Female Sex Determination Factors in Ceratitis capitata: Molecular and Structural Basis of TRA and TRA2 Recognition

Abstract

In the model system for genetics, Drosophila melanogaster, sexual differentiation and male courtship behavior are controlled by sex-specific splicing of doublesex (dsx) and fruitless (fru). In vitro and in vivo studies showed that female-specific Transformer (TRA) and the non-sex-specific Transformer 2 (TRA2) splicing factors interact, forming a complex promoting dsx and fru female-specific splicing. TRA/TRA2 complex binds to 13 nt long sequence repeats in their pre-mRNAs. In the Mediterranean fruitfly Ceratitis capitata (Medfly), a major agricultural pest, which shares with Drosophila a ~120 million years old ancestor, Cctra and Cctra2 genes seem to promote female-specific splicing of Ccdsx and Ccfru, which contain conserved TRA/TRA2 binding repeats. Unlike Drosophila tra, Cctra autoregulates its female-specific splicing through these putative regulatory repeats. Here, a yeast two-hybrid assay shows that CcTRA interacts with CcTRA2, despite its high amino acid divergence compared to Drosophila TRA. Interestingly, CcTRA2 interacts with itself, as also observed for Drosophila TRA2. We also generated a three-dimensional model of the complex formed by CcTRA and CcTRA2 using predictive approaches based on Artificial Intelligence. This structure also identified an evolutionary and highly conserved putative TRA2 recognition motif in the TRA sequence. The Y2H approach, combined with powerful predictive tools of three-dimensional protein structures, could use helpful also in this and other insect species to understand the potential links between different upstream proteins acting as primary sex-determining signals and the conserved TRA and TRA2 transducers.

Keywords: alpha fold; alternative splicing; autoregulation; development; gene regulation; pest control; protein–protein interactions; sex determination; structure; yeast two-hybrid.

Figure 1

Yeast two-hybrid assay of CcTRA and CcTRA2 interactions. Row 7 shows the interactions of CcTRA and CcTRA2. Row 9 shows the interaction of CcTRA2 with CcTRA2 itself. SD-W-L, Synthetic-Defined agar medium lacking tryptophan and leucine; SD-W-L-H, Synthetic-Defined agar medium lacking tryptophan, leucine, and histidine. n.d., not-diluted yeast liquid culture (10 µL); 1:10, 1:100, and 1:1000 are the dilutions of the yeast liquid culture.

Figure 2

Yeast two-hybrid assay of MOY potential interactions. SD-W-L, Synthetic-Defined agar medium lacking tryptophan and leucine; SD-W-L-H, Synthetic-Defined agar medium lacking tryptophan, leucine, and histidine. n.d., not diluted yeast liquid culture (10 µL); 1:10, 1:100, and 1:1000 are the dilutions of the yeast liquid culture.

Figure 3

Superimposition of the AF predicted model of the RRM domain of CcTRA2 (cyan) with the experimental model of the human TRA2β RRM domain (orange) (Protein Data Bank code 2RRB).

Figure 4

(A) Three-dimensional structure of CcTRA (green) and CcTRA2 (cyan) complex. (B) The contact region of the complex is highlighted. Hydrogen bonds are also shown.

Figure 5

(A) Three-dimensional structure of CcTRA (green) and DmTRA2 (magenta) complex. (B) The contact region of the complex is highlighted. Hydrogen bonds are also shown.