Abstract Pre-mRNA splicing ? the process of intron removal and exon ligation ?often occurs co-transcriptionally, i.e. during transcription by RNA polymerase II (Pol II). Cross-regulation between transcription and splicing represents an important gene regulatory pathway that influences how highly expressed a gene is, which alternative splice isoforms will be produced, and the efficiency of transcription elongation. Yet how the splicing and transcription machineries communicate is unknown. Our objective is to understand how splicing and transcription are coordinated. Accordingly, we will test the hypotheses that pausing of Pol II elongation within genes may be caused by splicing, specific gene characteristics, or incomplete splicing in the manner of a checkpoint. Our general strategy is to biochemically purify nascent RNA from chromatin and use next generation sequencing as a tool to precisely determine when during transcription splicing occurs. Our preliminary data on a small number of genes indicate that splicing occurs soon after the 3' splice site emerges from Pol II, much faster than predicted from indirect assays. We will investigate hundreds of endogenous genes in order to explore roles in co-transcriptional splicing for gene-specific features, such as sequence, exon-intron structure, promoter identity, nucleosome positioning and post-translational modifications on histones or Pol II. We will modify endogenous genes and employ mutants of the splicing and transcriptional machinery to probe mechanism. The simplicity of S. cerevisiae, with ~300 single-intron genes, allows us to investigate and experimentally alter transcription, splicing, and the architecture of genes. The relative complexity of S. pombe, with ~1000 genes harboring multiple introns, allows us to determine how splicing and transcription impact the order of intron removal, which must be regulated during alternative splicing. Our findings will help explain how cells control mRNA abundance and mRNA isoforms through splicing. Because mis-regulation of transcription and splicing are frequently associated with human diseases, such as cancer, a molecular understanding of their cross-regulation is significant for human health.