Proteome-wide quantitative RNA interactome capture (qRIC) identifies phosphorylation sites with regulatory potential in RBM20
bioRxiv (Cold Spring Harbor Laboratory)
Abstract
Summary 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. Highlights qRIC globally quantifies the fraction of RNA-binding proteins pulled down with mRNA Combining qRIC with phosphoproteomics identifies sites that affect RNA binding Phosphorylation sites in RBM20 regulate its function in splicing
Authors 4
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Max Delbrück Center · Humboldt-Universität zu Berlin
Affiliation as printed
Humboldt-Universität zu Berlin, Faculty of Life Sciences, Unter den Linden 6, 10099 Berlin, Germany
Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, D-13092 Berlin, Germany
Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 0, D-3092 Berlin, Germany
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Max Delbrück Center · Leiden University Medical Center
Affiliation as printed
Einthoven Laboratory, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, D-13092 Berlin, Germany
Einthoven Laboratory, Leiden University Medical Center, 2 ZA Leiden, The Netherlands
Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 0, D-3092 Berlin, Germany
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Max Delbrück Center · German Centre for Cardiovascular Research · Charité - Universitätsmedizin Berlin
Affiliation as printed
Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany
German Genter for Cardiovascular Research (DZHK) Partner Site Berlin
Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, D-13092 Berlin, Germany
Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 0, D-3092 Berlin, Germany
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Matthias Selbach corresponding
Max Delbrück Center · Charité - Universitätsmedizin Berlin
Affiliation as printed
Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany
Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, D-13092 Berlin, Germany
Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 0, D-3092 Berlin, Germany
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