Protein Interactions

At high concentrations, in the absence of DNA, both male and females proteins form tetramers; at low concentrations, they form dimers of highly asymmetric shape. A given DNA regulatory site binds to both monomers of the DSX dimer and to only two monomers of the DSX tetramer. Binding another DNA molecule to what is presumed to be the second and identical site in the tetramer dramatically shifts the equalibrium from tetramers to dimers. The oligomerization and DNA binding properties are indistinguishable between the male and female proteins. The unusual axial ratios of DSX monomers (18:1), based on the hydrodynamic shape, suggests that the sex-specific carboxyl-terminal domain is well separated from most of the shared sequence, and in particular, from the other known functional domain of the protein, the DNA binding domain (Cho, 1996).

A 66-amino-acid segment common to both proteins (amino acids 39-104) contains a sequence-specific DNA binding domain and an oligomerization domain (OD1). The OD1 domain oligomerizes up to at least a pentamer, but only dimers bound to a palindromic regulatory site of the yolk protein 1 gene. Another segment of each protein (amino acids 350 to 412 for the female protein and 350-427 for the male protein) contains a second oligomerization domain (OD2F and OD2M). The OD2 domains have both sex-specific and non-sex-specific sequences that are necessary for oligomerization. It is thought that the common function of OD1 and OD2 is to oligomerize the full-length proteins, whereas their specialized functions are to form a dimeric DNA binding unit and a sex-specific transcriptional activation or repression unit (An, 1996).

The doublesex (dsx) gene of Drosophila encodes both male-specific (DsxM) and female-specific (DsxF) polypeptides, which are required for normal differentiation of numerous sexually dimorphic somatic traits. The Dsx polypeptides are transcription factors that previously have been shown to bind through a zinc finger-like domain to specific sites within an enhancer; they regulate sex-specific expression of yolk protein genes. The consensus target sequence for this DNA binding domain is a palindromic sequence AGNNACTAAATGTNNTC composed of two half-sites around a central (A/T) base pair. As predicted by the symmetric nature of this site, the Dsx proteins exist as dimers in vivo. Two independent dimerization domains have been mapped by the yeast two-hybrid method: one in the non-sex-specific amino-terminal region of the protein and one that includes the partially sex-specific carboxy-terminal domains of both the male and female polypeptides. A missense mutation has been identified that eliminates dsx function in female flies. The same mutation prevents dimerization of DsxF in the yeast two-hybrid system, indicating a critical role for dimerization in dsx function in vivo (Erdman, 1996).

Genetic studies have indicated that intersex (ix) functions only in females and that it acts near the end of the sex determination hierarchy to control somatic sexual differentiation in Drosophila. ix has been cloned and characterized genetically, molecularly and biochemically. The ix pre-mRNA is not spliced, and ix mRNA is produced in both sexes. The ix gene encodes a 188 amino acid protein, which has a sequence similar to mammalian proteins thought to function as transcriptional activators, and a C. elegans protein that is thought to function as a transcription factor. Bringing together the facts -- (1) the ix phenotype is female-specific and (2) functions at the end of the sex determination hierarchy, yet (3) is expressed sex non-specifically and appears likely to encode a transcription factor with no known DNA-binding domain -- leads to the inference that ix may require the female-specific protein product of the doublesex (dsx) gene in order to function. Consistent with this inference, it has been found that for all sexually dimorphic cuticular structures examined, ix and dsx are dependent on each other to promote female differentiation. This dependent relationship also holds for the only known direct target of dsx, the Yolk protein (Yp) genes. Using multiple techniques (yeast 2-hybrid assay, immunoprecipitation of recombinant tagged Ix and Dsx proteins from Drosophila S2 cell extracts, and gel shifts with the tagged Ix and DsxF proteins) it has been demonstrated that Ix interacts with DsxF, but not DsxM. Taken together, the above findings strongly suggest that Ix and DsxF function in a complex, in which Ix acts as a transcriptional co-factor for the DNA-binding DsxF (Garrett-Engele, 2002).

