Fig. 4: Under-ubiquitylated RNAPII is cleared by the VCP segregase in the absence of TFIIH in the TCR complex.
From: Hierarchical mechanisms control the clearance of DNA lesion–stalled RNA polymerase II
a Top: Cartoon depicting the effect of UVSSAKO on TCR complex assembly. Bottom: Quantification of local DRB run-off assay as described for Fig. 1a-c RPE1 WT and UVSSAKO with or without VCPi treatment (NMS-873; 5 µM; 1 h pretreatment, then incubation for the indicated times after UV-C). b Representative images of RNAPII-S2P signal detected by western blot from whole cell lysates of primary fibroblasts from normal (48BR) or UVSSA-defective cells (Kps3) at 0, 2, 4, and 6 h after global UV-C (10 J/m2). Cells were incubated with VCPi (CB5083; 10 µM) for 1 h before UV-C and incubated in the same media. RNAPII-S2P was detected with an antibody against phosphorylated C-terminal domain (CTD) Ser2/5 (BioLegend, 920203 (H5)). Short and long exposures are shown (top and middle panels, respectively). SMC3 was used as loading control. This experiment was repeated three times. c Detection of RNAPII-S2P ubiquitylation (Cell Signaling, 3936) and TFIIH recruitment (p89 and p62) by western blot after immunoprecipitation of RNAPII-S2P (right) from chromatin fractions (left) of RPE1 WT and indicated TCR KOs. Cells were incubated with or without VCPi (NMS-873; 5 µM) for 1 h before 12 J/m2 UV-C. Then, cells were incubated in the same media and collected 1 h after UV-C. This experiment was repeated three times. d Detection of RNAPII-S2P ubiquitylation (Cell Signaling, 3936) and TFIIH recruitment (p89 and p62) by western blot after immunoprecipitation of RNAPII-S2P (right) from the chromatin fraction (left) of RPE1 WT cells or UVSSAKO with or without complementation of the indicated UVSSA mutants. Cells were treated with 12 J/m2 UV-C and collected after 1 h. This experiment was repeated three times. e Top: Cartoon depicting the effect of UVSSAΔTFIIH on TCR complex assembly. Bottom: Quantification of the local DRB run-off immunofluorescence assay as described for Fig. 1a-c in RPE1 WT, UVSSAKO, and UVSSAKO stably expressing the indicated UVSSA constructs and with or without VCPi treatment as in (a). f Top: Cartoon depicting the effect of STK19KO on TCR complex assembly. Bottom: Quantification of the local DRB run-off immunofluorescence assay as described for Fig. 1a-c in RPE1 WT and STK19KO with or without VCPi treatment as in (a).
