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Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease

Authors

  • A. Moeller
  • S.Q. Xie
  • F. Hosp
  • B. Lang
  • H.P. Phatnani
  • S. James
  • F. Ramirez
  • G.B. Collin
  • J.K. Naggert
  • M.M. Babu
  • A.L. Greenleaf
  • M. Selbach
  • A. Pombo

Journal

  • Molecular & Cellular Proteomics

Citation

  • Mol Cell Proteomics 11 (6): M111.011767

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, amongst 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.


DOI

doi:10.1074/mcp.M111.011767