to activate the zygotic function of F in females. Musca male development results whenever F cannot become active in the zygote. This happens when the male-determining M is present in the zygotic genome, or when maternal F is not functional because of either the presence of M or the mutational loss of function of F (Fman) in the germline. More interesting, embryonic RNAi against the Musca tra-2 homolog causes sex reversion of Musca XX adults into intersex and fertile males, although this gene is not sex-specifically expressed. These recent data in Musca and the results in Ceratitis support the idea that F of Musca functionally corresponds to the Ceratitis tra gene, which seems to autoregulate and maternally contribute to its own activation, rather than to the Drosophila tra gene (Pane, 2002).

These data show that a basic structure of sex determination is conserved in the two dipteran species, namely the flow of 'instructions' from tra to dsx. This confirms the model of 'bottom-up' evolution, suggesting that during evolution developmental cascades are built from bottom up and that the genes at the bottom are widely conserved, while further upstream new regulatory elements may be recruited. The results show that Ceratitis and Drosophila sex-determining cascade differ at the level of transformer as well as upstream of it. Indeed the gene has conserved its function during evolution, but it has female-specific positive autoregulation in Ceratitis, while in Drosophila it needs Sxl as upstream regulator to express its female determining function. More likely the sex-determining function of Sxl was co-opted after Drosophila and Ceratitis had separated more than 100 Myr ago. Furthermore, it is conceivable that the autoregulatory mechanism of Sxl could have been selected to overcome a mutation impairing the tra autoregulation. Hence, in both species the female pathway is maintained by a single gene positive-feedback mechanism through sex-specific alternative splicing. Single gene autoregulation by alternative splicing seems not to be infrequent in nature, especially in those genes encoding splicing regulators. Indeed, other genes encoding RNA-binding proteins are thought to autoregulate their expression by controlling the processing of their own pre-mRNAs. Such a single-gene network with positive regulation is capable of bistability. This suggests that the emergence of analogous positive autoregulation in different genes such as Drosophila Sxl and Ceratitis tra genes would have been selected, during evolution, to guarantee a similar ON/OFF-female/male bistable cell state (Pane, 2002).

Since Ceratitis capitata is a major agricultural pest in many areas of the world, the isolation of a key sex-determining gene such as Cctra will substantially aid the development of new strategies to optimize the efficacy of currently used male sterile techniques for pest control. It is expected that tra is also a key sex-determining gene in many other insect species. Hence, the isolation of corresponding tra genes will open new means to control not only agricultural pests but also medically relevant vectors of diseases such as Glossina palpalis and Anopheles gambiae (Pane, 2002).

Mouse and Human Tra2 homologs

htra-2alpha is a human homolog of tra2. Two alternative types of htra-2alpha cDNA clones have been identified than encode different protein isoforms with striking organizational similarity to Drosophila tra2. Comparison of the D. melanogaster and D. virilis genes reveals an unrecognized but highly conserved region of 19 amino acids immediately downstream of the RRM, referred to as the linker region. The linker region is conserved in humans. When expressed in flies, one hTRA-2alpha isoform partially replaces the function of Drosophila TRA2, affecting both female sexual differentiation and alternative splicing of DSX pre-mRNA. Like Drosophila TRA-2, the ability of hTRA-2alpha to regulate dsx is female specific and depends on the presence of the dsx splicing enhancer. These results demonstrate that htra-2alpha has conserved a striking degree of functional specificity during evolution and leads to the suggestion that the tra2 products of flies and humans have similar molecular functions, although they are likely to serve different roles in development (Dauwalder, 1996).

The SR protein B52 has been shown to be required for development in Drosophila; another SR protein, ASF/SF2, is required for viability of chicken DT40 cells, but Drosophila Tra2 is apparently nonessential. No functions for Tra2, outside sexual differentiation, have so far been discovered in Drosophila. Recently, two human homologs of Tra2, Tra2 and Tra2beta, have been identified. Using transgenic flies with nonfunctional Tra2, human Tra2 has been shown to be able to rescue Tra-dependent but not Tra-independent functions. The natural functions of mammalian Tra2 proteins are, however, unknown, as are their RNA binding specificities. Since the mechanisms of sexual differentiation are not conserved between mammals and flies, it is possible that human Tra2 proteins serve more general purposes than their Drosophila homolog (Tacke, 1998).

