NONO mitigates transcription-replication conflicts during replication stress by restraining BRD4-dependent transcription elongation.

Transcription-replication conflicts (TRCs) drive genomic instability and are linked to tumorigenesis and treatment response. Replication stress exacerbates TRCs, but how cells coordinate transcription and replication to limit these conflicts remains unclear. Here, we identify NONO as a key regulator that maintains transcription-replication balance via BRD4 under replication stress. Replication stress triggers NONO acetylation at K243, enhancing BRD4 binding. On single-stranded DNA, NONO-BRD4 form condensates that block paused RNA polymerase II (RNAP2) release, fine-tuning elongation. NONO loss causes aberrant accumulation of elongating RNAP2 at termination regions, promoting TRCs and genomic instability. Conditions that increase origin firing, including ATR inhibition or BRCA2 deficiency, further exacerbate this phenotype in NONO-deficient cells, leading to pronounced synthetic lethality. Together, these findings define a NONO-mediated surveillance mechanism that couples replication stress to transcriptional restraint via condensate-dependent regulation of BRD4, and identify NONO as a potential therapeutic target in replication stress-driven cancers.