What question did this study set out to answer?

The aim is to investigate how NONO's relocalisation in the nucleolus affects genome stability and DNA repair mechanisms.

March 26, 2026Open Access

The role of the RNA-binding protein NONO in the DNA damage response

Key Points

The aim is to investigate how NONO's relocalisation in the nucleolus affects genome stability and DNA repair mechanisms.
Utilized etoposide to induce double strand breaks in U2OS cells.
Conducted immunofluorescence to observe NONO relocalisation in response to DNA damage.
Employed mNET-seq to analyze RNA transcript changes following etoposide treatment.
Used CUT&RUN and 4sU-seq to assess NONO binding activity at gene transcription start sites.
NONO relocalises to the nucleolus in response to etoposide-induced DSBs.
Decreased NONO occupancy at the transcription start sites of highly expressed genes.
Formation of nucleolar antisense transcripts and R-loops was observed.
NONO binding to specific intron-containing transcripts was increased, which suggests its role in RNA detention.

Abstract

RNA metabolism factors such as RNA-binding proteins (RBPs) have emerged as critical regulators of the DNA damage response (DDR). NONO is a multifunctional RBP and a core component of paraspeckles and regulates numerous RNA metabolic processes. NONO expression levels are frequently deregulated in cancer and are associated with a poor survival prognosis in cancer patients. Along with the role of NONO in regulating the RNA metabolism, studies suggest that NONO also stimulates the Double strand break repair (DSBR). Depletion of NONO delays the clearance of γH2A.X foci and sensitises the cells to DNA damaging agents. However, the mechanism underlying the role of NONO in genome stability is unclear. In this study, I have investigated the relevance of NONO nucleolar relocalisation in promoting genome stability by using a wide range of methods. To understand the mechanism, I used etoposide, a topoisomerase II inhibitor to induce Double strand breaks (DSBs). Immunofluorescence experiments show that the induction of nucleoplasmic DSBs by etoposide promotes the nucleolar relocalisation of NONO. mNET-seq revealed that etoposide induces the formation of RNAPII-dependent nucleolar antisense transcripts at distinct intergenic spacer (IGS) loci. These transcripts form IGS R-loops and trigger nucleolar relocalisation of NONO in an RRM1 domain dependent manner. Interestingly, CUT&RUN and 4sU-seq showed that etoposide-induced NONO nucleolar relocalisation reduces both NONO occupancy at the transcription start site (TSS) of highly expressed genes, and pre-mRNA synthesis. Simultaneously, etoposide increases NONO binding to a subset of intron-containing transcripts and NONO mediates their detention in the nucleolus as revealed by eCLIP and APEX2-seq. Nucleolar relocalisation of NONO suppresses R-loops formation and mediates the recruitment of NHEJ factors to broken loci to promote efficient DSBR. Taken together, my data demonstrate that NONO couples the RNA metabolism with the DSBR to maintain genome stability in U2OS cells.

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