Human Tra2 proteins are present in HeLa cell nuclear extracts; they bind efficiently and specifically to a previously characterized pre-mRNA splicing enhancer element. The purine rich ESE, AS3, consists of three copies of a high affinity ASF/SF2 binding site. Human Tra2 proteins bind specifically to A3. Indeed, the two purified Tra2 proteins bind preferentially to RNA sequences containing GAA repeats, which are characteristic of many enhancer elements. Neither Tra2 protein functions in constitutive splicing in vitro, and neither can substitute for the essential splicing function of SR proteins, but both activate enhancer-dependent splicing in a sequence-specific manner and restore it after inhibition with competitor RNA. These findings indicate that mammalian Tra2 proteins are sequence-specific splicing activators that likely participate in the control of cell-specific splicing patterns (Tacke, 1998).

What are the specific functions of the two human Tra2 proteins in vivo? The data have not revealed functional differences between Tra2alpha and Tra2beta. Their RBDs are 85% identical, consistent with the finding that their RNA binding specificities are indistinguishable. The proteins differ mainly in the N-terminal 49 amino acids and in the position of a polyglycine stretch within their C-terminal RS domains, which otherwise are highly homologous. One possibility is that the two proteins are functionally redundant in vivo. Alternatively, the small differences in primary structure may account for potential differences in protein-protein interactions, including interaction with constitutive splicing factors such as SR proteins or with cell-specific factors yet to be discovered. An important aspect of the function of the Drosophila Tra protein in the regulation of dsx splicing appears to be its ability to influence the RNA binding properties of Tra2 through cooperative interaction. While Tra2 binds preferentially to the purine-rich element of the dsx enhancer in the absence of Tra, it can also bind to the dsx repeats in the presence of Tra. It is tempting to postulate a scenario in which cell-specific factors alter the RNA binding properties of human Tra2 proteins, analogous to the Tra/Tra2 cooperation in Drosophila. It is also noteworthy that Tra2beta was initially identified as a factor rapidly induced during reoxygenation of astrocytes after hypoxia and subsequently shown to display different mRNA expression levels in various mouse tissues. These examples raise the possibility that at least in some cases mammalian Tra2 proteins themselves might act as stage-specific regulators of pre-mRNA splicing. As in Drosophila, in vivo genetic studies may ultimately be required to elucidate the precise roles of mammalian Tra2 proteins in the control of cell- and stage-specific splicing patterns (Tacke, 1998).

Astrocytes have a critical role in the neuronal response to ischemia, sincetheir production of neurotrophic mediators can favorably impact on the extreme sensitivity of nervous tissue to oxygen deprivation. Using a differential display method, a novel putative RNA binding protein, RA301, was cloned from reoxygenated astrocytes. RA301 is the same as Tra2beta, a sequence-specific activator of pre-mRNA splicing and a homolog of Drosophila Tra2. Analysis of the deduced amino acid sequence shows two ribonucleoprotein domains and serine/arginine-rich domains, suggestive of a function as an RNA splicing factor. Northern analysis displays striking induction only in cultured astrocytes within 15 min of reoxygenation and reaches a maximum by 60 min after hypoxia/reoxygenation. Immunoblotting demonstrates expression of an immunoreactive polypeptide of the expected molecular mass, 36 kDa, in lysates of hypoxia/reoxygenated astrocytes. Induction of RA301 mRNA is mediated, in large part, by endogenously generated reactive oxygen species, as shown by diphenyl iodonium, an inhibitor of neutrophil-type nicotinamide adenine dinucleotide phosphate oxidase, which blocks oxygen-free radical formation by astrocytes. Similarly, increased expression of RA301 in supporting a neurotrophic function for astrocytes is suggested by inhibition of interleukin-6 elaboration, a neuroprotective cytokine, in the presence of antisense oligonucleotide for RA301. These studies provide a first step in characterizing a novel putative RNA binding protein, whose expression is induced by oxygen-free radicals generated during hypoxia/reoxygenation, and which may have an important role in redirection of biosynthetic events observed in the ischemic tissues (Matsuo, 1995).

