Biogenesis of eukaryotic messenger ribonucleoprotein complexes (mRNPs) involves the synthesis, splicing, and 3′ processing of pre-mRNA, and the assembly of mature mRNPs for nuclear export. We mapped 23 mRNP biogenesis factors onto the yeast transcriptome, providing 104–106 high-confidence RNA interaction sites per factor. The data reveal how mRNP biogenesis factors recognize pre-mRNA elements in vivo. They define conserved interactions between splicing factors and pre-mRNA introns, including the recognition of intron-exon junctions and the branchpoint. They also identify a unified arrangement of 3′ processing factors at pre-mRNA polyadenylation (pA) sites in yeast and human, which results from an A-U sequence bias at pA sites. Global data analysis indicates that 3′ processing factors have roles in splicing and RNA surveillance, and that they couple mRNP biogenesis events to restrict nuclear export to mature mRNPs.