PP1 PNUTS binds the “restrictor” and dephosphorylates pol II CTD Ser5 to stimulate RNA pol II transcription termination [nascent RNA-seq]

The restrictor complex, WDR82/ZC3H4, is the major termination factor for antisense transcription from bidirectional promoters, but its mechanism of action is poorly understood. We report that the PP1 phosphatase nuclear targeting subunit PNUTS binds to restrictor via its WDR82 subunit. AlphaFold predicts a quaternary complex, PPWZ, in which PP1-associated PNUTS and ZC3H4 both contact WDR82. A chimera comprising inactive PP1H66K fused to C-terminal PNUTS sequences is a dominant-negative inhibitor of antisense termination and CTD Ser5 dephosphorylation and both activities require the PNUTS WDR82 binding domain. PP1H66K-PNUTS is a substrate trap that also interacts with pol II large subunit and nuclear exosome components. Ser5 hyperphosphorylated pol II has increased processivity and pauses less frequently than total pol II. We propose that the elevated elongation efficiency of Ser5-P pol II antagonizes early termination and that Ser5 dephosphorylation by PP1/PNUTS/WDR82/ZC3H4 is coupled to termination of antisense transcription. Overall design: nascent RNA seq in Bromouridine pulse labelled HEK293 cells expressing doxycycline (dox) inducible PP1-PNUTS fusion proteins