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Translation-coupled resolution of RNA-protein crosslinks

ABSTRACT: The aim of the measurements was to investigate RNA damage and RNA-protein crosslinks (RPC) formation in the context of aldehyde-induced toxicity. For this, a photoactivatable-ribonucleoside-enhanced crosslinking (PAR-CL) strategy using 4-thiouridine (4-SU) and UVA-radiation was used. The submitted dataset contains data from LC-MS/MS measurements of crosslinks between proteins and poly-adenylated mRNAs (mRPCs). The measurements included the following six samples sets: Sample set 1 (9 runs = 3 conditions in 3 replicates): RPCs after formaldehyde (FA) and 4-SU + UVA treatment isolated using the protein-x-linked RNA extraction (XRNAX) protocol (doi:10.1016/j.cell.2018.11.004) Conditions: RPCs from untreated HAP1 =Ctrl RPCs from HAP1 treated with FA RPCs from HAP1 treated with 4-SU + UVA Sample set 2 (12 runs = 4 conditions in 3 replicates): mRPCs after 4-SU + UVA treatment using poly-A pulldown Conditions: mRPCs from untreated HAP1=Ctrl mRPCs from HAP1 treated with UVA but without 4-SU (0h after irradiation) mRPCs from HAP1 treated with 4-SU but without UVA mRPCs from HAP1 treated with 4-SU + UVA (0h after irradiation) Sample set 3 (24 runs = 6 conditions in 4 replicates): mRPCs after 4-SU + UVA and Ub-E1i treatment using poly-A pulldown Conditions: mRPCs from HAP1 treated with 4-SU + UVA (0h, 0.5h and 1h after irradiation) mRPCs from HAP1 treated with 4-SU + UVA and Ub-E1i (0h, 0.5h and 1h after irradiation) Sample set 4 (24 runs = 6 conditions in 4 replicates): mRPCs after 4-SU + UVA and MG132 treatment isolated using poly-A pulldown Conditions: mRPCs from HAP1 treated with 4-SU + UVA (0h, 0.5h and 1h after irradiation) mRPCs from HAP1 treated with 4-SU + UVA and MG132 (0h, 0.5h and 1h after irradiation) Sample set 5 (24 runs = 6 conditions in 4 replicates): mRPCs after 4-SU + UVA and ANS treatment using isolated poly-A pulldown Conditions: mRPCs from HAP1 treated with 4-SU + UVA (0h, 0.5h and 1h after irradiation) mRPCs from HAP1 treated with 4-SU + UVA and ANS (0h, 0.5h and 1h after irradiation) Sample set 6 (24 runs = 6 conditions in 4 replicates ): mRPCs after 4-SU+UVA in AAVS1 control and RNF14 KO cells using poly-A pulldown mRPCs from HAP1 AAVS1 control cells (clone #6) treated with 4-SU+UVA (0h, 0.5h and 1h after irradiation) mRPCs from HAP1 RNF15 KO cells (clone #15) treated with 4-SU+UVA (0h, 0.5h and 1h after irradiation) Abbreviations: 4-SU: 4-thiouridine ANS: Anisomycine, a translation elongation inhibitor FA: Formaldehyde MG132: proteasome-inhibitor mRPCs: crosslinks between proteins and poly-adenylated mRNAs isolated by Poly-A pulldown RPCs: crosslinks between proteins and RNA isolated by XRNAX Ub-E1i: ubiquitin E1 inhibitor (E1i, TAK-243) UVA: Ultraviolet A

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Thomas Fröhlich

LAB HEAD: Thomas Fröhlich

PROVIDER: PXD038332 | Pride | 2023-11-03

REPOSITORIES: Pride

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Project description:Long wavelength Ultraviolet (UVA-1) radiation causes oxidative stress that leads to the formation of noxious substances within the skin. As a defensive mechanism skin cells produce detoxifying enzymes and antioxidants when they detect modified molecules. We have recently shown that UVA-1 irradiation oxidizes the abundant membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which then induced the synthesis of the stress response protein heme oxygenase 1 (HO-1) in dermal fibroblasts. Here we examined the effects of UVA-1 and (UV-) oxidized phospholipids on the global gene expression in human dermal fibroblasts. We identified a cluster of genes that were co-induced by UVA-1-oxidized PAPC and UVA-1 radiation. The cluster included HO-1, glutamate-cysteine ligase modifier subunit (GCLM), aldo-keto reductases-1-C1 and -C2 (AKR1C1, AKR1C2), and interleukin 8 (IL8). These genes are members of the cellular stress response system termed âantioxidant responseâ or âPhase II detoxificationâ. Accordingly, the regulatory regions of all these genes contain binding sites for NF-E2-related factor 2 (Nrf2), a major regulator of the antioxidant response. Both UVA-1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation of Nrf2. Silencing expression of Nrf2 using siRNA or using cells and tissue from Nrf2-deficient mice, we show that the induction of the co-regulated genes was suppressed. Expression of other canonical UVA-1-induced genes, including cyclooxygenase 2 (Cox2) and interleukin 6 (IL6) was unaltered in the absence of Nrf2. Together, our data show that UVA-1-mediated lipid oxidation induces induction of antioxidant response genes, which is dependent on the redox-regulated transcription factor Nrf2. To activate Nrf2 is a major strategy for novel antioxidant drugs, the skin photo-adaptation (SPA) inducers. Our finding that specific uv-oxidized lipids act similar sheds a new (ultraviolet) light on the usually detrimental âimageâ of UV generated lipid mediators. Experiment Overall Design: we profiled global mRNA expression levels in human dermal fibroblasts that had been treated with either UVA-1 or oxidized lipids. To investigate the effect of oxidized phospholipids on gene regulation, we used two preparations, which differed in their degree of oxidation; the minimally oxidized UV-PAPC resulting from UVA-1 irradiation of PAPC, and air-oxidized PAPC (OxPAPC), which represents the full spectrum of oxidation products (Gruber 07) (Reis et al., 2005). We irradiated dermal fibroblasts with UVA-1 (40J/cmÂ²) or treated them with UV-PAPC, OxPAPC or native PAPC (100Âµg/ml each). We analyzed global gene expression four hours after stimulation with gene arrays (Affymetrix U133A Plus 2.0 Gene Chips).

			Action	DRS
	1_4SU_UVA_1.raw	Raw
	1_4SU_UVA_2.raw	Raw
	1_4SU_UVA_3.raw	Raw
	1_Ctrl_1.raw	Raw
	1_Ctrl_2.raw	Raw

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Translation-coupled resolution of RNA-protein crosslinks

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