Fig. 3: ZBP1 senses TERRA.
From: Telomere-to-mitochondria signalling by ZBP1 mediates replicative crisis
a,b, RT–qPCR analysis of TERRA transcripts. RNA from growing and pre-crisis IMR90E6E7 fibroblasts (a) and growing IMR90E6E7 fibroblasts expressing control shRNA or shRNA against TRF2 (b). RNA was collected at day 4 after transduction, normalized to growing (left) or shScramble (right). Data are mean ± s.d. from technical replicates. n values indicate the number of technical replicates. Statistical analysis was performed using two-tailed Student’s t-test. ***P < 0.001. Three independent experiments. c, fRIP–seq enrichment of ZBP1-associated RNA transcripts within 10 kb upstream of the subtelomere–telomere boundary. fRIP analysis was performed on growing (PD45) IMR90E6E7 fibroblasts expressing WT ZBP1(S)–Flag and transduced with either control shRNA or shTRF2. Immunoprecipitation at day 12 after shRNA transduction. Validation is shown in Extended Data Fig. 8c. fRIP and input samples were normalized to the same sequencing depth and average ratios of individual subtelomeres shown in Extended Data Fig. 8d. Data are log2-transformed. Two independent experiments were performed. d, RNA-dot blot from anti-Flag immunoprecipitates (fRIP) or total lysates (input) using 32P-dCTP-labelled probes targeting TERRA transcripts. fRIP analysis of growing (PD40) IMR90E6E7 fibroblasts expressing either WT ZBP1(S)–Flag or mutant ZBP1(S)–Flag lacking Zα2 with shTRF2. Immunoprecipitation was performed at day 12 after shRNA transduction. Validation is shown in Extended Data Fig. 8e. RNase A treatment was used as a contamination control. Two independent experiments were performed. e, Immunoblot analysis of growing (PD35) IMR90E6E7 fibroblasts (WT ZBP1(S)–Flag or ZBP1(S)–Flag lacking Zα2) with either TRF1(ΔN) or VP16–TRF1(ΔN). Extracts were collected as indicated after doxycycline (Dox) (1 μg ml−1) treatment. Two independent experiments were performed. f, IncuCyte images at day 50 (the growth curve is shown in Extended Data Fig. 10a) after 24 h of incubation (left). Cells are labelled as described in Fig. 1c. Scale bar, 200 μm. Three independent experiments were performed. Right, cell death by Cytotox green incorporation. Data are mean ± s.e.m. from technical replicates. Statistical analysis was performed using one-way ANOVA. ***P < 0.001. Three independent experiments were performed. g, ISG RNA heat maps. RNA was collected at day 10 after doxycycline treatment. Significantly upregulated ISGs (fold change > 3; FDR-adjusted P < 0.05) are shown. One experiment was performed. Chr., chromosome.
