Replication stress, a hallmark of pre-cancerous and cancerous lesions, is characterised by the slowing or stalling in replication fork progression and the consequent failure to preserve genomic stability. ZFP36L1, a member of the ZFP36
family of CCCH tandem zinc finger (ZF) proteins, is an RNA-binding protein that plays a crucial role in the post-transcriptional regulation of gene expression underlying
physiological processes and pathological diseases. Recent evidence has revealed that ZFP36L1, is significantly mutated or downregulated in certain human cancers, and exhibits tumour-suppressive properties across various cancer cell types. However, the precise molecular link between ZFP36L1 and cancer, particularly from the angle of genomic stability, remains poorly elucidated. Here, we report a novel role for ZFP36L1, characterised in human U2OS and U2OS H2B-GFP osteosarcoma cells, in suppressing replication stress-induced genomic instability. We found that, CRISPR/Cas9-mediated knockout of ZFP36L1 resulted in increased chromosome segregation defects including anaphase bridges, chromosome laggards and micronuclei formation, even in the absence of exogenous replication stress. Moreover, ZFP36L1-deficient cells exhibited an elevated accumulation of γH2AX, 53BP1 nuclear bodies (NBs) and RPA, which indicated an active role for ZFP36L1 in limiting replication stress-induced DNA damage. Furthermore, we demonstrated an unanticipated function for ZFP36L1 in potentially suppressing the expression of common fragile sites (CFSs) in response to replication stress, reflected by the increased mitotic CFS-characteristic chromosomal aberrations and MiDAS events at CFS loci observed in cells deficient in ZFP36L1. Finally, we also showed that a CRISPR/Cas9-mediated truncation in ZFP36L1, carrying a compromised lead-in sequence to its first ZF-domain, resulted in increased replication stress-associated phenotypes, similar to that of a ZFP36L1-knockout. Taken together, our data reveal, for the first time, a role for ZFP36L1 in contributing to the protection of genomic integrity against replication stress.