Journal: Cell Reports

Article Title: DROSHA, DICER, and damage-induced long ncRNA control BMI1-dependent transcriptional repression at DNA double-strand break

doi: 10.1016/j.celrep.2025.116605

Figure Lengend Snippet: BMI1, DICER, and DROSHA control ubH2A-K119 deposition in DIvA cells; RNA controls the proximity between BMI and DROSHA induced in damaged cells (A) Representative images of cells transfected with siCTRL, siBMI1, siDICER, and siDROSHA or siATM and stained for γH2AX and ubH2A-K119 in uncut and cut DIvA cells. DNA was counterstained with DAPI. Scale bar, 20 μm. (B) Quantification of ubH2A-K119 foci in cells treated as in (A). Data are presented as mean ± SEM of more than 150 cells from each of the three independent experiments. (C) Representative images of BMI1-DROSHA PLA signals in uncut and cut DIvA cells treated with siCTRL, siDROSHA, or siBMI1. Scale bar, 20 μm. (D) Quantification of DROSHA-BMI1 PLA signal in cells treated as in (C). Data are presented as mean ± SEM of more than 150 cells from each of the three independent experiments. (E) Quantification of DROSHA-BMI1 PLA signal in uncut and cut DIvA cells treated with DMSO, ATMi, or mirin. Data are presented as mean ± SEM of more than 150 cells from each of the three independent experiments. (F) Representative immunoblot of DROSHA and BMI1 from BMI1 immunoprecipitation (IP) in irradiated HEK-293 cells, either untreated or treated with RNase III during IP. (G) Representative immunoblot of DROSHA and BMI1 from BMI1 IP in irradiated HEK-293 cells treated with either DNase I or RNase III during IP. Statistical analyses in (B)–(E) were performed by one-way ANOVA. See also .

Article Snippet: Anti-BMI1 , rabbit, monoclonal , 1:4,000 , DI-PLA , Bethyl , A700-119 (BLR119H); RRID:AB_2891916.

Techniques: Control, Transfection, Staining, Western Blot, Immunoprecipitation, Irradiation

Journal: Cell Reports

Article Title: DROSHA, DICER, and damage-induced long ncRNA control BMI1-dependent transcriptional repression at DNA double-strand break

doi: 10.1016/j.celrep.2025.116605

Figure Lengend Snippet: BMI1 is recruited to the damaged site by DROSHA and DICER and interacts with dilncRNA and DDRNAs (A and B) ChIP-qPCR analysis at three AsiSI sites (DS1–DS3) and at an unrelated region, performed in cut and uncut DIvA cells treated with non-targeting siRNAs (siCTRL), siRNAs against DROSHA and DICER (siDICER/siDROSHA), or ATM (siATM) transcripts, for BMI1 (A) and ubH2A-K119 (B). DS1 corresponds to an AsiSI break site in the 3′ UTR of RBMXL1 gene; DS2 is located upstream of the CDS (promoter region) of CYB561D1 gene; DS3 is located at 30 bp upstream of the 5′ UTR of TRIM37 gene and 500 bp upstream of its CDS; and the unrelated region corresponds to chr22: 22,373,074; 22,373,287. Data are presented as mean ± SEM from three independent experiments. (C) Representative images of DI-PLA signals for BMI1 recruitment to sites of damage in cut and uncut DIvA cells treated with siCTRL, siDICER/siDROSHA, or siATM. Scale bar, 20 μm. (D) Quantification of DI-PLA signals for BMI1 recruitment in cells treated as in (C). Data are presented as mean ± SEM of more than 150 cells from three independent experiments. (E) RNA immunoprecipitation (RIP) of BMI1-associated RNAs followed by RT-qPCR analysis of dilncRNAs generated at the AsiSI site on Chr1 (DSB1), performed in uncut and cut DIvA cells. RPLP0 was used as a negative control. Data are presented as mean ± SEM from three independent experiments. (F) RIP of BMI1-associated RNAs followed by RT-qPCR analysis of dilncRNAs generated at the AsiSI site on Chr4, performed in uncut and cut DIvA cells. RPLP0 was used as a negative control. Data are presented as mean ± SEM from three independent experiments. (G) RIP of BMI1-associated RNAs followed by RT-qPCR analysis of dilncRNA generated at AsiSI site on Chr4, performed in uncut and cut DIvA cells treated with siCTRL or DICER/DROSHA co-KD. RPLP0 was used as a negative control. Data are presented as mean ± SEM from three independent experiments. Statistical analyses in (A), (B), (D), and (E)–(G) were performed by one-way ANOVA. See also and .

