RNase H1-dependent phosphorothioate oligonucleotides (PS-ASOs) have been developed to treat various diseases through specific degradation of target RNAs. Although many factors or features of RNA and PS-ASOs have been demonstrated to affect antisense activity of PS-ASOs, little is known regarding the roles of RNase H1-associated proteins in PS-ASO performance. In this study, we report that two nucleolar proteins, NAT10 and DDX21, interact with RNase H1 and affect the potency and safety of PS-ASOs. The interactions of these two proteins with RNase H1 were determined using BioID proximity labeling in cells and confirmed biochemically. Reduction of NAT10 and DDX21 decreased PS-ASO activity in cells, and purified NAT10 and DDX21 proteins enhanced RNase H1 cleavage rates, indicating that these two proteins facilitate RNase H1 endoribonuclease activity. Consistently, reduction of these proteins increased the levels of R-loops, and impaired pre-rRNA processing. In addition, reduction of the two proteins increased the cytotoxicity of toxic PS-ASOs, and treatment of toxic PS-ASOs also altered the localization of these proteins. Together, this study shows for the first time that NAT10 and DDX21 interact with RNase H1 protein and enhance its enzymatic activity, contributing to the potency and safety of PS-ASOs.

中文翻译：

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NAT10 和 DDX21 蛋白与 RNase H1 相互作用并影响硫代磷酸寡核苷酸的性能

RNase H1 依赖性硫代磷酸寡核苷酸 (PS-ASO) 已被开发用于通过靶 RNA 的特异性降解来治疗各种疾病。尽管已经证明 RNA 和 PS-ASO 的许多因素或特征会影响 PS-ASO 的反义活性，但关于 RNase H1 相关蛋白在 PS-ASO 性能中的作用知之甚少。在这项研究中，我们报告了两种核仁蛋白 NAT10 和 DDX21，与 RNase H1 相互作用并影响 PS-ASO 的效力和安全性。这两种蛋白质与 RNase H1 的相互作用是在细胞中使用 BioID 邻近标记确定的，并通过生化方法进行确认。NAT10 和 DDX21 的减少降低了细胞中 PS-ASO 的活性，而纯化的 NAT10 和 DDX21 蛋白提高了 RNase H1 的切割率，表明这两种蛋白质促进了 RNase H1 核糖核酸内切酶的活性。一致地，这些蛋白质的减少增加了R-loops的水平，并损害了pre-rRNA加工。此外，这两种蛋白质的减少增加了有毒 PS-ASO 的细胞毒性，并且有毒 PS-ASO 的处理也改变了这些蛋白质的定位。总之，这项研究首次表明 NAT10 和 DDX21 与 RNase H1 蛋白相互作用并增强其酶活性，有助于 PS-ASO 的效力和安全性。