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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE118795 | GEO | 2019/01/10

REPOSITORIES: GEO

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Project description:The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

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

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