Protein UFMylation regulates ribosomal DNA double-stranded break repair

Ribosomal DNA (rDNA) arrays are highly repetitive regions of the genome which encode essential genes required to produce ribosomes. DNA double-stranded breaks (DSBs) generated within rDNA genes elicit a unique cellular response involving robust transcriptional silencing and nucleolar reorganization into ‘cap’ structures at the nucleolar periphery. This process is coordinated by the nucleolar scaffolding protein TCOF1, which functions to recruit the DNA repair proteins NBS1 and TOPBP1 that activate the ATM and ATR kinases, resulting in ribosomal RNA (rRNA) transcriptional silencing and nucleolar segregation. However, the DNA damage and repair response at rDNA arrays remains incompletely understood. Here, we investigate the cellular response to rDNA DSBs using proteomics and genetic CRISPR-Cas9 screening. We show that the protein UFMylation pathway and the HUSH complex are important for cell viability and survival in response to rDNA DSBs, and that the E3 UFM1-ligase UFL1 and its heterodimer DDRGK1 are associated with TCOF1 at nucleolar caps. Loss of UFL1 leads to impaired ATM activation, reduced rRNA transcriptional silencing, and an overall reduction in nucleolar segregation. We identified ATM, UNC45A and SMC6 as UFMylated proteins, in which UFMylation may facilitate ATM activation and segregation of damaged rDNA to the nucleolar periphery. Altogether, our findings provide the first evidence for a role for UFMylation in rDNA DSB repair. To determine the genes essential for rDNA DSB repair, we conducted a genome-wide CRISPR-Cas9 screen in hTERT-RPE1 p53-/- cells expressing DD-HA-ER-I-PpoI. These cells were transdcued with the TKOv3 sgRNA library and split into techincal replicates after transduction and selection ("A" and "B"). Cells were treated with negaive control DMSO or Shield-1 + 4-OHT to induce rDNA DSBs 1 day after passaging over the course of 20 days. Sequencing timepoints correspond to T0, T16, and T24 (T=0 is 3 days after intial viral infection used to represent the intial transduced library).