Pol II Preferentially Regulates Ribosomal Protein Expression by Trapping Disassociated Subunits [ChIP-seq]

Pol II has been recognized as a passively regulated holoenzyme. However, whether Pol II plays specific regulatory roles remain unclear. Here, fractions containing disassociated RPB3 (dRPB3) were identified by size exclusion chromatography in various cells. Through a unique strategy, Specific Degradation of Disassociated Subunits (SDDS), we demonstrated that dRPB3 functions as a regulatory component of Pol II to enable the preferential control of 3’ end processing of ribosomal protein genes directly through N-terminal domain of RPB3. Machine learning analysis of large-scale genomic features revealed that the little elongation complex helps to specialize the functions of dRPB3. Mechanistically, dRPB3 facilitates CBC-PCF11 axis activity to increase the efficiency of 3’ end processing. Furthermore, RPB3 is dynamically regulated during development and diseases. These findings suggest that Pol II gains specific regulatory functions by trapping disassociated subunits. We performed ChAR-seq, TT-seq, RNA-seq, and ChIP-seq to investigate the roles of various specialized subunits in transcription and post-transcriptional regulation. The V6.5 mouse ES (mES) cell line was used for all the high-throughput analyses. Degron mES cells were pretreated with 1 μg/ml Doxycycline for 12h to induce TIR1 expression, and treated with or without 500 μM IAA for different hours; refer to individual Series. For TT-seq, ChAR-seq, and RNA-seq, the Drosophila S2 cells were used as the spike-in cells, and for ChIP-seq, the HEK293T cells were used as the spike-in cells. This dataset includes the ChIP-seq data.RNA聚合酶II（Pol II）已被认定为一种被动调控的全酶。然而，Pol II是否发挥特异性调控功能仍不明确。本研究通过尺寸排阻色谱法在多种细胞中鉴定到了含有解离型RPB3（dRPB3）的组分。通过一种名为解离亚基特异性降解法（Specific Degradation of Disassociated Subunits, SDDS）的独特策略，我们证实dRPB3作为Pol II的调控组分，可通过RPB3的N端结构域直接特异性调控核糖体蛋白基因的3'端加工过程。对大规模基因组特征开展的机器学习分析显示，小延伸复合物（little elongation complex）可协助特化dRPB3的功能。机制层面，dRPB3可增强CBC-PCF11信号轴的活性，从而提升3'端加工的效率。此外，RPB3的表达在发育与疾病进程中呈现动态调控特征。上述研究结果表明，Pol II可通过捕获解离亚基获得特异性调控功能。本研究开展了染色质关联RNA测序（Chromatin-associated RNA sequencing, ChAR-seq）、4-硫尿苷标记转录组测序（TT-seq）、RNA测序（RNA-seq）以及染色质免疫沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq），以探究各类特化亚基在转录及转录后调控中的作用。本研究所有高通量分析均采用V6.5小鼠胚胎干细胞（mouse embryonic stem, mES）系。降解标签胚胎干细胞先用1 μg/ml多西环素预处理12小时以诱导TIR1的表达，随后分别用或不用500 μM吲哚-3-乙酸（IAA）处理不同时长；具体细节参见各对应系列。针对TT-seq、ChAR-seq与RNA-seq实验，我们采用果蝇S2细胞作为外参对照细胞；针对ChIP-seq实验，则采用人胚肾293T（HEK293T）细胞作为外参对照细胞。本数据集包含ChIP-seq相关数据。