SIG41, a murine homolog of Drosophila TRA2, is a protein of 288 amino acids that is 45% identical to TRA2. There are four types of transcripts in mouse cells. Messenger RNA is present in virtually all cell lines and tissues studied, with remarkable levels in uterus and brain tissues. Differential stability of the SIG41 mRNAs is detected in mouse macrophage cells (Segade, 1996).

The role of SR proteins in splicing

SR proteins are essential splicing components that may participate in the maintenance and regulation of cell-specific splicing patterns. SR proteins are constitutive components of the splicing machinery that play important roles during spliceosome assembly by promoting splice site recognition and facilitating interactions between 5' and 3' splice site complexes. Individual SR proteins are able to complement splicing-deficient cytoplasmic S100 extracts, which lack SR proteins but contain other factors necessary for constitutive splicing. In addition, SR proteins can influence alternative splicing in a concentration-dependent manner, either when added to splicing-competent nuclear extracts or when transiently overexpressed in vivo. SR proteins have also been shown to participate in splicing activation of introns containing weak 5' or 3' splice sites by binding to purine-rich exonic splicing enhancers (ESE), frequently situated in downstream exons. Conversely, high-affinity binding sites for the SR proteins ASF/SF2 and SRp40 have been demonstrated to function as ESEs in vitro. These and other studies also show that SR proteins have distinct RNA binding specificities and display substrate specificity in vitro (Tacke, 1998).

The SR proteins constitute a family of splicing factors, highly conserved in metazoans, that contain one or two amino-terminal RNA-binding domains (RBDs) and a region enriched in arginine/serine repeats (RS domain) at the carboxyl terminus. Previous studies have shown that SR proteins possess distinct RNA-binding specificities that likely contribute to their unique functions, but it is unclear whether RS domains have specific roles in vivo. A genetic system was developed in the chicken B cell line DT40 to address this question. Expression of chimeric proteins generated by fusion of the RS domains of heterologous SR proteins, or a human TRA-2 protein, with the RBDs of ASF/SF2 allow cell growth following genetic inactivation of endogenous ASF/SF2, indicating that RS domains are interchangeable for all functions required to maintain cell viability. However, a chimera containing the RS domain from a related splicing factor, U2AF65, cannot rescue viability and is inactive in in vitro splicing assays, suggesting that this domain performs a distinct function. Depletion of ASF/SF2 affects splicing of specific transcripts in vivo. Although splicing of several simple constitutive introns is not significantly affected, the alternative splicing patterns of two model pre-mRNAs switch in a manner consistent with predictions from previous studies. Unexpectedly, ASF/SF2 depletion results in a substantial increase in splicing of an HIV-1 tat pre-mRNA substrate, indicating that ASF/SF2 can repress tat splicing in vivo. These results provide the first demonstration that an SR protein can influence splicing of specific pre-mRNAs in vivo (Wang, 1998).

A genomic clone encoding the Drosophila U1 small nuclear ribonucleoprotein particle 70K protein was isolated by hybridization with a human U1 small nuclear ribonucleoprotein particle 70K protein cDNA. Southern blot and in situ hybridizations show that this U1 70K gene is unique in the Drosophila genome, residing at cytological position 27D1,2. Polyadenylated transcripts of 1.9 and 3.1 kilobases were observed. While the 1.9-kilobase mRNA is always more abundant, the ratio of these two transcripts is developmentally regulated. Analysis of cDNA and genomic sequences indicated that these two RNAs encode an identical protein with a predicted molecular weight of 52,879. Comparison of the U1 70K proteins predicted from Drosophila, human, and Xenopus cDNAs reveals 68% amino acid identity in the most amino-terminal 214 amino acids, which include a sequence motif common to many proteins that bind RNA. The carboxy-terminal half is less well conserved but is highly charged and contains distinctive arginine-rich regions in all three species. These arginine-rich regions contain stretches of arginine-serine dipeptides like those found in Transformer, Transformer-2, and Suppressor-of-white-apricot proteins, all of which have been identified as regulators of mRNA splicing in Drosophila melanogaster (Mancebo, 1990).

