RNA Affinity Purification followed by sequencing (RAP-seq) allows identification of transcriptome-wide RBP binding sites using recombinant RBPs and purified total cellular RNA
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB92198 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:RAP-seq is a new method that provides in vitro derived RNA-Interactomes for any given RBP. In RAP-seq a recombinant RBP is produced as fusion with a HaloTag which is used to recover and purify the RBP of interest. The RBP-Halo fusion is then incubated with fragmented total RNA derived from any given sample of interest. The bound RNA fragments are subsequently eluted and cloned using a small RNA library preparation protocol for sequencing the pool of bound molecules on an Illumina NGS platform. In this study, RAP-seq was used to identify RNA-Interactomes of 26 novel RBPs (aka non-canonical RBPs) newly discovered in proteome-wide studies as RNA binders. RAP-seq was also used to profile vertebrate HuR orthologs and described the biochemical evolutionary differences and similarities of the 6 orthologs profiled. Cancer associated IGF2BP1, IGF2BP2 and IGF2BP3 variants were also profiled and transcriptome-wide changes in their RNA Interactomes with respect to the wild-type IGF2BPs were reported. Also a transcriptome-wide cooperative binding assay was perfomed to evaluate the cooperative roles of HuR and PTBP1 in binding to their native RNA targets. In addition, a tipical RAP-seq substrate (fragmented total RNA) if reverse-transcribed into cDNA and than in vitro transcribed again using a T7 RNA Polymerase can be depleted of any native endogenous RNA modifications and RAP-seq assyas perfomed in parallel with the native substrate and the T7 RNAP produced one allowed us to discern the m6A dependency in transcriptome-wide binding events for YTHDF1, hence with RAP-seq we also report T7-RAP-seq.
2023-09-13 | E-MTAB-10834 | biostudies-arrayexpress
Project description:Interactions between RNAs and RNA binding proteins (RBPs) regulate gene expression in eukaryotic cells. RNA-RBP affinities measured in vitro reveal diverse binding specificities, yet approaches to directly compare specificities across RBPs are lacking. Here, we introduce two quantitative metrics: inherent specificity, which measures how selectively an RBP distinguishes its strongest binding motif from all possible motifs, and mutational sensitivity, which assesses tolerance to single nucleotide variations within preferred motifs. Analyzing high-throughput sequencing datasets, we compared these metrics across 100 RBPs in vitro and 27 RBPs in cells, finding strong correlation between in vitro and cellular measurements for RBPs that bind RNA independently of a local structural context. Through CLIP experiments with swapped RNA recognition motifs between a low-specificity RBP (RBM25) and a high-specificity RBP (HNRNPC), we demonstrated that sequence specificity can be transferred between protein contexts. Using these insights, we developed mathematical models showing how RBPs with different specificity profiles compete for binding sites, revealing how variations in inherent specificity and mutational sensitivity influence target selection. Together, our results provide a quantitative framework for modeling RNA-RBP interactions and designing RBPs with targeted specificity.
2025-03-28 | GSE291358 | GEO
Project description:To identify RNAs specifically associated with potential RBPs, yeast cells expressing TAP-tagged RBP or the wild-type strain BY4741 (mock control) were grown to mid-log phase in rich medium and harvested by centrifugation. RNA affinity isolations were essentially performed as described (Gerber et al. 2004 PLoS Biol.; see protocol). In brief, TAP-tagged protein were captured from cell extracts with IgG coupled agarose beads (Sigma) and released by incubation with a site-specific protease (AcTEV, Sigma). from the extract (input) and from the affinity isolates was purified with the RNeasy Mini/ Micro Kit (Qiagen). RNAs were reverse transcribed using a mixture of oligo-dT and random nonamer oligos in the presence of amino-allyl dUTP/dNTP mixture. cDNAs were fluorescently labeled and hybridized on yeast oligo microarrays over night at 42 degrees in formamide-based hybridization buffer. Antigenic peptide used in IP: TAP-tag An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
2010-12-06 | E-GEOD-21864 | biostudies-arrayexpress
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.
2021-09-01 | E-MTAB-9612 | biostudies-arrayexpress
Project description:RNA binding proteins (RBPs) bind RNAs through specific RNA-binding domains, generating multi-molecular complexes known as ribonucleoproteins (RNPs). Various post-translational modifications (PTMs) have been described to regulate RBP structure, subcellular localization and interactions with other proteins or RNAs. Recent proteome-wide experiments showed that RBPs are the most representative group within the class of arginine (R)-methylated proteins; moreover, emerging evidence suggests that this modification plays a major role in the regulation of RBP-RNA interaction. Nevertheless, a systematic analysis of how changes in protein-R-methylation can affect globally RBPs-RNA interactions has not yet been carried out. We describe here a quantitative proteomics approach to profile global changes of RBP-RNA interactions upon the modulation of protein arginine methyltransferases PRMT1 and PRMT5. By coupling the recently described Orthogonal Organic Phase Separation (OOPS) with SILAC labelling and pharmacological modulation of PRMT1 and PRMT5 enzyme, we describe the RNA-binding protein dynamics in dependence of protein-R-methylation.
