McKetta Department of Chemical Engineering, The University of Texas at Austingrid.89336.37, Austin, Texas, USA.
Institute for Cellular and Molecular Biology, The University of Texas at Austingrid.89336.37, Austin, Texas, USA.
Microbiol Spectr. 2022 Aug 31;10(4):e0214022. doi: 10.1128/spectrum.02140-22. Epub 2022 Jul 20.
8-Oxo-7,8-dihydroguanine (8-oxoG) is a major RNA modification caused by oxidative stresses and has been implicated in carcinogenesis, neurodegeneration, and aging. Several RNA-binding proteins have been shown to have a binding preference for 8-oxoG-modified RNA in eukaryotes and protect cells from oxidative stress. To date, polynucleotide phosphorylase (PNPase) is one of the most well-characterized proteins in bacteria that recognize 8-oxoG-modified RNA, but how PNPase cooperates with other proteins to process oxidized RNA is still unclear. Here, we use RNA affinity chromatography and mass spectrometry to search for proteins that preferably bind 8-oxoG-modified RNA in Deinococcus radiodurans, an extremophilic bacterium with extraordinary resistance to oxidative stresses. We identified four proteins that preferably bind to oxidized RNA: PNPase (DR_2063), DEAD box RNA helicase (DR_0335/RhlB), ribosomal protein S1 (DR_1983/RpsA), and transcriptional termination factor (DR_1338/Rho). Among these proteins, PNPase and RhlB exhibit high-affinity binding to 8-oxoG-modified RNA in a dose-independent manner. Deletions of PNPase and RhlB caused increased sensitivity of D. radiodurans to oxidative stress. We further showed that PNPase and RhlB specifically reduce the cellular availability of 8-oxoG-modified RNA but have no effect on oxidized DNA. Importantly, PNPase directly interacts with RhlB in D. radiodurans; however, no additional phenotypic effect was observed for the double deletion of and compared to the single deletions. Overall, our findings suggest the roles of PNPase and RhlB in targeting 8-oxoG-modified RNAs and thereby constitute an important component of D. radiodurans resistance to oxidative stress. Oxidative RNA damage can be caused by oxidative stress, such as hydrogen peroxide, ionizing radiation, and antibiotic treatment. 8-oxo-7,8-dihydroguanine (8-oxoG), a major type of oxidized RNA, is highly mutagenic and participates in a variety of disease occurrences and development. Although several proteins have been identified to recognize 8-oxoG-modified RNA, the knowledge of how RNA oxidative damage is controlled largely remains unclear, especially in nonmodel organisms. In this study, we identified four RNA binding proteins that show higher binding affinity to 8-oxoG-modified RNA compared to unmodified RNA in the extremophilic bacterium , which can endure high levels of oxidative stress. Two of the proteins, polynucleotide phosphorylase (PNPase) and DEAD-box RNA helicase (RhlB), interact with each other and reduce the cellular availability of 8-oxoG-modified RNA under oxidative stress. As such, this work contributes to our understanding of how RNA oxidation is influenced by RNA binding proteins in bacteria.
8-氧代-7,8-二氢鸟嘌呤（8-oxoG）是一种主要的 RNA 修饰物，由氧化应激引起，并与致癌作用、神经退行性变和衰老有关。已经有研究表明，在真核生物中，几种 RNA 结合蛋白对 8-氧代 G 修饰的 RNA 具有结合偏好性，并能保护细胞免受氧化应激的影响。迄今为止，多核苷酸磷酸化酶（PNPase）是细菌中研究最深入的一种蛋白质，能够识别 8-氧代 G 修饰的 RNA，但 PNPase 如何与其他蛋白质合作来处理氧化的 RNA 仍不清楚。在这里，我们使用 RNA 亲和层析和质谱法，在一种具有极强抗氧化应激能力的极端微生物 中寻找优先结合 8-氧代 G 修饰 RNA 的蛋白质。我们鉴定出了四种优先结合氧化 RNA 的蛋白质：多核苷酸磷酸化酶（DR_2063）、DEAD 盒 RNA 解旋酶（DR_0335/RhlB）、核糖体蛋白 S1（DR_1983/RpsA）和转录终止因子（DR_1338/Rho）。在这些蛋白质中，PNPase 和 RhlB 以剂量不依赖的方式与 8-氧代 G 修饰 RNA 表现出高亲和力结合。PNPase 和 RhlB 的缺失会导致 对氧化应激的敏感性增加。我们进一步表明，PNPase 和 RhlB 特异性降低细胞中 8-氧代 G 修饰 RNA 的含量，但对氧化 DNA 没有影响。重要的是，PNPase 在 中与 RhlB 直接相互作用；然而，与单个缺失相比，和 的双缺失没有观察到额外的表型效应。总的来说，我们的研究结果表明 PNPase 和 RhlB 在靶向 8-氧代 G 修饰 RNA 方面的作用，这构成了 对氧化应激抗性的重要组成部分。
氧化的 RNA 损伤可能由氧化应激引起，如过氧化氢、电离辐射和抗生素治疗。8-氧代-7,8-二氢鸟嘌呤（8-oxoG）是一种主要的氧化 RNA，具有高度致突变性，参与多种疾病的发生和发展。尽管已经鉴定出几种能够识别 8-氧代 G 修饰 RNA 的蛋白质，但对于 RNA 氧化损伤的控制知之甚少，特别是在非模式生物中。在这项研究中，我们鉴定了四种 RNA 结合蛋白，它们在极端微生物 中与未修饰的 RNA 相比，对 8-氧代 G 修饰 RNA 具有更高的结合亲和力，该微生物可以耐受高水平的氧化应激。这两种蛋白质中的两种，多核苷酸磷酸化酶（PNPase）和 DEAD 盒 RNA 解旋酶（RhlB）相互作用，在氧化应激下降低细胞中 8-氧代 G 修饰 RNA 的含量。因此，这项工作有助于我们了解在细菌中 RNA 氧化如何受 RNA 结合蛋白的影响。
