Targeted protein degradation reveals Pol II heterogeneity and functional diversity [RNA-Seq]

RNA polymerase II (Pol II) subunits are thought to be involved in various transcription-associated processes, but it is unclear whether they play different regulatory roles in modulating gene expression. Here, we performed nascent and mature transcript sequencing after the acute degradation of 12 mammalian Pol II subunits and profiled their genomic binding sites and protein interactomes to dissect their molecular functions. We found that Pol II subunits contribute differently to Pol II cellular localization and transcription process and preferentially regulate RNA processing (such as RNA splicing and 3’ end maturation). Genes sensitive to the depletion of different Pol II subunits tend to be involved in diverse biological functions and show different RNA half-lives. Sequences, associated protein factors, and RNA structures are correlated with Pol II subunit-mediated differential gene expression. These findings collectively suggest that the heterogeneity of Pol II and different genes appear to depend on some of the subunits. We performed PRO-seq, ChAR-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 PRO-seq, ChAR-seq, and RNA-seq, the Drosophila S2 cells were used as the spike-in cells, and for ChIP-Rx, the ZBTB11-GFP expressing cells were used as the spike-in cells.