PROJECT SUMMARY The complexity of human splicing is daunting, yet intervention in splicing for treatment of diseases holds huge potential. Based on strong preliminary results, we propose three areas of investigation that leverage our group’s deep knowledge of splicing to address critical open questions, and to explore the potential for innovative engineering. The first area addresses the mechanism by which U2 snRNP captures the intron branchpoint early in spliceosome assembly, a step altered by recurrent cancer mutations and targeted in nature by antibiotic-producing bacteria. Using new reporters in which two branchpoints compete for recognition, we have identified a novel splicing fidelity mechanism we call “NO-BP decay,” in which U2 complexes that fail due to aberrant branchpoint selection are destroyed. We will characterize this process, applying a battery of candidate gene-based suppressor screens and biochemical tests in splicing extracts. The second area of investigation addresses how splicing is integrated with transcription and cell growth at the individual gene and cellular levels, an emerging area in need of innovation if splicing is to be successfully engineered. Preliminary results indicate that yeast cells have a limited capacity for splicing that creates competition for pre-mRNAs that is critical to cell function. We will measure both splicing capacity and the dynamics of competition, using RNA sequencing to develop a predictive model that explains how splicing is coordinated at a systems level. To understand the contribution of individual genes to this system we are applying synthetic biology approaches. We have engineered site-specific pauses of RNA polymerase II and shown that they alter splicing efficiency and alternative splicing, by unknown mechanism(s) that we will dissect. We will also explore in detail the role of splicing noise (stochastic variations in splicing output over time) on the ability of splicing to control stable homeostatic expre