School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China; Peking University First Hospital, Peking University Health Science Center, Beijing, 100034, China.
School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China.
Anal Chim Acta. 2021 Jul 11;1168:338609. doi: 10.1016/j.aca.2021.338609. Epub 2021 May 5.
As critical players in the regulation of gene expression, RNA-binding proteins (RBPs) play fundamental roles in cellular functions and diseases. In this study, we established an analytical strategy to characterize RBPs from different subcellular regions by combining subcellular fractionation, acidic guanidinium-thiocyanate-phenol-chloroform biphasic extraction, and quantitative mass spectrometry. Using this method, we identified 1775 and 2245 RBPs from the cell nucleus and cytoplasm. The data confirmed a large spectrum of known RBPs, revealed 614 novel ones that have never been reported before, and cataloged their subcellular localizations. Intriguingly, 200 metabolic enzymes from diverse metabolic pathways were observed as RBPs, some of which were further validated through western blotting following UV-mediated crosslinking and biphasic extraction. Furthermore, we characterized 2157 RNA-binding interfaces, providing structural information regarding the complex nature of RNA-protein interactions. Taken together, our data greatly expand the current reservoir of known RBPs and highlight the potential role of RNA-binding in the regulation of cellular metabolism.
作为基因表达调控的关键参与者，RNA 结合蛋白（RBPs）在细胞功能和疾病中发挥着基础性作用。在这项研究中，我们结合亚细胞组分分离、酸性胍盐-硫氰酸胍-酚-氯仿两相萃取和定量质谱分析，建立了一种从不同亚细胞区域鉴定 RBPs 的分析策略。利用该方法，我们从细胞核和细胞质中分别鉴定到 1775 种和 2245 种 RBP。这些数据不仅证实了大量已知 RBP，还揭示了 614 种以前从未报道过的新 RBP，并对它们的亚细胞定位进行了编目。有趣的是，我们观察到来自不同代谢途径的 200 种代谢酶作为 RBPs，其中一些通过紫外线介导的交联和两相萃取后进行 Western blot 进一步验证。此外，我们还鉴定到 2157 个 RNA 结合界面，为 RNA-蛋白质相互作用的复杂性质提供了结构信息。总之，我们的数据极大地扩展了已知 RBP 的储备库，并强调了 RNA 结合在调控细胞代谢中的潜在作用。
