Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts [BLISS]

RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localisation to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localisation is poorly understood. Here we show that the topoisomerase-II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-induced nucleolar antisense RNAs (diNARs) in human cancer cells. diNARs originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in ab RRM1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signaling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair. Overall design: Profiling of DSBs upon siCTR or siNONO, with or without etoposide treatment

RNA结合蛋白（RNA-binding proteins）作为DNA损伤应答（DNA damage response, DDR）的效应因子发挥功能。多功能非POU结构域包含八聚体结合蛋白（non-POU domain-containing octamer-binding protein）NONO/p54nrb在未受扰动的细胞中可标记核旁斑（nuclear paraspeckles），但在DNA双链断裂（DNA double-strand breaks, DSBs）诱导后，其会发生重定位并进入核仁。然而，目前对NONO的核仁重定位机制尚不清楚。本研究发现，拓扑异构酶II抑制剂依托泊苷（etoposide）可在人类癌细胞中促进RNA聚合酶II（RNA polymerase II）依赖的、DNA损伤诱导的核仁反义RNA（DNA damage-induced nucleolar antisense RNAs, diNARs）的生成。diNARs源自不同的核仁基因间隔区，并形成DNA-RNA杂合链（DNA-RNA hybrids），以依赖RRM1结构域（RRM1 domain）的方式将NONO锚定至核仁。依托泊苷会降低NONO在蛋白编码基因启动子（protein-coding gene promoters）处的结合占有率，该过程会减弱前体mRNA（pre-mRNA）合成、增强NONO与前体mRNA转录本（pre-mRNA transcripts）的结合，并伴随此类转录本子集的核仁滞留（nucleolar detention）。NONO的敲低或突变会干扰核仁滞留过程，并延长DSB信号的持续时间。综上，我们阐明了一条核仁DDR通路，该通路可在DSBs损伤中保护NONO与异常转录本，以促进DNA修复。实验整体设计：对经对照小干扰RNA（siCTR）或靶向NONO的小干扰RNA（siNONO）处理、并施以或不施以依托泊苷处理的样本进行DSBs特征分析。