In Vitro Transcription Recycling Assay Identifies PAF1 as a Driver of RNA Pol II Recycling

RNA Polymerase II transcriptional recycling is an underappreciated mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of novel RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from RNA Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed in vivo PAF1 complex cycling through gene bodies with RNA Pol II and recycling back to the transcription start sites, while PAF1 silencing triggered defective RNA Pol II recycling. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify RNA Pol II recycling as a potential target in cancer and demonstrate the assay’s applicability to characterize RNA Pol II recycling in cells and tissues from other disease states. We have performed time-resolved ChIP-seq of PAF1 and Pol II. PAF1 and Pol II ChIP-seq time course was generated during 0-, 10-, 20-, or 40-minute Flavopiridol treatment to block release of new Pol II into gene bodies and during 0-, 10-, 20- or 40-minute recovery time points after wash-out of Flavopiridol to release Pol II into the gene bodies in LNCaP-abl cells transfected with either non-targeting control siRNA or siPAF1.

RNA聚合酶II（RNA Polymerase II）转录再循环是一种长期未被充分重视的机制，其必需作用因子以及对整体基因表达水平的贡献迄今仍未被充分阐明。本研究报道了一种体外实验方法，可实现对新型RNA聚合酶II转录再循环因子的无偏鉴定，并对经再循环的转录复合物产生的转录输出进行定量检测。原理验证实验证实，PAF1复合体（PAF1 complex）组分属于转录再循环因子之一，并检测到PAF1敲低后RNA聚合酶II转录再循环的转录输出存在缺陷。动态染色质免疫沉淀测序（Dynamic ChIP-seq）结果证实，PAF1复合体可与RNA聚合酶II一同在基因体中循环，并重新回到转录起始位点；而PAF1沉默会引发RNA聚合酶II转录再循环缺陷。前列腺肿瘤组织中转录再循环活性增强，而基于抗体的PAF1敲低可削弱该异常激活现象。本研究结果将RNA聚合酶II转录再循环确立为癌症潜在治疗靶点，并证实该实验方法可用于表征其他疾病状态下细胞与组织中的RNA聚合酶II转录再循环。本研究已完成PAF1与RNA聚合酶II的时间分辨率染色质免疫沉淀测序（time-resolved ChIP-seq）。具体实验设计如下：在转染非靶向对照小干扰RNA（siRNA）或siPAF1的LNCaP-abl细胞中，设置两组时间进程样本：第一组为经夫拉平度（Flavopiridol）处理0、10、20、40分钟，以阻断新合成的RNA聚合酶II进入基因体；第二组为夫拉平度洗脱后恢复培养0、10、20、40分钟，以允许RNA聚合酶II进入基因体，最终获取PAF1与RNA聚合酶II的染色质免疫沉淀测序时间序列数据。