Molecular organisation of tra-2 in S. ocellaris and B. coprophila
The first step in the isolation of the S. ocellaris tra-2 gene (Sotra2) was to perform RT-PCR on total RNA from adults. Reverse transcription was performed using the primer oligo-dT, while two-nested PCR reactions were performed with three degenerated primers: Mar17, Mar26 and Tra2.B (the location and the sequences of the primers used in this work are shown in Additional file 1). The first PCR reaction was performed using the primer pair Mar26 plus Mar17, the second using Mar26 plus Tra2.B. An amplicon of 133 bp was amplified, cloned and sequenced. The conceptual amino acid sequence of this amplicon showed a high degree of similarity with the 3' region of the RRM domain of the D. melanogaster Tra2 protein, indicating that a fragment of the putative SoTra2 protein had been isolated.
Nested PCR reactions were then performed in 3' and 5' RACE analyses. The amplicons were then cloned and sequenced. A GenomeWalker library for S. ocellaris was then synthesised and used to perform PCR genome-walking on the genomic DNA of S. ocellaris from the initial amplicon towards the 5' and 3' directions. The genomic amplicons were cloned and sequenced. The sequences of the genomic fragments thus generated were compared with the cDNA sequences previously determined. In this way, the exon/intron junctions were unambiguously identified. Figure 1A shows the molecular organisation of Sotra2. Its transcription unit was made up of 4601 bp and was composed of six exons and five introns (Figure 1A). The transcription start site was located 94 bp upstream of the translation initiation codon.
Overlapping PCR on total RNA of S. ocellaris males and females showed that the gene Sotra2 mainly produced the transcript Sotra2-251, formed by exons E1-E2-E3-E4b-E5-E6 (1177 bp). This encoded a full-length putative Tra2 protein of 251 amino acids that included the RS1, RS2 and RRM domains characteristic of the Tra2 proteins. Two other less abundant transcripts were also detected: Sotra2-204 and Sotra2-130 (Figure 1A) (see Methods). The Sotra2-204 transcript differed from Sotra2-251 in its lack of exon 3, and it encoded a putative Tra2 protein of 204 amino acids that differed from the full Tra2 protein in its shorter RS1 domain. The Sotra2-130 transcript differed from Sotra2-251 by the inclusion of exon E4a. The latter carries translation stop codons causing the production of a truncated Tra2 protein 130 amino acids long and lacking both the RRM and RS2 domains.
To isolate the gene tra-2 of B. coprophila (Bctra2), the same strategy used for the isolation of this gene in S. ocellaris was followed, except that the initial step involved PCR amplification of the genomic DNA of adults using primers expSoT2.1 and expSoT2.2, corresponding to exons 5 and 6 of Sotra2. A genomic fragment of 1069 bp was amplified, cloned and sequenced. Its sequence showed 61% similarity to the corresponding region of Sotra2, suggesting that a genomic fragment of Bctra2 had been cloned. As for Sotra2, 5'RACE, 3'RACE and genome-walking methodologies were used to determine the molecular organisation of Bctra2 (Figure 1B). Its transcription unit was made up of 4255 bp and was composed of six exons and five introns.
Overlapping PCR performed on total RNA of males and females of B. coprophila showed that the gene Bctra2 mainly produced the transcript Bctra2-246, formed by exons E1-E2-E3-E4b-E5-E6 (1444 bp). This encoded a full putative Tra2 protein of 246 amino acids and included the RS1, RS2 and RRM domains characteristic of Tra2 proteins. Three other less abundant transcripts were also detected: Bctra2-203, Bctra2-99 and Bctra2-75 (Figure 1B) (see Methods). The Bctra2-203 transcript differed from Bctra2-246 in its lack of exon 3; it encoded a putative Tra2 protein of 203 amino acids. The Bctra2-99 transcript varied from Bctra2-246 by the inclusion of exon E4a, which carries translation stop codons. Thus, it produced a truncated Tra2 protein of 99 amino acids that lacked both the RRM and RS2 domains. Finally, the Bctra2-75 transcript, made up by exons E1, E2 and E6, encoded a truncated Tra2 protein with short RS1, RRM and RS2 domains.