A family of six highly conserved proteins that contain domains rich in alternating serine/arginine residues (SR proteins) function in the regulation of splice site selection and are required for splicing. More than 35 proteins were detected in nuclear extracts of HeLa cells that co-fractionate with the defined SR family. Many of these proteins were recognized by three monoclonal antibodies that bind to distinct phosphoepitopes on SR proteins. Two of these SR-related proteins were identified as the nuclear matrix antigens B1C8 and B4A11, which previously have been implicated in splicing. A subset of SR proteins, in their phosphorylated state, are associated with spliceosome complexes through both steps of the splicing reaction, remaining preferentially bound to complexes containing the exon-product. In contrast, other SR-related proteins appear to remain specifically associated with the intron-Lariat complex. The results indicate the existence of a potentially large group of SR-related proteins, and also suggest possible additional functions of SR proteins at a post-splicing level (Blencowe, 1996).

A monoclonal antibody (mAb 16H3) was generated against four SR proteins from a the family of six SR proteins, all known regulators of splice site selection and spliceosome assembly. In addition to the reactive SR proteins (SRp20, SRp40, SRp55, and SRp75), mAb 16H3 also binds approximately 20 distinct nuclear proteins in human, frog, and Drosophila extracts, whereas yeast do not detectably express the epitope. The antigens are shown to be nuclear, nonnucleolar, and concentrated at active sites of RNA polymerase II transcription, suggesting their involvement in pre-mRNA processing. Indeed, most of the reactive proteins observed in nuclear extract are detected in spliceosomes (E and/or B complex) assembled in vitro, including the U1 70K component of the U1 small nuclear ribonucleoprotein particle and both subunits of U2AF. Interestingly, the 16H3 epitope maps to a 40-amino acid polypeptide composed almost exclusively of arginine alternating with glutamate and aspartate. All of the identified antigens, including the human homolog of yeast Prp22 (HRH1), contain a similar structural element characterized by arginine alternating with serine, glutamate, and/or aspartate. These results indicate that many more spliceosomal components contain such arginine-rich domains. Because it is conserved among metazoans, it is proposed that the "alternating arginine" domain recognized by mAb 16H3 may represent a common functional element of pre-mRNA splicing factors (Neugebauer, 1995).

Phosphorylation and the function of SR proteins

ASF/SF2 is a member of a conserved family of splicing factors known as SR proteins. These proteins, which are necessary for splicing in vitro, contain one or two amino-terminal RNP-type RNA-binding domains and an extensively phosphorylated carboxy-terminal region enriched in repeating Arg-Ser dipeptides (RS domains). Previous studies have suggested that RS domains participate in protein-protein interactions with other RS domain-containing proteins. The RS domain of unphosphorylated recombinant ASF/SF2 is necessary, but not sufficient, for binding to the U1 snRNP-specific 70-kD protein (70K) in vitro. An apparent interaction of the isolated RS domain with 70K is observed if contaminating RNA is not removed, suggesting a nonspecific bridging among the basic RS domain, RNA, and 70K. In vitro phosphorylation of recombinant ASF/SF2 significantly enhances binding to 70K and also eliminates the RS domain-RNA interaction. Providing evidence that these interactions are relevant to splicing, ASF/SF2 can bind selectively to U1 snRNP in an RS domain-dependent, phosphorylation-enhanced manner. Conditions are described that reveal for the first time a phosphorylation requirement for ASF/SF2 splicing activity in vitro (Xiao, 1997).

