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.
Steps to reproduce
Flp-In TRex 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. For quantification of the cellular distribution of RBM20 and colocalization analysis cells were immuno stained by incubation for 2 hour at room temperature with a specific antibody against FLAG (1:100 dilution, Sigma, cat# F3165), MOV10 (1:100 dilution, Sigma, cat# PLA0195. Gift from Dr. Marina Chekulaeva at the Max-Delbrueck Center for Molecular Medicine). Sample were washed three times with dPBS before further incubation with secondary antibodies Alexa Fluor 488 (1:500, Invitrogen cat# A11001 or cat# A11008) or 594 (1:500, Invitrogen cat# A11032or cat# A11012). Nucleus was stained with DAPI (Sigma-Aldrich, cat# D9564). Images were acquired with an inverted AxioObserver Z1 at a confocal laser scanning microscope LSM 980 (Zeiss) using an C Plan-Apochromat 63x/NA 1.40 objective. Confocal z-stacks covering the whole cell layer were acquired with a lateral and axial scaling of 0.071 µm and 0.190 µm, respectively, using photomultiplier tubes with a gain between 500 and 700V without oversaturation of pixels and at 16-bit data depth, with a pixel time of 0.41 µs, averaging 2 and a pinhole set close to 1 AU to ensure the same optical section for all channels. Fluorescence signals were acquired by sequential image acquisition using a 405nm laser at 1 % and 420 to 470 nm emission for DAPI, 488 nm laser at 1 % for AlexaFluor488 with 500 to 550 nm emission and 561nm laser at 1 % with 590 to 650 nm emission for AlexaFluor594. All image acquisition parameters were kept constant for all images, 3 to 5 positions containing each on average more than 30 cells were acquired from each sample.