Expression pattern of tra-2 in S. ocellaris and B. coprophila
The expression of tra2 in S. ocellaris was studied by performing RT-PCR on total RNA from a mixture of male plus female embryos, from a mixture of male plus female larvae at different developmental stages, from the heads plus thoraces of male and female adults (separately), from the abdomens of male and female adults (separately), and from adult ovaries and testis (separately). The expression of tra2 in B. coprophila was similarly analysed, although in this case it was possible to distinguish male from female embryos as well as male from female larvae (see Methods). The primers used were expSoT2.1 from exon 5 and expSoT2.2 from exon 6 (Figure 2A), which have the same sequence in both Sciara species. The expression of the constitutive gene rpL10, which encodes the ribosomal protein L10, was used as a control in RT-PCR. In all cases, a fragment of 273 bp was amplified (Figure 2B,C). This was cloned and sequenced, confirming that it corresponded to the expected Sotra2 or Bctra2 fragment. Negative controls in all these PCR reactions produced no amplicons (see Methods). These results indicate that the genes Sotra2 and Bctra2 are expressed at all developmental stages and during adult life in both sexes, including in the gonads of males and females.
Comparison of the molecular organisation of Sciara tra-2gene with that of other insects
The gene tra-2 of D. melanogaster gives rise to three mRNAs by alternative splicing pathways and alternative promoters, which encode three distinct isoforms of the Tra2 protein [27, 28]. In B. mori, tra-2 produces six different transcripts by alternative splicing pathways, which encode six distinct isoforms of the Tra2 protein [24]. In the case of other dipterans such as C. capitata [20, 21], Anastrepha species [22], M. domestica [19] and L. cuprina [23], only a single tra-2 mRNA was detected.
Figure 3 compares the molecular organisation of Sciara tra-2 with tra-2 of Drosophila, Ceratitis, Musca and Bombyx. The number of exons varies: six in Sciara, seven in Drosophila, eight in Ceratitis and Musca, and nine in Bombyx. In Sciara, all the exon/intron junctions agree with the consensus GT/AG. These joints are found in the same position in S. ocellaris and B. coprophila. Similarly, the connection between exons 5 and 6 is in the same position in all these species. The RS1 and RRM domains of the putative Tra2 proteins are encoded by the exons E2-E4 and exons E5-E6 respectively in all species. However, the RS2 domain is encoded by exon E6 in Sciara, by exons E6-E7 in Drosophila, by exons E6b-E7 in Ceratitis, by exons E6-E7 in Musca, and by exons E7-E8 in Bombyx. The gene tra-2 has a single promoter except in Drosophila, which carries two promoters.
Comparison of the Tra2 protein of Sciarawith that of other insects
The conceptual translation of the Sotra2-251 and Bctra2-246 mRNAs showed them to encode a polypeptide with the main structural features characteristic of the SR protein family, i.e., the RNA-binding motif (RRM) and two RS-domains. These are rich in serine-arginine dipeptides and confer upon these proteins the capacity to interact with others.
The putative Tra2 protein of the dipterans S. ocellaris and B. coprophila (Sciaridae, suborder Nematocera) were compared to those of the dipterans belonging to the suborder Brachycera, i.e., D. melanogaster (Drosophilidae), C. capitata (Tephritidae), M. domestica (Muscidae) and L. cuprina (Calliphoridae), and that of the lepidopteran B. mori. Figure 4 shows their alignment. The number of amino acids in these Tra2 proteins varied: S. ocellaris 251, B. coprophila 246, D. melanogaster 264, C. capitata 251, M. domestica 232, L. cuprina 271, and B. mori 274. These differences are due to changes throughout the Tra2 protein except in the RRM domain and the linker region (72 and 19 amino acids respectively) in all these species. The highest degree of similarity (measured as identical plus conserved amino acids) to S. ocellaris was shown by B. coprophila (86%) (as expected for species belonging to the same Family), followed by Musca (46.9%), Bombyx (44.6%), Ceratitis (42.6%), Lucilia (39,4%) and Drosophila (36.3%). The highest degree of similarity was observed in the RRM (55.5-94.4%) and the linker region (68.4-100%). In fact, the linker region is a signature motif of Tra2 proteins [29]. This conservation agrees with the fundamental role of RRM in the function of the Tra-Tra2 complex, conferring upon the complex its capacity to interact with the tra and dsx pre-mRNAs and thus regulate its sex-specific splicing. The similarity of the RS1 (21.8-87.6%) and RS2 (28.2-81.6%) domains was lower and a variable number of SR dipeptides were seen, ranging from 11 to 18 for RS1 and 3 to 6 for RS2. Variation in the content of RS dipeptides seems to be a feature of the SR proteins whenever they are maintained enough to preserve their function [30].
