PP1/PNUTS phosphatase binds the restrictor complex and stimulates RNA Pol II transcription termination

The restrictor ZC3H4/WDR82 terminates antisense transcription from bidirectional promoters, but its mechanism is poorly understood. We report that ZC3H4/WDR82 immunoprecipitates with PP1 phosphatase and its nuclear targeting subunit, PP1 phosphatase nuclear targeting subunit (PNUTS), which binds to WDR82. AlphaFold predicts a complex of PP1/PNUTS with the restrictor where both PNUTS and ZC3H4 contact WDR82. A substrate trap, PP1^H66K-PNUTS, comprising inactive PP1 fused to the PNUTS C terminus, antagonizes restrictor-mediated termination, whereas PP1^WT-PNUTS has less of an effect, suggesting that phosphatase activity is required for termination. One PP1/PNUTS substrate implicated in termination by the restrictor is RNA polymerase II (RNA Pol II) CTD Ser5-P. PP1^H66K-PNUTS induces Ser5-P hyperphosphorylation at 5' ends, presumably by inhibiting dephosphorylation. NET-seq analysis suggests that CTD Ser5 dephosphorylation would promote termination by increasing RNA Pol II pausing. Both inhibition of termination and CTD hyperphosphorylation require the WDR82 binding domain of PP1^H66K-PNUTS, which mediates restrictor binding. In summary, the PP1/PNUTS phosphatase associated with the restrictor via WDR82 promotes efficient transcription termination.