The transformer gene regulates female somatic sexual differentiation and has no known function in males. It gives rise to two sizes of RNA, one non-sex-specific and one female-specific. These two RNAs are shown to be present throughout the life cycle, and related by the use of alternative first intron splice acceptor sites. The non-sex-specific RNA has a 73 base first intron, while that in the female-specific RNA is 248 bases. The non-sex-specific RNA has no long open reading frame, while the female-specific RNA has a single long open reading frame beginning at the first AUG. Substitution of a heat shock promoter for the tra promoter still leads to female-specific differentiation of otherwise tra-females. A mechanism is suggested by which Sex-lethal controls itself and tra (Boggs, 1987).
Sex-specific alternative splicing of RNA from the Drosophila transformer gene involves competition between two 3' splice sites. In the absence of Sex-lethal activity (as in males), only one site functions; in the presence of Sex-lethal activity (as in females), both sites function. Information for sex-specific splice site choice is contained within the intron itself. Deletions of the splice site used in males lead to Sex-lethal-independent use of the otherwise female-specific site. The relative amounts of unspliced and spliced RNA derived from these mutant genes do not change with changes in Sex-lethal activity. Specific nucleotide changes in the non-sex-specific splice site do not affect splicing activity but eliminate Sex-lethal-induced regulation. A deletion removing material between the two splice sites does not eliminate sex-specific regulation, while a deletion of the female splice site leads to a female-specific increase in unspliced RNA. These results are consistent with a model in which female-specific factors block the function of the non-sex-specific 3' splice site (Sosnowski, 1989).
Somatic sexual differentiation in Drosophila melanogaster is accomplished by a hierarchy of genes of which one, Sex-lethal, is required for the functional female-specific splicing of the transcripts of the immediately downstream regulatory gene, transformer (tra). The first exon of the tra primary transcript is spliced to one of two acceptor sites. Splicing to the upstream site yields a messenger RNA which is neither sex-specific nor functional, but that produced after splicing to the downstream acceptor site yields a functional female-specific mRNA. This study addresses the question of how the Sxl gene product determines the alternative splicing of tra primary transcripts. One suggestion is that non-sex-specific splicing to the upstream acceptor is blocked in female flies by sex-specific factors, but neither the identity of the female-specific factors nor the mechanism of the blockage has been specified. Co-transfection experiments were performed in which Sxl complementary DNA and the tra gene are expressed in Drosophila Kc cells. Moreover, it was found that female Sxl-encoded protein binds specifically to the tra transcript at or near the non-sex-specific acceptor site, implying that the female Sxl gene product is the trans-acting factor that regulates the alternative splicing (Inoue, 1990).
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).
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).
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).
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).
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).
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).
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 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).
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).
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).
Sex specific splicing of Dsx mRNA is determined by the splice site acceptor sequences. The three splice donor sites following the common exons and the site following the first male-specific exon agree with a consensus donor sequence. The consensus acceptor sequence is n(T/C),NCAG, where the length of the pyrimidine-rich stretch is somewhat ill-defined. The two common acceptors of the Dsx introns plus one female-specific, and two male-specific acceptors all match the last four nucleotides of this consensus. However, of the next 12 nucleotides upstream, the common and male-specific acceptors have pyrimidines at from 9 to 11 positions, but the female-specific acceptor has pyrimidines at only six positions. Thus, the female specific acceptor is a very poor acceptor by this criterion. Since use of the female-specific acceptor depends on the activity of the tra and tra-2 loci, the products of these two genes may act to redirect the normal preference of the splicing machinery of the cell from the stronger 'consensus' male acceptor sequence to the weaker female acceptor (Burtis, 1989).
Sex-specific alternative processing of doublesex (dsx) precursor messenger RNA (pre-mRNA) regulates somatic sexual differentiation in Drosophila melanogaster. Cotransfection analyses in which the dsx gene and the female-specific transformer and transformer-2 complementary DNAs were expressed in Drosophila Kc cells revealed 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 showed 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 doublesex precursor messenger RNA (pre-mRNA) is one of the key steps that regulates somatic sexual differentiation in Drosophila melanogaster. By transfection analyses using dsx minigene constructs, six copies of the 13-nucleotide sequences TC(T/A)(T/A)C(A/G)ATCAACA were identified in the female-specific fourth exon that act as the cis elements for the female-specific splicing of dsx pre-mRNA. UV-crosslinking experiments revealed that both female-specific transformer and transformer-2 products 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 binding of these proteins to the 13-nucleotide sequences (Inoue, 1992).