Effect of the gene tra-2 of Sciara on the somatic sexual development of Drosophila
Outside Drosophila, the function of tra-2 in sex determination has been unambiguously demonstrated in M. domestica [19], in C. capitata [21] and in Anastrepha [22] using the interference-RNA technique, which permits functional studies of genes in genetically less amenable organisms. An imperative of this technique is to have markers that allow one to determine whether male survivors really do correspond to XX females that have been transformed into pseudomales by the destruction of the endogenous tra-2 gene function, or to normal XY males. In the case of the insects mentioned above, this distinction was possible thanks to the existence of molecular markers located on the Y chromosome [19, 21] and to the different morphology of the X and Y chromosomes [22]. However, the lack of molecular makers, plus the fact that Sciara females are XX and males are X0, together with the extreme fragility of their tiny eggs, makes this RNAi procedure unfeasible for these insects at the present time. Thus, direct proof of the role of tra-2 in Sciara sex determination remains elusive.
Notwithstanding, we were able to study whether the Sciara Tra2 protein shows conserved sex-determination function in Drosophila. The rationale of the experiment was to express transgenic SoTra2 protein in Drosophila XX pseudomales lacking the tra-2 gene function and checked whether these pseudomales showed feminisation. The GAL4-UAS system was used to analyse the effect of the Sciara tra-2 gene in Drosophila.
The systemic expression of SoTra2 with the ubiquitous-expression da-GAL4 or hs-GAL4 drivers was found to be lethal to both male and female flies. The same lethality has been observed in Drosophila males and females that ectopically express their own Tra2 protein [31]. Therefore, the rn-GAL4 local expression driver was used. This driver expresses GAL4 in agreement with the expression domain of the gene rotund (rn), which is expressed in the tarsal region of the foreleg imaginal disc [32], a well-characterised sexually dimorphic region of Drosophila. For details of the experimental design see Methods.
Figure 5 shows the effect of expressing the SoTra2 protein on the sexually dimorphic development of the foreleg basitarsus in XX pseudomales and in their brother XY males, both mutant for tra-2 and carrying Sotra2-UAS, rn-GAL4 and Tub-GAL80ts . The foreleg basitarsus contained several transversal rows, the last one forming the sex comb structure (SC) in males and in XX pseudomales mutants for tra-2. This is composed of dark, thick bristles, and is rotated to lie parallel to the proximal-distal leg axis (Figure 5C). Females lack the sex comb (Figure 5B). A significant reduction (P < 0.0001, one-way ANOVA) was seen in the number of bristles forming the male sex comb structure in the foreleg basitarsus of XX pseudomales raised at 25°C (expressing the SoTra2 protein) (see Figure 5D and the enlargement in Figure 5E) compared to those raised at 18°C (no expression of the SoTra2 protein) (see Figure 5C). The sex comb size of these latter pseudomales was the same as the sex comb of their XY brothers whether raised at 18 or 25°C (Figure 5A). Thus, the Sciara Tra-2 protein supplies tra-2 function in Drosophila.
This reversion of the male towards the female phenotype of the foreleg basitarsus in XX pseudomales mutant for tra-2 and expressing the SoTra2 protein is probably caused by the presence of the endogenous Drosophila DsxF protein. As mentioned in the Introduction, the Tra-Tra2 complex controls the female-specific splicing of the dsx pre-mRNA. If the Sciara Tra2 protein is capable of forming a complex with the endogenous Drosophila Tra protein, then this complex could bind to specific sequences in the female-specific exon of dsx pre-mRNA. This would promote its inclusion in mature dsxF mRNA, which encodes the DsxF protein that establishes female development. This expectation was confirmed at the molecular level.
