Journal: PLoS ONE

Article Title: TODRA , a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51 , and Enhances RAD51-Dependent DSB (Double Strand Break) Repair

doi: 10.1371/journal.pone.0134120

Figure Lengend Snippet: A. TODRA transcript: Top: Schematic representation of the predicted TODRA ( AK125393 ) gene , as described in the UCSC genome browser. Light grey shaded rectangles depict TODRA exons, the dark grey rectangle depicts RAD51 exon 1, transcribed in the opposite direction. Bottom: Results of TODRA transcript analysis. 5’RACE using capped HeLa mRNA, identified one transcription start site (full arrowhead, +1 corresponds to chr. 15: 40987374, hg19), and 3’RACE identified several possible transcription termini (arrows). The most 5’ end of RAD51 identified using 5’RACE is also shown (empty arrowhead, +1 corresponds to chr. 15: 40987303, hg19). Black bars beneath the diagram indicate confirmed regions of unidirectional transcription determined using strand specific primers for reverse transcription from both HeLa and MCF7 cells. B. Splicing of TODRA exons 1 and 2 is demonstrated in the representative gel. Lane M: pUC Mix Marker, (Fermentas), Lanes 1&2: TODRA strand specific RT-PCR products (F primer located in exon 1, R primer in exon 2). Expected size of product in genomic DNA: 696bp, Expected size of spliced transcript: 480bp, as observed in lanes 1 (cDNA prepared from HeLa cells) and 2 (cDNA prepared from MCF7 cells). C. The RAD51/TODRA region supports transcription in both directions. Top: Schematic representation of the RAD51 and TODRA promoter regions and the fragments cloned into luciferase promoter constructs. Bottom: TODRA putative promoter activity . MCF7 cells were co-transfected with the promoter-less pGL3-basic, pRAD51-UTR or pTODRA and pRL-TK (to normalize for transfection efficiency). Results are shown as fold increase in RLA (relative luciferase activity), compared to pGL3-basic. Values are means ± SE of 4–5 independent transfections performed in duplicates. * p< 0.002, ** p< 0.0001.

Article Snippet: The following primary antibodies were used in this study: polyclonal rabbit anti-E2F1 (C-20; Santa Cruz Biotechnology Cat# sc-193, RRID:AB_631394); monoclonal mouse anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Millipore Cat# 05–636, RRID:AB_309864); and polyclonal rabbit anti-Rad51 (Ab-1; Merck Cat# PC130-100UL, RRID:AB_10684676).

Techniques: Marker, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Luciferase, Construct, Activity Assay, Transfection

Journal: PLoS ONE

Article Title: TODRA , a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51 , and Enhances RAD51-Dependent DSB (Double Strand Break) Repair

doi: 10.1371/journal.pone.0134120

Figure Lengend Snippet: A. Top: Diagram of the core TODRA promoter region cloned into the luciferase reporter vector. Bottom: Effect of mutagenesis of the E2F binding site and E2F1 induction on the TODRA reporter. Wild type TODRA luciferase (reporter) construct, or an E2F binding site mutant (E2F site mut) construct were transfected into MCF7 and U2OS cells. A TODRA luciferase (reporter) construct was also co-transfected with either an E2F1 expression vector or an empty vector control into serum-starved MCF7 and U2OS cells. All experiments included co-transfection with pRL-TK (to normalize for transfection efficiency). Results are depicted as the fold change in RLA compared to the WT construct transfection. Values in all experiments are means ± SE of 3–4 independent transfections performed in duplicate. ** p≤ 0.007, * p≤ 0.02, ^ p = 0.00001. B. Top: Diagram of the core RAD51 promoter region cloned into the luciferase reporter vector. Bottom: Effect of mutagenesis of the E2F binding site and E2F1 induction on the RAD51 reporter. Wild type RAD51 luciferase (reporter) construct, or an E2F binding site mutant (E2F site mut) construct were transfected into MCF7 and U2OS cells. A RAD51 luciferase (reporter) construct was also co-transfected with either an E2F1 expression vector or an empty vector control into serum-starved MCF7 and U2OS cells. All experiments included co-transfection with pRL-TK (to normalize for transfection efficiency). Results are depicted as the fold change in RLA compared to the WT construct transfection. Values in all experiments are means ± SE of 3–4 independent transfections performed in duplicate. ** p≤ 0.007, * p≤ 0.02. C. and D. Top: Diagram of the RAD51/TODRA bidirectional promoter region cloned between the firefly and Renilla luciferase reporter genes (pBDP). C. Mutagenesis of the E2F binding site . E2F site mutant (pBDP E2F site mut) or wild type bidirectional promoter constructs (pBDP) were transfected into MCF7 and U2OS cells. Results are depicted as the fold change in the mutant compared to the WT in the ratio of Firefly/Renilla luciferase activities, which represents the ratio of RAD51/TODRA promoter activities. Values are means ± SE of 3–6 independent transfections performed in duplicate. ** p< 0.0001. D. E2F1 overexpression . pBDP activity was examined in MCF7 and U2OS cells co-transfected with the pBDP construct and either an E2F1 WT, an E2F1 trans-activation domain deletion mutant (ΔTA), or an empty expression vector. Results are depicted as the fold change between each E2F1 expression vector and the empty vector control, in the ratio of Firefly/Renilla luciferase activities, which represents the ratio of RAD51/TODRA promoter activities. Values are means ± SE of 3–6 independent transfections performed in duplicate. ** p< 0.0001. Additional comparisons are indicated above the bars. * p≤ 0.003.

