Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled 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 function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability.

Department of Molecular Mechanisms of Disease University of Zurich 8057 Zurich Switzerland

Department of Molecular Mechanisms of Disease University of Zurich 8057 Zurich Switzerland; Life Science Zurich Graduate School 8057 Zurich Switzerland

Institute of Molecular Cancer Research University of Zurich 8057 Zurich Switzerland; Institute of Molecular Genetics Academy of Sciences of the Czech Republic Videnska 1083 143 00 Czech Republic

Institute of Molecular Cancer Research University of Zurich 8057 Zurich Switzerland; Life Science Zurich Graduate School 8057 Zurich Switzerland

Institute of Molecular Genetics Academy of Sciences of the Czech Republic Videnska 1083 143 00 Czech Republic

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