Analysis of databases using the gapped Blast program identified mammalian ESTs and predicted proteins with significant similarity to the Ix protein. The functions of the genes represented by these ESTs are unknown. From amino acids 15 to the C terminus of Ix, the longest EST, a mouse EST (AA388092), is 37% identical and 52% similar to the Ix protein; this mouse EST does not show similarity to the N-terminal 15 amino acids of Ix. This similarity is highest in a 35 amino acid region of these proteins from amino acid 95 to amino acid 129. The sequence in the 35 amino acid region is 55% identical and 74% similar between the Ix protein and either the mouse EST or a very similar human EST (U46237). Comparison with sequences in various databases using the PSI Blast program with aa position 3 to 47 in the N-terminal region of the Ix protein has revealed sequence similarity to the human synovial sarcoma translocation (SYT) protein, mouse SYT protein and the C. elegans suppressor of ras protein (SUR-2). In the 44 amino acid region of similarity, the Ix protein is 45% identical and 51% similar to the human SYT protein; 50% identical and 52% similar to the mouse SYT protein, and 42% identical and 47% similar to SUR-2 (Garrett-Engele, 2002).

The sur-2 gene was identified as a suppressor of the ras multivulva phenotype. Genetic epistasis analysis placed sur-2 at the same position as transcription factors in the vulval signal transduction pathway, suggesting that the sur-2 protein may function as a transcription factor (Garrett-Engele, 2002).

The SYT protein is proposed to act as a transcriptional activator. In vitro analysis of SYT indicates that the 155 amino acid region of SYT with the highest transcriptional activation function contains the 44 amino acid sequence with similarity to Ix. The sequence similarity of the Ix protein to a region of the SYT protein that is capable of activating transcription raises the possibility that ix may function as a transcriptional activator (Garrett-Engele, 2002).

Because the ix phenotype is female specific and some genes in the somatic sex-determination hierarchy are regulated at the level of splicing, it was conceivable that the ix pre-mRNA would be sex-specifically spliced. However, no introns were identified by comparing the genomic sequence with the ix cDNA sequence, and Northern analysis did not detect sex-specific transcripts. In both males and females, a single hybridizing RNA species of approximately 750 bp was observed, consistent with the expected transcript size as determined by 5' and 3' RACE, which is 734-766 bases (start position 612-626, end position 1332, tail 28-46 bases). The start position determined by 5' RACE is variable in both males and females but does not show a sex-specific difference. The ix transcript is ~8.7 times as abundant in wild-type females as in wild-type males. Preliminary data from ix mutant germline clones in females, and from RNA analysis of females lacking a germline, suggest that the difference in transcript levels between females and males may be due to high ix expression in ovaries. The Northern hybridization, cDNA analysis and 5' RACE results suggest that the ix transcript is not sex specific and is not spliced (Garrett-Engele, 2002).

However, sequence analysis of the genomic region just upstream of the transcription start site of the ix gene identified by 5' RACE revealed a potential exon and intron. The putative exon would encode 33 amino acids and contain a consensus donor splice site. RT-PCR experiments, using a 5' PCR primer that begins upstream of and extends into the putative exon, detected products that were of the size expected from the genomic DNA and smaller, apparently spliced, products were sometimes observed. The RT-PCR result raises the possibilities that the transcription start determined by 5' RACE is not correct or that a transcript initiating from an upstream start site is also expressed but at a much lower level, and was not detected by Northern analysis or in the cDNAs isolated (Garrett-Engele, 2002).

