3'READS+RIP defines differential Staufen1 binding to alternative 3'UTR isoforms and reveals structures and sequence motifs influencing binding and polysome association
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ABSTRACT:
PROVIDER: PRJNA625690 | ENA |
REPOSITORIES: ENA
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Project description:Using 3ʹ region extraction and deep sequencing coupled to ribonucleoprotein immunoprecipitation (3’READS+RIP), together with reanalyzing previous STAU1 binding and RNA structure data, we delineate STAU1 interactions transcriptome-wide, including binding differences between alternative polyadenylation (APA) isoforms. Consistent with previous reports, RNA structures are dominant features for STAU1 binding to CDSs and 3ʹUTRs. Overall, relative to short 3ʹUTR counterparts, longer 3ʹUTR isoforms of genes have stronger STAU1 binding, most likely due to a higher frequency of RNA structures, including specialized IRAlus. However, a sizable fraction of genes express transcripts showing the opposite trend, attributable to AU-rich sequences in their alternative 3'UTRs, possibly recruiting antagonistic RBPs and/or destabilizing STAU1-binding RNA structures. Using STAU1-knockout cells, we show that strong STAU1 binding to mRNA 3'UTRs generally enhances polysome association. However, IRAlus have little impact on STAU1-mediated polysome association despite having strong interactions with the protein.
2020-11-05 | GSE148771 | GEO
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Project description:Oocyte maturation, fertilization, and early embryonic development occur in the absence of gene transcription. Therefore, it is critical to understand at a global level the post-transcriptional events that are driving these transitions. Here, we have used a systems approach by combining polysome mRNA profiling and bioinformatics to identify RNA binding motifs in mRNAs that either enter or exit the polysome pool during mouse oocyte maturation. Association of mRNA with the polysomes correlates with active translation.
2012-01-14 | GSE35106 | GEO
Project description:Oocyte maturation, fertilization, and early embryonic development occur in the absence of gene transcription. Therefore, it is critical to understand at a global level the post-transcriptional events that are driving these transitions. Here, we have used a systems approach by combining polysome mRNA profiling and bioinformatics to identify RNA binding motifs in mRNAs that either enter or exit the polysome pool during mouse oocyte maturation. Association of mRNA with the polysomes correlates with active translation. Forty-eight hours (h) after PMSG injection, mice were stimulated with hCG for 0, 4, or 14 h, and GV-, MI- and MII-stage oocytes were collected. Polysome-bound mRNAs were purified, reverse-transcribed and linearly amplified with the WT-Ovation FFPE RNA Amplification System V2 (NuGEN). 5µg cDNA were fragmented and hybridized with Affymetrix Mouse Genome 430.2 array chips. Experiments were done using 3 independent sample sets.
2012-01-14 | E-GEOD-35106 | biostudies-arrayexpress
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Project description:RNA binding proteins are key regulators of gene expression, yet only a small fraction of these proteins has been functionally characterized. Here, we report the first large-scale analysis of the RNA motifs recognised by RNA binding proteins, encompassing 205 distinct proteins from 24 diverse eukaryotes. The sequence specificities of RBPs display deep evolutionary conservation, such that the recognition preferences for a large fraction of metazoan RNA binding proteins can be inferred from the sequences of their binding domains. The motifs we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA binding proteins in both normal physiology and in human disease. These data provide an unprecedented global view of RBPs and their targets and constitute an invaluable resource for defining post-transcriptional regulatory mechanisms in eukaryotes. Here, we analyze the RNA-binding preferences of 205 distinct RNA-binding proteins from 24 different eukaryotes, using RNAcompete. In this assay, purified GST-tagged RBPs are incubated with an excess of RNA pool and bound RNA from individual pulldowns are directly labeled, hybridized to a custom Agilent 244K microarray, and analyzed computationally to identify RNA-binding motifs.
