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RNA interactome capture in Escherichia coli globally identifies RNA-binding proteins

ABSTRACT: RNA-binding proteins (RPBs) are deeply involved in many 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 the poly(A) polymerase I, we drastically increase the frequency of polyadenylated RNA in E. coli, allowing us to pull down RNA-protein complexes using immobilized oligo-d(T) as bait. With this approach, we identified 167 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of several proteins previously unknown to interact with RNA, including the uncharacterized protein YhgF. YhgF is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF in vivo RNA targets using CLIP-seq, two of which were verified using electromobility shift assays. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lays the foundation for further studies of the strongly conserved RBP Yhg

INSTRUMENT(S): Synapt MS

ORGANISM(S): Escherichia Coli

SUBMITTER: Erik Jansson

LAB HEAD: Erik Jansson

PROVIDER: PXD032939 | Pride | 2023-03-30

REPOSITORIES: Pride

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RNA interactome capture in E. coli to identify RNA-binding proteins

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

2022-11-13 | GSE217569 | GEO

Global discovery of bacterial RNA-binding proteins by RNase-sensitive gradient profiles reports a new FinO domain protein

Project description:RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinO-domain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element for building a comprehensive catalogue of microbial RBPs.

2020-07-16 | PXD018422 | Pride

Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis

Project description:RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture(RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of two melanoma cell lines with different aggressiveness revealed prevalent changes in RNA binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five differentin vitroassays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression and map its RNA-binding domain, which we find to be required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novelvulnerabilities of cancer cells.

2022-07-19 | PXD029927 | Pride

Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis

Project description:RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of two melanoma cell lines with different aggressiveness revealed prevalent changes in RNA binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression and map its RNA-binding domain, which we find to be required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells

2022-07-11 | GSE174821 | GEO

Global analysis of the RNA-protein interaction and RNA secondary structure landscapes of the Arabidopsis nucleus

Project description:Post-transcriptional regulation in eukaryotes requires cis- and trans-acting features and factors including RNA secondary structure, and RNA-binding proteins (RBPs). However, a comprehensive view of the structural and RBP interaction landscape of RNAs in the nucleus has yet to be compiled for any organism. Here, we use our ribonuclease-mediated structure and RBP binding site mapping approach on Arabidopsis seedling nuclei in vivo to globally profile these features within the nuclear compartment. We reveal opposing patterns of secondary structure and RBP binding levels throughout native messenger RNAs that demarcate alternative splicing and polyadenylation. We also uncover a collection of protein bound sequence motifs, and identify their structural contexts, co-occurrences in transcripts encoding functionally related proteins, and interactions with putative RBPs. Finally, we identify a nuclear role for the chloroplast RBP, CP29A. In total, we provide the first simultaneous view of the RNA secondary structure and RBP interaction landscapes in a eukaryotic nucleus. Protein interaction profile sequencing (PIP-seq) in Arabidopsis seedling nuclei. These are crosslinked with formaldehyde and treated with two RNases (ssRNase and dsRNase) with two replicates

2014-12-16 | E-GEOD-58974 | biostudies-arrayexpress

S. cerevisiae Nab2-PrA interactomes in exosome, transport and ribosome biogenesis mutant backgrounds

Project description:RNA-binding proteins (RBPs) are key mediators of RNAbiogenesis and metabolism. An RBP can be dedicated to a specific transcript class (e.g. mRNAs) or function broadly across both coding and non-coding RNAs. Consequently, nuclear RNA homeostasis requires maintenance of bothRBP binding specificity and the proper distribution of RBP activity across multiple transcript classes. In S. cerevisiae, mutation of an RBP that functions in ribosome biogenesis, Enp1, was previously found to have terminal phenotypes similar to exosome or TRAMP mutants that extended beyond ribosomal RNA processing defects. Here, we show through proteomicand RNomic analyses that enp1-1 cells have stabilized polyadenylated pre-rRNAs that bind and sequester RBPs in the nucleolus, including the poly(A)-binding protein (PABP) Nab2. Changes in nucleolar polyadenylated RNA levels and PABP availability are further accompanied by transcriptome-wide changes in RNA biogenesis in enp1-1 cells, including increased levels of pervasive transcripts and snoRNA processing defects. The observed changes in RNA processing can be mitigated if enp1-1 cells are provided excess PABP activity through overexpression of Nab2 or Pab1. Together, these findings indicate that generation of excess nucleolar poly(A)-RNA is toxic to the cell, disrupting nuclear RNA processing homeostasis through sequestering RBPs, including the PABP Nab2.

