RNA oxidation, predominantly through the accumulation of 8-oxo-7,8-dihydroguanosine (8-oxo-rG), represents an important biomarker for cellular oxidative stress. Polynucleotide phosphorylase (PNPase) is a 3′-5′ exoribonuclease that has been shown to preferentially recognize 8-oxo-rG-containing RNA and protect E scherichia coli cells from oxidative stress. However, the impact of 8-oxo-rG on PNPase-mediated RNA degradation has not been studied. Here, we show that the presence of 8-oxo-rG in RNA leads to catalytic stalling of E. coli PNPase through in vitro RNA degradation experiments and electrophoretic analysis. We also link this stalling to the active site of the enzyme through resolution of single-particle cryo-EM structures for PNPase in complex with singly or doubly oxidized RNA oligonucleotides. Following identification of Arg399 as a key residue in recognition of both single and sequential 8-oxo-rG nucleotides, we perform follow-up in vitro analysis to confirm the importance of this residue in 8-oxo-rG-specific PNPase stalling. Finally, we investigate the effects of mutations to active site residues implicated in 8-oxo-rG binding through E. coli cell growth experiments under H 2 O 2 -induced oxidative stress. Specifically, Arg399 mutations show significant effects on cell growth under oxidative stress. Overall, we demonstrate that 8-oxo-rG-specific stalling of PNPase is relevant to bacterial survival under oxidative stress and speculate that this enzyme might associate with other cellular factors to mediate this stress.

中文翻译：

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在降解过程中，多核苷酸磷酸化酶 （PNPase） 的催化核心中发生选择性 8-oxo-rG 停滞


RNA 氧化主要通过 8-氧代-7,8-二氢鸟苷 （8-oxo-rG） 的积累，是细胞氧化应激的重要生物标志物。多核苷酸磷酸化酶 （PNPase） 是一种 3′-5′ 核糖核酸外切酶，已被证明可优先识别含 8-oxo-rG 的 RNA 并保护大肠杆菌细胞免受氧化应激。然而，尚未研究 8-oxo-rG 对 PNPase 介导的 RNA 降解的影响。在这里，我们通过体外 RNA 降解实验和电泳分析表明 RNA 中存在 8-oxo-rG 导致大肠杆菌 PNPase 的催化停滞。我们还通过分辨 PNPase 与单氧化或双氧化 RNA 寡核苷酸复合物中的单颗粒冷冻电镜结构，将这种停滞与酶的活性位点联系起来。在将 Arg399 鉴定为识别单个和连续 8-oxo-rG 核苷酸的关键残基后，我们进行后续体外分析以确认该残基在 8-oxo-rG 特异性 PNPase 停滞中的重要性。最后，我们在 H 2 O 2 诱导的氧化应激下，通过大肠杆菌细胞生长实验研究了突变对 8-oxo-rG 结合的活性位点残基的影响。具体来说，Arg399 突变在氧化应激下对细胞生长有显著影响。总体而言，我们证明 PNPase 的 8-oxo-rG 特异性停滞与氧化应激下的细菌存活有关，并推测这种酶可能与其他细胞因子结合以介导这种应激。