PROJECT SUMMARY/ABSTRACT This MIRA award supports our studies of how RNA binding proteins regulate alternative splicing and other posttranscriptional steps in mammalian gene expression. The previous funding period was very productive with multiple publications. We developed new methods and we discovered new interactions and new functions for several proteins and RNAs. We propose to continue these studies applying our strategy for isolating splicing regulatory complexes and snRNPs from the chromatin compartment of cells, characterizing their components, determining their structures, and testing their impact on splicing regulation or other processes. We will complete our structure by cryo-electron microscopy of chromatin-isolated U2 snRNP’s associated with the epitope-tagged RBM5 regulator and bound to intron branchpoints. We will go on to determine the structures of U2 particles associated with tagged SF3A2 and other factors. These structures will provide new insight into how regulators like RBM5/10 and the RNA Helicase DHX15 alter splicing at a late stage of spliceosome assembly. We will apply our method of chromatin extraction to new molecules including the U1 snRNP and polypyrimidine tract binding protein 1. We expect to identify new interactions of these molecules by isolating them bound to their nascent RNA substrate. These particles also will be examined by EM to assess their suitability for structure determination. We will continue our studies of the Rbfox family of splicing regulators. Earlier studies of chromatin associated Rbfox1 demonstrated its association with a complex of other RNA binding proteins (LASR) and its higher order assembly through homomeric interactions of its disordered C-terminal domain. We will continue our analysis of the residues that mediate this homomeric interaction and define a minimal segment conferring self-assembly. Results from this analysis will enable manipulations of Rbfox/LASR assembly in vivo and studies of the role of thes