The SR proteins constitute a large family of nuclear phosphoproteins required for constitutive pre-mRNA splicing. These factors also have global, concentration-dependent effects on alternative splicing regulation; this activity is antagonized by members of the hnRNP A/B family of proteins. Whereas some human SR proteins are confined to the nucleus, three of them (SF2/ASF, SRp20, and 9G8) shuttle rapidly and continuously between the nucleus and the cytoplasm. By swapping the corresponding domains between shuttling and nonshuttling SR proteins, it has been shown that the carboxy-terminal arginine/serine-rich (RS) domain is required for shuttling. This domain, however, is not sufficient to promote shuttling of an unrelated protein reporter, suggesting that stable RNA binding mediated by the RNA-recognition motifs may be required for shuttling. Consistent with such a requirement, a double point-mutation in RRM1 of SF2/ASF that impairs RNA binding prevents the protein from shuttling. Phosphorylation of the RS domain affects the shuttling properties of SR proteins. These findings show that different SR proteins have unique intracellular transport properties and suggest that those SR family members that shuttle may have roles not only in nuclear pre-mRNA splicing but also in mRNA transport, cytoplasmic events, and/or processes that involve communication between the nucleus and the cytoplasm (Caceres, 1998).

Mammalian Clk/Sty is the prototype for a family of dual specificity kinases (termed LAMMER kinases) that have been conserved in evolution, but whose physiological substrates are unknown. In a yeast two-hybrid screen, the Clk/Sty kinase specifically interacted with RNA binding proteins, particularly members of the serine/arginine-rich (SR) family of splicing factors. Clk/Sty itself has an serine/arginine-rich non-catalytic N-terminal region that is important for its association with SR splicing factors. In vitro, Clk/Sty efficiently phosphorylated the SR family member ASF/SF2 on serine residues located within its serine/arginine-rich region (the RS domain). Tryptic phosphopeptide mapping demonstrates that the sites on ASF/SF2 that phosphorylate in vitro overlap with those that phosphorylate in vivo. A catalytically inactive form of Clk/Sty co-localizes with SR proteins in nuclear speckles. Overexpression of the active Clk/Sty kinase causes a redistribution of SR proteins within the nucleus. These results suggest that Clk/Sty kinase directly regulates the activity and compartmentalization of SR splicing factors (Colwill, 1996).

SR protein subnuclear localization

If pre-mRNA splicing begins during RNA synthesis, then transcriptionally active genes may be expected to contain high concentrations of pre-mRNA splicing factors. However, previous studies have localized splicing factors to a network of "speckles," regions distinct from individual sites of gene transcription where pre-mRNA is spliced. Speckles have been detected with antibodies specific for splicing snRNPs and members of the SR family of splicing factors. Dilution of these probes results in the visualization of hundreds of sites throughout the HeLa cell nucleus, the size and distribution of which are consistent with transcription units viewed with light microscopy. Analysis of the particles detected at varying concentrations of anti-SR reveals that particle dimensions are fairly uniform at a variety of low concentrations of anti-SR, but the diameters increase at higher concentrations. These sites of highest SR protein concentration frequently coincide in three-dimensional space with active sites of RNA polymerase II transcription. A newly developed reagent specific for a single member of the SR family, SRp20, detects a subset (~20%) of these sites (suggesting the gene-specific accumulation of these splicing regulators) that have distinct functions in pre-mRNA splicing. These observations question the view that the nucleus and its functions are highly compartmentalized; instead, they support a model in which the localization of these and possibly other gene regulators is determined primarily by their function (Neugebauer, 1997).

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). The role of the individual domains of these closely related proteins has been studied in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. Striking differences are observed in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, it was found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that has altered specificity in 5' splice site selection, although it is active in alternative splicing. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo (Caceres, 1997).

