Exp Mol Med.  2016 Nov;48(11):e271. 10.1038/emm.2016.101.

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

• Han J
• Lee JH
• Park S
• Yoon S
• Yoon A
• Hwang DB
• Lee HK
• Kim MS
• Lee Y
• Yang WJ
• Youn HD
• Kim H
• Chung J

• KMID: 2362295
• DOI: http://doi.org/10.1038/emm.2016.101

Abstract

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)â‚‚ 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.

MeSH Terms

Antibodies
Chromatin Immunoprecipitation
Clone Cells
Complementarity Determining Regions
DNA-Directed RNA Polymerases*
Exons*
Peptides
Phosphopeptides
Phosphorylation*
Phosphoserine
Receptor, Epidermal Growth Factor
RNA Polymerase II*
RNA*
Sensitivity and Specificity
Serine
Antibodies
Complementarity Determining Regions
DNA-Directed RNA Polymerases
Peptides
Phosphopeptides
Phosphoserine
RNA
RNA Polymerase II
Receptor, Epidermal Growth Factor
Serine