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

In the model system for genetics, , sexual differentiation and male courtship behavior are controlled by sex-specific splicing of () and (). 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 and female-specific splicing. TRA/TRA2 complex binds to 13 nt long sequence repeats in their pre-mRNAs. In the Mediterranean fruitfly (Medfly), a major agricultural pest, which shares with Drosophila a ~120 million years old ancestor, and genes seem to promote female-specific splicing of and , which contain conserved TRA/TRA2 binding repeats. Unlike Drosophila , 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.