title: Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease.
abstract: RNA polymerase II (RNAPII) transcribes protein-coding genes in eukaryotes and interacts with factors involved in chromatin remodeling, transcriptional activation, elongation, and RNA processing. Here, we present the isolation of native RNAPII complexes using mild extraction conditions and immunoaffinity purification. RNAPII complexes were extracted from mitotic cells, where they exist dissociated from chromatin. The proteomic content of native complexes in total and size-fractionated extracts was determined using highly sensitive LC-MS/MS. Protein associations with RNAPII were validated by high-resolution immunolocalization experiments in both mitotic cells and in interphase nuclei. Functional assays of transcriptional activity were performed after siRNA-mediated knockdown. We identify >400 RNAPII associated proteins in mitosis, among these previously uncharacterized proteins for which we show roles in transcriptional elongation. We also identify, as novel functional RNAPII interactors, two proteins involved in human disease, ALMS1 and TFG, emphasizing the importance of gene regulation for normal development and physiology.
authors.affiliation: MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, London W12 0NN, United Kingdom. andre.moeller.rnapii@gmail.com
authors.forename: André
authors.initials: A
authors.lastname: Möller
authors.forename: Sheila Q
authors.initials: SQ
authors.lastname: Xie
authors.forename: Fabian
authors.initials: F
authors.lastname: Hosp
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lang
authors.forename: Hemali P
authors.initials: HP
authors.lastname: Phatnani
authors.forename: Sonya
authors.initials: S
authors.lastname: James
authors.forename: Francisco
authors.initials: F
authors.lastname: Ramirez
authors.forename: Gayle B
authors.initials: GB
authors.lastname: Collin
authors.forename: Jürgen K
authors.initials: JK
authors.lastname: Naggert
authors.forename: M Madan
authors.initials: MM
authors.lastname: Babu
authors.forename: Arno L
authors.initials: AL
authors.lastname: Greenleaf
authors.forename: Matthias
authors.initials: M
authors.lastname: Selbach
authors.forename: Ana
authors.initials: A
authors.lastname: Pombo
mesh_terms: D002850:Chromatography, Gel; D004194:Disease; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007399:Interphase; D008938:Mitosis; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D025941:Protein Interaction Mapping; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020543:Proteome / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics; D034622:RNA Interference; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D009687:Nuclear Proteins; D021122:Protein Subunits; D020543:Proteome; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins; D012319:RNA Polymerase II