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

SIMPLE SUMMARY: In insects, sex determination is generated using intricate and variegate biological processes that may be effectively described as variations on a common theme. In the model system Drosophila melanogaster, genetic and biochemical studies have shown that the female-specific Transformer (TRA) and the non-sex-specific Transformer2 (TRA2) are RNA-binding proteins that physically interact to promote female differentiation by female-specific alternative splicing of downstream genes. This tra gene responds and transduces different primary sex-determining signals, and its master function is widely conserved in Diptera, Coleoptera, and Hymenoptera. Here, combining yeast two-hybrid and computational methodologies, we demonstrate that the TRA and TRA2 orthologs of the agricultural pest Ceratitis capitata physically interact through a molecular mechanism that could be evolutionarily conserved in other species. These technical approaches can be helpful to verify or to identify other proteins interacting with TRA and TRA2, for example, those promoting male sex determination in this and other species, as well as to design new compounds that could induce masculinization of XX individuals in applications of the Sterile Insect Technique. 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.