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.
Highlights
The transcription of protein coding genes is a highly regulated process that depends on the enzymatic activity of RNA polymerase II (RNAPII)1, a complex comprised of 12 subunits
RNAPII is Present in Ն2 MDa Complexes During Mitosis—To enhance our understanding of the protein interaction landscape of RNAPII, we purified RNAPII complexes under mild conditions from mitotic cells, where RNAPII is mostly excluded from the condensed chromosomes and distributed throughout the mitotic cytoplasm (Fig. 1A)
To investigate the size of RNAPII complexes in mitosis, we performed size fractionation of the mitotic lysate followed by Western blotting using various antibodies directed against subunits of RNAPII (RPB1, RPB2, and RPB3) and carboxy-terminal domain (CTD) phospho-epitopes (S2p and S5p; Fig. 1D)
Summary
Mitotic Cell Collection, Lysis, Gel Filtration and Immunoprecipitation—Mitotic cells were collected and frozen in conditions that preserve viability. False discovery rate was set to 1% at both the peptide and protein level. SiRNA results were analyzed by ANOVA (using SAS, version 9.1), testing for all main effects (siRNA treatment, day of transfection and Br-UTP labeling set) of which only the siRNA treatment and day of transfection were significant (both p Ͻ 0.0001). Confocal microscopy images from cryosections were collected using settings calibrated on the negative control (no antibody) samples without saturation of the intensity signal. ANOVA estimates were used to compare the blocking effects of antibodies to proteins of interest relative to control blocking antibodies using Student’s t test. Full experimental details and primary antibodies used are available in Supplementary Text
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