Project description:The primary mRNA sequence determines its secondary structure and the repertoire of interacting RNA-binding proteins (RBPs). The resulting mRNA ribonucleoprotein complex (mRNP) then influences all stages of the life of an mRNA. Here, we determined the mRNP composition of individual Kaposi Sarcoma Herpesviral (KSHV) mRNAs. In KSHV, the viral RNA regulator ORF57 ensures the translation of viral mRNAs by increasing mRNA stability and nuclear export. By optimizing an LNA/DNA mixmer RNA capture protocol to both transfection and virus replication settings, we identified the RBPome of specific ORF57-dependent viral transcripts. Both capture and eCLIP experiments robustly detected ORF57 as a direct RNA binder to an AU-rich motif, which may enable ORF57 to discriminate viral from cellular RNAs. Furthermore, we identified the RNA processing factor SRSF3 as a key regulator of viral replication. This work facilitates RNA-interactome studies of specific mRNAs and sheds light on how the mRNP composition orchestrates gene expression.
Project description:RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to globally identifying bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down mRNA-protein complexes. By overexpressing poly(A) polymerase I, we drastically increase the fraction of polyadenylated RNA in Escherichia coli, allowing us to pull-down RNA-protein complexes using immobilized oligo-d(T) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding
Project description:RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to direct and global identification of bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down RNA-protein complexes. Overexpressing poly(A) polymerase I in Escherichia coli drastically increased transcriptome-wide RNA polyadenylation, enabling pull-down of crosslinked RNA-protein complexes using immobilized oligo(dT) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of a number of uncharacterized RBPs, including YhgF, which is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF RNA targets in vivo using CLIP-seq, verified specific binding in vitro, and reveal a putative role for YhgF in regulation of gene expression. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lay the foundation for further studies of the highly conserved RBP YhgF.
Project 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, 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.
Project description:Pathogen components, such as lipopolysaccharides of Gram-negative bacteria that activate Toll-like receptor 4, induce mitogen activated protein kinases and NFκB through different downstream pathways to stimulate pro- and anti-inflammatory cytokine expression. Importantly, post-transcriptional control of the expression of Toll-like receptor 4 downstream signaling molecules contributes to the tight regulation of inflammatory cytokine synthesis in macrophages. Emerging evidence highlights the role of RNA-binding proteins (RBPs) in the post-transcriptional control of the innate immune response. To systematically identify macrophage RBPs and their response to LPS stimulation, we employed RNA interactome capture in LPS-induced and untreated murine RAW 264.7 macrophages. This combines RBP-crosslinking to RNA, cell lysis, oligo(dT) capture of polyadenylated RNAs and mass spectrometry analysis of associated proteins. Our data revealed 402 proteins of the macrophage RNA interactome including 91 previously not annotated as RBPs. A comparison with published RNA interactomes classified 32 RBPs uniquely identified in RAW 264.7 macrophages. Of these, 19 proteins are linked to biochemical activities not directly related to RNA. From this group, we validated the HSP90 cochaperone P23 that was demonstrated to exhibit cytosolic prostaglandin E2 synthase 3 (PTGES3) activity, and the hematopoietic cell-specific LYN substrate 1 (HCLS1 or HS1), a hematopoietic cell-specific adapter molecule, as novel macrophage RBPs. Our study expands the mammalian RBP repertoire, and identifies macrophage RBPs that respond to LPS. These RBPs are prime candidates for the post-transcriptional regulation and execution of LPS-induced signaling pathways and the innate immune response. Macrophage RBP data have been deposited to ProteomeXchange with identifier PXD002890.
Project 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, 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.