Cellular sensitivity to UV-irradiation is mediated by RNA polymerase I transcription

Abstract

The nucleolus has long been considered to be a pure ribosome factory. However, over the last two decades it became clear that the nucleolus is involved in numerous other functions besides ribosome biogenesis. Our experiments indicate that the activity of RNA polymerase I (Pol I) transcription monitors the integrity of the DNA and influences the response to nucleolar stress as well as the rate of survival. Cells with a repressed ribosomal DNA (rDNA) transcription activity showed an increased and prolonged p53 stabilisation after UVC-irradiation. Furthermore, p53 stabilisation after inhibition and especially after UVC-irradiation might be due to abrogation of the HDM2-p53 degradation pathway by ribosomal proteins (RPs). Apoptosis mediated by highly activated p53 is a typical hallmark of Cockayne syndrome cells and transcriptional abnormalities and the following activation of the RP-HDM2-p53 pathway would be a possible explanation.

Fig 1

UVC-irradiation repressed the expression of full length transcripts (A) but stimulated Pol I transcription initiation (B). Young foreskin fibroblasts (FF95) with a cumulative population density under 25 were irradiated with 10J/m² UVC and isolated RNA after the indicated time points was used for northern blot analysis (A) as well as qPCR amplifying the initiation (5`ETS) and elongation (5.8 ITS) sites of the pre-rRNA (B). (C) Whole cell lysates of irradiated (10J/m²) FF95 have been analysed for p53 stabilisation by western blot. Values are mean ± SD of three independent experiments (* = ρ<0.05, ** = ρ<0.01, *** = ρ<0.001). Blots are representatives of at least three independent experiments.

Fig 2. Repression of Pol I transcription by inhibitors rendered cells more sensitive to UVC-mediated DNA damage.

Survival rate of cells with different Pol I transcription activity after UVC-irradiation. FF95 and CS cells were UVC-irradiated (10J/m²) and cell survival was measured by counting (A). Primary fibroblasts were treated with low-dose (10ng/ml) actinomycin D (B) or HCT116 cells with 150nM CX-5461 (C) in combination with UVC-irradiation (10J/m²). (D) Cells with a repressed Pol I transcription undergo apoptosis more frequently after UVC-irradiation. Identification of a hypodiploid DNA content of FF95, CS3BE (CSA) and CS1AN (CSB) after UVC-irradiation (Nicoletti). CS cells showed a significant increase in apoptosis after UVC-irradiation (10J/m²) (D). Inhibition of rDNA transcription in HCT116 cells by knockdown of TIF-IA (E) as well as by the specific compound CX-5461 (F) increased apoptotic cell death significantly. (G-I) HCT116 cells transfected with TIF-IA (G) show a significant increase in rRNA synthesis (H) and less apoptosis (I) after UVC-irradiation. (10J/m²). Values are mean ± SD of three independent experiments (* = ρ<0.05, ** = ρ<0.01, *** = ρ<0.001).

Fig 3

(A) TIF-IA silencing by shRNA led to increased and prolonged p53 levels after UVC-irradiation. Stable transfected HCT116 cells have been irradiated with 10J/m² UVC and analysed for p53 stabilisation by western blot. (B) P53 levels in whole cell lysates of HCT116 cells treated with the specific Pol I inhibitor CX-5461 and irradiated with 10J/m² UVC. (C, D) CoIP experiments of HCT116 cells treated with either CX-5461 or UVC-irradiation or a combination of both. Cells were first incubated with 5μM of MG132 for 12h. Afterwards one group was treated with 150nM CX-5461, a second group was irradiated with 10J/m² UVC and the third group was treated with 150nM CX-5461 as well as irradiated with 10J/m² UVC. Whole cell lysates were prepared 24h after UVC-irradiation and subsequently used for CoIP. Precipitated and co-precipitated proteins were analysed on western blots. (C) IP of p53 and CoIP of HDM2. (D) IP of HDM2 and CoIP of ribosomal protein L11 (rp L11). Blots are representatives of at least three independent experiments.