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

Item Type:Article
Title:Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease
Creators Name:Moeller, A. and Xie, S.Q. and Hosp, F. and Lang, B. and Phatnani, H.P. and James, S. and Ramirez, F. and Collin, G.B. and Naggert, J.K. and Babu, M.M. and Greenleaf, A.L. and Selbach, M. and Pombo, A.
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.
Keywords:Gene Expression, Mass Spectrometry, Molecular Biology, siRNA, Transcriptional Regulation, RNA Interference, RNA Polymerase II, In Vitro Transcription, Mitosis
Source:Molecular & Cellular Proteomics
ISSN:1535-9476
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:11
Number:6
Page Range:M111.011767
Date:June 2012
Official Publication:https://doi.org/10.1074/mcp.M111.011767
PubMed:View item in PubMed

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