Genome-wide mapping of formaldehyde-induced DNA-protein crosslinks reveals unique patterns of formation and transcription-coupled removal in mammalian cells
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PROVIDER: PRJNA1147654 | ENA |
REPOSITORIES: ENA
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Project description:DNA-protein crosslinks (DPCs) form following exposure to various alkylating agents including environmental carcinogens, cancer chemotherapeutics, and reactive aldehydes. If not repaired, DPCs can interfere with key biological processes such as transcription and replication and activate programmed cell death. A growing body of evidence implicates nucleotide excision repair (NER), homologous recombination, and other mechanisms in the removal of DPCs. However, the effects of genomic context on DPC formation and removal have not been comprehensively addressed. Using a combination of next generation sequencing and DPC enrichment via protein precipitation, we show that DPCs induced following exposure to formaldehyde are non-randomly distributed across the human genome, based on chromatin state. The data further show that the efficiency of DPC removal correlates with transcription at loci transcribed by RNA polymerase II. Data presented herein indicate that efficient removal of chromosomal DPCs requires both the Cockayne syndrome group B gene as well as ‘downstream’ TC-NER factor xeroderma pigmentosum group A gene. In contrast, loci transcribed by RNA Polymerase I showed no evidence of transcription-coupled DPC removal. Taken together, our results indicate that complex interactions between chromatin organization, transcriptional activity, and numerous DNA repair pathways dictate genomic patterns of DPC formation and removal.
2025-07-26 | GSE274678 | GEO
Project description:Reactive aldehydes are produced during cellular metabolism and can accumulate in specific tissues particularly when aldehyde clearance mechanisms are impaired. Reactive aldehydes induce DNA-DNA and DNA-protein crosslinks (DPCs) that are repaired by different DNA repair pathways. Cellular toxicity of endogenous formaldehyde has been attributed to the damage of the genomic DNA and consequent inhibition of transcription. However, whether damage to other cellular macromolecules and interference with additional metabolic processes contributes to formaldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces toxic RNA-protein crosslinks (RPCs) in mRNA that inhibit translation and induce a specific RPC stress response pathway that is characterized by linkage-specific ubiquitylation. RPCs in the mRNA are recognized by stalling ribosomes and marked by K6-linked ubiquitylation that promotes their clearance by the ubiquitin-dependent unfoldase VCP.
2023-09-01 | GSE225044 | GEO
Project description:PxP-MS (Purification of x-linked Proteins coupled to Mass Spectrometry) was used to assess the role of CSB in DNA-protein crosslink repair. The CSB protein is a sensor that can detect stalled RNA polymerases at sites of DNA damage, thereby triggering transcription-coupled repair mechanisms. DPCs were induced in WT and CSB knock-out RPE1 cells using a pulse of formaldehyde. To identify crosslinked proteins that specifically require CSB for repair, DNA-protein crosslinks were isolated from cells using PxP either directly after formaldehyde exposure or following a chase in drug-free media and identified by mass spectrometry.
2024-02-15 | PXD047668 | Pride
Project description:Translation-coupled sensing and degradation of RNA-protein crosslinks
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Project description:Atypical K6-linked ubiquitylation marks toxic RNA-protein crosslinks induced by formaldehyde
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Project description:Reactive aldehydes are abundant cytotoxic metabolites, which challenge homoeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA-DNA crosslinks cause cancer and bone marrow failure in Fanconi anemia, while covalent DNA-protein crosslinks require proteolytic repair to prevent liver tumours and premature ageing. Whether RNA damage contributes to the toxicity of aldehydes and whether cells possess mechanisms to resolve RNA-protein crosslinks (RPCs) in particular is unknown. Studying the specific consequences of aldehyde-induced RNA damage is challenging due to confounding induction of DNA damage. Here, we establish photoactivatable ribonucleosides as a tractable model system to study aldehyde-mimicking RNA damage in the absence of DNA damage. We find that RNA crosslinking damage causes translational stress by stalling elongating ribosomes, which causes cell death upon ZAKα-dependent activation of the ribotoxic stress response (RSR) and GCN2-dependent activation of the integrated stress response (ISR). Moreover, we discover the principles of a translation-coupled cellular quality control mechanism that targets RPCs. Collisions between translating ribosomes and crosslinked mRNA-binding proteins trigger their ubiquitylation and subsequent proteasomal degradation. Our findings reveal RNA damage and RPC formation as a central aspect of aldehyde-induced toxicity and establish a framework to study the cellular responses to these threats in mechanistic detail.
2023-11-13 | GSE240135 | GEO
Project description:Reactive aldehydes are abundant cytotoxic metabolites, which challenge homoeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA-DNA crosslinks cause cancer and bone marrow failure in Fanconi anemia, while covalent DNA-protein crosslinks require proteolytic repair to prevent liver tumours and premature ageing. Whether RNA damage contributes to the toxicity of aldehydes and whether cells possess mechanisms to resolve RNA-protein crosslinks (RPCs) in particular is unknown. Studying the specific consequences of aldehyde-induced RNA damage is challenging due to confounding induction of DNA damage. Here, we establish photoactivatable ribonucleosides as a tractable model system to study aldehyde-mimicking RNA damage in the absence of DNA damage. We find that RNA crosslinking damage causes translational stress by stalling elongating ribosomes, which causes cell death upon ZAKα-dependent activation of the ribotoxic stress response (RSR) and GCN2-dependent activation of the integrated stress response (ISR). Moreover, we discover the principles of a translation-coupled cellular quality control mechanism that targets RPCs. Collisions between translating ribosomes and crosslinked mRNA-binding proteins trigger their ubiquitylation and subsequent proteasomal degradation. Our findings reveal RNA damage and RPC formation as a central aspect of aldehyde-induced toxicity and establish a framework to study the cellular responses to these threats in mechanistic detail.
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Project description:Compromised enzymatic actions, reactive metabolites, and various chemotherapeutics cause toxic covalent DNA-protein crosslinks (DPCs). Failure to repair DPCs results in genomic instability, premature aging, and tumorigenesis. However, understanding the principles underlying DPC formation and repair has been hampered by a lack of methodologies to study DPCs in human cells. Here, we developed a technique for the Purification of x-linked Proteins (PxP), which allows identification and tracking of enzymatic and nonenzymatic DPCs. By combining PxP with quantitative proteomics, we investigated the nature of DPCs induced by formaldehyde.
2023-01-20 | PXD026654 | Pride
Project description:Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA–protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-Between-RING E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.
2023-11-13 | PXD038655 | Pride
Project description:Translation-coupled sensing and degradation of RNA-protein crosslinks [Ribo-Seq: dataset_2]
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