Proteome-wide quantitative RNA interactome capture (qRIC) identifies phosphorylation sites with regulatory potential in RBM20. Vieira-Vieira et al Figure S5A
Description
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.
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Steps to reproduce
HEK293 cells stably expressing RBM20 variants were seeded on coverslips coated with poly-L-lysine. Variant protein expression was induced in media containing 1 µg/mL tetracycline for 24 hours before cells were fixed for 15 min with 4 % paraformaldehyde at room temperature. Fixed cells were permeabilized for 10 min with 0.5 % Triton in PBS at room temperature and nonspecific protein binding was blocked by incubation in 1.5 % BSA PBS solution for 1 hours with low shaking. Cells were immuno stained by incubation for 1 hour at room temperature with a specific antibody against FLAG (1:1000 dilution) conjugated to Alexa 488 (Cell Signaling, cat# 5407S). Nucleus was stained with DAPI (Sigma-Aldrich, cat# D9564). Immunoflourescence images were acquired by Leica DM5000b microscope with an HCX PL FL 20x/0.50 objective.