Variation of C-terminal domain governs RNA polymerase II genomic locations and alternative splicing in eukaryotic transcription (ChiP-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. Overall design: To investigate genomic locations of RPB1 with different CTD length or charges, we overexpressed RPB1_52xCTD, RPB1_26xCTD or RPB1_26xCTD T4E/S7E mutants with YFP tag in HEK 293T cells, then performed Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) by GFP antibody with 2 replicates respectively. IP IgG from wild-type HEK 293T cells as control.