Exonic sequences are involved in proper splicing of the female-specific acceptor site. The activation of a female-specific 3' splice site by Transformer and Transformer-2 proteins involves their binding to an essential exon sequence. Nuclear proteins in addition to TRA and TRA-2 have been found to bind specifically to this exon sequence. Therefore, TRA and TRA-2 may act by promoting the assembly of a multiprotein complex on the exon sequence. This complex may facilitate recognition of the adjacent 3' splice site by the splicing machinery (Tian, 1992).
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).
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 alternatively spliced transcripts from the distal promoter are generated by use of 5' splice sites 1590 nucleotides apart. Splice site selection is sex-specific. A downstream splice site for the transcript of the distal promoter is present in fruitless mRNA in female, but not male heads, indicating that this specific splice site is used only in females. Analysis of the upstream 5' splice site reveals that the transcript class missing the repeats region is only detected in males. Thus, the upstream 5' splice site is used only in males and the downstream 5' splice site is used only in females. In chromosomally XX females mutant for tra or tra-2, the male form of FRU transcripts is produced, while the FRU splice pattern is unaffected in XY flies mutant for either tra or tra-2. These results demonstrate that sex-specific splicing of FRU transcripts is indeed controlled by tra and tra-2. A genetic test also reveals that FRU mRNA splicing is controlled by tra and tra-2. If FRU splicing is controlled by tra and tra-2, then XX flies double mutant for fru and tra (or tra-2) should exhibit the mutant fruitless Muscle of Lawrence and behavioral phenotypes. Double mutants have an abnormal MOL muscle like that of fruitless mutant males, and demonstrate abnormal behavior as well (Ryner, 1996).
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 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).
A gal4-containing enhancer-trap called C309, which is expressed up broadly in central-brain and VNC regions, has been shown to cause subnormal courtship of Drosophila males toward females and courtship among males when driving a conditional disrupter of synaptic transmission (shiTS). These manipulations have been extended to analyze all features of male-specific behavior, including courtship song, which was almost eliminated by driving shiTS at high temperature. In the context of singing defects and homosexual courtship affected by mutations in the fru gene, a tra-regulated component of the sex-determination hierarchy, C309/traF combination was also found to induce high levels of courtship between pairs of males and 'chaining' behavior in groups; however, these doubly transgenic males sang normally. Because production of male-specific FRUM protein is regulated by Tra, it was hypothesized that a fru-derived transgene encoding the male (M) form of an Inhibitory RNA (fruMIR) would mimic the effects of traF; but C309/fruMIR males exhibited no courtship chaining, although they courted other males in single-pair tests. Double-labeling of neurons in which GFP was driven by C309 revealed that 10 of the 20 CNS clusters containing FRUM in wild-type males included coexpressing neurons. Histological analysis of the developing CNS could not rationalize the absence of traF or fruMIR effects on courtship song, because C309 was found to be coexpressed with FRUM within the same 10 neuronal clusters in pupae. Thus, it is hypothesized that elimination of singing behavior by the C309/shiTS combination involves neurons acting downstream of FRUM cells (Villella, 2005).
Various portions of the CNS in Drosophila are inferred to control separate elements of normal male courtship, in part by analysis of abnormal behavior. Some such studies have involved brain-behavioral analyses of the fruitless (fru) gene and its mutants. Different fru mutants exhibit courtship subnormalities to varying degrees and at separate stages of the courtship sequence, depending on the mutant allele. Most fru mutants court other males substantially above levels normally exhibited by pairs or groups of wild-type males. The original fruitless mutation leads to spatially nonrandom decreases of fru-product presence within particular subsets of the normal CNS expression pattern, which may be causally connected with the breakdown of recognition that is a salient effect of fru1 on male behavior. fru-like courtship can be induced by the effects of a transgene that encodes GAL4 (a transcription factor derived from yeast). When this broadly expressed C309 enhancer trap was combined with a GAL4-drivable factor containing a dominant-negative, conditionally expressed variant of the shibire gene (shiTS), heat treatment of doubly transgenic males caused them to court females subnormally and to court other males vigorously. Although this strain had been termed a mushroom body enhancer trap in terms of the gal4 sequence it contains, being expressed 'predominantly' within that dorsal-brain structure, C309 drives marker expression in a widespread manner. Therefore, attempts were made to correlate various CNS regions in which this transgene is expressed with its effects on male behavior, emphasizing a search for 'C309 neurons' that might overlap with elements of the FRUM pattern (Villella, 2005).
