The spliceosome is a highly dynamic ribonucleoprotein that assembles upon nascent pre-mRNA transcripts to remove non-coding introns prior to export of the mature mRNA from the nucleus for translation. Importantly, the spliceosome must remove introns quickly to ensure timely protein production, but must balance efficiency with accuracy, as incorrectly spliced mRNAs can result in non-functional or deleterious proteins. One known regulator of splicing speed and fidelity is Prp8, a large, highly conserved protein component of the spliceosome. Within the structure of Prp8 there exists a domain which closely resembles the structure of a RNaseH molecule, but which differs by the presence of a short extension in Prp8. Interestingly the extension within this RNaseH-like domain of Prp8 has been shown to exist in two mutually exclusive conformations – a structured beta hairpin and an open loop – and altering the protein in a way which preferentially stabilizes one or the other of these conformations has been shown to affect splicing speed and fidelity in an antagonistic way. Specifically, work in Saccharomyces cerevisiae showed that biasing the extension to the open loop conformation resulted in faster, less accurate splicing, while biasing the extension to the hairpin conformation resulted in slower, more accurate splicing, leading to a model wherein ‘toggling’ of this extension region between these two conformations reflected a switch between proofreading and catalytically active states of the spliceosome. To further characterize the mechanistic basis by which this extension of Prp8 regulates speed and fidelity of splicing, I sought to determine if Multiplexed Primer Extension Sequencing (MPE-seq), a modified version of RNA seq developed in the Pleiss lab, could be used to detect the changes in splicing efficiency seen using other assays in a hairpin biased mutant and to better understand how this amino acid change affects splicing outcomes. I also generated a four amino acid mutant in S. pombe, a yeast species with a more complex intron landscape, and similarly tested if MPE-seq measured a change in splicing efficiency. Here I share my progress on this work.