title: Distribution of different phosphorylated forms of RNA polymerase II in relation to Cajal and PML bodies in human cells: an ultrastructural study.
abstract: The mammalian nucleus is a highly organised organelle that contains many subcompartments with roles in DNA replication and repair, gene expression and RNA processing. Cajal and promyelocytic leukaemia (PML) bodies are discrete nuclear structures with specific molecular signatures. RNA polymerase II and many transcription factors have been identified within these compartments by immunofluorescence microscopy, suggesting a role in polymerase II assembly or transcriptional activity. Here, we have examined the presence of different phosphorylated forms of polymerase II and newly made RNA in Cajal and PML bodies using high-resolution imaging of ultrathin cryosections (approximately 120 nm thick) with fluorescence and electron microscopes. We show that Cajal bodies contain polymerase II phosphorylated on Ser5, and not the Ser2-phosphorylated (active) form or newly made RNA. The presence of polymerase II in the absence of transcriptional activity suggests that Cajal bodies have roles in polymerase assembly or transport, but not in gene transcription. PML bodies contain no detectable polymerase II or nascent RNA in HeLa cells, at the resolution achieved by electron microscopy, but are often surrounded by these markers at distances>25 nm. These results support the view that although PML bodies are present in transcriptionally active areas of the nucleus, they are not generally sites of polymerase II assembly, transport or activity.
authors.affiliation: MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.
authors.forename: Sheila Q
authors.initials: SQ
authors.lastname: Xie
authors.forename: Ana
authors.initials: A
authors.lastname: Pombo
mesh_terms: D000906:Antibodies; D020541:Coiled Bodies / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D015473:Leukemia, Promyelocytic, Acute / Q000201:enzymology*; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D010766:Phosphorylation; D012319:RNA Polymerase II / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000096:biosynthesis; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D000906:Antibodies; D012334:RNA, Neoplasm; C018009:5-bromouridine triphosphate; D012319:RNA Polymerase II; D014544:Uridine Triphosphate