Article Snippet: The following primary antibodies were used in this study: polyclonal rabbit anti-E2F1 (C-20; Santa Cruz Biotechnology Cat# sc-193, RRID:AB_631394); monoclonal mouse anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Millipore Cat# 05–636, RRID:AB_309864); and polyclonal rabbit anti-Rad51 (Ab-1; Merck Cat# PC130-100UL, RRID:AB_10684676).

Techniques: Clone Assay, Luciferase, Plasmid Preparation, Mutagenesis, Binding Assay, Construct, Transfection, Expressing, Cotransfection, Over Expression, Activity Assay, Activation Assay

Journal: PLoS ONE

Article Title: TODRA , a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51 , and Enhances RAD51-Dependent DSB (Double Strand Break) Repair

doi: 10.1371/journal.pone.0134120

Figure Lengend Snippet: A. E2F1 induction results in E2F1 binding of the RAD51/TODRA promoter . E2F1 expression was induced in serum starved ER-E2F1 U2OS cells (stably transfected with a constitutively expressed ER-E2F1 fusion protein which upon ligand-dependent activation translocates from the cytoplasm to the nucleus) by treatment with OHT for 8 hours. RAD51/TODRA promoter occupancy was measured with a ChIP assay using E2F1 antibodies (Ab) in lysates of either OHT treated or untreated cells. Real-time PCR was performed to quantitate the RAD51/TODRA template captured by the E2F1 Ab. Promoter occupancy is expressed as fold change relative to that observed in untreated cells. Values are means ± SE of 3 ChIP independent experiments. Real-time reactions were performed in triplicates. ^ p = 0.01. A representative gel of the promoter region PCR amplification products is shown on the left . B. E2F1 induction and endogenous RAD51 and TODRA transcription . RAD51 and TODRA transcript levels were determined after E2F1 induction (see above), using quantitative real-time RT-PCR normalized to GAPDH . Results are depicted as the fold change in either RAD51 or TODRA transcript levels compared to non-treated cells (time 0). Values are means ± SE of 3–4 independent experiments. Real-time reactions were performed in duplicates. + p≤ 0.04, * p≤ 0.004.

Article Snippet: The following primary antibodies were used in this study: polyclonal rabbit anti-E2F1 (C-20; Santa Cruz Biotechnology Cat# sc-193, RRID:AB_631394); monoclonal mouse anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Millipore Cat# 05–636, RRID:AB_309864); and polyclonal rabbit anti-Rad51 (Ab-1; Merck Cat# PC130-100UL, RRID:AB_10684676).

