Restrictor synergizes with Symplekin and PNUTS to terminate extragenic transcription

Transcription termination pathways mitigate the detrimental consequences of unscheduled promiscuous initiation occurring at hundreds of thousands of genomic cis-regulatory elements, thus representing cornerstones of genomic regulation in eukaryotes. Restrictor, a complex conserved from worms to humans and composed of the RNA-binding protein ZC3H4 and of WDR82, a Pol II-carboxyterminal repeat domain (CTD)-interacting protein, is required to suppress long-range, processive transcription at thousands of extragenic sites, with instead comparatively limited effects on gene transcription. Restrictor contains no RNA cleavage subunit and does not coopt known cleavage complexes, indicating alternative termination mechanisms. Here we show that Restrictor-driven termination involves Symplekin, a protein associated with RNA cleavage complexes but also involved in cleavage-independent, phosphatase-dependent termination of non-coding RNAs in yeast. Competition among Restrictor, the cleavage and polyadenylation specificity complex (CPSF) and the histone cleavage complex for a limited pool of Symplekin resulted in mutual regulation, thus revealing unexpected regulatory interactions among multiple transcription termination pathways and a general role of Symplekin in both genic and extragenic cleavage-independent termination pathways. Transcriptome analysis to measure transcription in HeLa TREx Flp-In cells, overexpressed by NH2, COOH and ZC3H4 full length

转录终止通路可缓解数十万基因组顺式调控元件（cis-regulatory elements）处发生的非计划性杂乱转录起始所带来的有害后果，因此是真核生物基因组调控的核心基石。Restrictor是一类从线虫保守至人类的复合物，由RNA结合蛋白（RNA-binding protein）ZC3H4以及RNA聚合酶II（Pol II）羧基末端重复结构域（CTD）相互作用蛋白WDR82组成，其功能为抑制数千个基因外位点处的长距离持续性转录，而对基因转录的影响相对有限。Restrictor复合物不含RNA切割亚基，也不征用已知的切割复合物，这提示其存在独特的转录终止机制。本研究发现，Restrictor介导的终止过程涉及Symplekin蛋白（Symplekin）——该蛋白既可与RNA切割复合物结合，也参与酵母中非编码RNA的不依赖切割、依赖磷酸酶的转录终止过程。Restrictor、切割与多聚腺苷酸化特异性复合物（CPSF）以及组蛋白切割复合物之间，针对有限Symplekin蛋白池的竞争会产生相互调控效应，由此揭示了多种转录终止通路间此前未被发现的调控互作，以及Symplekin蛋白在基因内与基因外两类不依赖切割的转录终止通路中的通用功能。本研究通过转录组分析，对经NH₂端、COOH端及全长ZC3H4过表达的HeLa TREx Flp-In细胞中的转录水平进行了检测。