To confirm the ix pre-mRNA is not sex-specifically processed, RNase protection assays of polyA+ RNA isolated from males and females were performed using a probe that could distinguish between the spliced and unspliced products. RNase protection assays depend on neither reverse transcription nor amplification of the RNA as RT-PCR does, and RNase protection assays are more sensitive than Northern analysis and could detect a rare transcript. The major protected fragment is approximately 200 bp, as expected for an unspliced transcript that begins at the site indicated by 5' RACE. Additionally, no qualitative difference between male and female protected fragments was observed. These results agree with the Northern data, cDNA analysis and 5' RACE results, and indicate the ix pre-mRNA is not spliced. Therefore, alternative processing of the ix transcript is not responsible for the female-specific ix phenotype, suggesting that ix functions together with one or more female-specific proteins to achieve the sex-specificity of the ix phenotype (Garrett-Engele, 2002).

Since ix functions at approximately the same position in the sex determination hierarchy as dsx, genetic experiments were carried out to ascertain whether ix cooperates with, or functions independently of, dsx to control female sexual differentiation. An examination was carried out of how ix and dsx function relative to one another in controlling Yp gene expression and the development of an array of sexually dimorphic cuticular structures (Garrett-Engele, 2002).

Focus was first placed on the role of ix in controlling Yp gene expression. Previous studies identified the Fat Body Enhancer (FBE) in the Yp1 and Yp2 intergenic region as necessary and sufficient for the sex-specific expression of both Yp1 and Yp2. Dsx regulates Yp gene expression through three Dsx binding sites in the FBE and Northern analysis suggests that ix+ is required for DsxF mediated activation of Yp1 transcription. To confirm that ix regulates Yp gene expression and to determine whether ix activates Yp expression through the same regulatory region as dsx, the expression of Yp reporter gene constructs was assayed in wild-type and ix mutant females (Garrett-Engele, 2002).

Analysis of the expression levels of the pML-58 Yp reporter construct, which contains the FBE and 196 bp of the Yp1 and Yp2 intergenic region fused to the lacZ gene, indicates that this region is sufficient for ix regulation of the Yp genes in females. Chromosomal females either homozygous or heterozygous for an ix mutation and either carrying or not carrying an ix+ transgene were compared. Including the transgene in the analysis allows definitive assignment of an effect on reporter expression to ix and not to a linked locus. A 1.9-3.5-fold reduction in lacZ activity from pML-58 reporter-construct expression was observed comparing homozygous and heterozygous ix-mutant females. Therefore there is a significant effect of the ix genotype on expression of the Yp reporter construct regardless of the presence or absence of the ix transgene. Additionally, the effect of the ix transgene on expression of the Yp gene reporter construct is to increase lacZ activity. There is no significant interaction between the ix genotype and the presence of the ix transgene, indicating that adding one wild-type copy of the ix gene, either at the ix locus or via the transgene, increases Yp reporter gene expression equivalently. Since the ix-mutant females assayed are heteroallelic (ix2/ix3) and one copy of the ix transgene rescues the decreased Yp reporter construct expression observed in these ix-mutant females, the reduction in Yp gene expression is due to the ix mutation and not another mutation on the second chromosome. These results indicate that the Ix protein activates transcription of the Yp gene reporter construct in females through a region that contains the DsxF DNA-binding sites, raising the possibility that Ix interacts with DsxF to regulate expression of the Yp genes. Results indicate that Ix protein only functions in females and cooperates with DsxF to activate Yp gene expression (Garrett-Engele, 2002).

In addition to regulating Yp expression, DsxF controls the development of sexually dimorphic cuticular structures. Because the ix phenotype is indistinguishable from the dsx female phenotype, Ix may also interact with DsxF to regulate these aspects of female differentiation. However, her, another gene with a phenotype similar to ix and dsx, cooperates with dsx to control female differentiation of foreleg bristles and tergites 5 and 6, but functions independently of dsx to regulate development of vaginal teeth and anal plates in females. If ix acts independently of dsx to regulate some aspect of terminal sexual differentiation, then in ix; dsx mutant females that aspect of sexual differentiation would be masculinized compared with the individual mutants. However, if the genes function together then the phenotype of ix; dsx double mutants would be the same as that of the single mutants. To test these possibilities, the phenotypes of five sexually dimorphic cuticular structures in ix, dsx and ix; dsx mutant flies were assayed (Garrett-Engele, 2002).