Techniques: Binding Assay, Expressing, Stable Transfection, Transfection, Activation Assay, Real-time Polymerase Chain Reaction, Amplification, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: TODRA , a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51 , and Enhances RAD51-Dependent DSB (Double Strand Break) Repair

doi: 10.1371/journal.pone.0134120

Figure Lengend Snippet: A. Schematic representation of the HRind cell system . The mCherry- ISce I-GR (Glucoroticoid Receptor) endonuclease is cytoplasmic. Upon addition of Dexamethasone, it rapidly translocates into the nucleus generating a DSB at the ISce I site in the DR-GFP cassette. The DSB can be repaired either by NHEJ (non-homologous end-joining) or HR, but only HR repair reconstitutes functional GFP (green nucleus) from DR-GFP. B. Overexpression of TODRA induces RAD51-dependent HR. HRind cells were transfected with an empty vector (EV) or TODRA minigene and induced with Dexamethasone for 48 hours. GFP expression was measured by FACS. Results are depicted as the fold change in observed HR (as indicated by the number of GFP-positive cells) compared to the empty vector. Values are means ± SE of 3 independent experiments performed in triplicate. * p< 0.04. C. Overepxression of TODRA elevates DNA damage-induced RAD51 foci formation. U2OS cells were transfected with an empty vector (EV) or the TODRA minigene. 48 hrs. post transfection half of each culture was treated for 1 hr. with the DNA damaging agent phleomycin (10μg/ml). Medium was then replaced in all cultures, releasing treated cells from phleomycin exposure. γH2AX and RAD51 foci were imaged either immediately (0 hours) or 6 hours after removal of phleomycin and medium exchange. Top: A representative image of γH2AX (green) and RAD51 (red) foci in empty vector (EV) and TODRA transfected cells 6 hours after removal of phleomycin. DAPI (blue signal in merged images) was used for counterstaining. Scale bars = 10 μm. Bottom: The number of RAD51-positive foci was normalized as the fraction of γH2AX-positive foci per cell and averaged across all samples in each condition. Cells were treated with phleomycin, as indicated, and fixed at the indicated time points post-treatment. Results are depicted as the fold change in the fraction of RAD51 foci in cells overexpressing TODRA compared to the empty vector. Values are means ± SE of 3 independent experiments. * p≤ 0.03.

Article Snippet: The following primary antibodies were used in this study: polyclonal rabbit anti-E2F1 (C-20; Santa Cruz Biotechnology Cat# sc-193, RRID:AB_631394); monoclonal mouse anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Millipore Cat# 05–636, RRID:AB_309864); and polyclonal rabbit anti-Rad51 (Ab-1; Merck Cat# PC130-100UL, RRID:AB_10684676).

Techniques: Non-Homologous End Joining, Functional Assay, Over Expression, Transfection, Plasmid Preparation, Expressing

Journal: PLoS ONE

Article Title: TODRA , a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51 , and Enhances RAD51-Dependent DSB (Double Strand Break) Repair

doi: 10.1371/journal.pone.0134120

Figure Lengend Snippet: A. TPIP co-activates E2F1 induction of RAD51 . pRAD51-UTR with an E2F1 expression vector or an empty vector were co-transfected into serum starved MCF7 cells together with either PTEN, TPIPα, TPIPβ or an empty pEGFP-C2 based expression vector and pRL-TK (to normalize for transfection efficiency). Results are depicted as fold change in RLA compared to pRAD51-UTR alone (left bar). Values are means ± SE of 3 independent transfections performed in duplicates. ** p< 0.0001. Additional comparisons are indicated above the bars. * p = 0.002. B. E2F1 induction and endogenous TPIP expression . Endogenous TPIP mRNA levels were determined using quantitative real-time RT-PCR normalized to GAPDH , with and without E2F1 induction in serum starved ER-E2F1 U2OS cells. E2F1 was induced by treatment with OHT for 4 hours. Values are means ± SE of 4 independent experiments. Real-time reactions were performed in duplicates. ** p< 0.00001. C. Overexpression of TPIP reduces HR. HRind cells were transfected with an mOrange2 control vector (CV) or TPIP expression vector (tagged with mOrange2) and induced with Dexamethasone for 48 hours. GFP expression was measured by FACS. Results are depicted as the fold change in observed HR (as indicated by the number of GFP-positive cells among the transfected population [mOrange2 positive cells]) compared to the control vector. Values are means ± SE of 3 independent experiments performed in triplicate. ** p<0.002.

Article Snippet: The following primary antibodies were used in this study: polyclonal rabbit anti-E2F1 (C-20; Santa Cruz Biotechnology Cat# sc-193, RRID:AB_631394); monoclonal mouse anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Millipore Cat# 05–636, RRID:AB_309864); and polyclonal rabbit anti-Rad51 (Ab-1; Merck Cat# PC130-100UL, RRID:AB_10684676).

