BioProject

he RNA polymerase II carboxy-terminal domain (CTD) consists of conserved repeats of the consensus sequence Y1-S2-P3-T4-S5-P6-S7, which can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. More...

he RNA polymerase II carboxy-terminal domain (CTD) consists of conserved repeats of the consensus sequence Y1-S2-P3-T4-S5-P6-S7, which can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. Although affinity purification coupled with mass spectrometry has defined CTD-associated proteins, phospho-dependent CTD interactions have remained largely elusive. Proximity-dependent biotinylation (PDB) provides an alternative approach to identify protein-protein associations in the native cellular environment. Here we present a PDB-based map of the fission yeast RNAPII CTD in live cells. The proteomic screen identified known CTD-associated proteins, but also captured new and unexpected CTD proximal proteins. We also used PDB to identify phospho-dependent CTD interactions by using a mutant in which Ser2 was replaced by alanine in every repeat of the fission yeast CTD. Surprisingly, CTD-mediated biotinylation of most 3’ end processing factors was not affected in the S2A mutant, consistent with RNA-seq and ChIP-seq analysis indicating that CTD Ser2 phosphorylation is not required for 3’ end processing and transcription termination. Conversely, we found that CTD Ser2 phosphorylation is critical for the association between RNAPII and the histone methyltransferase Set2 during transcription elongation. We show that loss of CTD Ser2 phosphorylation disables the Set2-Clr6(II) axis, resulting in a global increase in cryptic antisense transcription that correlates with elevated levels of histone acetylation in gene bodies. Our findings reveal that the fundamental role of CTD Ser2 phosphorylation is to establish a chromatin-based repressive state that prevents cryptic intragenic transcription initiation. Overall design: Gene expression, chromatin modification (H3K36me3 and H3K9ac), RNAPII occupancy, and RNAPII-interacting proteins (Set2 and Pcf11) occupancy, in wild-type and Rpb1 CTD mutants. Less...