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. 2020 Jan 14;9:e49817. doi: 10.7554/eLife.49817

Figure 3. TRF1 depletion causes TERRAs upregulation.

(A) RNA dot blot analysis in wt and TRF1 deleted MEFs. The blot was revealed with a DIG-Tel-C-rich probe or 18 s rRNA as a control. TERRA signals were normalised to 18 s rRNA and GFP control ± SEM of at least three independent biological replicates. P values, two-tailed student t-test (****, p<0.0001). (B) TERRA detection by Northern blotting upon TRF1 deletion. The blot was revealed with a DIG-Tel-C-rich probe (upper part). Ethidium bromide (EtBr) staining (bottom) of rRNAs was used as loading control. TERRA signals were normalized to 28 s rRNA signal from EtBr staining ± SEM of 2 independent biological replicates. P values, two-tailed student t-test (**, p<0.01). (C) Representative images of TERRA-FISH experiment (top panel) showing the difference between cells stained with TERRA (red), negative control with RNAse A treatment and positive control after denaturation. TERRA-FISH quantification (bottom panel) in wt (+GFP) and TRF1-/- (+CRE) conditions. Violin plots are representing the number of TERRA foci (left) and TERRA intensity (right) (n = 250) per nucleus, where the median is underlined in red and quartiles in white, two-tailed student t-test (****, p<0.0001); Mann-Whitney test used for TERRA intensity quantification (*, p<0.05).

Figure 3—source data 1. Quantification of Telomeric RNA molecules by dot-blot in TRF1F/F MEFs.
Related to Figure 3A.
Figure 3—source data 2. Quantification of TERRAs by Northern.
Related to Figure 3B.
elife-49817-fig3-data2.xlsx (761.2KB, xlsx)
Figure 3—source data 3. Quantification of number of TERRA foci.
Related to Figure 3C.

Figure 3.

Figure 3—figure supplement 1. TRF1 deletion and APH replication stress cause increased TERRA levels in immortalised MEFs (day4) and also in primary MEFs (day 6).

Figure 3—figure supplement 1.

(A) Western blotting showing protein expression in wt and TRF1 deficient primary MEFs (P4) 6 days post-infection with GFP- or CRE-Adenovirus (left panel) and in SV40-immortalised MEFs, 4 days post-infection (right panel). (B) RNA dot-blot analysis upon TRF1 deletion showing increased TERRA signals in CRE-infected conditions compared to control GFP-. The blot was revealed with a DIG-Tel-C-rich probe or 18 s rRNA as a control. (C) Quantification of B. Data are shown as TERRA signal relative to GFP condition ± SEM of 4 independent biological replicates. P values, two-tailed student t-test (*, p<0.05; ****, p<0.0001) (D) TERRA detection by Northern blotting upon TRF1 deletion showing increased High Molecular Weights (HMW) RNA molecules upon alkaline treatment (left blot). In native conditions, HMW-TERRAs are not detected and no significative difference is observed for low molecular weight species (right blot). The blots were revealed with a DIG-Tel-C-rich probe (upper part). Ethidium bromide (EtBr) staining (bottom) of rRNAs was used as loading control. Source data are provided as a Source Data File. (E) RNA dot-blot analysis in wt MEFs cells upon Aphidicolin treatment showing increased TERRA signals compared to control DMSO. The blot was revealed with a DIG-Tel-C-rich probe or 18 s rRNA as a control (top panel). Quantification of Data are shown as TERRA signal relative to DMSO condition ± SEM of 4 independent biological replicates (bottom panel). P values, two-tailed student t-test (*, p<0.05). (F) TERRA detection by Northern blotting upon Aphidicolin treatment showing increased High Molecular Weights (HMW) RNA molecules upon alkaline treatment. The blot was revealed with a DIG-Tel-C-rich probe (upper part). Ethidium bromide (EtBr) staining (bottom) of rRNAs was used as loading control. Source data are provided as a Source Data File.
Figure 3—figure supplement 1—source data 1. WB of TRF1F/F primary MEFs and immortalized after 4 days of CRE.
Related to Figure 3—figure supplement 1A.
Figure 3—figure supplement 1—source data 2. Telomeric RNA molecules by dot-blot in primary TRF1F/F MEFs and immortalized after 4 days of CRE.
Related to Figure 3—figure supplement 1B.
Figure 3—figure supplement 1—source data 3. Quantification of Telomeric RNA molecules by dot-blot in primary TRF1F/F MEFs and immortalized after 4 days of CRE.
Related to Figure 3—figure supplement 1C.
Figure 3—figure supplement 1—source data 4. TERRAs by Northern in TRF1F/F MEFs.
Related to Figure 3—figure supplement 1D.
Figure 3—figure supplement 1—source data 5. Quantification of Telomeric RNA molecules by dot-blot in wt MEFs treated with APH.
Related to Figure 3—figure supplement 1E.
Figure 3—figure supplement 1—source data 6. TERRAs by Northern in wt MEFs treated with APH.
Related to Figure 3—figure supplement 1F.
Figure 3—figure supplement 2. TRF1 deficient MEFs present normal telomere distribution, telomerase activity and no c-circles.

Figure 3—figure supplement 2.

(A) Terminal Restriction Fragments (TRF) blot showing no telomere length heterogeneity upon TRF1 deletion (+CRE, 7 days post-infection) compared to control TRF1F/F +GFP MEFs. The blot was revealed with a DIG-Tel-C-rich probe (right). Ethidium bromide (EtBr) staining (left) is used as loading control. (B) Quantification of telomerase activity levels by TRAP assay showing no changes in telomerase activity after TRF1 deletion in MEFs. Values are normalised to the control HT1080-ST cells (100%) and are represented as mean ± SEM of 4 independent biological replicates. (C) C-circle assay showing no c-circle formation upon TRF1 deletion (+CRE, 7 days post-infection) compared to control TRF1F/F +GFP MEFs. Phi polymerase amplification products were spotted on the membrane and revealed using DIG-TelC probe. TRF1F/F MEFs treated with aphidicolin (APH) are used as negative control for telomere fragility not inducing C-circles, while U2OS, ALT positive cell line, is used as positive control. Source data are provided as a Source Data File.
Figure 3—figure supplement 2—source data 1. Telomere length by Southern in TRF1F/F MEFs.
Related to Figure 3—figure supplement 2A.
Figure 3—figure supplement 2—source data 2. Telomerase activity by TRAP in TRF1F/F MEFs.
Related to Figure 3—figure supplement 2B.
Figure 3—figure supplement 2—source data 3. c-circle amplification assay in TRF1F/F MEFs and U2OS (+ctl).
Related to Figure 3—figure supplement 2C.