LAUSR

Post-translational modifications (PTMs) within arginine (Arg)-rich RNA-binding proteins, such as phosphorylation and methylation, regulate multiple steps in RNA metabolism. However, the identification of PTMs within Arg-rich domains with complete trypsin digestion is extremely challenging due to the high density of Arg residues within these proteins. Here, we report a middle-down proteomic approach coupled with electron transfer dissociation (ETD) mass spectrometry to map previously unknown sites of phosphorylation and methylation within the Arg-rich domains of U1-70K and structurally similar RNA-binding proteins from nuclear extracts of HEK293T cells. Notably, the Arg-rich domains in RNA-binding proteins are densely modified by methylation and phosphorylation compared with the remainder of the proteome, with methylation and phosphorylation favoring RSRS motifs. Although they favor a common motif, analysis of combinatorial PTMs within RSRS motifs indicate that phosphorylation and methylation do not often co-occur, suggesting they may functionally oppose one another. Furthermore, we show that phosphorylation may modify interactions between Arg-rich proteins, as SRSF2 has stronger association with U1-70K and LUC7L3 upon dephosphorylation. Collectively, these findings suggest that the level of PTMs within Arg-rich domains may be among the highest in the proteome, and a possible unexplored regulator of RNA-binding protein interactions.