ABSTRACT It has long been known that pre-messenger RNA (pre-mRNA) splicing is an essential component of gene expression in eukaryotic organisms, yet the past decade has seen a dramatic increase in our appreciation for its role in regulating gene expression1. Most higher eukaryotes, including humans, regulate alternative splicing as a tool for proteome expansion, and an ever-increasing number of human diseases are associated with mutations in this pathway2,3. The mechanisms by which the spliceosome, which catalyzes pre-mRNA splicing, enacts this regulation is a complex problem whose solution remains poorly understood yet will be critical to understanding the etiology of many diseases. Proper regulation requires the spliceosome to faithfully assemble upon and activate ‘cognate’ splice site sequences in the background of scores of aberrant, ‘near-cognate’ splice sites, yet the spliceosome must balance this high fidelity splice site selection with the need for rapid, efficient splicing. At the simplest level, improved knowledge of how the spliceosome achieves this balance will require understanding both: (1) the landscape of cis-regulatory elements at splice sites that enable them to be distinguished as either ‘cognate’ or ‘non-cognate’; and (2) the mechanisms by which the spliceosome discriminates between such sites. In the work described here, we seek to better understand basic mechanisms of pre-mRNA splicing regulation by leveraging a powerful methodology recently developed in my lab called Multiplexed Primer Extension sequencing, or MPE-seq. Our approach is unique in that it allows for the genome-wide detection of pre-mRNA splicing intermediates. By combining this technique with rapid metabolic RNA labeling techniques developed by others, my group has now determined the in vivo rates of both chemical steps of pre-mRNA splicing across the complement of spliced transcripts in budding yeast. Remarkably, these data reveal a wide variation among the rates, both between the two steps for individual transcripts and between different transcripts. The goals of the work described here are to leverage the information derived from these experiments to push our understanding of the principles that underlie this regulation.