Techniques: Expressing, Plasmid Preparation, Transfection, Quantitative RT-PCR, Over Expression

Journal: PLoS ONE

Article Title: TODRA , a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51 , and Enhances RAD51-Dependent DSB (Double Strand Break) Repair

doi: 10.1371/journal.pone.0134120

Figure Lengend Snippet: The RAD51-TODRA regulatory pathway in breast cancer tumors.

Article Snippet: The following primary antibodies were used in this study: polyclonal rabbit anti-E2F1 (C-20; Santa Cruz Biotechnology Cat# sc-193, RRID:AB_631394); monoclonal mouse anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Millipore Cat# 05–636, RRID:AB_309864); and polyclonal rabbit anti-Rad51 (Ab-1; Merck Cat# PC130-100UL, RRID:AB_10684676).

Techniques: Expressing, Amplification

Journal: PLoS ONE

Article Title: TODRA , a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51 , and Enhances RAD51-Dependent DSB (Double Strand Break) Repair

doi: 10.1371/journal.pone.0134120

Figure Lengend Snippet: E2F1 induction enhances RAD51 expression (thin green arrow) while simultaneously reducing lncRNA TODRA expression. While E2F1 induction of RAD51 is synergistically enhanced by TPIP (thick green arrow), E2F1 induction also reduces TPIP expression, possibly by affecting TODRA expression, as TODRA expression can increase TPIP expression. This feedback regulation of RAD51 expression can fine-tune RAD51 expression and HR-DSB repair. Green: Enhancement of expression/activity. Red: Suppression of expression/activity.

Article Snippet: The following primary antibodies were used in this study: polyclonal rabbit anti-E2F1 (C-20; Santa Cruz Biotechnology Cat# sc-193, RRID:AB_631394); monoclonal mouse anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Millipore Cat# 05–636, RRID:AB_309864); and polyclonal rabbit anti-Rad51 (Ab-1; Merck Cat# PC130-100UL, RRID:AB_10684676).

Techniques: Expressing, Activity Assay

Journal: Nucleus

Article Title: CiRS-7 Enhances the Liquid-liquid Phase Separation of miRISC and Promotes DNA Damage Repair

doi: 10.1080/19491034.2023.2293599

Figure Lengend Snippet: ciRS-7 promoted AGO2-mediated DNA damage repair. a. Detection of γ-H2A.X by immunofluorescence assay in HeLa-ciRS-7 or HeLa-EV cells at different time points (0.5 h, 6 h, 20 h) after irradiation. Quantitative counts of γ-H2A.X foci per cell or cells with more than 5 γ-H2A.X foci were presented. For γ-H2A.X foci number, EV: n = 97 (-), 107 (0.5 h), 84 (6 h), 93 (20 h); ciRS-7: n = 110 (-), 102 (0.5 h), 99 (6 h), 84 (20 h). Data were presented as mean ± SD from 3 replications. b. Detection of RAD51 by immunofluorescence assay in HeLa-ciRS-7 or HeLa-EV cells at different time points (0.5 h, 6 h, 20 h) after irradiation. Quantitative counts of RAD51 foci per cell or cells with more than 5 RAD51 foci were presented ( n = 3). For RAD51 foci number, EV: n = 101 (-), 116 (0.5 h), 117 (6 h), 88 (20 h); ciRS-7: n = 117 (-), 150 (0.5 h), 135 (6 h), 112 (20 h). Data were presented as mean ± SD from 3 replications. c. Detection of γ-H2A.X by immunofluorescence assay in HeLa cells transfected with NC or siciRS-7 at 20 h after irradiation. Quantitative counts of γ-H2A.X foci per cell were presented ( n = 50 for NC; n = 50 for siciRS-7). Data were presented as mean ± SD. d. Detection of γ-H2A.X by immunofluorescence assay in HeLa cells treated with anti-NC or anti-miR-7 at 20 h after irradiation. Quantitative counts of γ-H2A.X foci per cell were presented ( n = 47 for anti-NC; n = 47 for anti-miR-7). Data were presented as mean ± SD. HeLa cells were exposed to 3 Gy X-ray. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance.

Article Snippet: The primary antibodies, rabbit anti-AGO2 (ab186733, Abcam, diluted in 1:200), rabbit anti-γ-H2A.X (9718S, CST, diluted in 1:500), and rabbit anti-RAD51 (ET1705–96, HuaBio, Hangzhou, China, diluted in 1:100), were used in this study.

Techniques: Immunofluorescence, Irradiation, Transfection