What's all the phos about? Insights into the phosphorylation state of the RNA polymerase II C-terminal domain via mass spectrometry

Abstract

RNA polymerase II (RNAP II) is one of the primary enzymes responsible for expressing protein-encoding genes and some small nuclear RNAs. The enigmatic carboxy-terminal domain (CTD) of RNAP II and its phosphorylation state are critically important in regulating transcription in vivo. Early methods of identifying phosphorylation on the CTD heptad were plagued by issues of low specificity and ambiguous signals. However, advancements in the field of mass spectrometry (MS) have presented the opportunity to gain new insights into well-studied processes as well as explore new frontiers in transcription. By using MS, residues which are modified within the CTD heptad and across repeats are now able to be pinpointed. Likewise, identification of kinase and phosphatase specificity towards residues of the CTD has reached a new level of accuracy. Now, MS is being used to investigate the crosstalk between modified residues of the CTD and may be a critical technique for understanding how phosphorylation plays a role in the new LLPS model of transcription. Herein, we discuss the development of various MS techniques and evaluate their capabilities. By highlighting the pros and cons of each technique, we aim to provide future investigators with a comprehensive overview of how MS can be used to investigate the complexities of RNAP-II mediated transcription.