The possibility was also entertained that the C309/shiTS combination causes a mere caricature of fruitless-like behavior. Therefore, what would be the courtship effects of C309 driving a transgene that produces the female form of the transformer gene product? This Tra protein participates in posttranscriptional control of fru's primary 'sex transcript,' so that FRUM protein is not produced in females. If C309 and traF are naturally coexpressed in a subset of the to-be-analyzed neurons, feminization of the overlapping cells should eliminate this protein. These transgenic experiments were extended to target fruitless expression specifically by gal4 driving of an inhibitory RNA (IR) construct, which was generated with fru DNA. Their experiments furnish one object lesson as to how 'enhancer–trap mosaics' can delve into the neural substrates of a complex behavioral process, an approach commonly taken to manipulate brain structures and functions in courtship experiments. Because few genetic loci putatively identified by such transposons have been specified, the tactics applied are in the context of CNS regions in which expression of a 'real gene' is hypothesized to underlie well defined behaviors (Villella, 2005).
Mutations at the fruitless locus and the C309/UAS-shiTS transgene combination each cause similar courtship subnormalities and anomalies. In this context, the C309 enhancer trap is expressed in many CNS neurons that contain male-specific FRUM protein. One element of the courtship effects of C309/UAS-shiTS involves subnormal interactions between males and females. From correlating C309/FRUM coexpression with the fact that fruitless mutations lead to lower-than-normal male–female courtship, it is speculated that FRUM brain regions 2, 8, 13, and 14 are connected with the deleterious effects of shiTS. As to why two additional neuronal groups that coexpress FRUM and C309 are not noted here (groups 5 and 7), see below (Villella, 2005).
With respect to males courting females, special attention might be paid to clusters 13 and 14. These posteriorly located groups of FRUM neurons are likely to include brain regions within which genetic maleness is required if sex mosaics are to exhibit orientation toward and following of females. A problem with this interpretation is that feminizing substantial proportions of the C309/FRUM coexpressing cells led to no decrements in male-female interactions, despite the 7- to 10-fold coexpression reductions caused by XY/C309/UAS-traF within clusters 13 and 14. Perhaps the relevant 'overlap percentages' would have had to drop from 47 and 31 to 0 for both of these clusters if a traF-affected courtship decrement were to be realized. An alternative to viewing this matter in the context of C309/FRUM coexpression is that certain neurons in which this gal4 driver is active could be anatomically downstream of the fru-expressing brain cells that influence a male's ability to initiate and sustain courtship of a female (Villella, 2005).
This conception is relevant to the striking elimination of courtship song in recordings of C309/UAS-shiTS males. Once again, UAS-traF has no such effect. Neurogenetic findings pertinent to this matter are that C309 is expressed in imaginal thoracic ganglia; fru is 'song-involved'; this gene makes its products within several regions of the ventral nerve cord, coexpressing C309 within most of them; and genetic maleness within Drosophila's VNC has been implicated in song control. Thus, turning off synaptic transmission emanating from one or more subsets of the C309/UAS-shiTS neurons in the thoracic ganglia could be the etiology of heat-induced songlessness exhibited by these doubly transgenic males. Regarding the absence of a traF effect, what if C309 was not expressed in any FRUM-containing song-relevant neurons during metamorphosis? In other words, C309 expression in VNC neurons underlying song control could be activated late in the life cycle, allowing for the shiTS effect to take hold after adult males are heated; however, the progenitors of such cells might not express C309 during an earlier 'feminization-relevant' stage, so that post-metamorphic activation of traF would occur too late to affect singing ability. However, substantial coexpression of FRUM and C309 was found within the pupal VNC. In this respect, it is submitted that assessing the C309's expression pattern throughout the life cycle is a valuable object lesson as to what must be done properly to interpret the biological effects of a given enhancer trap (Villella, 2005).