Article Snippet: Anti-BMI1 , rabbit, monoclonal , 1:4,000 , DI-PLA , Bethyl , A700-119 (BLR119H); RRID:AB_2891916.

Techniques: ChIP-qPCR, RNA Immunoprecipitation, Quantitative RT-PCR, Generated, Negative Control

Journal: Cell Reports

Article Title: DROSHA, DICER, and damage-induced long ncRNA control BMI1-dependent transcriptional repression at DNA double-strand break

doi: 10.1016/j.celrep.2025.116605

Figure Lengend Snippet: Functional inhibition of the dilncRNA:DDRNA axis by ASOs and its targeting via Cas13d abolishes DISC and BMI1 recruitment to the break (A) RT-qPCR analysis of GFP mRNA levels in HeLa cells bearing an integrated GFP gene (HeLa-GFP) treated with control siRNAs (siCTRL) or siRNAs against DROSHA and DICER transcripts (siDROSHA/siDICER). Data are relative to uncut cells for each knockdown condition. Data are presented as mean ± SEM from three independent experiments. (B) RT-qPCR analysis of GFP mRNA levels in cut and uncut HeLa-GFP cells treated with siCTRL or siRNAs against BMI1 transcript (siBMI1). Data are presented as mean ± SEM from three independent experiments. Data are relative to uncut cells for each condition. (C) Representative images of DI-PLA signals for BMI1 recruitment to sites of damage in cut and uncut DIvA cells treated with siCTRL, siDICER/siDROSHA, or siATM. Scale bar, 20 μm. (D) RT-qPCR analysis of GFP mRNA levels in cut and uncut HeLa-GFP cells treated with control ASOs or ASOs targeting dilncRNAs generated at the GFP locus. Data are presented as mean ± SEM from three independent experiments. Data are relative to uncut cells for each condition. (E) ChIP-qPCR analysis for BMI1 at the GFP locus and an unrelated region, performed in uncut (scramble-guide-Cas9) and cut (GFP-guide-Cas9) HeLa-GFP cells treated with control ASOs or dilncRNAs-targeting ASOs. Data are relative to uncut cells after the subtraction of mock values for each treated condition. Data are presented as mean ± SEM from three independent experiments. (F) RT-qPCR analysis of LIN54 (DSB at Chr4) mRNA levels in cut (GFP-guide-Cas9) and uncut (scramble-guide-Cas9) DIvA cells stably expressing an inducible Cas13d. Cells were treated with a control Cas13 RNA guide (CTRL Cas13g) or guides targeting dilncRNAs generated downstream the AsI cut site on Chr4 (anti-dilncRNA Cas13g). Data are presented as mean ± SEM from three independent experiments. Statistical analyses in (A), (B), and (D)–(F) were performed by one-way ANOVA. Statistical analysis in (C) was performed using paired Student’s t test. See also .

Article Snippet: Anti-BMI1 , rabbit, monoclonal , 1:4,000 , DI-PLA , Bethyl , A700-119 (BLR119H); RRID:AB_2891916.

Techniques: Functional Assay, Inhibition, Quantitative RT-PCR, Control, Knockdown, Generated, ChIP-qPCR, Stable Transfection, Expressing