Fixation-induced redistribution of hyperphosphorylated RNA polymerase II in the nucleus of human cells.
RNA polymerase II (pol II) transcribes the most varied group of genes and is present in hypo- and hyperphosphorylated forms, with residues Ser(2) and Ser(5) of the C-terminal domain (CTD) of the largest subunit as main targets of phosphorylation. The elongating (active) form is phosphorylated on Ser(2) and can be specifically recognized with the H5 antibody. It has been found in different nuclear distributions: in discrete sites throughout the nucleoplasm, consistent with a role in transcription, and/or concentrated in "splicing speckles", a nuclear compartment mostly devoid of transcriptional activity. Here, we assess the effects of cell fixation and permeabilization on the distribution of polymerase II and correlate its distribution with the preservation of cellular ultrastructure. We show that phospho-Ser(2) polymerase II can redistribute to, or be differentially retained in, "speckles" in conditions that do not preserve cellular ultrastructure. The fixation protocols that disrupt polymerase II distribution also cause partial or total loss of TATA-binding protein, Sm antigen and PML staining in PML bodies, and have no noticeable effect in the labeling of SC35 in "splicing speckles" or coilin in Cajal bodies. When nuclear ultrastructure is preserved, phospho-Ser(2) polymerase II is found in discrete sites throughout the nucleoplasm, without visible enrichment within splicing speckles. A minor proportion of the total amount of the phospho-Ser(2) form is present in these domains.
Guillot
Pascale V
PV
MRC-Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, London W12 0NN, UK.
Xie
Sheila Q
SQ
Hollinshead
Michael
M
Pombo
Ana
A
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001324:Autoantigens; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D005404:Fixatives; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D010755:Organophosphates / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D055517:snRNP Core Proteins
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D005404:Fixatives; D010755:Organophosphates; D017411:Ribonucleoproteins, Small Nuclear; D055517:snRNP Core Proteins; D012694:Serine; C009524:tributyl phosphate; D012319:RNA Polymerase II