Project Summary/Abstract Pre-mRNA splicing is a critical step in eukaryotic gene expression, controlled by a large set of RNA- binding proteins (RBPs) that recognize short sequence motifs on pre-mRNA. This process is largely coupled with transcription and many introns are removed while the primary transcript is this being associated with chromatin via the RNA polymerase II complex. We recently demonstrated that the pre- mRNA-bound RNA-binding Fox (Rbfox) proteins are integrated in a large protein complex with a defined set of additional splicing regulators, termed LASR. The LASR complex recognizes composite sites determined by the individual sequence preferences of several of its members. We also observed regulatory activities of LASR, separate from those of the free subunits of this complex. We now have evidence of additional complexes containing different sets of RNA-binding proteins including hnRNP A1, hnRNP A2/B1, SRSF1, SRSF2, ELAVL1, and RBM10. We further find RBM10 and its paralogous protein RBM5 to participate in novel interactions with components of the 17S U2 snRNP in the nucleus. Our findings offer new insights into the RBP interactions that drive the combinatorial assembly of regulatory complexes on pre-mRNA. We propose to purify these new protein complexes, characterize their composition, and probe their internal structure. We will also define the natural targets of these complexes in the human transcriptome and will examine how their RNA-recruitment sites differ from those of the individual RBP subunits. Finally, we will determine the activities of these complexes in mRNA splicing and the contributions of their protein constituents. Successful completion of the proposed research will have far-reaching implications for both deciphering the 'splicing code', and understanding the molecular mechanisms behind genetic disorders affected by splicing regulators.