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RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome

ABSTRACT: RNAs are continuously associated with RNA-binding proteins (RBPs), and these interactions are necessary for many key cellular processes ranging from splicing to chromatin regulation. Although numerous approaches have been developed to map RNA-binding sites of individual RBPs, few methods exist that allow assessment of global RBP-RNA interactions. Here, we describe a universal, high-throughput, ribonuclease-mediated protein footprint sequencing approach that reveals RNA-protein interaction sites throughout a transcriptome of interest. We apply this method to the HeLa transcriptome and compare RBP binding sites found using different cross-linkers and ribonucleases. From this analysis, we identify numerous putative RBP binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites.

ORGANISM(S): Homo sapiens

PROVIDER: GSE49309 | GEO | 2013/11/06

SECONDARY ACCESSION(S): PRJNA213617

REPOSITORIES: GEO

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RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome

Project description:RNAs are continuously associated with RNA-binding proteins (RBPs), and these interactions are necessary for many key cellular processes ranging from splicing to chromatin regulation. Although numerous approaches have been developed to map RNA-binding sites of individual RBPs, few methods exist that allow assessment of global RBP-RNA interactions. Here, we describe a universal, high-throughput, ribonuclease-mediated protein footprint sequencing approach that reveals RNA-protein interaction sites throughout a transcriptome of interest. We apply this method to the HeLa transcriptome and compare RBP binding sites found using different cross-linkers and ribonucleases. From this analysis, we identify numerous putative RBP binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites. Protein interaction profile sequencing (PIP-seq) in HeLa cells. Two crosslinkers (formaldehyde and UV) with two RNases (dsRNase and ssRNase) each, as well as a no-crosslink sample. Performed with and without proteins. Three replicates for formaldehyde, two replicates for UV, single replicate for no crosslinker.

2013-11-06 | E-GEOD-49309 | biostudies-arrayexpress

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.

2021-09-01 | E-MTAB-9612 | biostudies-arrayexpress

The kinetic landscape of an RNA binding protein in cells

Project description:The binding and dissociation of RNA Binding Proteins (RBPs) to their cognate sites on cellular RNAs are critical for the regulation of gene expression. Yet, association and dissociation kinetics of RBPs at individual cellular binding sites have not been experimentally accessible. Inaccessibility of binding and dissociation kinetics of RBPs in cells severely limits or even precludes the establishment of quantitative connections between RBP-RNA interactions and cellular RBP function. Here, we describe an approach to measure binding and dissociation kinetics of the RBP Dazl at thousands of individual binding sites in cells. We then show how these kinetic parameters inform a quantitative understanding of the cellular function of Dazl.

2020-12-16 | GSE150214 | GEO

Profiling of RNA-binding protein binding sites by in-situ reverse transcription-based sequencing

Project description:RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA substrates are still lacking, especially when the interaction is dynamic or materials are limited. Here we present an assay of reverse transcription-based RBP binding sites sequencing (ARTR-seq), which relies on in-situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites. ARTR-seq avoids ultraviolet cross-linking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section. Taking advantage of rapid formaldehyde fixation, ARTR-seq enables capturing the dynamic binding of RBPs over a short period of time, as demonstrated by the discovery of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 min.

2023-11-05 | GSE226161 | GEO

Tranposable elements modulate human mRNAs and lncRNAs via specific RNA-protein interactions.

Project description:Transposable elements (TEs) have significantly influenced the evolution of transcriptional regulatory networks in the human genome. Post-transcriptional regulation of human genes by TE-derived sequences has been observed in specific contexts, but has yet to be systematically and comprehensively investigated. Here, studied a collection of CLIP-Seq (CrossLinked ImmunoPrecipitation) experiments mapping the RNA binding sites for a diverse set of 46 human proteins across 68 experiments to explore the role of TEs in post-transcriptional regulation genome-wide via RNA-protein interactions. We detected widespread interactions between RNA binding proteins (RBPs) and various families of TE-derived sequence in the CLIP-Seq data. Alignment coverage clustered on specific positions of the TE consensus sequences, illuminating a diversity of TE-specific motifs for many RBPs. Evidence of binding and conservation of these motifs in the nonrepetitive transcriptome suggest that TEs have appropriated existing sequence preferences of the RBP. Upon depletion of the RBPs, transcripts possessing TE-derived binding sites were similarly regulated as those bound in nonrepetitive sequence. However, in a few cases the effect of RBP binding depended on the specific TE family boundM-bM-^@M-^Te.g., the ubiquitously expressed RBP HuR conferred opposite effects on stability to transcripts when bound to Alu elements versus other families. Our meta-analysis suggests a widespread role for TEs in shaping RNA-protein regulatory networks in the human genome. HuR formaldehyde RIP-Seq in K562 cells, with RIP and input sequenced in triplicate.

