The nucleolus is the site of RNA polymerase I (Pol I) transcription of ribosomal RNA (rRNA) genes and ribosome subunit assembly. Disruptions in RNA Pol I transcription and ribosome biogenesis are associated with significant perturbations in the nucleolus that lead to a unique form of cellular stress, known as nucleolar stress (1). This form of stress triggers the nucleolar stress response, which involves the activation of multiple signalling pathways that are not yet fully characterised. The nucleolar stress response controls and regulates several critical processes, such as DNA replication, cell cycle control, and apoptosis.
The nucleolar DNA damage response (n-DDR), a component of the nucleolar stress response, is triggered by DNA damage at the highly active and repetitive rRNA genes. Our studies have highlighted the potential of activating n-DDR, using the novel inhibitor of RNA Pol I transcription CX-5461, as a promising approach in cancer therapy (2,3). Therefore, characterizing the molecular response and the functional impact of the n-DDR and will open new avenues for the development of new targeted treatments.
To gain a better understanding of n-DDR, we have established an inducible model called TRINSIC (Targeted Induction of Double-Strand DNA Breaks at rRNA Genes using CRISPR-Cas9). We have confirmed that the induction of n-DDR causes global stalling of DNA replication leading to replication stress and growth arrest in high-grade serous ovarian cancer cells. We propose that activation of n-DDR by CX-5461 and TRINSIC leads to a unique replication stress via the release of nucleolar factors to the nucleoplasm that stall DNA replication. We aim to analyse the changes in the distribution of nucleolar proteins following n-DDR and the changes in the proteome of stalled replication forks to provide a new understanding of the nucleolar stress response and uncover a novel class of DDR therapeutics. Altogether, our work highlights the crosstalk between nucleolar and global DDR pathways and aims to define the pathways linking n-DDR with DNA repair and replication processes, as a basis for novel therapy design in ovarian cancer cells.