Proteome-wide quantitative RNA interactome capture (qRIC) identifies phosphorylation sites with regulatory potential in RBM20. Vieira-Vieira et al Figure S5A

RNA-binding proteins (RBPs) are major regulators of gene expression at the post-transcriptional level. While many posttranslational modification sites in RBPs have been identified, little is known about how these modifications regulate RBP function. Here, we developed quantitative RNA-interactome capture (qRIC) to quantify the fraction of cellular RBPs pulled down with polyadenylated mRNAs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphorylated and non-phosphorylated forms of RBPs. Almost 200 phosphorylation events increased or decreased pull-down efficiency compared to the unmodified RBPs 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 RNP granules and alternative splicing. Together, we show that qRIC in conjunction with phosphoproteomics is a scalable method to identify functional posttranslational modification sites in RBPs.