Middle-Down Proteomics Reveals Dense Sites of Methylation and Phosphorylation in Arginine-Rich RNA-Binding Proteins

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.

富含精氨酸（Arg）的RNA结合蛋白内部的翻译后修饰（PTMs），如磷酸化与甲基化，可调控RNA代谢的多个步骤。然而，由于此类蛋白质内部精氨酸残基密度极高，采用完全胰蛋白酶消化的方法对富含精氨酸结构域内的翻译后修饰进行鉴定，极具挑战性。本研究报道了一种结合电子转移解离（ETD）质谱的中分段蛋白质组学方法，用于定位人胚肾（HEK）-293T细胞核提取物中U1-70K及结构相似RNA结合蛋白的富含精氨酸结构域内此前未被发现的磷酸化与甲基化位点。值得注意的是，与蛋白质组其余区域相比，RNA结合蛋白中的富含精氨酸结构域被甲基化与磷酸化修饰的密度更高，且两类修饰更倾向于富集于RSRS基序。尽管二者共享偏好的基序，但对RSRS基序内组合式翻译后修饰的分析显示，磷酸化与甲基化很少同时发生，提示二者可能在功能上相互拮抗。此外，本研究证实磷酸化可调控富含精氨酸蛋白之间的相互作用：丝氨酸-精氨酸剪接因子2（SRSF2）在去磷酸化后，与U1-70K及LUC7L3的结合能力显著增强。综上，上述研究结果表明，富含精氨酸结构域内的翻译后修饰水平可能是蛋白质组中最高的修饰类型之一，同时也是一类尚未被探索的RNA结合蛋白相互作用调控因子。