Fig 2: Loss of RPA32 inhibits rRNA transcription and increases PAPAS and pRNA expression in an R-loop-dependent manner. (A) RT-qPCR quantification of 47S pre-rRNA levels after siRPA32 KD in V5-RNaseH1-D210N or V5-RNaseH1-WTcells. SE shown. n = 3. Statistical analysis was performed using a Student's t-test. (*) P < 0.05. Schematics of rRNA locus are shown and positions of probes are indicated by red bars. (B) R-loop distribution along rDNA locus was determined using V5 ChIP in V5-RNaseH1-D210N cells, with probes designed to recognize the UCE, H42.9, 5'ETS, H4, and H27 regions. (C) RNAP I occupancy along the rDNA; RNAP I was immunoprecipitated by RPA194 antibody. SE shown. n = 3. Statistical analysis was performed using a Student's t-test. (*) P < 0.05, (**) P < 0.01, (n.s) no significance. RT-qPCR quantification of antisense rRNA PAPAS (D) or pRNA (E) levels by strand-specific qPCR. Positions of probes are indicated by red bars in the schematic above. Cells were transfected with two different siRNAs targeting RPA32 (siRPA32 and siRPA32-2). Values were normalized to GAPDH in V5-RNaseH1-WT and V5-RNaseH1-D210N cells. Data are presented as mean and SE (n = 3). (*) P < 0.05, (**) P < 0.01.

Fig 3: RNaseH1 depletion leads to RPA activation at ALT telomeres.(a) HeLa and U2OS cells were transfected with the indicated siRNAs and 48 and 72 h later protein extracts were prepared. Western blot analysis was performed using antibodies against RNaseH1, RPA32 phosphorylated at Serine 33 (pSer33), total RPA32 and KAP1 (loading control). The asterisk indicates a cross-reacting band. Cells treated for 6 h with 5 mM hydroxyurea (HU) were used as controls for pSer33 activation. (b) SiRNA transfected cells were subjected to indirect immunofluorescence using antibodies against TRF2 (to visualize telomeres; red) and pSer33 (green). DNA was counterstained with DAPI (blue). Arrows point to examples of pSer33 foci co-localizing with TRF2 (TIFs). Scale bar, 9 µm. Cells treated for 6 h with 5 mM HU were used as controls for pSer33 activation.

Fig 4: TERRA transcription inhibition alleviates telomere instability.a Examples of RPA32, pSer33, or ?H2AX IF (green) combined with telomeric DNA FISH (teloDNA) or TRF2 IF (red). DAPI-stained DNA is in blue. The indicated cell lines were treated with dox for 24 h for RPA32 and pSer33 or 72 h for ?H2AX. Arrowheads in the merge panels point to co-localization events. Scale bar: 5 µm. b Quantifications of experiments as in a. Boxplots represent the medians (middle lines) and the first and third quartiles (boxes), the whiskers extend to the 90th percentile of the values. A total of at least 300 nuclei from three independent experiments were analyzed for each sample. P-values were calculated with a two-tailed Mann–Whitney U test. P1 = 0.0008; P2 = 0.0010; P3 = 0.0511; P4 = 0.0080; P5 < 0.0001; P6 < 0.0001. Source data are provided as a Source Data file.

Fig 5: Induced dimerization of mini-ETAA1 restores function in ETAA1-deficient cells.A, HEK293T, HEK293T ?ETAA1, or HEK293T ?ETAA1 cells complemented with the indicated cDNAs were preincubated with 100 nM AP20187 for 2 h before incubation with 300 nM CPT ± 100 nM AP20187 for 6 h as indicated. Cells were lysed and proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. B, quantification of RPA32 S33 phosphorylation in HEK293T ?ETAA1 cells expressing FK-mini-ETAA1 treated with 300 nM CPT and/or 100 nM AP20187 as indicated. Statistical significance was calculated with a one-way ANOVA and Tukey’s multiple comparisons test (mean ± SD, n = 3). C, U2OS cells expressing HA-FK-mini-ETAA1 were treated with 300 nM CPT for 3 h or pretreated with 100 nM AP20187 for 1 h before treatment with 300 nM CPT and 100 nM AP20187 for 3 h. Cells were fixed and stained for RPA70 and HA-FK-mini-ETAA1. Representative images for each condition are shown. Scale bar is 10 µm. D, U2OS ?ETAA1 cells expressing HA-FK, HA-FK-ETAA1, or HA-FK-mini-ETAA1 were generated by lentiviral infection. HA-FK-ETAA1 expression is below limit of detection. E, micronuclei quantification and representative images in the indicated cell types incubated in the absence or presence of 100 nM AP20187 for 24 h. Scale bar is 10 µm. Statistical significance was calculated with a one-way ANOVA and Tukey’s multiple comparisons test (Mean ± SD, n = 3). F, representative images of a clonogenic survival assay of the indicated cell types treated with 1 mM HU ± 100 nM AP20187 or 5 nM CPT ± 100 nM AP20187 for 24 h. G, quantification of survival assay shown in F. Percent viability for each condition is normalized to the corresponding untreated control. Statistical significance was calculated with a one-way ANOVA and Tukey’s multiple comparisons test (mean ± SD, n = 3 technical triplicate).