The effect of Sciara Tra2 protein on the splicing control of Drosophila dsx pre-mRNA was studied in transgenic Drosophila XX flies mutant for tra-2 and expressing the Sciara Tra2 protein (Figure 6). The inducible hs-GAL4 driver was used to express the Sotra2 transgene. XX pseudomales yw/w; Df(2R)trix,tra-2[-] /tra-2B; Sotra2/hs-GAL4 were produced at 25°C (see cross in the legend to Figure 6). After the hatching of the adults the flies were divided into two populations; one was maintained at 25°C (control flies) and one subjected to heat-shock pulses (experimental flies) to induce the expression of the Sotra2 transgene. Total RNA was extracted and used in RT-PCR to determine the splicing pattern of the endogenous dsx primary transcript. rp49, which codes for the constitutive ribosomal protein 49 [33], was used as an RT-PCR control. At 25°C, the four transgenic lines only expressed the male dsx mRNA isoform (data not shown); this was to be expected since they lack the endogenous tra-2 function and do not express the Sotra2 transgene. After the heat shocks, however, these transgenic lines expressed the female dsx mRNA isoform (Figure 6B). Two amplicons were detected. The smaller one (646 bp) corresponded to the female dsxF mRNA. The larger amplicon (758 bp) was to be expected if the intron 3 were retained (Figure 6A,B). The cloning and sequencing of both fragments confirmed these suppositions. These results were not the consequence of the heat-shocks since their brothers (males yw/Y; Df(2R)trix,tra2[-]/tra-2B; Sotra2/hs-GAL4) expressing the Sotra2 transgene did not express the female dsx mRNA isoform (Figure 6C). Negative controls for all these PCR reactions produced no amplicons (see Methods). Thus, the Sciara Tra2 protein is able to promote the female-specific splicing of the Drosophila dsx pre-mRNA.
Whereas the expression of Sciara Tra2 protein in the XX pseudomales produced their feminisation, its expression in their XY normal brothers mutant for tra-2 did not affect their normal male development. This different effect is explained by the presence of Tra protein in the XX pseudomales and its absence in XY normal males. The Drosophila Tra protein seems to lack an RNA binding domain, thus its influence in female development is exerted at the level of its interaction (via SR domains) with other proteins carrying RNA-binding domains, such as Tra2 (reviewed in [34]). Therefore, the Sciara Tra2 protein would form a complex with the endogenous Drosophila Tra protein to promote the female-specific splicing of the Drosophila dsx pre-mRNA.
The feminisation produced by the Sciara Tra2 protein was, however, partial, indicating that the function of this protein in Drosophila was incomplete. There are two possible explanations for this. It might be due to an insufficient quantity of Sciara Tra2 protein being produced in the Drosophila transgenic flies; it was necessary to restrict the amount of Sciara Tra2 protein that was expressed since the production of any greater amount is lethal. However, although this possibility cannot be rejected outright it seems unlikely since Drosophila XX flies with a single dose of tra-2 develop as normal females; i.e., a single dose of the endogenous tra-2 gene supplies enough Drosophila Tra2 protein for normal female development to be followed. Alternatively, the interaction between the endogenous Drosophila Tra protein and the transgenic Sciara Tra2 protein might be affected such that the DrosophilaTra-SciaraTra2 complex is less efficient than the DrosophilaTra-Tra2 complex at inducing the female-specific splicing of the endogenous Drosophila dsx pre-mRNA. This explanation agrees with the presence of the aberrant spliced dsxF mRNA isoform in XX pseudomales expressing the Sciara Tra2 protein in addition to the normally spliced dsxF mRNA. Note that this isoform contains intron 3, the target where the Tra-Tra2 complex binds to promote its inclusion into mature dsxF mRNA [7–10]. Further, the retention of intron 3 does not appear to be the consequence of any general trouble in the splicing of dsx pre-mRNA since intron 2 (and probably also intron 1) is normally spliced.
This aberrant dsxF mRNA isoform has been also found in Drosophila XX pseudomales mutant for the endogenous tra gene and expressing the Anastrepha Tra protein, whereas Drosophila XX flies with different doses of the endogenous tra and tra-2 genes do not show this abnormally spliced dsxF mRNA isoform [35]. With respect to the proposed inefficient interaction between the endogenous Drosophila Tra protein and the transgenic Sciara Tra2 protein, the high degree of divergence between the Sciara and the Drosophila Tra2 proteins should be noted. This divergence was mainly observed in the RS domains, which are involved in protein-protein interactions. Hence, the interaction between the Sciara Tra2 protein and the Drosophila Tra protein might be impeded as a consequence of changes accumulated in these proteins after the Sciara and Drosophila phylogenetic lineages separated. These results suggest that Tra and Tra2 proteins co-evolved to exert their functions in sex determination. To this respect, it is worth mentioning that the D. virilis tra-2 gene can fully replace the endogenous tra-2 function of D. melanogaster for normal female sexual development [18]. The similarity between the D. melanogaster and D. virilis Tra-2 proteins is 51,5% [22], whereas the similarity between the D. melanogaster and S. ocellaris Tra-2 proteins is 36.3% [this work].