Middle-down proteomics reveals dense sites of methylation and phosphorylation in arginine-rich RNA-binding proteins

Abstract

Abbreviations:MS: mass spectrometry ETD: electron transfer dissociation PTM: post-translational modification AD: Alzheimer's disease LC: liquid chromatography RBP: RNA-binding proteins PSM : peptide spectral match Briefs:Middle-down proteomics reveals arginine-rich RNA-binding proteins contain many sites of methylation and phosphorylation. AbstractArginine (Arg)-rich RNA-binding proteins play an integral role in RNA metabolism. Posttranslational modifications (PTMs) within Arg-rich domains, 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 HEK293 cells. Remarkably, the Arg-rich domains in RNA-binding proteins are densely modified by methylation and phosphorylation compared with the remainder of the proteome, with di-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. 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 metabolism. These data also serve as a resource to facilitate future mechanistic studies of the role of PTMs in RNA-binding protein structure and function.Key proteins that carry out specialized biological processes such as RNA splicing, polyadenylation and transport, contain domains disproportionately enriched with arginine [1,2].RNA-binding proteins (RBPs) that harbor Arginine (Arg)-rich domains may be broadly classified into two subsets based on residue composition. One class of these RBPs contains highly repetitive complementary repeats of basic (K/R) and acidic (D/E) residues, that we have previously referred to as Basic Acidic Dipeptide (BAD) domains [3]. Importantly, BAD domains facilitate protein aggregation, and in the context of Alzheimer's disease (AD), facilitate interactions with pathological Tau protein [3]. A second subset, related to the BAD proteins, are the Arginine/serinerich (RS) domains that are ubiquitous in the Serine/Arginine (SR) family of proteins [2]. Upon serine phosphorylation, RS domains mimic BAD domains with a similarly alternating basic-acidic dipeptide sequence pattern. The RS domains are commonly found in splicing factors and are essential for alternative splicing, protein-protein interactions, and localization [4-9]. The functional and structural diversity of the proteome is markedly increased through posttranslational modifications (PTMs), which...