Ultraviolet radiation alters the phosphorylation of RNA polymerase II large subunit and accelerates its proteasome-dependent degradation

It has been shown that ultraviolet (UV) radiation induces the ubiquitinatio n of the large subunit of RNA polymerase II (RNAP II-LS) as well as its pro teasomal degradation. Studies in mammalian cells have indicated that highly phosphorylated forms of RNAP II-LS are preferentially ubiquitinated, but s tudies in Saccharomyces cerevisiae have provided evidence that unphosphoryl ated 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 th e phosphorylation status of its C-terminal domain (CTD), was utilized to ev aluate the degradation of total cellular RNAP II-LS in human fibroblasts un der basal conditions or after UV-C (10 J/m(2)) irradiation. It was found th at UV radiation rapidly shifted the phosphorylation profile of RNAP II-LS f rom 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 whi ch have been implicated in the cellular UV response. In addition to shiftin g 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 d egradation is proteasome-dependent. However, the UV-induced loss of hypopho sphorylated RNAP II-LS was proteasome-independent. Lastly, UV radiation ind uced a similar shift to all hyperphosphorylated RNAP II-LS in Cockayne synd rome (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 we re not significantly altered when comparing CSA or CSB to repair competent control cells. The implications for the cellular Uv response are discussed. (C) 2001 Elsevier Science B.V All rights reserved.