Transcriptional Pause Sites Delineate Stable Nucleosome-Associated Premature Polyadenylation Suppressed by U1 snRNP [2P-seq, Pabpn1 CKO]. Transcriptional Pause Sites Delineate Stable Nucleosome-Associated Premature Polyadenylation Suppressed by U1 snRNP [2P-seq, Pabpn1 CKO]

Regulation of RNA polymerase II (Pol II) elongation is a critical step in gene regulation. Here, we report that U1 snRNP recognition and transcription pausing at stable nucleosomes are linked through premature polyadenylation signal (PAS) termination. By generating RNA exosome conditional deletion mouse embryonic stem cells, we identified a large class of polyadenylated short transcripts in the sense direction destabilized by the RNA exosome. These PAS termination events are enriched at the first few stable nucleosomes flanking CpG islands and suppressed by U1 snRNP. Thus, promoter-proximal Pol II pausing consists of two processes: TSS-proximal and +1 stable nucleosome pausing, with PAS termination coinciding with the latter. While pausing factors NELF/DSIF only function in the former step, flavopiridol-sensitive mechanism(s) and Myc modulate both steps. We propose that premature PAS termination near the nucleosome-associated pause site represents a common transcriptional elongation checkpoint regulated by U1 snRNP recognition, nucleosome stability, and Myc activity. Overall design: 3'-End sequencing (2P-seq) in Pabpn1 conditional KO mESCs

RNA聚合酶II（Pol II）的延伸调控是基因调控的关键环节。本研究发现，U1小核糖核蛋白颗粒（U1 snRNP）的识别与稳定核小体处的转录暂停，可通过提前聚腺苷酸化信号（PAS）介导的终止过程形成关联。我们通过构建RNA外切酶体（RNA exosome）条件性敲除的小鼠胚胎干细胞，鉴定得到一类由RNA外切酶体介导发生不稳定降解的正义链方向多聚腺苷酸化短转录本。这类PAS终止事件富集于CpG岛（CpG islands）侧翼的前数个稳定核小体区域，并可被U1 snRNP所抑制。由此可见，启动子近端的Pol II暂停包含两个过程：转录起始位点（Transcription Start Site，TSS）近端暂停与+1位稳定核小体暂停，其中PAS终止与后者相契合。尽管暂停因子NELF/DSIF仅在前一过程中发挥功能，但黄酮哌啶醇敏感的调控机制及Myc可同时调控两个步骤。我们提出，核小体相关暂停位点附近的提前PAS终止，代表了一类由U1 snRNP识别、核小体稳定性及Myc活性共同调控的常见转录延伸检查点。整体实验设计：在Pabpn1条件性敲除小鼠胚胎干细胞中开展3'端测序（2P-seq）。