Efficient mRNA processing prevents gene gating-associated replication stress in mammalian cells

Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify novel cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry, and ranked candidate genes according to multivariate read-outs reflecting viability, proliferative capacity, replisome integrity and DNA damage signaling. This revealed new regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR kinase activity. Surprisingly, while excessive formation of RNA:DNA-hybrids under these conditions appears mainly as consequence rather than cause of DNA damage, inhibition of transcription rescued fork speed, origin activation and alleviated replication catastrophe. Importantly, uncoupling pre-mRNA processing from nuclear export by depleting the THO complex also protected cells from DNA damage, suggesting that efficient pre-mRNA cleavage provides a mechanism to prevent gene gating-associated genomic instability. RNA Polymerase II phospho-S2 ChIP in U-2 OS cells treated with control siRNA or siRNA against WDR33