title: A phosphorylation pattern-recognizing antibody specifically reacts to RNA polymerase II bound to exons.
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)2of 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)2is a mediator of exon definition.
authors.affiliation: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.|Department of Biomedical science, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Jungwon
authors.initials: J
authors.lastname: Han
authors.affiliation: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.|Department of Biomedical science, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Jong-Hyuk
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Sunyoung
authors.initials: S
authors.lastname: Park
authors.affiliation: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Soomin
authors.initials: S
authors.lastname: Yoon
authors.affiliation: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Aerin
authors.initials: A
authors.lastname: Yoon
authors.affiliation: Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Do B
authors.initials: DB
authors.lastname: Hwang
authors.affiliation: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Hwa K
authors.initials: HK
authors.lastname: Lee
authors.affiliation: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Min S
authors.initials: MS
authors.lastname: Kim
authors.affiliation: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Yujean
authors.initials: Y
authors.lastname: Lee
authors.affiliation: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Won J
authors.initials: WJ
authors.lastname: Yang
authors.affiliation: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.|Department of Biomedical science, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Hong-Duk
authors.initials: HD
authors.lastname: Youn
authors.affiliation: Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea.
authors.forename: Hyori
authors.initials: H
authors.lastname: Kim
authors.affiliation: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.|Department of Biomedical science, Seoul National University College of Medicine, Seoul, Republic of Korea.|Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Junho
authors.initials: J
authors.lastname: Chung
mesh_terms: D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D005091:Exons; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D012319:RNA Polymerase II / Q000276:immunology / Q000378:metabolism*
chemical_list: D000906:Antibodies; D012319:RNA Polymerase II