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 immunoprecipitate with PP1 phosphatase and its nuclear targeting subunit, PNUTS, which binds to WDR82. AlphaFold predicts a complex of PP1/PNUTS with restrictor where both PNUTS and ZC3H4 contact WDR82. A substrate trap, PP1H66K-PNUTS, comprising inactive PP1 fused to the PNUTS C-terminus antagonizes restrictor mediated termination whereas PP1WT-PNUTS has less effect suggesting that phosphatase activity is required for termination. One PP1/PNUTS substrate implicated in termination by restrictor is pol II CTD Ser5-P. PP1H66K-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 pol II pausing. Both inhibition of termination and CTD hyperphosphorylation require the WDR82 binding domain of PP1H66K-PNUTS that mediates restrictor binding. In summary, the PP1/PNUTS phosphatase associated with restrictor via WDR82 promotes efficient transcription termination.