Dataset Information

Quantitative RNA interactome capture (qRIC) identifies phosphorylation sites with regulatory potential - RBM20 BioID

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, less is known about how these modifications regulate RBP function. Here, we develop quantitative RNA-interactome capture (qRIC) to quantify the fraction of cellular RBPs pulled down with polyadenylated mRNAs. Applying qRIC to HEK293T cells quantified pulldown efficiencies of over 300 mRBPs. Combining qRIC with phosphoproteomics allowed us to systematically compare pulldown efficiencies of phosphorylated and non-phosphorylated forms of mRBPs. Over hundred phosphorylation sites show increased or decreased pull-down efficiency compared to their host 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 shuttling, association with cytosolic RNA granules and alternative splicing. Together, we show that qRIC is a scalable method to identify the function of posttranslational modifications in RBPs.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Carlos Henrique Vieira e Vieira

LAB HEAD: Matthias Selbach

PROVIDER: PXD027138 | Pride | 2022-09-05

REPOSITORIES: Pride

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Proteome-wide quantitative RNA-interactome capture identifies phosphorylation sites with regulatory potential in RBM20.

Vieira-Vieira Carlos Henrique CH Dauksaite Vita V Sporbert Anje A Gotthardt Michael M Selbach Matthias M

Molecular cell 20220414 11

Cellular mRNA-binding proteins (mRBPs) are major posttranscriptional regulators of gene expression. Although 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 mRBPs pulled down with polyadenylated mRNAs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphoryla ...[more]

PMID: 35427468

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Project description:Global transcriptomic alterations of both coding and non-coding RNA species are a ubiquitous feature associated with human cancers including hepatocellular carcinoma (HCC). Dysregulation of RNA-binding proteins (RBPs), the key regulators of RNA processing, is one mechanism in which cancer cells select to promote tumorigenesis. We analyzed genomic alterations amongst a family of more than 800 mRNA RBPs (mRBPs) in 1,225 clinical specimens from HCC patients and found that RBPs are significantly activated through gene amplification in a subset of tumors with poor prognosis, suggesting their potential oncogenic roles in HCC progression. Amongst the top candidates, RD binding protein (RDBP) was further characterized for its oncogenic role and effects on the HCC transcriptome. While the activation of RDBP induced an oncogenic phenotype, the abrogation of RDBP in HCC cells significantly decreased cancer associated phenotypes such as cell proliferation, migration/invasion and tumorigenicity in vivo. Further microarray analyses revealed that RDBP-dependent genes were tumor-related with a significant enrichment for c-Myc targets, suggesting interplay between RDBP and c-Myc signaling. Similar data were also found in HCC clinical specimens where c-Myc amplification was uncommon. Consistently, the RDBP-dependent c-Myc target gene signature was able to predict HCC patient survival in two independent cohorts of more than 400 patients. Taken together, our results suggest that oncogenic activation of RDBP is a novel mechanism that contributes to global transcriptome imbalance that is selective for the activation of c-Myc oncogenic signaling in HCC. We used microarray analysis to determine the affects of siRNA mediated RDBP knockdown in HCC transcriptome in cell lines. Hep3b and Huh1 cells were transfected with RDBP or scramble control siRNA for 48 hours in quadruplicates. Quality control using Spearman or Pearson correlation removes outliers resulting in triplicates for each group

Dataset Information

Quantitative RNA interactome capture (qRIC) identifies phosphorylation sites with regulatory potential - RBM20 BioID

Publications

Proteome-wide quantitative RNA-interactome capture identifies phosphorylation sites with regulatory potential in RBM20.

Similar Datasets

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