PROJECT SUMMARY/ABSTRACT RNA splicing is a key feature of human gene expression and a major contributor to expansion of genetic information by alternative splicing. Splicing is carried out by a large and dynamic cellular machine called the spliceosome. Spliceosomes are composed of small nuclear ribonucleoproteins (snRNPs) that assemble on precursor transcripts (pre-mRNAs) to remove introns and splice together exons. This process must occur precisely in order to preserve the genetic information carried in the mRNA. Critical for splicing is the correct identification of the sites of RNA bond cleavage and formation [the 5' and 3' splice sites (SS) and the branch site (BS)]. A number of different ATPases contribute to the fidelity of SS and BS recognition as well as carry out extensive compositional and conformational remodeling of the spliceosome. Recently, biochemical studies of splicing have been transformed by determination of dozens of different structures of yeast and human spliceosomes by cryo-EM. Despite this structural revolution, much remains unknown about central features of the splicing reaction. The goal of my laboratory’s research is to elucidate mechanisms of spliceosome assembly and regulation in biochemical depth using a variety of techniques. We often use single molecule fluorescence microscopy to deconvolute the complex and heterogeneous reaction pathways employed by the splicing machinery. In recent work, we have studied mechanisms of 5'SS and BS recognition, assembly and dynamics of the U6 snRNP, and developed methods for fluorescently-labeling, purifying, and inhibiting RNAs and RNPs. Our vision for the next five years is to merge the insights obtained from structures of spliceosomes with single molecule, biochemical, computational, and genetic experiments to address outstanding gaps in our knowledge of splicing. These gaps include fundamental principles of RNP folding and assembly, the mechanisms of regulated splicing, and the scarcity of specific and effective chemical inhibitors of the spliceosome. As part of this vision, we will answer the following questions using multi-disciplinary approaches: 1) How do RNA and protein co-fold to assemble the U6 snRNP? 2) How is the spliceosome remodeled during creation of its active site? 3) How do regulatory proteins promote splicing at weak 5'SS? 4) How can we block ATPase-dependent transitions during splicing with small molecule inhibitors? 5) How do we quantitatively analyze, compare, and integrate cryo-EM structures of spliceosomes?