title: Ultraviolet radiation alters the phosphorylation of RNA polymerase II large subunit and accelerates its proteasome-dependent degradation.
abstract: It has been shown that ultraviolet (UV) radiation induces the ubiquitination of the large subunit of RNA polymerase II (RNAP II-LS) as well as its proteasomal degradation. Studies in mammalian cells have indicated that highly phosphorylated forms of RNAP II-LS are preferentially ubiquitinated, but studies in Saccharomyces cerevisiae have provided evidence that unphosphorylated RNAP II-LS is an equally suitable substrate. In the present study, an antibody (ARNA-3) that recognizes all forms of RNAP II-LS, regardless of the phosphorylation status of its C-terminal domain (CTD), was utilized to evaluate the degradation of total cellular RNAP II-LS in human fibroblasts under basal conditions or after UV-C (10J/m(2)) irradiation. It was found that UV radiation rapidly shifted the phosphorylation profile of RNAP II-LS from a mixture of dephosphorylated and phosphorylated forms to entirely more phosphorylated forms. This shift in phosphorylation status was not blocked by pharmacologic inhibition of either the ERK or p38 pathways, both of which have been implicated in the cellular UV response. In addition to shifting the phosphorylation profile, UV radiation led to net degradation of total RNAP II-LS. UV-induced degradation of RNAP II-LS was also greatly reduced in the presence of the transcriptional and CTD kinase inhibitor DRB. Using a panel of protease inhibitors, it was shown that the bulk of UV-induced degradation is proteasome-dependent. However, the UV-induced loss of hypophosphorylated RNAP II-LS was proteasome-independent. Lastly, UV radiation induced a similar shift to all hyperphosphorylated RNAP II-LS in Cockayne syndrome (CS) cells of complementation groups A or B (CSA or CSB) when compared to appropriate controls. The UV-induced degradation rates of RNAP II-LS were not significantly altered when comparing CSA or CSB to repair competent control cells. The implications for the cellular UV response are discussed.
authors.affiliation: Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Luo
authors.forename: J
authors.initials: J
authors.lastname: Zheng
authors.forename: Y
authors.initials: Y
authors.lastname: Lu
authors.forename: D B
authors.initials: DB
authors.lastname: Bregman
mesh_terms: D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism / Q000528:radiation effects; D006801:Humans; D015151:Immunoblotting; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism*; D010766:Phosphorylation / Q000528:radiation effects; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D004004:Dichlororibofuranosylbenzimidazole; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D012319:RNA Polymerase II; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex