Fig. 4: The UV-induced degradation of promoter-bound Pol II is regulated in trans.
a, b Left panels: Representative image of a GFP-RPB1 KI cell expressing XPC-mCherry as a live-cell damage marker within 1 h (a) or 1 to 2 h (b) after UV irradiation through a porous filter to induce local DNA damage. Cells containing local UV-damage (top images) are marked by local accumulation of XPC-mCherry (outlined with dotted circle) or without local damage (bottom images), or non-treated cells (NT) were used for FRAP analysis. GFP-RPB1 FRAP was analyzed outside the local UV damaged, indicated by box. Middle panels: FRAP analysis of GFP-RPB1 cells that were non-treated (NT) or 0–1 h (a) or 1–2 h (b) after local UV irradiation through a porous filter. Right panels: GFP-RPB1 pre-bleach fluorescence intensity (FI) ± SD of cells analysed by FRAP as a measure for Pol II protein levels. N = 23 cells for ‘NT’, n = 10 cells for ‘no local damage 0–1 h’, n = 29 cells for ‘local UV damage 0–1 h’, n = 35 cells for ‘no local damage 1–2 h’, n = 30 cells for ‘local UV damage 1–2 h’. Cells were measured in 3 independent experiments. c Western blots after cellular fractionation of GFP-RPB1 knock in (KI) cells after control siRNA transfection (siCTRL), or siRNA-mediated gene knockdown of the indicated E3 ubiquitin ligases in non-perturbed conditions (0 J) or 1 h after irradiation with 4 J/m2 (4 J). NTD = RPB1 N-terminal domain, P-Ser = phospho-serine. Experiment has been performed two times with similar results. d Fluorescence recovery after photo-bleaching (FRAP) of GFP-RPB1 in wild type (WT) or in NER knock out (KO) MRC5 cells without UV irradiation (0 J) or 1 h after irradiation with 4 J/m2 (4 J 1 h). GFP-RPB1 was bleached and fluorescence intensity was measured every 0.4 sec, background-corrected and normalized to pre-bleach fluorescence intensity (FI), which was set to 100. RFI = relative FI. Right panel: Mean ±SD of pre-bleach GFP-RPB1 FI as a measure for Pol II protein levels in nuclei analysed by FRAP. N = 32 cells for ‘WT 4 J 1–2 h’ and ‘CSB KO 0 J 1–2 h’ measured in 4 independent experiments, n = 16 cells for all other conditions measured in 2 independent experiments. ***P ≤  0.001, ****P ≤  0.0001, analyzed by ordinary one-way ANOVA using Dunnett’s multiple comparisons test versus NT (a, b) or respective 0 J condition (d).