2020-10-08 | PXD021855 | Pride

System-wide profiling of RNA-binding proteins uncovers key regulators of virus infection

Project description:The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed ‘comparative RIC’ and applied it to cells challenged with an RNA virus, called sindbis (SINV). Over two hundred RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localisation and function in SINV-infected cells.

2019-02-21 | GSE125182 | GEO

Serial interactome capture of the human cell nucleus

Project description:Novel RNA-guided cellular functions are paralleled by an increasing number of RNA binding proteins (RBPs). We present “serial interactome capture” (serIC), a multiple purification procedure of UV-crosslinked poly(A)-RNA-protein complexes that enables global RBP detection with maximal specificity. We apply serIC to nuclei of proliferating K562 cells to obtain the first human nuclear interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including fewer factors without known RNA binding domains that are in better agreement with computationally predicted RNA binding. serIC extends the number of DNA-RNA binding proteins (DRBPs), and reveals a network of RBPs involved in p53 signaling and double strand break repair. serIC is an effective tool to couple global RBP capture with additional selection or labelling steps for specific detection of highly purified RBPs. The nuclear interactome presented here is a stepping-stone towards deciphering of the functional RNA-protein network in the mammalian nucleus.

2016-10-21 | MSV000080265 | MassIVE

RNA-interactome capture in human embryonic stem cells (hESCs)

Project description:Embryonic stem cell (ESC) self-renewal and cell-fate decisions are driven by a broad array of molecular signals. While transcriptional regulators have been extensively studied in human ESCs (hESCs), the extent to which RNA-binding proteins (RBPs) contribute to human pluripotency remains unclear. Here, we carried out a proteome-wide screen and identified 810 proteins that directly bind RNA in hESCs. We determined the RBP catalog by using RNA-interactome capture (RIC), a method based on UV light-mediated cross-linking (CL) of RNAs to proteins in living cells, followed by oligo(dT) purification of poly(A)-RNA-protein complexes and mass spectrometry analysis of captured proteins. As control, we applied a similar strategy to non-cross-linked (non-CL) samples. To uncover the identity of the eluted proteins, we performed in-solution tryptic digestion of CL and non-CL eluates and analyzed their contents by a high-resolution mass spectrometer (Q-Exactive Plus). We then performed differential proteome analysis between CL and non-CL eluates, resulting in a set of 810 high-confidence protein groups, defined as the hESC RNA-interactome. RIC was carried out in four independent biological replicates. This data accompanies the manuscript: "Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells".

2023-05-05 | PXD025194 | Pride

rec-Y2H matrix in S. cerevisiae screening reveals a vast potential for direct protein-protein interactions among RNA binding proteins

Project description:RNA-binding proteins (RBPs) are crucial factors of post-transcriptional gene regulation and their modes of action are intensely investigated. At the center of attention are RNA motifs that guide where RBPs bind. However, sequence motifs recognized by RBPs are typically a poor predictor of RBP-RNA interactions in vivo. It is hence believed that many RBPs recognize RNAs as complexes, to increase specificity and regulatory potential. To probe the potential for RBP–RBP complex formation, we assembled a library of 978 mammalian RBPs and used rec-Y2H screening to detect direct interactions between RBPs, sampling >1M possible interactions. We discovered 1994 new interactions and demonstrate that our interaction screening discovers RBP pairs that bind RNAs adjacently. We further find that the mRNA binding region preferences of an RBP can deviate, depending on its adjacently binding interaction partner. Finally, we reveal novel RBP–RBP interaction networks among major RNA processing steps and show that RBP mutations observed in cancer rewire spliceosomal interaction networks.

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