Fig. 5: Elevated TRF1 levels prevent telomere fusions in NMD-deficient cells.

a, Schematic of Myc-tagged TRF1^FL and TRF1^ΔE8 DOX-inducible constructs. Stop codons resulting from exon 8 skipping events are indicated in the Trf1^ΔE8 construct. b,c, Expression of TRF1 and, as a loading control, tubulin in NMD-proficient cells (b) and SMG6-deficient cells (c). DOX treatment results in the expression of full length TRF1 in iTRF1^FL cells and of the TRF1^ΔE8 variant in the iTRF1^ΔE8 cells. d, Metaphases derived from Ctrl or Trf2^−/− ES cells expressing TRF1^FL or TRF1^ΔE8 ± DOX. Percentage of telomere fusions were calculated from a total of >17 metaphases per genotype. More than 678 chromosomes per genotype (n = 3 biological replicates) were scored. For details on the exact chromosome number per genotype, see the Source Data. Mean ± s.d., one-way ANOVA; n.s., not significant (P ≥ 0.9950), **P = 0.0013. e, Metaphases from Smg6^−/− or Smg6^−/− Trf2^−/− ES cells expressing TRF1^FL ± DOX. The percentage of telomere fusions was calculated from a total of >19 metaphases per genotype. More than 880 chromosomes per genotype (n = 3 biological replicates) were scored. For details on the exact chromosome number per genotype, see the Source Data. Mean ± s.d., one-way ANOVA; ****P = 0.0001. f, Expression of TRF1^FL and iTRF1^ΔE8 without (−) and upon DOX treatment (+DOX), with actin as a loading control. g, ChIP assay performed on TRF1^dTAG ES cells (Ctrl.), untreated TRF1^dTAG iTRF1^ΔE8 (−) and treated with DOX (+DOX). Input DNA (1% of the total DNA used), as well as DNA pulled down with FLAG, was hybridized with a radio-labelled telomeric probe (TelC). A representative image from three independent experiments per genotype is shown. h, ChIP and input signals from g were quantified using ImageJ, the ChIP signal was normalized with the corresponding input signal for FLAG. i, Model: NMD activity restrict the expression of the Trf1^ΔE8 splice variant. Upon NMD inhibition, the Trf1^ΔE8 transcript accumulates, producing TRF1^ΔE8 protein, which binds full-length TRF1, and interferes with its binding to telomeres, resulting in telomere deprotection. Source numerical data, unprocessed blots and images are available in the Source data.