Yang Zhixiang, Zhou Junwei, Li Zhuang, Guo Jiahui, Fang Liurong, Xiao Xun, Xiao Shaobo
National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
The Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China.
RNA Biol. 2024 Jan;21(1):32-45. doi: 10.1080/15476286.2024.2386498. Epub 2024 Aug 8.
Interactions between double-stranded RNA (dsRNA) and proteins play an important role in cellular homeostasis by regulating the editing, stability, and splicing of intracellular RNA. The identification of dsRNA-binding proteins (dsRBPs) is key; however, it has long been challenging to purify dsRBPs from cells. In this study, we developed a novel method, dsRBPC (dsRNA-binding protein capture), to purify cellular dsRBPs based on classic phase separation purification procedures. A global dsRNA-binding proteome of LLC-PK1 cells was obtained, and we identified 1326 dsRBPs, including 1303 putative novel dsRBPs. Functional analyses suggested that these enriched dsRBPs are mainly associated with rRNA processing, RNA splicing, transcriptional regulation, and nucleocytoplasmic transport. We also found that the ARM (armadillo/beta-catenin-like repeats) motif is a previously unknown dsRNA-binding domain, as demonstrated by biochemical experiments. Collectively, this study provides a useful approach for dsRBP identification and the discovery of a global dsRNA-binding proteome to comprehensively map the dsRNA - protein interaction network.
双链RNA（dsRNA）与蛋白质之间的相互作用通过调节细胞内RNA的编辑、稳定性和剪接，在细胞内稳态中发挥重要作用。鉴定双链RNA结合蛋白（dsRBPs）是关键；然而，长期以来，从细胞中纯化dsRBPs一直具有挑战性。在本研究中，我们基于经典的相分离纯化程序，开发了一种新型方法dsRBPC（双链RNA结合蛋白捕获法）来纯化细胞dsRBPs。我们获得了LLC-PK1细胞的全基因组双链RNA结合蛋白质组，并鉴定出1326种dsRBPs，其中包括1303种推定的新型dsRBPs。功能分析表明，这些富集的dsRBPs主要与rRNA加工、RNA剪接、转录调控和核质运输有关。我们还发现，通过生化实验证明，ARM（犰狳/β-连环蛋白样重复序列）基序是一个以前未知的双链RNA结合结构域。总的来说，本研究为dsRBP鉴定提供一种有用的方法，并为全面绘制双链RNA-蛋白质相互作用网络发现了一个全基因组双链RNA结合蛋白质组。