In all cases examined in which ix and dsx regulate female differentiation of cuticular structures – formation of vaginal teeth, development of the dorsal anal plate, development of the LTRB and pigmentation of the sixth tergite – the phenotype of the ix; dsx double mutant females was not masculinized compared with the ix and dsx single mutant phenotypes. Although the analysis of vaginal teeth differentiation, pigmentation of the sixth tergite, and formation of LTRB revealed that elimination of wild type dsx activity weakly masculinized ix mutant females, this effect probably represents the elimination of residual ix activity because the ix alleles may not be complete loss of function alleles. Therefore, the results of the phenotypic analysis of sexually dimorphic cuticular structures and the Yp gene reporter constructs indicate that ix and dsx act interdependently to regulate all aspects of female terminal differentiation (Garrett-Engele, 2002).

Thus, phenotypic analysis of ix; dsx mutant females demonstrated that ix and dsx also cooperate to regulate female-specific differentiation of sexually dimorphic cuticular structures. The ix mutation failed to masculinize the dsx mutant females, indicating that dsx is dependent on ix activity in the precursor cells that differentiate into the vaginal teeth, dorsal anal plates, last transverse row of bristles on the basitarsus and sixth tergite pigment-producing cells. Additionally, the phenotypic analysis of ix mutant males confirmed that ix does not function in males. The possibility remains to be tested that ix also functions with her to control female-specific differentiation of some sexually dimorphic structures. The tight interdependence of DsxF and Ix suggests that the relationship between Her and Ix is likely to be the same as that between Her and Dsx in females (Garrett-Engele, 2002).

Understanding of the role of the sex determination hierarchy in sex-specific differentiation has been substantially revised and enhanced by recent studies that have begun to illuminate how information from the sex determination hierarchy is integrated with information from other developmental hierarchies. In particular, it had been thought that dsx played a mainly permissive role in the development of the internal and external genitalia. These structures develop from the genital imaginal disc, which is composed of three primordia deriving from embryonic abdominal segments A8, A9 and A10. The classical view of the genital disc was that the A8-derived primordium differentiated into female genital structures in females and was repressed in males, whereas the A9-derived primordium differentiated into male genital structures in males and was repressed in females; the A10-derived primordium differentiates into anal structures appropriate to the sex of the individual. Thus, whereas the differentiation of the anal primordium requires an instructive cue from the sex hierarchy, the differentiation of the appropriate genital primordium was inferred to require only a permissive function of the sex hierarchy, with segmental identity determining the structures that ultimately developed. This classical view was overturned by the finding that the 'repressed' genital primordium in each sex actually develops into adult structures: the 'repressed' female (A8) primordium produces a miniature eighth tergite in males and the 'repressed' male (A9) primordium produces the parovaria in females (Keisman, 2001a). Consistent with its instructive role, the sex hierarchy actively modulates the regulation by other developmental pathways of sex-specifically deployed genes. The dachshund (dac) gene is differentially expressed in the male and female genital discs, and the sex hierarchy mediates this sex-specific deployment by determining cell-autonomously whether dac is activated by wingless signaling (in females) or by decapentaplegic signaling (in males) (Keisman, 2001a). Fibroblast growth factor (FGF) signaling in the genital disc is also regulated cell-autonomously by the sex hierarchy (Ahmad, 2002). DsxF represses the FGF-encoding branchless (bnl) gene, thus restricting bnl-expressing cells to the male genital disc. FGF signaling from these cells recruits into the disc mesodermal cells expressing the FGF receptor encoded by the breathless (btl) gene. Once inside the male genital disc, these btl-expressing cells become epithelial and eventually give rise to the paragonia and vas deferens, components of the internal male genitalia. An instructive role for the sex hierarchy is also evident in an adult tissue not derived from the genital imaginal disc. The bric à brac (bab) locus (Kopp, 2000) integrates signals from the homeotic genes, as well as the sex hierarchy, to repress pigmentation of tergites 5 and 6 in females (Garrett-Engele, 2002).

