Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. Defects in splicing lead to human genetic disease and splicing mutations in several genes involved in growth control have been implicated in multiple types of cancer. Insights into the basic mechanisms of pre-mRNA splicing and splice site recognition are therefore fundamental to understanding regulated gene expression and human disease. The control of alternative splicing is a highly combinatorial process, where many inputs dictate the splicing outcome for each exon. A critical feature of these regulatory mechanisms is the specific interaction of trans-acting splicing factors with cis-acting RNA elements. We use a highly integrated approach to investigate the molecular mechanisms that regulate pre-mRNA splicing. This includes knockout and knock-in tissue culture models, reconstitution assays using radioligands, transcriptomics, bioinformatics, kinetics, structure- function and biochemical techniques. In the next five years, we aim to address several outstanding challenges in the field, pursuing the following novel research directions. (1) Our demonstration that splicing regulatory proteins display highly position-dependent activities that negatively or positively influence splice site choice changed the way we think about the classical splicing activators (SR proteins) and the classical splicing repressors (hnRNPs). It is now appreciated that the context-dependent activation or repression of U1 snRNP serves as a gateway to allow the abundant U1 snRNP to fulfill its splicing function and its role in protecting the pre-mRNA from premature degradation. However, how splicing regulators achieve activation or repression of U1snRNP at the 5’ splice site is not understood. We aim to dissect the mechanisms of splicing repression by embracing multi-system approaches and by understanding the role of U1 snRNP conformers in mediating spliceosomal assembly. (2) Intron retention is an important alternative splicing pathway that has eluded extensive study. Thus, its regulation is not well understood. The existence of inefficiently spliced introns within coding exons (exitrons) further highlights the biological importance of understanding when introns are removed efficiently and when they are not. Using synthetic biology approaches, we will decipher the rules of efficient intron removal and investigate the impact of cis-acting elements in this process. The argument is that the depth of the sequence variation tested in massively parallel reporter assays is far greater than the testing landscape that the human genome offers. Here, we will use our expertise in experimental molecular biology and bioinformatics. (3) It has become widely appreciated that gene expression events are highly integrated, with evidence suggesting that most pre-mRNA processing occurs co-transcriptionally. Defects in any one of these steps have been linked to disease. However, most published studies ...