Repeat-Specific Functions for the C-Terminal Domain of RNA Polymerase II in Budding Yeast.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) is required to regulate transcription and to integrate it with other essential cellular processes. In the budding yeast Saccharomyces cerevisiae, the CTD of Rpb1p consists of 26 conserved heptad repeats that are post-translationally modified to orchestrate protein factor binding at different stages of the transcription cycle. A long-standing question in the study of the CTD is if there are any functional differences between the 26 repeats. In this study, we present evidence that repeats of identical sequence have different functions based on their position within the CTD. We assembled plasmids expressing Rpb1p with serine to alanine substitutions in three defined regions of the CTD and measured a range of phenotypes for yeast expressing these constructs. Mutations in the beginning and middle regions of the CTD had drastic, and region-specific effects, while mutating the distal region had no observable phenotype. Further mutational analysis determined that Ser5 within the first region of repeats was solely responsible for the observed growth differences and sequencing fast-growing suppressors allowed us to further define the functional regions of the CTD. This mutational analysis is consistent with current structural models for how the RNAPII holoenzyme and the CTD specifically would reside in complex with Mediator and establishes a foundation for studying regioselective binding along the repetitive RNAPII CTD.

Comprehensive RNA Polymerase II Interactomes Reveal Distinct and Varied Roles for Each Phospho-CTD Residue.

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.