Extended Data Fig. 3: Validation of the DNA damage-inducing effects of BRCA1 or Dicer loss and sdRNA destruction. | Nature

Extended Data Fig. 3: Validation of the DNA damage-inducing effects of BRCA1 or Dicer loss and sdRNA destruction.

From: BRCA1 and RNAi factors promote repair mediated by small RNAs and PALB2–RAD52

Extended Data Fig. 3

a, A Venn diagram showing a common set of deregulated sdRNAs after depletion with the indicated siRNAs in HeLa cells. bd, Heat maps centred around the sdRNA peaks (±0.8 kb) showing the sdRNA changes in abundance after BRCA1 depletion in HMECs (b) and in HeLa cells before and after exposure to α-amanitin (c) and DRB (d).e, A correlation between the abundance of ACTB sdRNA and the nascent ACTB RNA (detected using intron 1) is shown. f, Top, schematic of the CASP16P genomic locus. Primers used for BELD on the forward (PE2-F) and reverse (PE2-R) strands and the qPCR amplicons. Bottom, BELD quantification in T98G cells of the CASP16P Pause2 site reverse strand. Representative experiment showing the relative mean ± s.d. abundance of the qPCR replicates. gj, DNA damage quantification at the CASP16P pause site performed in G1 or S/G2 BRCA1-depleted HeLa FUCCI cells (representative graph) (g), in BRCA1-depleted, synchronized T98G cells (h; n = 3 biological replicates) or in BRCA1 (i; n = 4 biological replicates) or Dicer (j; n = 3 biological replicates) rescue experiments. k, Top left, quantification of ACTB and CASP16P sdRNA in DROSHA-depleted cells (siDrosha). mir191, positive control. Bottom, genome-wide heat maps centred around sdRNA peaks, showing that DROSHA depletion in HeLa cells does not affect sdRNA abundance. Data are mean ± s.e.m. qPCR values. Top right, DROSHA depletion did not induce DNA damage at the ACTB or CASP16P pause site. CCNB1 CoTC is a negative control (n = 3 biological replicates for both graphs). l, sdRNA quantification showing endogenous sdRNA subcellular localization (right) (n = 2–3 biological replicates) and the level of sdRNA overexpression (around tenfold) (left) (representative). m, Benzonase effect on immunoblotted proteins co-immunoprecipitated with BRCA1, Representative blot from n = 3 experiments. n, Efficacy and specificity of depletion of ACTB and CASP16P sdRNAs using separate LNA GapmeRs (n = 3–4 biological replicates). o, Heat maps centred around the sdRNA peaks (±0.8 kb) showing no genome-wide effect after depletion of ACTB or CASP16P sdRNAs. γ-H2AX ChIP analyses shown as an mean fold change compared to relevant control cells. Data were analysed by one-way (g) or two-way (hk, n) ANOVA with post hoc Tukey HSD test or multiple/unpaired t-test (l) and compared to an undamaged locus or relevant control cells. ns, not significant.

Back to article page