Characterization of RNA-binding proteins in the cell nucleus and cytoplasm

Abstract

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.

Keywords: Enzyme; LC-MS/MS; Mass spectrometry; RNA binding protein; RNA binding site.