Native elongating transcript sequencing (NET-seq), nascnet RNA-seq, and total RNA-seq of wild type and RNA polymerase II C-terminal domain mutants and ChIP-nexus of RNA polymerase II C-terminal domains phosphoisoforms and splicing factors in S. cerevisiae

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 novel role for threonine-4 in 3’ end processing through controlling 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 through serving as a mark of spliceosomal 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. NET-seq of WT, rai1, rtt103 and Pol II CTD threonine-4 mutants. Nascent RNA-seq of WT and Pol II CTD threonine-4 mutant. RNA-seq of WT and Pol II CTD threonine-4 mutants. ChIP-nexus analysis of phospho-Ser5 and phospho-Thr4 of the Pol II CTD and ChIP-nexus of splicing factors.