2021-08-25 | PXD024601 | Pride
Project description:Tumor metastasis is a major factor contributing to high mortality in patients with hepatocellular carcinoma (HCC). RNA binding protein (RBP)-mediated post-transcriptional regulation is involved in the precise control of signal transduction; however, the role of RBPs in HCC metastasis remains to be elucidated. To identify the key RNA binding protein (RBP) genes involved in hepatocellular carcinoma (HCC) metastasis, we conducted an RNA-seq analysis in the human HCC cell lines with high (MHCC97H) and low (MHCC97L) spontaneous metastatic potentials.
2025-07-12 | GSE208148 | GEO
Project description:The construction of complex gene regulatory networks requires both inhibitory and up-regulatory modules. However, the vast majority of RNA-based regulatory “parts” are inhibitory. Using a synthetic biology approach combined with SHAPE-Seq, we explored the regulatory effect of RBP-RNA interactions in bacterial 5’-UTRs. By positioning a library of RNA hairpins upstream of a reporter gene and co-expressing them with the matching RBP, we observed a set of regulatory responses, including translational stimulation, translational repression, and cooperative behavior. Our combined approach revealed three distinct states in-vivo: in the absence of RBPs, the RNA molecules can be found either in a molten state that is amenable to translation, or a structured phase that inhibits translation. In the presence of RBPs, the RNA molecules are in a semi-structured phase with partial translational capacity. Our work provides new insight into RBP-based regulation and a blueprint for designing complete gene regulatory circuits at the post-transcriptional level.
2019-04-03 | GSE129163 | GEO
Project description:RNA-binding proteins (RBPs) are critical regulators of gene expression and elucidating the interactions of RBPs with their RNA targets is necessary to understand how combinations of RBPs control transcriptome expression. The Quaking-related (QR) sub-family of STAR domain RBPs includes developmental regulators and tumor suppressors such as C. elegans GLD-1, which functions as a master regulator of germ line development. To understand how GLD-1 interacts with the transcriptome, we identified GLD-1 associated mRNAs by a ribonomic approach. The scale of GLD-1 mRNA interactions allowed us to determine rules governing GLD-1 target selection and to derive a predictive model where GLD-1 association with mRNA is based on the number and strength of 7-mer GLD-1 binding elements (GBEs) within UTRs. GLD-1/mRNA interactions were quantified, and predictions were verified both in vitro and in live animals, including by M-bM-^@M-^XtransplantationM-bM-^@M-^Y experiments where M-bM-^@M-^XweakM-bM-^@M-^Y and M-bM-^@M-^XstrongM-bM-^@M-^Y GBEs imposed translational repression of increasing strength on a non-target mRNA.Importantly, this study provides a unique quantitative picture of how an RBP interacts with its mRNA targets. As combinatorial regulation by multiple RBPs is thought to regulate gene expression, quantification of RBP/mRNA interactions should be a way to predict and potentially modify biological outcomes of complex posttranscriptional regulatory networks, and our analysis suggests that such an approach is possible. Wild-type (N2) or gld-1(q485); ozIs2 [gld-1::gfp::flag] worms were synchronized and harvested as young adults. To identify GLD-1 associated transcripts, two independent IP strategies were performed: 1. Comparison of anti-FLAG IP to anti-MYC IP on the gld-1(q485); ozIs2 [gld-1::gfp::flag] worm extract, 2. Comparison of anti-FLAG IP on the gld-1(q485); ozIs2 [gld-1::gfp::flag] worm extract to anti-FLAG IP on wild type extract. All IPs were performed in triplicate (12 IPs).
2011-02-04 | E-GEOD-21591 | biostudies-arrayexpress
Project description:RNA-binding proteins (RBPs) form highly diverse and dynamic ribonucleoprotein complexes, whose functions determine the molecular fate of the bound RNA. In the model organism S. cerevisiae, the number of proteins identified as RBPs has largely increased over the last decade. However, the cellular function for most of these novel RBPs still remains largely unexplored. We use mass spectrometry-based quantitative proteomics to systematically identify protein-protein interactions (PPIs), and RNA-dependent interactions (RDIs) to create a novel data set for 40 RBPs associated with the mRNA life cycle. Domain, functional, and pathway enrichment analysis revealed an overrepresentation of RNA functionalities among the enriched interactors. With the creation of extensive PPI and RDI networks, we revealed putative new members within RNA-associated pathways, as well as highlighted potential new roles of several RBPs, such as metabolism. Our RBP interactome resource is available through an online interactive platform as a community tool to guide further in-depth functional studies and RBP network analysis.
2023-07-20 | PXD035979 | Pride
Project description:RNA-binding proteins (RBPs) form highly diverse and dynamic ribonucleoprotein complexes, whose functions determine the molecular fate of the bound RNA. In the model organism S. cerevisiae, the number of proteins identified as RBPs has largely increased over the last decade. However, the cellular function for most of these novel RBPs still remains largely unexplored. We use mass spectrometry-based quantitative proteomics to systematically identify protein-protein interactions (PPIs), and RNA-dependent interactions (RDIs) to create a novel data set for 40 RBPs associated with the mRNA life cycle. Domain, functional, and pathway enrichment analysis revealed an overrepresentation of RNA functionalities among the enriched interactors. With the creation of extensive PPI and RDI networks, we revealed putative new members within RNA-associated pathways, as well as highlighted potential new roles of several RBPs, such as metabolism. Our RBP interactome resource is available through an online interactive platform as a community tool to guide further in-depth functional studies and RBP network analysis.
2023-07-20 | PXD035971 | Pride