Transcription controls splicing and other gene regulatory processes, yet mechanisms remain obscure due to our fragmented knowledge of the molecular connections between the dynamically phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD) and regulatory factors. By systematically isolating phosphorylation states of the CTD heptapeptide repeat (Y1S2P3T4S5P6S7), we identify hundreds of protein factors that are differentially enriched, revealing unappreciated connections between the Pol II CTD and co-transcriptional processes. These data uncover a role for threonine-4 in 3′ end processing through control of the transition between cleavage and termination. Furthermore, serine-5 phosphorylation seeds spliceosomal assembly immediately downstream of 3′ splice sites through a direct interaction with spliceosomal subcomplex U1. Strikingly, threonine-4 phosphorylation also impacts splicing by serving as a mark of co-transcriptional spliceosome release and ensuring efficient post-transcriptional splicing genome-wide. Thus, comprehensive Pol II interactomes identify the complex and functional connections between transcription machinery and other gene regulatory complexes. Display omitted •Pol II phospho-CTD residues interact with unique sets of protein factors•CTD threonine-4 and Rtt103 regulate Pol II pausing after polyadenylation sites•CTD Phospho-serine-5 recruits the spliceosome and regulates splicing•CTD threonine-4 is required for efficient post-transcriptional splicing Many RNA-processing events are coupled to transcription. To gain insight into the mechanisms linking these processes, Harlen et al. determine interactomes for each RNA polymerase II phospho-CTD residue, linking CTD Thr4 to transcription termination and CTD Thr4 and Ser5 to splicing.