Project Summary The biogenesis of mRNA requires relatively small exons to be identified and stitched together from within an expanse of intronic sequence. The majority of intron removal occurs co-transcriptionally on the moving target of the nascent RNA complex. Adding to this complexity, introns are not always removed in the same way. Rather, alternative splicing can give rise to proteins with distinct functions arising from the same gene. Over the last few decades, a great deal has been uncovered about the mechanisms by which complex molecular machineries regulate splicing. However, we still know very little about the kinetics of splicing on its in vivo substrate, nascent elongating pre-mRNA. The co-transcriptional nature of most splicing underlies why transcriptome-wide splicing kinetics have remained a barrier to progress in the field. The rate of intron removal from nascent RNA is obfuscated by the transcriptional dynamics of the substrate itself. In order, to determine co-transcriptional splicing rates, the kinetic parameters of transcription (e.g. initiation rate, elongation rates and transcript cleavage rates) must also be determined. Here, we unveil a novel technique called SKaTER-seq (Splicing Kinetics and Transcription Elongation Rates through sequencing) and a sophisticated computational pipeline which together can simultaneously determine the critical parameters of transcription and splicing rates transcriptome-wide. Our overall goal is to apply the SKaTER-seq method in order to gain further mechanistic understanding of how splicing kinetics are regulated. The first aim will determine transcriptional and splicing kinetics transcriptome-wide and determine their relation to genomic, chromatin and trans-acting factors. The second aim will determine the role of three chromatin components (H3K36me3, linker histone H1, histone variant macroH2A1) in the establishment of transcriptional and splicing kinetics. This data will illuminate key unanswered questions in the field about the role of chromatin, transcriptional elongation, and spliceosomal perturbation in directing splicing outcomes.