Journal: bioRxiv

Article Title: rDNA breaks activate dsRNA pattern recognition through sense-antisense transcription

doi: 10.64898/2026.05.26.728043

Figure Lengend Snippet: (A) Representative images of γH2AX immunostaining with following I-PpoI- or AsiSI-mediated DNA double-strand breaks. Scale bar, 20 μm. (B) Immunoblots confirming DNA damage and immune activation induced by I-PpoI or AsiSI. (C, D) Immunofluorescence of nucleolar RNA synthesis visualized by EU-click labeling with representative images and quantification. For EU analysis, at least 150 cells were quantified per experiment. Three independent experiments were performed, and one representative was displayed. Bars represent mean ± SEM. Statistical significance was determined by an unpaired two-tailed Student’s t -test. **** p < 0.0001. Scale bar, 20 μm. (E, F) Immunoblots of whole-cell lysates showing STAT1 phosphorylation and ISG56 expression 48 h after I-PpoI-mediated rDNA breaks in the presence of ATM, ATR or DNA-PK inhibition in hTERT RPE-1 p53 KO cells. (G) (Top) Schematic of the human rDNA showing the 47S pre-rRNA transcription unit and the positions of sgRNAs targeting the 5’ETS, 18S, ITS2, and 28S regions, along with the I-PpoI recognition site. (Bottom) Immunoblots of whole-cell lysates following transfection of rDNA-targeting sgRNAs in Cas9-expressing hTERT RPE-1 p53 KO cells. (H) Immunoblots of whole-cell lysates assessing the dependence of the cytosolic RNA sensors MDA5 and RIG-I on I-PpoI-induced immune activation in the presence or absence of ATM inhibition.

Article Snippet: hTERT RPE-1 p53 KO cell line was generated by transfecting pSpCas9(BB)-2A-GFP(PX458) vector targeting p53 into hTERT RPE-1 cell line (ATCC).

Techniques: Immunostaining, Western Blot, Activation Assay, Immunofluorescence, Labeling, Two Tailed Test, Phospho-proteomics, Expressing, Inhibition, Transfection

Journal: bioRxiv

Article Title: rDNA breaks activate dsRNA pattern recognition through sense-antisense transcription

doi: 10.64898/2026.05.26.728043

Figure Lengend Snippet: (A) Schematic diagram of proximity ligation assay (PLA) between biotin-labeled nascent RNA and the cytosolic RNA pattern recognition receptors MDA5 and RIG-I following rDNA breaks. Nascent RNA was labeled with 5’-ethynyl uridine (EU) and biotinylated by click chemistry prior to PLA detection. ( B ) Experimental scheme for detecting cytosolic nascent RNA sensing by MDA5 or RIG-I. (C) Immunoblots of p-STAT1 and ISG56 following I-PpoI induction, camptothecin (CPT), or etoposide treatment. (D) Representative images of PLA following EU labeling and biotin click reaction after DNA damage. (Top) PLA signals indicate proximity between biotin-labeled nascent RNA (EU) and MDA5 or RIG-I. (Bottom) Nucleolar RNA synthesis visualized by EU-click labeling. Scale bar, 20 μm. (E-G) Quantification of cytosolic PLA signals EU-labeled nascent RNA and MDA5 (E), RIG-I (F), and nucleolar EU signal intensity ( G ). ( H ) Immunoblots of p-STAT1 and ISG56 following I-PpoI-mediated rDNA breaks in the presence or absence of RNA polymerase I inhibitor BMH-21. (I) Representative images of cytosolic PLA foci between biotin-labeled nascent RNA (EU) and MDA5 or RIG-I following BMH-21 treatment. Scale bar, 20 μm. (J, K) Quantification of cytosolic PLA signals between biotinylated EU-labeled nascent RNA and MDA5 (J) or RIG-I (K). ( C-K ) Experiments were performed in hTERT RPE-1 p53 KO cells with inducible expression of I-PpoI. ( E, F, J, K ) Each dot represents the mean value of at least 50 cells. ( G ) For EU analysis, at least 150 cells were quantified per experiment. Three independent experiments were performed, and one representative result is displayed. ( E-G, J, K ) Bars represent mean ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t -test. **** p < 0.0001, *** p <0.001, ** p < 0.01.

Article Snippet: hTERT RPE-1 p53 KO cell line was generated by transfecting pSpCas9(BB)-2A-GFP(PX458) vector targeting p53 into hTERT RPE-1 cell line (ATCC).

Techniques: Proximity Ligation Assay, Labeling, Western Blot, Expressing, Two Tailed Test