SR protein interactions

Although transcription and pre-mRNA processing are colocalized in eukaryotic nuclei, molecules linking these processes have not previously been described. Four novel rat proteins have been identified by their ability to interact with the repetitive C-terminal domain (CTD) of RNA polymerase II in a yeast two-hybrid assay. A yeast homolog of one of the rat proteins has also been shown to interact with the CTD. These CTD-binding proteins are all similar to the SR (serine/arginine-rich) family of proteins that have been shown to be involved in constitutive and regulated splicing. In addition to alternating Ser-Arg domains, these proteins each contain discrete N-terminal or C-terminal CTD-binding domains. SR-related proteins have been identified in a complex that can be immunoprecipitated from nuclear extracts with antibodies directed against RNA polymerase II. In addition, in vitro splicing is inhibited either by an antibody directed against the CTD or by wild-type but not mutant CTD peptides. Thus, these results suggest that the CTD and a set of CTD-binding proteins may act to physically and functionally link transcription and pre-mRNA processing (Yuryev, 1996).

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are abundant nuclear polypeptides, most likely involved in different steps of pre-mRNA processing. Protein A1 (34 kDa), a prominent member of the hnRNP family, seems to act by modulating the RNA secondary structure and by antagonizing some splicing factors (SR proteins) in splice-site selection and exon skipping/inclusion. A role of A1 in the nucleo-cytoplasmic transport of RNA has also been proposed. These activities might depend not only on the RNA-binding properties of the protein but also on specific protein-protein interactions. A1 can indeed selectively interact, in vitro, both with itself and with other hnRNP basic "core" proteins. Such selective binding is mediated exclusively by the Gly-rich C-terminal domain, where a novel protein-binding motif constituted by hydrophobic repeats can be envisaged. The same domain is necessary and sufficient to promote specific interaction in vivo, as assayed by the yeast two-hybrid assay. Moreover, an in vitro interaction with some SR proteins is also observed. These observations suggest that diverse and specific protein-protein interactions might contribute to the different functions of the hnRNP A1 protein in mRNA maturation (Cartegni, 1996).

An in vitro splicing assay has been devised in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. This assay was used to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB can be partially reversed if pre-mRNAs are preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affects the joining of exons 1-3 with no dramatic increases in 1-2 splicing detected. Preincubation of pre-mRNAs with SR proteins leads to small increases in 1-2 splicing. However, if pre-mRNAs are preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there is a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus multiple pairwise and sometimes antagonizing interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection (Lin, 1995).

SR phosphorylation and activity

Serine/arginine-rich splicing factors (SR proteins) are substrates for serine phosphorylation that can regulate SR protein function. Changes in the phosphorylation of splicing components occurs at the level of the individual reaction as well as in a broader, cell-wide manner. Phosphatase inhibitors have been used to demonstrate that dephosphorylation is important for single splicing reactions. Specific phosphatases including protein phosphatase 1 (PP1) have been shown to affect in vitro splicing. In addition, thiophosphorylation of splicing factors interferes with pre-mRNA splicing. More directed experiments have demonstrated that the state of phosphorylation of SR proteins in particular can be linked to splicing activity in vitro. Several SR protein kinases have been described, including SR protein kinase 1 (SRPK1), Clk/Sty, SRPK2, and a kinase activity of DNA topoisomerase I. Hyperphosphorylation with SRPK1 can inhibit splicing, as can dephosphorylation of SR proteins; thus, an intermediate level of phosphorylation is required for splicing in vitro. In addition, dephosphorylation of SR proteins appears to be required during constitutive splicing reactions but not for their ability to act as splicing activators in regulated splicing. Broad changes in SR protein phosphorylation are also seen in response to events that affect the entire cell. Hyperphosphorylation of SR proteins occurs during M phase of the cell cycle, in which splicing is quiescent. Large-scale dephosphorylation of SR proteins is observed in late adenovirus-infected HeLa cells. In Drosophila, defects in SR protein phosphorylation have been correlated with discrete phenotypes in the sex determination pathway (Du, 1998). Changes in SR protein phosphorylation have also altered SR protein subnuclear localization, with increased expression of either SRPK1 or Clk/Sty leading to diffusion of speckles. PP1 also alters the speckled morphology. In addition, hyperphosphorylation of SF2/ASF leads to its accumulation in the cytoplasm (Sanford, 1999 and references).

