THE ROLE OF SPECIFIC PROTEIN-RNA AND PROTEIN-PROTEIN INTERACTIONS IN POSITIVE AND NEGATIVE CONTROL OF PRE-MESSENGER-RNA SPLICING BY TRANSFORMER-2

We have investigated the function of different structural domains of t he Drosophila splicing regulator Transformer 2 (Tra2). We find that th e ribonucleoprotein consensus sequence (RNP-CS) of Tra2 is required fo r male fertility and positive and negative control of alternative spli cing in transgenic flies, as well as for in vitro binding of recombina nt Trail to doublesex and tra2 pre-mRNAs. Thus, all of the known funct ions of Tra2 require specific protein-RNA interactions. We also show t hat one of the two arginine-serine (RS)-rich domains of Tra2 is dispen sable, while the other is essential for all of the in vivo functions. Part of this domain is also required for RNA binding in vitro. Signifi cantly, the essential RS domain is also required for specific protein- protein interactions. We find that Tra2 interacts with itself, with th e splicing regulator Transformer, and with the general splicing factor SF2 in vitro and in the yeast two-hybrid system. These results demons trate that both protein-RNA and protein-protein interactions are invol ved in tra2-dependent activation and repression of alternative splicin g.