Although the Yp genes, which are activated by DsxF and repressed by DsxM, are the only known direct target of dsx, it is likely that DsxF acts in some cases to repress transcription and that DsxM acts in some cases to activate transcription. Indeed, if the examples above represent cases of direct regulation, then it is clear that the effect of DsxF or DsxM is dependent upon both the cellular context and the promoter organization of the target gene. Such context-dependent duality of function finds precedent in several well characterized transcription factors. The mechanisms that determine whether a bi-functional transcription factor is in an activating or repressing state are diverse, and include binding of ligand co-factors, differential organization of binding sites in promoters, interaction with other DNA-binding factors, and concentration-dependent structural changes. The Dsx proteins provide an especially interesting case of dual regulatory activity because not only are DsxF and DsxM each capable of activating some target genes and repressing others, but the two isoforms often have opposite effects, with DsxF repressing those genes that DsxM activates and vice versa. It may be that Ix, functioning as a co-factor for DsxF, plays a key role in effecting this symmetry of dual regulatory activities (Garrett-Engele, 2002).

Splicing to generate male and female transcripts.

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).

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).

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).

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).

Six copies of the 13-nucleotide sequences TC(T/A)(T/A)C(A/G)ATCAACA have been identified in the female-specific fourth exon that act as cis elements for the female-specific splicing of DSX pre-mRNA. UV-crosslinking experiments revealed that both female-specific TRA and TRA-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 the binding of these proteins to the 13-nucleotide sequences (Inoue, 1992).

TRA and TRA-2 act by recruiting general splicing factors to a regulatory element located downstream of a female-specific 3' splice site. Remarkably, either TRA or TRA-2, as well as any of the members of the serine/arginine-rich (SR) family of general splicing factors are sufficient to commit DSX pre-mRNA to female-specific splicing; 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, TRA-2, SR proteins, and additional unidentified components (Tian, 1993).

The Drosophila proteins Transformer and Transformer-2 regulate the sex-specific alternative splicing of Drosophila DSX pre-mRNA by specifically binding to a splicing enhancer (dsx repeat element; dsxRE) located 300 nucleotides downstream from a female-specific 3' splice site. The dsxRE can function as a TRA and TRA-2-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 TRA-2, 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 TRA-2 allow the dsxRE to function at a distance from the intron by stabilizing the interactions between these proteins and the dsxRE (Tian, 1994).

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 were found within two regions of the DSX pre-mRNA that are important for the regulation of DSX alternative splicing: 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).

SR proteins are essential for pre-mRNA splicing in vitro, act early in the splicing pathway, and can influence alternative splice site choice. B52, a gene for a Drosophila SR protein alters the pattern of sex-specific splicing of Doublesex mRNA under sensitized conditions, so that the male-specific splice is favored (Peng, 1995).

The Doublesex splice enhancer functions by assembling specific SR protein complexes. Transformer (Tra and Transformer 2 (Tra2) 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).

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).

Exonic splicing enhancer (ESE) sequences are important for the recognition of splice sites in pre-mRNA. These sequences are bound by specific serine-arginine (SR) repeat proteins that promote the assembly of splicing complexes at adjacent splice sites. A splicing 'coactivator', SRm160/300, has been identified that contains SRm160 (the SR nuclear matrix protein of 160 kDa) and a 300-kDa nuclear matrix antigen. SRm160/300 is required for a purine-rich ESE to promote the splicing of a pre-mRNA derived from the Drosophila doublesex gene. The association of SRm160/300 and U2 small nuclear ribonucleoprotein particle (snRNP) with this pre-mRNA requires both U1 snRNP and factors bound to the ESE. Independent of pre-mRNA, SRm160/300 specifically interacts with U2 snRNP and with a human homolog of the Drosophila alternative splicing regulator Transformer 2, which binds to purine-rich ESEs. The results suggest a model for ESE function in which the SRm160/300 splicing coactivator promotes critical interactions between ESE-bound 'activators' and the snRNP machinery of the spliceosome (Eldridge, 1999).

