Proteome-wide quantitative RNA interactome capture (qRIC) identifies phosphorylation sites with regulatory potential in RBM20. Vieira-Vieira et al. Figure 5A. WT RBM20

Cellular mRNA-binding proteins (mRBPs) are major regulators of gene expression at the post-transcriptional level. While many posttranslational modification sites in mRBPs have been identified, little is known about how these modifications regulate mRBP function. Here, we developed quantitative RNA-interactome capture (qRIC) to quantify the fraction of cellular mRBPs pulled down with polyadenylated mRNAs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphorylated and non-phosphorylated forms of mRBPs. Almost 200 phosphorylation events increased or decreased pull-down efficiency compared to the unmodified mRBPs and thus have regulatory potential. Our data captures known regulatory phosphorylation sites in ELAVL1, SF3B1 and UPF1 and identifies new potentially regulatory sites. Follow-up experiments on the cardiac splicing regulator RBM20 revealed that multiple phosphorylation sites in the C-terminal disordered region affect nucleo-cytoplasmic localization, association with cytoplasmic ribonucleoprotein granules and alternative splicing. Together, we show that qRIC in conjunction with phosphoproteomics is a scalable method to identify functional posttranslational modification sites in mRBPs.细胞内mRNA结合蛋白（mRBPs）是转录后水平基因表达的核心调控因子。尽管目前已鉴定出mRBPs上诸多翻译后修饰位点，但对于这些修饰如何调控mRBP的功能仍知之甚少。本研究开发了定量RNA相互作用组捕获技术（qRIC），用于定量可与多聚腺苷酸化mRNA共沉淀的细胞内mRBPs比例。将qRIC与磷酸化蛋白质组学相结合，我们得以系统性对比磷酸化与非磷酸化形式mRBPs的共沉淀效率。近200个磷酸化事件相较于未修饰的mRBPs，可提升或降低其共沉淀效率，因此具备潜在调控功能。本研究的数据不仅捕捉到ELAVL1、SF3B1与UPF1中已报道的调控性磷酸化位点，还鉴定出全新的潜在调控位点。针对心脏剪接调控因子RBM20的后续实验显示，其C端无序区域内的多个磷酸化位点可影响核质定位、与细胞质核糖核蛋白颗粒的结合以及可变剪接。综上，本研究证明qRIC与磷酸化蛋白质组学联用是一种可规模化的方法，用于鉴定mRBPs中具有功能的翻译后修饰位点。