2014-09-10 | E-GEOD-61238 | biostudies-arrayexpress

Global Profiling of RNA-Binding Protein Target Sites by LACE-seq

Project description:RNA-binding proteins (RBPs) have essential functions during germline and early embryo development. However, current methods are unable to identify the in vivo targets of an RBP in those rare cells. Here, by coupling RBP-mediated reverse transcription termination with linear amplification of cDNA ends and sequencing, we present the LACE-seq method for identifying RBP-regulated RNA networks at or near the single-oocyte level. We determined the binding sites and regulatory mechanisms for several RBPs, including Argonaute 2 (Ago2), Mili, Ddx4, and PTBP1, in mature oocytes. Unexpectedly, transcriptomic and proteomic analysis of Ago2-/- oocytes revealed that Ago2 interacted with endogenous small interfering RNAs (endo-siRNAs) to repress mRNA translation globally. Furthermore, the Ago2 and endo-siRNA complexes could fine-tune the transcriptome by slicing long terminal repeat retrotransposon-derived chimeric transcripts. The precise mapping of RBP binding sites in a few cells opens the door for studying the roles of RBPs in embryonic development and reproductive diseases.

2021-04-25 | GSE137925 | GEO

SONAR discovers RNA binding proteins from analysis of large-scale protein-protein interactomes.

Project description:RNA metabolism is controlled by an expanding yet incomplete catalog of RNA binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull-down of twelve nuclear and cytoplasmic RBPs followed by quantitative mass-spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1923 human, 489 fly and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins.

2016-10-06 | GSE86035 | GEO

Global signatures of protein binding on structural RNAs in Saccharomyces cerevisiae

Project description:Protein binding is essential to the transport, decay and regulation of almost all RNA molecules. However, the structural preference of protein binding on RNAs and their cellelar functions and dynamics upon changing environmental condictions are poorly understood. Here, we integrated various high-throughput data and introduced a computational framework to describe the global interactions between RNA binding proteins (RBPs) and structured RNAs in yeast at single-nucleotide resolution. We found that on average, in terms of percent total lengths, ~15% of mRNA untranslated regions (UTRs), ~37% of canonical ncRNAs and ~11% of long ncRNA (lncRNAs) are bound by proteins. The RBP binding sites, in general, tend to occur at single-stranded loops, with evolutionarily conserved signatures, and often facilitate a specific RNA structure conformation in vivo. We found that four nucleotide modifications of tRNA are significantly associated with RBP binding. We also identified various structural motifs bound by RBPs in the UTRs of mRNAs, associated with localization, degradation and stress responces. Moreover, we identified >200 novel lncRNAs bound by RBPs, and about half of them contain conserved secondary structures. We present the first ensemble pattern of RBP binding sites in the structured noncoding regions of a eukaryotic genome, emphasizing their structural context and cellular functions. Duplicate gPAR-CLIP libraries were sequenced from yeast strains for each of three conditions: log-phase growth, growth after 2 hour glucose starvation, and growth after 2 hour nitrogen starvation. polyA RNAs were isolated for all conditions. Total RNA were isolated from log phase growth conditions. Sucrose gradient fractionation was performed: some RNAs were isolated from the "light" fraction (lighter than 40S ribosome) and some from the "heavy" fraction. gPAR-CLIP libraries were used to determine regions of RNA bound by proteins.

2013-07-15 | E-GEOD-48888 | biostudies-arrayexpress

Exonuclease assisted mapping of protein-RNA interactions (ePRINT)

Project description:RNA processing is a fundamental mode of gene regulation that is perturbed in a variety of diseases including cancer and neurodegenerative disorders. RNA-binding proteins (RBPs) regulate key aspects of RNA processing including alternative splicing, mRNA degradation and localization by physically binding RNA molecules. Current methods to map these interactions such as CLIP rely on purifying single proteins at a time. Methods to identify transcriptome wide binding of RBPs without purifying individual RBPs are gaining interest. We have developed a new method (ePRINT) to map RBP-RNA interaction networks on a global scale in an RBP agnostic manner. Our method allows precise mapping of the 5’ end of the RBP binding site and uncovers RBPs that are differentially activated during cell fate transitions.

2024-03-04 | GSE230097 | GEO

Quantifying RNA-Protein Interactions Characterizes Recurrent Mutations in RNA Binding Proteins in Cancer

Project description:Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP “target” RNAs. To address this, an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP, was developed. easyCLIP, provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize unamplified libraries. 213 cross-link measurements were performed and found that RBPs could be distinguished from non-RBPs by RNA cross-link rates and that target RNAs could be defined as those with a complex frequency unlikely for a random protein. easyCLIP was used to study the 33 most recurrent cancer mutations across 28 RBPs. This demonstrated increased RNA binding per RBP molecule for KHDRBS2 and A1CF cancer mutants, and showed that the PCBP1L100P/Q cancer mutation also dramatically increased RNA binding. Quantitating RBP-RNA interactions can thus categorize proteins as RBPs or not and define the impact of specific disease-associated RBP mutations on RNA binding.

2020-12-01 | GSE162366 | GEO

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