As to the divergent effects C309-driven shiTS vs. traF, recall that the former factor seems broadly to impinge on VNC functioning, in that the fly's general ability to vibrate its wings is shut down by the synaptic disruptor; in contrast, songless fruitless mutants fly normally. Thus, consider a scenario in which C309 neurons would include those that mediate wing vibrations during flight, and that this transgene is expressed in separate VNC cells hypothetically dedicated to such vibrations during courtship. Therefore, it is speculated that the expression domain of C309 includes inter- or motor-neurons functioning within and downstream of a 'command center' for flight as well as neurons located in relatively distal regions of a separate anatomical pathway. The latter would originate where fru-expressing cells exert the gene's crucial regulation of courtship song (Villella, 2005).
Turning to anomalous courtship interactions among males, focus shifts back to the brain: FRUM clusters 5 and 7, where fru1 causes an apparent absence of this protein. This mutation minimally affects the gene's expression in other brain regions. It is notable that the C309/UAS-traF combination knocked down driver/FRUM coexpression to ~10% of normal in cluster 5. Cluster 7 was similarly affected, but special attention should be paid to cluster 5. One reason that this group was thought to be the etiology of frantic courtship among fru1 males is that cluster 5 is located near the antennal lobes; and transgenically mediated feminization of a brain region near these structures induces intermale courtships, although none of the gal4 drivers in that study included C309. Therefore, if proper male-specific structure or function of cluster 5 is involved in normal sex recognition, the mutation's demasculinizing effect on this brain region, or transgene-effected feminization of it, could cause this aspect of courtship to break down (Villella, 2005).
Elements of the current findings suggest that abnormal formation of the brain region in question is not necessary for it to mediate anomalous interactions between males; this is because deactivating synaptic transmission in cluster 5, after CNS development has been completed in a male manner, is sufficient to induce intermale courtship. Perhaps this behavioral effect of driving UAS-shiTS involves removal of inhibitory neurotransmission relevant to the functioning of this brain region, which in normal males would block their wherewithal to sustain courtship between males. Therefore, the fru1 effect on cluster 5 and that of driving Tra production in this region might not involve the formation of a sex recognition center (such that a hypothetical circuit involved in inhibiting intermale courtship is not present or miswired), but instead the intracellular quality and function of neurons in the mature brain (Villella, 2005).
Considering further that certain cluster 5 neurons comprise the subset of FRUM's spatial domain for shiTS- or traF-induced intermale courtship, the relevant cells would be those in which both fruitless and C309 generate their gene products (20% of the 35 neurons within this group). One problem with this supposition is that C309/UAS-traF flies elicit fairly high levels of courtship. Thus, groups of C309/UAS-traF males may form chains for reasons extending beyond a given fly's inappropriate 'motivation' to court another male: The extent to which a C309/UAS-traF fly is feminized could include self-stimulation that might contribute to intermale courting. However, recall the case of C309/UAS-traF/Cha-gal80 males, a transgenic type that is similarly feminine externally and elicits courtship. The diminished extent to which C309's gal4 is effective when combined with Cha-gal80 led to weakened homosexual courtship in single-pair tests and dramatically reduced chaining behavior, although there was essentially no effect of Cha-gal80 on the basic courtship ability of these triply transgenic males. Thus, the effects of this 'neurons-only' manipulation suggest that hypothetical self-stimulation, which did not cause C309/UAS-traF/Cha-gal80 males vigorously to court other ones, is minimally operating to induce the homosexual courtships performed by XY/C309/UAS-traF flies. Males carrying C309 and UAS-fruMIR are also not feminized externally; however, they courted other males robustly in single-pair tests, an effect that was diminished by adding Cha-gal80. Therefore, it is surmised that flies carrying a given fruitless-affecting transgene exhibit intermale courtship because the relevant CNS neurons are demasculinized (Villella, 2005).
