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

阻遏复合物（restrictor complex）WDR82/ZC3H4是双向启动子反义转录的主要终止因子，但其作用机制尚未阐明。本研究发现，蛋白磷酸酶1（PP1）的核靶向亚基PNUTS可通过其WDR82亚基与该阻遏复合物结合。AlphaFold预测得到四聚体复合物PPWZ，其中结合PP1的PNUTS与ZC3H4均可与WDR82产生相互作用。将失活型PP1H66K融合至PNUTS C端序列所构建的嵌合蛋白，可作为反义转录终止及羧基末端结构域（CTD）丝氨酸5位去磷酸化的显性负效抑制剂，且这两种生物学活性均依赖于PNUTS的WDR82结合结构域。PP1H66K-PNUTS属于底物陷阱蛋白，同时可与RNA聚合酶II（pol II）大亚基及核外酶体组分发生相互作用。丝氨酸5位高度磷酸化的pol II相较于总pol II，具有更高的持续合成能力且停顿频率更低。本研究据此提出：丝氨酸5位磷酸化的pol II其延伸效率提升可拮抗早期终止过程，而PP1/PNUTS/WDR82/ZC3H4介导的丝氨酸5位去磷酸化与反义转录终止存在偶联关系。实验整体设计：对表达强力霉素（doxycycline，dox）诱导型PP1-PNUTS融合蛋白的溴尿苷脉冲标记HEK293细胞进行新生RNA测序（nascent RNA-seq）。