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Translation-coupled sensing and degradation of RNA-protein crosslinks

ABSTRACT: Reactive aldehydes are abundant cytotoxic metabolites, which challenge homoeostasis by crosslinking cellular macromolecules. 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. The aim of this phosphoproteome measurement is to elucidate the changes in phosphorylation sites upon RPC induction using the treatment with a photoactivatable-ribonucleoside-enhanced crosslinking (PAR-CL) by incubation with 4-thiouridine (4-SU) and subsequent crosslinking by UVA irradiation.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Petra Beli

LAB HEAD: Petra Beli

PROVIDER: PXD038398 | Pride | 2023-11-13

REPOSITORIES: Pride

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			Action	DRS
	20220330_SFB1_PBE_ASR_TiO2_TMT16_Hela_RPC_ZAKKO_GCNKO_4Reps_pH025.raw	Raw
	20220330_SFB1_PBE_ASR_TiO2_TMT16_Hela_RPC_ZAKKO_GCNKO_4Reps_pH028.raw	Raw
	20220330_SFB1_PBE_ASR_TiO2_TMT16_Hela_RPC_ZAKKO_GCNKO_4Reps_pH032.raw	Raw
	20220330_SFB1_PBE_ASR_TiO2_TMT16_Hela_RPC_ZAKKO_GCNKO_4Reps_pH035.raw	Raw
	20220330_SFB1_PBE_ASR_TiO2_TMT16_Hela_RPC_ZAKKO_GCNKO_4Reps_pH038.raw	Raw

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Translation-coupled sensing and degradation of RNA-protein crosslinks [Ribo-Seq: dataset_1]

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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