However, what about neural structures not analyzed in the current study that could be involved in the behavioral effects of C309 driving either traFor fruMIR? Thus, consider that Tra affects the primary transcript emanating from the doublesex (dsx) gene and that dsx null mutations cause XY flies to exhibit modest levels of intermale courtships. C309 driving of traF could lead to the female (F) form of DSX (thus, no DSXM, as in dsx–) within brain cells connected to sex recognition other than those analyzed. Indeed, dsx+ is expressed in the brain; however, the functional significance of these cells is unknown, let alone whether any of them also express fru+. In this regard, it was important to home in on disruption of fruitless's CNS expression alone by combining C309 with the UAS-fruMIR transgenes; this was sufficient to induce courtship between a given pair of doubly transgenic males but led to no chaining. Thus, anomalously high levels of courtship between two males has been disconnected from courtship chaining. [The same disconnect between these different kinds of intermale courtship occurred when Cha-gal80 was added to the C309/UAS-traF combination. It is as if the broad neural effects of a genetic abnormality such as a fruitless mutation, or combining C309 with UAS-traF, is necessary to cause sustained courtship among several variant males; however, if the impingement on fru+ expression is more limited, only courtship between a pair of males can occur (Villella, 2005).
In this regard, the C309/UAS-fruMIR flies were substantially less affected in terms of numbers of brain neurons within which FRUM became undetectable, compared with the effect of the same driver combined with UAS-traF. This brings us to the matter of additional neurons that are potentially relevant to courtship and should be analyzed in context of the C309 effects. Here, the many PNS cells recently discovered to express fruitless in external sensory structures are referred to. It is unknown whether any of these neurons coexpress C309, such that sensory inputs relevant to courtship may have been impinged upon by combining that transgene with UAS-shiTS or with the sex-affecting transgenes. However, fru+ expression in external appendages is not required for a fly to recognize, follow, and perform wing extension at a female: when these structures are genetically female in certain gynandromorphs, maleness within the brain is sufficient to trigger mosaic-with-female courtship (Villella, 2005).
The current study aimed to delve into various regions of the male CNS in which the fruitless gene is expressed: Do certain subsets of the spatial pattern govern a male's ability to perform a discrete feature of the reproductive sequence? Using the gal4-containing C309 enhancer trap was valuable, because it leads to impersonations of certain fru-mutant behaviors when this driver is combined with a shiTS-containing factor that broadly disrupts neural functioning. By limiting C309's efficacy to disrupt by causing it to drive sex-related transgenes succeeded in provisionally partitioning fru-related 'sex recognition' neurons to a subset of the normal brain pattern. By subtraction, the partitioning was further delimited by knocking out the driver's efficacy in a subset C309's spatial domain: adding a neurally driven gal80 transgene substantially attenuated anomalous intermale courtships. A pleasant surprise occurred when the C309/UAS-fruMIR combination was found not to mimic the effects on courtship among males of combining the driver with UAS-traF. Thus, the broader pattern of FRUM expression, unaffected by the IR compared with the substantial decrement caused by traF, takes the analysis a further step. For example, the manner by which fru mutations and related factors influence courtship between two males, as opposed to the much more complicated behavioral dynamics that can occur in a group of such Drosophila, are now being teased out (Villella, 2005).
However, inferences about the potentially relevant subsets of a given brain cluster do not approach specifically identifiable neurons. For this, it will be necessary to do more than quantify the cells in which a transgene driver and fruitless are coexpressed. Further brain-behavioral dissections will require assessing the differential connectivity patterns defining a given class of FRUM neurons, along with variations of cellular content that are likely to discriminate one category of such neurons from another. The relevant object lessons stem from analyses of, so far, only the posterior-most component of fruitless's expression domain in the male CNS: partitioning certain abdominal-ganglion neurons that differentially connect with either a male-specific muscle or with internal reproductive organs, and discovering that the latter type of FRUM cells uniquely contain serotonin. Neurons containing another neurotransmitter, acetylcholine, are on point; but not all of the C309 effects can be ascribed to neurons affected by Cha-gal80, because certain courtship defects were found to remain when analyzing males that carried this transgene along with C309 and UAS-shiTS. This finding reinforces that notion that additional neuronal qualities must be uncovered with regard to cells expressing this enhancer-trap, the fruitless gene, or both (Villella, 2005).
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