Journal: Nature

Article Title: RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops

doi: 10.1038/s41586-020-2815-6

Figure Lengend Snippet: a , TRF analysis of HeLa clones used for transient expression of TERRA b, d, TERRA expression levels measured by RT-qPCR and compared to the levels of TERRA in the clone with the shortest telomeres. n=3 biologically independent experiments, data are mean ± s.d. c , TRF analysis of HeLa clones expressing transgenic TERRA from the AAVS1 locus. e , HeLa clones of different telomere length were transiently transfected with the PP7-15q TERRA construct and co-localization was assessed by IF-FISH. The percentage of TERRA foci that co-localized with telomeres was plotted as a function of telomere length. White arrows indicate co-localization of PP7 foci with the telomeric signal. Random colocalization events (~3%) were not subtracted. f , Co-localization events as in e but for PP7-15q TERRA expressed from the AAVS locus on chromosome 19. n=3 biologically independent experiments, data are mean ± s.d. Significant differences are indicated. Two-tailed unpaired t test was used to calculate the P values. (*P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001).

Article Snippet: The PP7 stem loops were amplified from Addgene plasmid # 61762 (kind gift from Daniel Larson) and introduced into the pTRE2puro through In-Fusion cloning.

Techniques: Clone Assay, Expressing, Quantitative RT-PCR, Transgenic Assay, Transfection, Construct, Two Tailed Test

Journal: Nature

Article Title: RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops

doi: 10.1038/s41586-020-2815-6

Figure Lengend Snippet: a , Schematic of chimeric TERRA construct and assay. The plasmids were transfected into HeLa cells constitutively expressing the GFP-PCP protein. After 24 hours TERRA transcription was induced with doxycycline. Chimeric TERRA, that is recognized and bo und by the GFP-PCP protein, was analyzed 24 hours upon induction. b , Fluorescence in situ hybridization (FISH) for telomeric DNA (red) and immunofluorescence (IF) for GFP (green) was employed to assess the co-localization of transiently expressed PP7 constructs with telomeres. Confocal images are shown. White arrows indicate co-localization of PP7 foci with telomeric signals. c , Schematic representation of siRNA transfection and TERRA transfection sequence. HeLa clones with long (10 kb average) and short (3 kb) telomeres were transfected with the corresponding siRNA pools, followed by chimeric TERRA transfection. The percentage of co-localizing TERRA foci was assessed by telomeric FISH combined with GFP IF. n=2 biologically independent experiments, > 40 nuclei were analyzed per condition, data are mean ± s.d. One-way ANOVA with Dunnett’s multiple comparisons test was used, comparing all conditions to siControl (*P < 0.05; **P < 0.01; ***P < 0.001). d , RAD51 enzymatic activity is required for TERRA recruitment. Endogenous RAD51 was depleted with siRNA and WT RAD51 or RAD51 II3A mutant was expressed from plasmid containing cDNA. TERRA colocalization with telomeres was assessed as in c. n=3 biologically independent experiments, > 80 nuclei were analyzed per condition, data are mean ± s.d. Two-tailed unpaired t test was used to calculate the P values. (*P < 0.05; **P < 0.01; ***P < 0.001).

Article Snippet: The PP7 stem loops were amplified from Addgene plasmid # 61762 (kind gift from Daniel Larson) and introduced into the pTRE2puro through In-Fusion cloning.

Techniques: Construct, Transfection, Expressing, Fluorescence, In Situ Hybridization, Immunofluorescence, Sequencing, Clone Assay, Activity Assay, Mutagenesis, Plasmid Preparation, Two Tailed Test

Journal: Nature

Article Title: RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops

doi: 10.1038/s41586-020-2815-6

Figure Lengend Snippet: a , Detection of telomeric hybrids on dot-blots. Telomeric hybrids were isolated by DNA-RNA immunoprecipitation with S9.6 antibody from cells with long or short telomeres. Cells expressed either the PP7 stem loops or the PP7-15q TERRA and had been transfected with control siRNA (siC), siRNAs against RNaseH1 (RNH1), or with a plasmid that overexpressed RNH1. In vitro treatment with RNaseH1 served as a negative control. Quantification of signals is shown on the right. b , Schematic representation of PP7-15q TERRA expressed from a plasmid and forming R-loops in trans at telomeres 1q, 10q and 13q. Telomeric hybrids arising at the indicated chromosome ends were quantified by qPCR with primers amplifying specific subtelomeric DNA next to the telomeric tracts in cells with long telomeres. c , Telomere fragility induced upon expression of TERRA from a plasmid. The fraction of fragile telomeres was quantified on metaphase chromosomes stained with a telomeric FISH probe. d , Effects of RNaseH1 and RAD51 on telomere fragility. In cells expressing PP7+15qTERRA from a plasmid, telomere fragility was quantified upon depletion (si) or overexpression (OE) of RNaseH1 (RNH1), or depletion of RAD51. e , Detection of endogenous telomeric R-loops in siC, siRNH1 and siRAD51 transfected cells. For all data n=3 biologically independent experiments, data are mean ± s.d. Two-tailed unpaired t test was used to calculate the P values. (*P < 0.05; **P < 0.01; ***P < 0.001). For Fig. c and d the number of metaphases scored are indicated for each condition in .

Article Snippet: The PP7 stem loops were amplified from Addgene plasmid # 61762 (kind gift from Daniel Larson) and introduced into the pTRE2puro through In-Fusion cloning.

Techniques: Isolation, RNA Immunoprecipitation, Transfection, Control, Plasmid Preparation, In Vitro, Negative Control, Expressing, Staining, Over Expression, Two Tailed Test

Journal: Nature

Article Title: RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops

doi: 10.1038/s41586-020-2815-6

Figure Lengend Snippet: a , Representative examples of metaphase chromosomes stained with FISH to visualize telomeres. DNA is stained with DAPI. Fragile telomeres are indicated by white arrowheads. b , Quantification of telomere fragility. c , Quantification of telomere fragility in cells expressing PP7-15qTERRA in which expression of RNaseH1 (RNH1) and RAD51 was manipulated as indicated. For Fig. b and c , the number of metaphases scored over 3 biologically independent experiments are indicated for each condition as n.

Article Snippet: The PP7 stem loops were amplified from Addgene plasmid # 61762 (kind gift from Daniel Larson) and introduced into the pTRE2puro through In-Fusion cloning.

Techniques: Staining, Expressing

Journal: Nature

Article Title: RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops

doi: 10.1038/s41586-020-2815-6

Figure Lengend Snippet: Oligonucleotides used in this study

Article Snippet: The PP7 stem loops were amplified from Addgene plasmid # 61762 (kind gift from Daniel Larson) and introduced into the pTRE2puro through In-Fusion cloning.

Techniques: Construct, Amplification