Fig 1: CSA interacts with chaperonin TRiC. a A SILAC-mass spectrometry approach identified all TRiC subunits as CSA-interacting proteins. CSA-deficient CS3BE-SV40 cells expressing FLAG or CSA-FLAG were cultured in medium containing light or heavy lysine and arginine isotopes, respectively. FLAG- and CSA-FLAG-interacting proteins were pulled down and samples were processed and analyzed by mass spectrometry. The table shows the number of unique peptides found for the top ranked interactors, as well as the ratio of the interactor in the CSA-FLAG pulldown to that in the control FLAG pulldown (ratio H/L). b FLAG pulldowns confirm the UV-independent interaction between CSA-FLAG and TCP1. CS3BE-SV40 cells expressing FLAG or CSA-FLAG were mock-treated or UV-C irradiated (20 J/m2). After 1 h of recovery cells were lysed and fractionated into soluble or solubilized chromatin. FLAG pulldowns using both fractions were followed by western blot analysis for the indicated proteins. c CSA co-immunoprecipitation confirms the interaction between endogenous CSA and TCP1. As in b, except that VH10-hTert cells were used and that endogenous CSA was immunoprecipitated. d GFP pulldowns confirm the interaction between CSA and TRiC subunits CCT4 and CCT5. GFP or CSA-GFP was pulled down from CS3BE-SV40 cells. e Tandem FLAG and GFP pulldowns show preferential binding of TRiC to DDB1/CUL4A/RBX1-free CSA. CSA-FLAG, GFP, and GFP-DDB1 were expressed in U2OS cells as indicated. Enrichment of CSA-interacting proteins by means of FLAG pulldowns confirmed interactions between CSA and DDB1 and CUL4A, as well as the TRiC subunits CCT4 and CCT7. Subsequently, eluted protein complexes were subjected to pulldown of GFP-DDB1, revealing an interaction with CUL4A, but not CCT4 and CCT7. Full-size scans of western blots are provided in Supplementary Fig. 7
Fig 2: Quantitative interaction proteomics reveal UVSSA interaction partners and required UVSSA-domains. (A) Scatter plot of log2 SILAC ratios of proteins isolated by GFP-pulldown in UVSS-A cells stably expressing either GFP-UVSSA or GFP (non-specific binding control). The experiment was conducted in duplicate with a label swap. The log2 SILAC ratios of proteins identified in the forward experiment (GFP-UVSSA versus GFP, H/L, x-axis) are plotted against the log2 SILAC ratios of proteins identified in the reversed experiment (GFP-UVSSA versus GFP, L/H, y-axis). Proteins were classified as specific UVSSA interactors (marked in blue) when log2 SILAC ratio >0.6 (indicated by gray dotted line) in both replicates. (B) GO-term analysis of the 66 proteins identified as specific interactors of UVSSA. A selection of the top 10 enriched biological process pathways is shown. GC: gene count; FDR: false discovery rate. (C) Scatter plot of log2 SILAC ratios of proteins identified in the GFP-pulldowns of wt UVSSA versus ?VHS, only proteins that were also identified in the GFP-UVSSA versus GFP proteomics experiment are depicted. The experiment was conducted in duplicate, including a label swap. The log2 SILAC ratios of proteins identified in the forward experiment (wt versus ?VHS, H/L, x-axis) are plotted against the log2 SILAC ratio of proteins identified in the reversed experiment (wt versus ?VHS, L/H, y-axis). The majority of proteins have similar binding ability to the ?VHS mutant compared to the wt (log2 SILAC ratio <0.6, proteins marked in gray). Proteins marked in blue represent proteins whose interaction with UVSSA is decreased in the absence of the VHS domain. (D) Scatter plot of log2 SILAC ratios of proteins identified in the GFP-pulldowns of wt UVSSA versus ?DUF only proteins that were also identified in the GFP-UVSSA versus GFP proteomics experiment are depicted. The experiment was conducted in duplicate, including a label swap. The log2 SILAC ratios of proteins identified in the forward experiment (wt versus ?DUF, H/L, x-axis) are plotted against the log2 SILAC ratio of proteins identified in the reversed experiment (wt versus ?DUF, L/H, y-axis). Proteins marked in blue have a reduced interaction with UVSSA?DUF compared to wt (proteins are marked in blue, log2 SILAC ratio >0.6, gray dotted line marks the threshold). (E) Cross-linked nuclear extracts of UVSS-A patient cell line (TA24), stably expressing the indicated constructs were subjected to GFP immunoprecipitation. Non-complemented UVSS-A patient cell line (-) was used as negative binding control. WCE: whole-cell extract, IP: Immunoprecipitation. Western blot analysis of the co-immunoprecipitated proteins was performed for GFP, Spt16, SSRP1, USP7 and CSA.
