Sequencing of the human genome and those of other organisms has revealed that a large percentage of pre-mRNAs are spliced in multiple patterns to produce mRNAs encoding distinct proteins. Although alternate splicing is widespread and must regulate the expression of thousands of gene products, the fundamental mechanisms that control it are not yet well understood. The RNA binding protein Tra2 has been shown to affect the regulated alternative splicing of several pre-mRNAs. In humans these include splice site choices in disease-associated genes such as SMN, tau and CD44. In Drosophila, Tra2 is required for the alternate processing of multiple mRNAs that are necessary for the regulation and realization of sexual differentiation. These include those of the doublesex, fruitless and exuperantia genes as well as tra2 itself. While the splicing activation function of Tra2 in doublesex RNA processing has been well studied and has served as an important paradigm for developmental control of exonic splicing enhancers, little is known about how Tra2 affects splicing of other pre-mRNAs. In this project the mechanism by which Tra2 represses splicing of a specific intron in its own pre-mRNA will be investigated and compared with regulatory elements used in the activation of doublesex splicing. Using an in vitro splicing assay in which recombinant Tra2 specifically blocks splicing of the M1 intron by Drosophila nuclear extracts, the regulatory elements through which Tra2 represses splicing will be identified and the hypothesis that Tra2 blocks the activity of a required exonic splicing enhancer upstream of the intron will be tested. Regulatory complexes that form on the Tra2 pre-mRNA will be compared to those that it associates with in the dsx splicing enhancer to define key elements that determine activation or repression of splicing. The veracity of mechanistic models developed from in vitro splicing studies will be tested by generating transgenic fly strains in which pre-mRNA and regulators with altered sequences are expressed in the tissue and stage of development where Tra2-dependent repression normally occurs. In further studies genetic approaches will be used to define the requirements for SR proteins and other splicing factors that cooperate with Tra2 in the control splice site recognition. These studies will elucidate the fundamental mechanisms by which individual splicing regulators can alter splicing in different ways for different pre-mRNAs.