The gene transformer of anastrepha fruit flies (Diptera, tephritidae) and its evolution in insects

Abstract

In the tephritids Ceratitis capitata and Bactrocera oleae, the gene transformer acts as the memory device for sex determination, via an auto-regulatory function; and functional Tra protein is produced only in females. This paper investigates the evolution of the gene tra, which was characterised in twelve tephritid species belonging to the less extensively analysed genus Anastrepha. Our study provided the following major conclusions. Firstly, the memory device mechanism used by this gene in sex determination in tephritids likely existed in the common ancestor of the Ceratitis, Bactrocera and Anastrepha phylogenetic lineages. This mechanism would represent the ancestral state with respect to the extant cascade seen in the more evolved Drosophila lineage. Secondly, Transformer2-specific binding intronic splicing silencer sites were found in the splicing regulatory region of transformer but not in doublesex pre-mRNAs in these tephritids. Thus, these sites probably provide the discriminating feature for the putative dual splicing activity of the Tra-Tra2 complex in tephritids. It acts as a splicing activator in dsx pre-mRNA splicing (its binding to the female-specific exon promotes the inclusion of this exon into the mature mRNA), and as a splicing inhibitor in tra pre-mRNA splicing (its binding to the male-specific exons prevents the inclusion of these exons into the mature mRNA). Further, a highly conserved region was found in the specific amino-terminal region of the tephritid Tra protein that might be involved in Tra auto-regulatory function and hence in its repressive splicing behaviour. Finally, the Tra proteins conserved the SR dipeptides, which are essential for Tra functionality.

Figure 1. Comparison of the molecular organisation of the gene tra of C. capitata, B. oleae and A. obliqua (A) and the transcripts encoded by the A. obliqua tra gene (B).

Exons (boxes) and introns (lines) are not drawn to scale. The numbers inside the boxes indicate the number of the exon; ms1, ms2 and ms3 stand for the male-specific exons. The beginning and the end of the ORF are indicated by ATG and TAA respectively. The longest female mRNA is shown. The male-specific transcripts show the stop codons in the mature mRNA; these depend on the male-specific exons incorporated.

Figure 2. Expression of A. obliqua tra.

RT-PCR analyses of total RNA from male plus female larvae (L), female adult (F), male adult (M), female soma (head plus thorax), male soma (head plus thorax), and ovaries (O). The sequence of the primers used and their locations are shown.

Figure 3. Comparison of the predicted Tra polypeptides of C. capitata , B. oleae and A. obliqua (taken as the reference for the twelve Anastrepha species here studied).

The shadowed regions correspond to the domains that show 100% similarity among the three species (similarity refers to identical plus conservative amino acids)

Figure 4. Comparison of the molecular organisation of the tra genomic region (encompassing the male-specific exons and their flanking introns) involved in sex-specific splicing regulation of the tra pre-mRNA, in the twelve Anastrepha species, in C. capitata (unpublished sequence) and in B. oleae (accession number AJ715414). Boxes represent exons, lines represent introns (not drawn to scale).

In B. oleae, exon 1 is split into exons 1A and exon 1B . The male-specific exons are denoted by ms1, ms2 and ms3, and the introns corresponding to the compared tra genomic region by is1, is2 and is3. The locations of the Tra-Tra2, RBP1 and Tra2-ISS binding sites are shown, along with their consensus sequences, together with those found in D. melanogaster (bottom of the Figure). The numbers and the letters (a) and (b) underneath the small rectangle and ellipsoids representing these binding sequences refer to the exact same sequences described in the Supporting Material.

Figure 5. (A) Phylogenetic tree reconstructed from 22 protein TRA sequences belonging to different insect species (taxonomic groups indicated on the right hand side of the tree).

The tree was built using the neighbour-joining method. The confidence levels for the groups are indicated in the corresponding nodes as bootstrap (BP, normal type) and interior branch test results (CP, bold type) based on 1000 replications. Values are shown only when either the BP or CP values are higher than 50%. (B) Proportion of nucleotide sites at which two sequences being compared are different (p, nucleotide substitutions per site) and ratio between the numbers of non-synonymous (pN) and synonymous (pS) substitutions per site across the coding regions of tra in the tephritids. These values were calculated using a sliding-window approach with a window length of 40 bp and a step size of 10 bp. The relative positions of the RS and SR dipeptides across tra are represented in the white boxes below the graph.

Figure 6. Proposed explanation of the dual role-played by the tephritid Tra protein in sex determination.

For the sake of simplification, the tra pre-mRNA is shown as containing a single male-specific exon (yellow box); the white boxes represent common exons. The dsx pre-mRNA shows only the one common exon (white box), a female-specific exon (grey box) and a male-specific exon (yellow box). The lines represent introns. AAA stands for polyadenylation. The black, yellow and red rectangles represent the Tra-Tra2, the RBP1 and the Tra2-ISS binding sites respectively. The X-SR factor refers to the unknown factor mentioned in the text. The green part of the Tra protein corresponds to the amino terminal region of the tephritid Tra protein, which is not present in the Tra protein of the drosophilids. The complex made up by Tra, Tra2, RBP1 and X-SR inhibits splicing, whereas the complex formed by Tra, Tra2 and RBP1 promotes splicing. For details see text.