Abstract
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 human embryonic kidney (HEK)-293T 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 indicates that phosphorylation and methylation do not often co-occur, suggesting that they may functionally oppose one another. Furthermore, we show that phosphorylation may modify interactions between Arg-rich proteins, as serine-arginine splicing factor 2 (SRSF2) has a 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.
Highlights
Key proteins that carry out specialized biological processes such as RNA splicing, polyadenylation and transport, contain domains disproportionately enriched with arginine [1, 2]
The LC1/Basic Acidic Dipeptide (BAD) domain of U1 small nuclear ribonucleoprotein kDa (U1-70K) is of particular biological interest due to its central role in U1-70K nuclear localization, granule formation, and co-aggregation with Tau in Alzheimer’s disease [3, 35, 43]
We describe a method to examine the post-translational modification AD (PTM) diversity of Arg-rich domains, re-purposing a technique first conceptualized by others to explore the biology of histone tails [16, 24, 32, 78]
Summary
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]. 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]. RS domains mimic BAD domains with a 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 [4,5,6,7,8,9]
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