2013-05-10 | E-GEOD-41235 | biostudies-arrayexpress
Project description:Purpose: 224 GSM Samples form GSE32970 and GSE29692 was reanalyzed to find the binding sites of 542 TFs. Methods: 1. iFORM (incorporating Find Occurrence of Regulatory Motifs) is a tool we designed to scan through sequences in search of binding sites that match given motifs. 2. We mapped motif-binding protein information found in TRANSFAC, JASPAR, and UniPROBE databases to 542 coding genes, using GeneCards (Rebhan et al., 1997) and UniProt Knowledgebase (Magrane and Consortium, 2011). The position-specific weight matrices of the 542 TFs, corresponding to 796 motif models, were collected from these databases. 3.We used a uniform set of open chromatin elements (DNaseI hypersensitive sites, DHSs) in 133 ENCODE cell types, based on DNase-seq data produced by the "Open Chromatin" (Duke/UNC/UT-A)(GSE32970) and University of Washington (UW) ENCODE groups (GSE29692)from the project inception in 2007 through the ENCODE January 2011 data freeze. Processing pipeline: Combined all replicates for a given cell type; Subsampled the result at a level of 30 million tags; Ran results through the Stam Lab hotspot pipeline. Hotspots were identified using a relaxed threshold. NarrowPeaks were generated by first thresholding hotspots at FDR 1%, and then locating local maxima of the tag density within the hotspots. The 133 narrowPeaks are base sequences for the given motifs scan. Result: We used our iFORM with a custom library of all 796 motifs to scan for motif instances in 133 ENCODE DHSs cell types separately. Result in 133 files correspond to 133 cell types, each file contains binding sites of 542 TFs in a cell type.
2014-10-12 | GSE53962 | GEO
Project description:G-quadruplex structures in the 5’ UTR of mRNAs are widely considered to suppress translation without affecting transcription. The current study describes the comprehensive analysis of proteins binding to four different G-quadruplex motifs located in mRNAs of the cancer-related genes Bcl-2, NRAS, MMP16, and ARPC2. Following metabolic labeling (Stable Isotope Labeling with Amino acids in Cell culture, SILAC) of proteins in the human cell line HEK293, G-quadruplex binding proteins were enriched by pull-down assays and identified by LC-orbitrap mass spectrometry. We found different patterns of interactions for the G-quadruplex motifs under investigation. While the G-quadruplexes in the mRNAs of NRAS and MMP16 specifically interacted with a small number of proteins, the Bcl-2 and ARPC2 G-quadruplexes exhibited a broad range of proteinaceous interaction partners with 99 and 82 candidate proteins identified in at least two replicates, respectively. Among the interaction partners were many proteins known to bind to RNA, including multiple heterogenous nuclear ribonucleoproteins (hnRNPs). The identified ribosomal proteins are likely to reflect stalling of the ribosome by RNA G-quadruplex structures. In addition, several proteins were identified that have not previously been described to interact with RNA. Gene ontology analysis of the set of candidate proteins revealed that many interaction partners are known to be tumor related. The majority of the identified RNA G-quadruplex interacting proteins are thought to be involved in post-transcriptional processes, particularly in splicing. These findings indicate that protein-G-quadruplex interactions may be relevant to the regulation of mRNA maturation and may play an important role in tumor biology.
2017-11-14 | PXD005761 | Pride
Project description:RNA binding proteins are key regulators of gene expression, yet only a small fraction of these proteins has been functionally characterized. Here, we report the first large-scale analysis of the RNA motifs recognised by RNA binding proteins, encompassing 205 distinct proteins from 24 diverse eukaryotes. The sequence specificities of RBPs display deep evolutionary conservation, such that the recognition preferences for a large fraction of metazoan RNA binding proteins can be inferred from the sequences of their binding domains. The motifs we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA binding proteins in both normal physiology and in human disease. These data provide an unprecedented global view of RBPs and their targets and constitute an invaluable resource for defining post-transcriptional regulatory mechanisms in eukaryotes.
2013-05-10 | GSE41235 | GEO