Department of Core Antisense Research, Ionis Pharmaceuticals, Inc., Carlsbad, California, USA.
Nucleic Acid Ther. 2022 Aug;32(4):280-299. doi: 10.1089/nat.2021.0107. Epub 2022 Jul 18.
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.
RNase H1 依赖性硫代磷酸酯寡核苷酸（PS-ASO）已被开发用于通过特异性降解靶 RNA 来治疗各种疾病。尽管许多 RNA 和 PS-ASO 的因素或特征已被证明会影响 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。这些两种蛋白与 RNase H1 的相互作用是通过细胞中的 BioID 邻近标记法确定的，并通过生化方法得到证实。NAT10 和 DDX21 的减少降低了 PS-ASO 在细胞中的活性，而纯化的 NAT10 和 DDX21 蛋白增强了 RNase H1 的内切核酸酶活性，表明这两种蛋白促进了 RNase H1 的内切酶活性。一致地，这些蛋白的减少增加了 R 环的水平，并损害了 pre-rRNA 的加工。此外，两种蛋白的减少增加了毒性 PS-ASO 的细胞毒性，而毒性 PS-ASO 的处理也改变了这些蛋白的定位。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，从而提高了 PS-ASO 的效力和安全性。