Gross changes have been observed in SR protein phosphorylation during early development coincident with major zygotic gene activation in the nematode Ascaris lumbricoides. These differences correlate with large-scale changes in SR protein activity in the promotion of both trans- and cis-splicing. Importantly, inactive early stage extracts can be made splicing competent with the addition of later stage SR proteins. To assess their phosphorylation state, SR proteins at these different stages were phosphorylated with the SR protein-specific kinase SRPK1 and [32Pgamma]ATP. Over this developmental span, the SR proteins become progressively better substrates for SRPK1. SR proteins that are hyperphosphorylated do not incorporate label, whereas the same proteins that have undergone partial dephosphorylation are labeled more efficiently. To demonstrate that this difference is the result of limited dephosphorylation and not a mass effect, phosphorylation reactions were performed after first dephosphorylating the SR preparations with PP1. Under these conditions, there is an equivalent amount of phosphate incorporated into SR proteins prepared from each of three developmental stages. In summary, each of these assays indicates that SR proteins undergo dephosphorylation concomitant with the major activation of gene expression. These data suggest that changes in SR protein phosphorylation have a role in the activation of pre-mRNA splicing during early development (Sanford, 1999).

The splicing reaction that removes introns from pre-messenger RNAs requires the assembly of the spliceosome on the nascent transcript, proper folding of the substrate-enzyme complex, and finally, two transesterification reactions. These stages in the splicing reaction must require careful orchestration. The sequential phosphorylation and dephosphorylation of SR proteins marks the transition between stages in the splicing reaction. Much evidence has already led to the idea that phosphorylation of SR proteins could modulate their activity. Dephosphorylation of these proteins abrogates their activity in a reaction measuring conversion of pre-spliceosomes to spliceosomes Phosphorylated ASF/SF2, but not mock-phosphorylated ASF/SF2, activates the splicing of HIV tat pre-mRNA in reactions challenged with excess random RNA. These two findings are confirmed and extended by this study. Phosphorylated ASF/SF2 efficiently complements an SR protein-deficient HeLa S100 extract in promoting the splicing of an adenovirus-2-derived pre-messenger RNA, whereas unphosphorylated ASF/ SF2 does not. Whereas unphosphorylated ASF/SF2 inhibits splicing in HeLa nuclear extracts, phosphorylation of the ASF/SF2 reverses the inhibition and enhances splicing. Data is presented that shows that dephosphorylation of ASF/SF2 is required for the first transesterification reaction once the spliceosome has assembled. Thiophosphorylated ASF/SF2, which cannot be readily dephosphorylated, can promote spliceosome assembly, but cannot promote the first transesterification reaction. These data, together with other observations, indicate for the first time a requirement for SR protein dephosphorylation in pre-messenger RNA splicing in vitro (Cao, 1997).

SR proteins are a family of essential splicing factors required for early recognition of splice sites during spliceosome assembly. They also function as alternative RNA splicing factors when overexpressed in vivo or added in excess to extracts in vitro. SR proteins are highly phosphorylated in vivo, a modification that is required for their function in spliceosome assembly and splicing catalysis. SR proteins purified from late adenovirus-infected cells are inactivated as splicing enhancer or splicing repressor proteins by virus-induced dephosphorylation. The virus-encoded protein E4-ORF4 activates dephosphorylation by protein phosphatase 2A of HeLa SR proteins and converts their splicing properties into those of SR proteins purified from late adenovirus-infected cells. Taken together, these results suggest that E4-ORF4 is an important factor controlling the temporal shift in adenovirus alternative RNA splicing. It is concluded that alternative pre-mRNA splicing, like many other biological processes, is regulated by reversible protein phosphorylation (Kanopka, 1998).


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