A phosphorylation pattern-recognizing antibody specifically reacts to RNA polymerase II bound to exons

The C-terminal domain of RNA polymerase II is an unusual series of repeated residues appended to the C-terminus of the largest subunit and serves as a flexible binding scaffold for numerous nuclear factors. The binding of these factors is determined by the phosphorylation patterns on the repeats in the domain. In this study, we generated a synthetic antibody library by replacing the third heavy chain complementarity-determining region of an anti-HER2 (human epidermal growth factor receptor 2) antibody (trastuzumab) with artificial sequences of 7–18 amino-acid residues. From this library, antibodies were selected that were specific to serine phosphopeptides that represent typical phosphorylation patterns on the functional unit (YSPTSPS)2 of the RNA polymerase II C-terminal domain (CTD). Antibody clones pCTD-1stS2 and pCTD-2ndS2 showed specificity for peptides with phosphoserine at the second residues of the first or second heptamer repeat, respectively. Additional clones specifically reacted to peptides with phosphoserine at the fifth serine of the first repeat (pCTD-1stS5), the seventh residue of the first repeat and fifth residue of the second repeat (pCTD-S7S5) or the seventh residue of either the first or second repeat (pCTD-S7). All of these antibody clones successfully reacted to RNA polymerase II in immunoblot analysis. Interestingly, pCTD-2ndS2 precipitated predominately RNA polymerase II from the exonic regions of genes in genome-wide chromatin immunoprecipitation sequencing analysis, which suggests that the phosphoserine at the second residue of the second repeat of the functional unit (YSPTSPS)2 is a mediator of exon definition. Antibodies that bind to an enzyme that allows genes to make the proteins they encode could prove a valuable research tool. The ‘RNA polymerase II’ enzyme copies a gene into messenger RNAs – molecules that carry genetic information from the cell nucleus to direct protein synthesis out in the cytoplasm. The enzyme has an unusual pattern of amino acids carrying phosphate groups at one end of its protein chain. Hyori Kim at the University of Ulsan in South Korea, with Junho Chung and co-workers at Seoul National University, created a series of antibodies that selectively bind to these ‘phosphorylated’ regions of the enzyme depending on the phosphorylation pattern. The antibodies are being used to explore how different phosphorylation patterns control the binding of this vital enzyme to molecules required for gene activity, and to specific sites on DNA like exon and intron.