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Serial interactome capture of the human cell nucleus.

ABSTRACT: Novel RNA-guided cellular functions are paralleled by an increasing number of RNA-binding proteins (RBPs). Here we present 'serial RNA interactome capture' (serIC), a multiple purification procedure of ultraviolet-crosslinked poly(A)-RNA-protein complexes that enables global RBP detection with high specificity. We apply serIC to the nuclei of proliferating K562 cells to obtain the first human nuclear RNA interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including few factors without known RNA-binding domains that are in good 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 signalling 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.

SUBMITTER: Conrad T

PROVIDER: S-EPMC4822031 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Progress toward resolving the controversy of positive Vs. negative interstitial fluid pressure.

Brace R A RA

Circulation research 19810801 2

PMID: 7018732

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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.

2016-03-31 | PXD003664 | Pride

Serial interactome capture of the human cell nucleus

2016-10-21 | MSV000080265 | MassIVE

RNA interactome capture in yeast.

Project description:RNA-binding proteins (RBPs) are key players in post-transcriptional regulation of gene expression in eukaryotic cells. To be able to unbiasedly identify RBPs in Saccharomyces cerevisiae, we developed a yeast RNA interactome capture protocol which employs RNA labeling, covalent UV crosslinking of RNA and proteins at 365nm wavelength (photoactivatable-ribonucleoside-enhanced crosslinking, PAR-CL) and finally purification of the protein-bound mRNA. The method can be easily implemented in common workflows and takes about 3days to complete. Next to a comprehensive explanation of the method, we focus on our findings about the choice of crosslinking in yeast and discuss the rationale of individual steps in the protocol.

| S-EPMC5421583 | biostudies-literature

Driving integrative structural modeling with serial capture affinity purification.

Project description:Streamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here we describe serial capture affinity purification (SCAP), in which two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multistep affinity enrichment of specific protein complexes. The multifunctional capabilities of this protein-tagging system also permit in vivo validation of interactions using acceptor photobleaching Förster resonance energy transfer and fluorescence cross-correlation spectroscopy quantitative imaging. By coupling SCAP to cross-linking mass spectrometry, an integrative structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC protein complex, culminating in a structural model with two SPINDOC molecules docked on one SPIN1 molecule. In this model, SPINDOC interacts with the SPIN1 interface previously shown to bind a lysine and arginine methylated sequence of histone H3. Our approach combines serial affinity purification, live cell imaging, and cross-linking mass spectrometry to build integrative structural models of protein complexes.

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Identification of RNA-binding Proteins in Macrophages by Interactome Capture.

Project description:Pathogen components, such as lipopolysaccharides of Gram-negative bacteria that activate Toll-like receptor 4, induce mitogen activated protein kinases and NF?B 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.

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Capture and identification of the eIF4E:mRNA interactome in human cells

Project description:Here we develop a novel methodology, capCLIP, to capture and identify mRNA interactions with the major cellular cap-binding protein eIF4E.

2020-12-31 | GSE138473 | GEO

Comprehensive Identification of mRNA-Binding Proteins of Leishmania donovani by Interactome Capture.

Project description:Leishmania are unicellular eukaryotes responsible for leishmaniasis in humans. Like other trypanosomatids, leishmania regulate protein coding gene expression almost exclusively at the post-transcriptional level with the help of RNA binding proteins (RBPs). Due to the presence of polycystronic transcription units, leishmania do not regulate RNA polymerase II-dependent transcription initiation. Recent evidence suggests that the main control points in gene expression are mRNA degradation and translation. Protein-RNA interactions are involved in every aspect of RNA biology, such as mRNA splicing, polyadenylation, localization, degradation, and translation. A detailed picture of these interactions would likely prove to be highly informative in understanding leishmania biology and virulence. We developed a strategy involving covalent UV cross-linking of RBPs to mRNA in vivo, followed by interactome capture using oligo(dT) magnetic beads to define comprehensively the mRNA interactome of growing L. donovani amastigotes. The protein mass spectrometry analysis of captured proteins identified 79 mRNA interacting proteins which withstood very stringent washing conditions. Strikingly, we found that 49 of these mRNA interacting proteins had no orthologs or homologs in the human genome. Consequently, these may represent high quality candidates for selective drug targeting leading to novel therapeutics. These results show that this unbiased, systematic strategy has the promise to be applicable to study the mRNA interactome during various biological settings such as metabolic changes, stress (low pH environment, oxidative stress and nutrient deprivation) or drug treatment.

| S-EPMC5279761 | biostudies-literature

Capture Hi-C identifies the chromatin interactome of colorectal cancer risk loci.

Project description:Multiple regulatory elements distant from their targets on the linear genome can influence the expression of a single gene through chromatin looping. Chromosome conformation capture implemented in Hi-C allows for genome-wide agnostic characterization of chromatin contacts. However, detection of functional enhancer-promoter interactions is precluded by its effective resolution that is determined by both restriction fragmentation and sensitivity of the experiment. Here we develop a capture Hi-C (cHi-C) approach to allow an agnostic characterization of these physical interactions on a genome-wide scale. Single-nucleotide polymorphisms associated with complex diseases often reside within regulatory elements and exert effects through long-range regulation of gene expression. Applying this cHi-C approach to 14 colorectal cancer risk loci allows us to identify key long-range chromatin interactions in cis and trans involving these loci.

| S-EPMC4346635 | biostudies-literature

Validation and classification of RNA binding proteins identified by mRNA interactome capture.

Project description:RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines, and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we used a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RBPs. Half of the analyzed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster end binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1's RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather nonspecific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

| S-EPMC8456996 | biostudies-literature

Complementary proteomics strategies capture an ataxin-1 interactome in Neuro-2a cells.

Project description:Ataxin-1 mutation, arising from a polyglutamine (polyQ) tract expansion, is the underlying genetic cause of the late-onset neurodegenerative disease Spinocerebellar ataxia type 1 (SCA1). To identify protein partners of polyQ-ataxin-1 in neuronal cells under control or stress conditions, here we report our complementary proteomics strategies of proximity-dependent biotin identification (BioID) and affinity purification (via GFP-Trap pulldown) in Neuro-2a cells expressing epitope-tagged forms of ataxin-1[85Q]. These approaches allowed our enrichment of proximal proteins and interacting partners, respectively, with the subsequent protein identification performed by liquid chromatography-MS/MS. Background proteins, not dependent on the presence of the polyQ-ataxin-1 protein, were additionally defined by their endogenous biotinylation (for the BioID protocol) or by their non-specific interaction with GFP only (in the GFP-Trap protocol). All datasets were generated from biological replicates. Following the removal of the identified background proteins from the acquired protein lists, our experimental design has captured a comprehensive polyQ-ataxin-1 proximal and direct protein partners under normal and stress conditions. Data are available via ProteomeXchange, with identifier PXD010352.

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