Variation of C-terminal domain governs RNA polymerase II genomic locations and alternative splicing in eukaryotic transcription (RNA-Seq)

The C-terminal domain of RPB1 (CTD) orchestrates transcription by recruiting regulators to RNA Pol II upon phosphorylation. Recent insights highlight CTD’s pivotal role in driving condensate formation on gene loci. Yet, the molecular mechanism behind how CTD-mediated recruitment of transcriptional regulators influences condensates formation remains unclear. Our study unveils that phosphorylation reversibly dissolves phase separation induced by the unphosphorylated CTD. Phosphorylated CTD, upon specific association with transcription regulatory proteins, forms distinct condensates from unphosphorylated CTD. Function studies demonstrate CTD variants with diverse condensation properties in vitro exhibit difference in promoter binding and mRNA co-processing in cells. Notably, varying CTD lengths lead to alternative splicing outcomes impacting cellular growth, linking the evolution of CTD variation/length with the complexity of splicing from yeast to human. These findings provide compelling evidence for a model wherein post-translational modification enables the transition of functionally specialized condensates, highlighting a co-evolution link between CTD condensation and splicing. To investigate gene expression and RNA splicing of different CTD length, we overexpressed RPB1_52xCTD and RPB1_26xCTD in HEK 293T cells, upon α-amanitin administration, we then performed gene expression and alterative splicing profiling using data obtained from RNA-seq of 2 conditions (HEK 293T_RPB1_52xCTD vs HEK 293T RPB1_26xCTD) with 2 replicates respectively.RPB1的C端结构域（C-terminal domain, CTD）可在磷酸化后招募调控因子至RNA聚合酶II（RNA Pol II），从而调控转录进程。近期研究揭示，CTD在基因位点驱动凝聚体形成的过程中发挥关键作用。然而，CTD介导的转录调控因子招募如何影响凝聚体形成的分子机制仍不明确。本研究发现，磷酸化可可逆地解离未磷酸化CTD诱导的相分离（phase separation）。磷酸化CTD在与转录调控蛋白特异性结合后，会形成与未磷酸化CTD截然不同的凝聚体。功能研究表明，体外具备不同凝聚特性的CTD变体，在细胞内的启动子结合与mRNA共加工过程中存在差异。值得注意的是，CTD长度的差异会导致可变剪接（alternative splicing）结果改变，进而影响细胞生长；这一现象将CTD变异/长度的演化与从酵母到人类的剪接复杂性关联起来。本研究结果为"翻译后修饰（post-translational modification）介导功能特化凝聚体转化"的模型提供了有力证据，同时揭示了CTD凝聚与剪接之间的共演化关联。为探究不同CTD长度下的基因表达与RNA剪接情况，本研究在HEK 293T细胞中过表达RPB1_52xCTD与RPB1_26xCTD；在施加α-鹅膏蕈碱（α-amanitin）处理后，利用两个实验组（每组均设置2个生物学重复）的RNA测序（RNA-seq）数据，完成了基因表达与可变剪接谱分析，两个实验组分别为HEK 293T_RPB1_52xCTD与HEK 293T_RPB1_26xCTD。