Exonic splicing enhancer (ESE) sequences are important for the recognition of adjacent splice sites in pre-mRNA and for the regulation of splice site selection. It has been proposed that ESEs function by associating with one or more serine/arginine-repeat (SR) proteins which stabilize the binding of the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor (U2AF: U2 small nuclear riboprotein auxiliary factor 50 see ) to the polypyrimidine tract upstream of the 3' splice site. This model by analyzing the composition of splicing complexes assembled on an ESE-dependent pre-mRNA derived from the doublesex gene of Drosophila. Several SR proteins and hTra2beta, a human homolog of the Drosophila alternative splicing regulator Transformer-2, associate with this pre-mRNA in the presence, but not in the absence, of a purine-rich ESE. By contrast, the 65-kDa subunit of U2AF (U2AF-65 kDa) binds equally to the pre-mRNA in the presence and absence of the ESE. Time course experiments revealed differences in the levels and kinetics of association of individual SR proteins with the ESE-containing pre-mRNA, whereas U2AF-65 kDa binds prior to most SR proteins and hTra2beta and its level of binding does not change significantly during the course of the splicing reaction. Binding of U2AF-65 kDa to the ESE-dependent pre-mRNA is, however, dependent on U1 snRNP. The results indicate that an ESE promotes spliceosome formation through interactions that are distinct from those required for the binding of U2AF-65 kDa to the polypyrimidine tract (Li, 1999).

All animals exhibit innate behaviors that are specified during their development. Drosophila melanogaster males (but not females) perform an elaborate and innate courtship ritual directed toward females (but not males). Male courtship requires products of the fruitless (fru) gene, which is spliced differently in males and females. Alleles of fru have been generated that are constitutively spliced in either the male or the female mode. Male splicing is essential for male courtship behavior and sexual orientation. More importantly, male splicing is also sufficient to generate male behavior in otherwise normal females. These females direct their courtship toward other females (or males engineered to produce female pheromones). The splicing of a single neuronal gene thus specifies essentially all aspects of a complex innate behavior (Demir, 2005).

Male courtship behavior performed by fruM and fruΔtra females is a remarkable mimic of courtship by wild-type or control fruC males. Some courtship steps, such as initiation, orientation, following, and wing extension, are indistinguishable in fruM (and fruΔtra) females and fruC males. Other steps are clearly abnormal. fruM females do not, for obvious reasons, copulate. But licking, which should be anatomically possible, is also significantly reduced. Qualitatively, this pattern of courtship resembles that of dsx males. This is perhaps not surprising, since fruM females resemble dsx males in that they lack male-specific Dsx isoforms (DsxM) and hence are anatomically female, yet they express the male-specific Fru isoforms (FruM) (Demir, 2005).

The distinct roles of fru and dsx in sexual development are clearly illustrated by the differences between animals that produce either only FruM or only DsxM. Animals that express DsxM but not FruM (either fruF males or dsxM females) resemble normal males but do not court. Conversely, animals that express FruM but not DsxM (either fruM females or dsx males) do court, even though they resemble normal females. Thus, FruM is both necessary and sufficient for male courtship, whereas DsxM is neither necessary nor sufficient. The role of DsxM in courtship may simply be to provide the gross male anatomy needed for its optimal execution. This anatomical contribution of DsxM includes the formation of male reproductive organs and external genitalia, the generation of the neurons that innervate these organs, and the formation of male-specific taste sensilla on the forelegs that may house pheromone-detecting neurons (Demir, 2005).

