Department of Core Antisense Research, Ionis Pharmaceuticals, Inc., Carlsbad, CA, USA.
Nucleic Acids Res. 2019 Nov 18;47(20):10865-10880. doi: 10.1093/nar/gkz771.
The rapid RNase H1-dependent mislocalization of heterodimer proteins P54nrb and PSF to nucleoli is an early event in the pathway that explains the effects of most toxic phosphorothioate ASOs (PS-ASOs). Using a recently developed NanoLuciferace (NLuc)-based structural complementation reporter system which allows us to observe ASO/protein interactions in real time in live cells, we have determined that safe and toxic PS-ASOs associate with these proteins with kinetics and impact on subcellular localization that differ. Toxic PS-ASOs interact in a complex that includes RNase H1, P54nrb and PSF; but RNase H1/P54nrb complexes were observed in only the cells treated with toxic, but not safe PS-ASOs. In addition, experiments performed in vitro suggest that RNA is also a required component of the complex. The protein-protein interaction between P54nrb and RNase H1 requires the spacer region of RNAse H1, while the P54nrb core domains are required for association with RNase H1. In addition, we have determined that PS-ASOs bind P54nrb via RRM1 and RRM2, while they bind RNase H1 primarily via the hybrid binding domain, however catalytic domain interactions also contribute to overall affinity. These ASO-protein interactions are highly influenced by the chemistry of the PS-ASO binding environment, however little correlation between affinity for specific proteins and PS-ASO toxicity was observed.
异二聚体蛋白 P54nrb 和 PSF 依赖于快速的 RNase H1 向核仁的错误定位是解释大多数毒性硫代磷酸酯 ASO（PS-ASO）作用途径的早期事件。使用最近开发的基于 NanoLuciferase（NLuc）的结构互补报告系统，我们可以实时观察活细胞中的 ASO/蛋白相互作用，我们已经确定安全和毒性 PS-ASO 与这些蛋白的结合动力学和对亚细胞定位的影响不同。毒性 PS-ASO 与包括 RNase H1、P54nrb 和 PSF 在内的复合物相互作用；但仅在使用毒性而非安全 PS-ASO 处理的细胞中观察到 RNase H1/P54nrb 复合物。此外，体外实验表明 RNA 也是复合物的必需成分。P54nrb 和 RNase H1 之间的蛋白-蛋白相互作用需要 RNase H1 的间隔区，而 P54nrb 核心结构域需要与 RNase H1 结合。此外，我们已经确定 PS-ASO 通过 RRM1 和 RRM2 结合 P54nrb，而它们主要通过杂交结合域结合 RNase H1，但催化域相互作用也有助于整体亲和力。这些 ASO-蛋白相互作用受到 PS-ASO 结合环境化学性质的高度影响，但观察到对特定蛋白的亲和力与 PS-ASO 毒性之间几乎没有相关性。
