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Proteome-wide quantitative RNA interactome capture (qRIC) identifies phosphorylation sites with regulatory potential in RBM20

Item Type:Preprint
Title:Proteome-wide quantitative RNA interactome capture (qRIC) identifies phosphorylation sites with regulatory potential in RBM20
Creators Name:Vieira-Vieira, C.H. and Dauksaite, V. and Gotthardt, M. and Selbach, M.
Abstract: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. Applying qRIC to HEK293T cells quantified pull-down efficiencies of over 300 RBPs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphorylated and non-phosphorylated forms of RBPs. Over hundred 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 cytosolic RNA granules and alternative splicing. Together, we show that qRIC is a scalable method to identify functional posttranslational modification sites in RBPs.
Keywords:RNA-Binding Proteins, RNA-Interactome Capture, Cell Signaling, Phosphorylation, Proteomics, RBM20, Alternative Splicing, Cardiomyopathy
Source:bioRxiv
Publisher:Cold Spring Harbor Laboratory Press
Article Number:2021.07.12.452044
Date:12 July 2021
Official Publication:https://doi.org/10.1101/2021.07.12.452044

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