PROJECT SUMMARY Precursor messenger RNA (pre-mRNA) processing is an essential aspect of gene expression that occurs concurrently with transcription by RNA polymerase II. One essential step in pre-mRNA processing is the removal of non-coding introns and ligation of coding exons together (pre-mRNA splicing) by the spliceosome. The spliceosome assembles on the nascent RNA during transcription, and splicing is completed soon after the intron has been transcribed. Thus, the splicing and transcription machineries are spatially and temporally coupled – working in concert to ensure timely and accurate expression of cellular mRNAs. Mutations or perturbations of either process change gene output and are frequently associated with human disease. Remarkably, the mechanisms coordinating splicing with transcription are poorly understood. The proposed work combines established techniques and novel approaches to elucidate how the two cellular processes regulate one another. The Neugebauer lab recently developed single-molecule intron tracking (SMIT) and other RNAseq-based approaches to measure the in vivo kinetics of splicing relative to transcription. Specific Aim 1 investigates the contribution of intron sequence and other RNA features to the splicing reaction and determines how co- transcriptional splicing can influence gene output. Specific Aim 2 combines SMIT and other RNAseq-based approaches with Pol II mutant backgrounds to reveal the impact of the C-terminal domain and post-translational modifications of RNA polymerase II on splicing. In particular, this aim asks how the CTD contributes to the efficient splicing of multiple introns in a single transcript. These experiments will generate new mechanistic insights into how RNA polymerase interacts with the spliceosome to promote efficient RNA splicing. Specific Aim 3 investigates the influence of the splicing machinery on transcriptional dynamics and polymerase pausing using potent splicing inhibitors and genetic tools that cause the spliceosome to remain associated with the nascent transcript. These approaches and aims will provide an entry point for developing expertise in transcription, bioinformatics, RNA-seq based methods and other computational approaches. In addition, the proposed work will generate unprecedented molecular insight into the cross-talk between essential processes in gene expression and provide fundamental knowledge that will be vital in future studies on how splicing and transcription are altered in disease.