Fig 3: Loss of TRiC reduces RRS and protection against UV damage. a TCP1 loss reduces RNA synthesis recovery following UV-C irradiation. VH10-hTert cells were transfected with the indicated siRNAs and UV-C irradiated (10 J/m2). RNA synthesis was measured by means of EU incorporation at the indicated time points after UV. RNA synthesis levels were normalized to those in non-irradiated cells, which were set to 100%. Data represent the mean ± SEM of four independent experiments. b Expression of CSA-FLAG 8M shows reduced RNA synthesis recovery as compared to expression of CSA-FLAG WT. As in a, except that CS3BE-SV40 cells expressing CSA-FLAG WT or CSA-FLAG 8M were used. Data represent the mean ± SEM of four independent experiments. c TCP1 loss renders cells hypersensitive to UV damage. VH10-hTert cells were transfected with the indicated siRNAs, UV-C irradiated at the indicated doses and 72 h later assayed for viability using alamarBlue®. Data represent mean ± SEM of four independent experiments. d Expression of CSA-FLAG 8M in CS3BE-SV40 cells fails to rescue UV-sensitivity. CS3BE-SV40 cells stably expressing CSA-FLAG WT or CSA-FLAG 8M were UV-C irradiated and clonogenic survival was measured. Data represent mean ± SEM of three independent experiments. e CSA WT, but not CSA ?N, complements the Illudin S sensitivity of CSA knockout (KO) U2OS cells. The indicated cells were treated with different concentrations of Illudin S and clonogenic survival was determined. Data represent mean ± SEM of three independent experiments
Fig 4: CSA interaction and recruitment to DNA damage is mediated by distinct UVSSA domains. (A) Schematic overview of the protein domains present in UVSSA and the used UVSSA deletion mutants that either lack the VHS domain (?VHS) or the DUF2043 domain (?DUF). NLS: nuclear localization signal. (B) UV sensitivity of UVSS-A cells (-) and UVSS-A cells complemented with GFP-UVSSA (wt), GFP-UVSSA ?DUF (?DUF) or UVSSA ?VHS-GFP (?VHS) was determined by their colony-forming ability, following irradiation with the indicated UV-C doses. The percentage of surviving colonies is plotted against the UV-C dose. The number of colonies counted at 0 J/m2 is set as 100% survival. Data represents the experiment conducted in triplicate and error bars represent SEM. (C) Whole-cell extracts (WCE) of UVSS-A patient cells stably expressing the indicated constructs were subjected to GFP immunoprecipitation. Western blot analysis of the immunoprecipitated proteins was performed using GFP, CSA or USP7 antibodies. WCE: whole-cell extract, IP: Immunoprecipitate. (D) Representative images of live cell imaging analysis of GFP-UVSSA or ?DUF and ?VHS mutants following local UV-C laser (266 nm) induced damage (indicated by a white arrow); scale bar: 7.5 µm. Right panel: 4× zoomed image to visualize accumulation at 3 s post-damage induction. (E) GFP fluorescence intensity of the indicated constructs at LUD was quantified over time and normalized to pre-damage intensity set at 100 at t = 0 (n = 30 cells of two independent experiments, mean ± SEM). A one-way Anova test was performed and P-values < 0.001 (***) are depicted. The moment of damage induction is indicated with a black arrow.
Fig 5: CSB depletion is an early event in replicative senescence. a RT-qPCR of CSB and CSA. n = 3 independent experiments; mean ± SD; one-way ANOVA (CSB: F = 19.40, DFn = 5, DFd = 12, p < 0.0001; CSA: F = 2.55, DFn = 5, DFd = 12, p = 0.855) with post-hoc Tukey’s test vs. PN16 (p-values on the top of scatter plots/columns) when not specifically indicated. b WB of CSB and CSA, each with the loading control GAPDH (lower blot, GAPDH F-c staining). Upper band in CSB blot is non-specific67. c Representative confocal acquisitions of cells immunostained for CSB (green) and counterstained with Hoechst (blue) after maximum intensity projection with Imaris, scale bar = 50 µM, and d quantification of the CSB mFI/cell. n = 30–50 cells from three independent experiments; mean ± SEM; one-way ANOVA (F = 13.76, DFn = 5, DFd = 189, p < 0.0001) with post-hoc Tukey’s test vs. PN16 when not specifically indicated. IF measurements include normalization to cell size. Source data are provided as Source Data files.
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