Heptad-Specific Phosphorylation of RNA Polymerase II CTD

• Published in: Molecular cell 2016-01, Vol.61 (2), p.305-314
• Main Authors: Schüller, Roland, Forné, Ignasi, Straub, Tobias, Schreieck, Amelie, Texier, Yves, Shah, Nilay, Decker, Tim-Michael, Cramer, Patrick, Imhof, Axel, Eick, Dirk
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Animals; Cell Line, Tumor; Cyclin-Dependent Kinase 9 - antagonists & inhibitors; Cyclin-Dependent Kinase 9 - metabolism; Humans; Mammals; Mass Spectrometry; Molecular Sequence Data; Peptide Library; Phosphorylation; Repetitive Sequences, Amino Acid; Reproducibility of Results; RNA Polymerase II - chemistry; RNA Polymerase II - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - metabolism; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Structure-Activity Relationship
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2015.12.003

The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5, and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD. •CTD variants make the entire CTD sequence accessible to mass spectrometry analysis•Valid CTD phosphosite mapping is based on a synthetic CTD peptide library•Predominant phosphorylation signatures prevail within neighboring CTD residues•Inhibition of CDK9 strongly reduces Ser2-P phosphorylation in the CTD A combined genetic and mass spectrometric approach offers comprehensive insights into CTD phosphorylation patterns and changes in CTD heptad-specific phosphorylation states in mammals and yeast.