In vivo commitment to yeast cotranscriptional splicing is sensitive to transcription elongation mutants

<jats:p>Spliceosome assembly in the budding yeast <jats:italic>Saccharomyces cerevisiae</jats:italic> was recently shown to occur at the site of transcription. However, evidence for cotranscriptional splicing as well as for coupling between transcription and splicing is still lacking. Using modifications of a previously published chromatin immunoprecipitation (ChIP) assay, we show that cotranscriptional splicing occurs ∼1 kb after transcription of the 3′ splice site (3′SS). This pathway furthermore protects most intron-containing nascent transcripts from the effects of cleavage by an intronic hammerhead ribozyme. This suggests that a high percentage of introns are recognized cotranscriptionally. This observation led us to screen a small deletion library for strains that sensitize a splicing reporter to ribozyme cleavage. Characterization of the <jats:italic>Δmud2</jats:italic> strain indicates that the early splicing factor Mud2p functions with U1 snRNP to form a cross-intron bridging complex on nascent pre-mRNA. The complex helps protect the transcript from ribozyme-mediated destruction and suggests an intron-definition event early in the spliceosome assembly process. The transcription elongation mutant strains <jats:italic>Δdst1</jats:italic> and <jats:italic>Δpaf1</jats:italic> show different cotranscriptional splicing phenotypes, suggesting that different transcription pathways differentially impact the efficiency of nascent intron definition.</jats:p>