Abstract: The splicing machinery associates with genes to facilitate efficient cotranscriptional mRNA processing. We have mapped these associations by genome localization analysis to ascertain how splicing is achieved and regulated on a system-wide scale. Our data show that factors important for intron recognition sample nascent mRNAs and are retained specifically at intron-containing genes via RNA-dependent interactions. Spliceosome assembly proceeds cotranscriptionally but completes posttranscriptionally in most cases. Some intron-containing genes were not bound by the spliceosome, including several developmentally regulated genes. On this basis, we predicted and verified regulated splicing and observed a role for nuclear mRNA surveillance in monitoring those events. Finally, we present evidence that cotranscriptional processing events determine the recruitment of specific mRNA export factors. Broadly, our results provide mechanistic insights into the coordinated regulation of transcription, mRNA processing, and nuclear export in executing complex gene expression programs.