The RNA binding protein (RBP) ZFP36L1, which binds to adenylate/uridylate (AU)-rich elements (AREs) (AU-RBP) in the 3’ untranslated region of many messenger RNAs, has been extensively characterised for its role in post-transcriptional control of gene expression and is reported as a newly identified cancer driver gene. Replication stress (RS) threatens DNA replication fidelity and stability of the genome. Recently, a small number of AU-RBPs have emerged as key figures in the maintenance of genome integrity through mechanisms that govern the replication stress response and DNA repair. Herein, we report that treatment with low doses of aphidicolin results in hallmarks of RS-associated genomic instability in a cellular model depleted of ZFP36L1 using CRISPR/Cas9. We find that loss of ZFP36L1 results in defects in mitosis leading to chromosome segregation errors and genomic instability. Remarkably, we also identify loss of ZFP36L1 increases the prevalence of FANCD2-associated anaphase ultra-fine bridges indicating chromatid non-disjunction at intrinsically labile common fragile site loci. Furthermore, we detected an increase in RPA and γH2AX foci in S/G2 cells indicative of replication stress-induced DNA damage potentially indicating chronic replication fork stalling and double-strand break formation as demonstrated by increased γH2AX foci colocalising with 53BP1. Surprisingly, chromatin enrichment of U-2OS, HCT116 and Hela cells demonstrated that ZFP36L1 is physically bound to chromatin fractions. Here we also demonstrated the specificity of CRISPR-Cas9 mediated ablation of ZFP36L1 through the inducible expression of ZFP36L1 that demonstrated suppression of 53BP1 nuclear bodies (NBs) and micronuclei formation. Importantly, we demonstrate, by overexpression of a catalytically inactive mutant of human RNase H1 tagged with GFP that loss of ZFP36L1 induces R-loop formation. We also implicate unscheduled R-loop formation as a potential cause for replication stress associated genomic instability through the expression of wild-type RNase H1 which was able to limit the occurrence of 53BP1 NBs in G1 phase cells and RPA in S/G2 phase cells. Finally, we highlight potential ZFP36L1 interactions through mass spectrometry that uncover proteins involved in the maintenance of genome integrity and R-loop resolution. Taken together, our work highlights an important, yet previously unidentified role, for ZFP36L1 in preserving genomic stability including limiting the formation of R-loops in response to replication stress