Splicing of doublesex: Identification of sex-specific transcripts of the Anopheles gambiae doublesex gene

The understanding of the molecular mechanisms of sex differentiation in the mosquito Anopheles gambiae could identify important candidate genes for inducing selective male sterility in transgenic lines or for sex-controlled expression of lethal genes. In many insects, doublesex is the double-switch gene at the bottom of the somatic sex-determination cascade that determines the differentiation of sexually dimorphic traits. The dsx homolog has been identified in A. gambiae and its sex-specific transcripts have been identified. Agdsx consists of seven exons, distributed over an 85 kb region on chromosome 2R, which are sex-specifically spliced to produce the female and male AgdsxF and AgdsxM transcripts. AgdsxF contains a 795 bp ORF, coding for a protein of 265 amino acids, while AgdsxM comprises a much longer (1866 bp) ORF, coding for a 622 aa protein. Differences in the exon/intron organization suggest that Agdsx sex-specific splicing results from a different mechanism from Drosophila melanogaster dsx. These findings represent an important step towards the understanding of sex differentiation in Anopheles and will facilitate the use of gene transfer technologies to manipulate sex ratios for vector control programs based on the sterile insect technique (Scali, 2005).

Apart from the highly divergent sequence of the male-specific region, the major difference between Agdsx and Dmdsx resides in the splicing mechanism of the female-specific exon. In D. melanogaster, the inclusion or excision of the female-specific exon depends on the presence of a weak 3' acceptor site in the preceding intron. Activation of this splice site in females is brought about by TRA and TRA2, which form a multiprotein complex with RNA-binding protein 1 and bind to cis-regulatory elements (dsxREs and PRE) present in the 3' UTR of the female-specific exon immediately downstream of the 3' splice acceptor site. In males, the absence of TRA leads to a default male-specific splicing in which the weak 3' splice site is not recognized and the next downstream acceptor site is chosen by the splicing machinery, resulting in the splicing of the female-specific exon. In contrast, in A. gambiae, the retention of exon 5 in females seems to depend on the activation of the 5' donor site of the downstream intron 5. This scenario would resemble the splicing of fruitless in D. melanogaster, where the TRA/TRA2 enhancer complex activates a female-specific 5' splice site. Similarly to Agdsx, fru contains repeat elements (fruREs) nearly identical to the DmdsxREs but located immediately upstream of this alternative 5' splice donor site, approximately 1.3 kb downstream of the 3' acceptor site of the preceding intron. The remarkable similarity between Agdsx and Dmfru suggests that splicing of Agdsx follows the same mechanism, with the female mode of splicing occurring though the activation of the 5' splice site of intron 5 following the binding of TRA and TRA2 to the AgdsxREs. In contrast, the female transcript may represent the default splicing mode, with the 5' donor site of intron 5 repressed in males. The region preceding intron 5 shows the presence of putative silencer-binding sites such as guanosine-rich motifs (GGGG and UAGG), which are involved in the regulation of a cassette exon in the glutamate NMDA R1 receptor (GRIN1). Alternatively, a decoy 3' acceptor site could engage the 5' donor site of intron 5 in a non-productive interaction, conferring in turn a competitive advantage to the skipping of exon 5, as found in the caspase-2 pre-mRNA (Scali, 2005).

Conservation of the exon/intron structure and of the functional regions (DBD/OD1, OD2) found in all known dsx homologues suggests a role for Agdsx in dimorphic differentiation in A. gambiae. The identification of female- and male-specific transcripts of Agdsx represents an important step towards the understanding of the sex differentiation process in A. gambiae and will facilitate the development of genetic tools to induce male sterility or manipulate sex ratios in mosquitoes, for instance by constitutively expressing the female-specific form of dsx in the male gonads or by inducing the sex-specific splicing of a dominant lethal (Scali, 2005).

doublesex: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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