RNA polymerase II activity revealed by GRO-seq and pNET-seq in Arabidopsis

RNA polymerase II (Pol II) play an essential role in gene expression. Here, we adapted plant Native Elongation Transcript sequencing and Global Run On sequencing to profile nascent RNA genome wide in Arabidopsis. We found Pol II tends to accumulate downstream of transcription start site and pausing at proximal promoter is an important regulatory step for Pol II transcription although loosely controlled. Furthermore, the Pol II with unphosphorylated carboxyl-terminal domain (CTD) mainly accumulates downstream the TSS, while the Ser5P CTD Pol II associates with spliceosome, and the Ser2P CTD Pol II presents a sharp peak 250 base pair downstream of polyadenylation site indicating a stringent control of termination for protein coding genes; whilst the termination of noncoding genes is not. Active expressed genes can be classified into three clusters according to the distribution patterns of different Pol II isoforms. In summary, we demonstrated the modified plant GRO-seq and pNET-seq are suitable to study RNA Pol II dynamics in planta. Although transcription is conserved among high eukaryotes, Pol II has its feature in Arabidopsis. Transcriptional profiling by GRO-seq and pNET-seq

RNA聚合酶II（RNA polymerase II，Pol II）在基因表达过程中发挥不可或缺的核心作用。本研究对植物原生延伸转录测序（Native Elongation Transcript sequencing，NET-seq）与全局运行-on测序（Global Run On sequencing，GRO-seq）技术进行适配改造，以在全基因组范围内对拟南芥的新生RNA进行图谱分析。我们发现，Pol II倾向于在转录起始位点（transcription start site，TSS）下游区域富集，而近端启动子处的Pol II暂停是Pol II转录调控的重要步骤，尽管该调控过程相对松散。进一步研究表明，携带未磷酸化羧基末端结构域（carboxyl-terminal domain，CTD）的Pol II主要富集于TSS下游；携带Ser5P修饰CTD的Pol II可与剪接体（spliceosome）相结合；而携带Ser2P修饰CTD的Pol II则在多聚腺苷酸化位点（polyadenylation site）下游250碱基对位置呈现尖锐富集峰，这提示蛋白编码基因的转录终止受到严格调控，而非编码基因的转录终止则无此特征。活性表达的基因可依据不同Pol II亚型的分布模式划分为三类聚类。综上，本研究证实经改造的植物GRO-seq与pNET-seq技术适用于在植物体内研究RNA Pol II的动态调控过程。尽管转录过程在高等真核生物中具有保守性，但Pol II在拟南芥中具有其物种特异性特征。借助GRO-seq与pNET-seq开展的转录谱分析