HuR (ELAVL1) RIP-chip and knockdown exon array
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE29778 | GEO | 2011/08/19
SECONDARY ACCESSION(S): PRJNA153883
REPOSITORIES: GEO
Similar Datasets
Project description:Integrative regulatory mapping indicates that the RNA-binding protein HuR (ELAVL1) couples pre-mRNA processing and mRNA stability In this dataset, we employed two distinct experiments. 1) HuR RIP-chip to identify mRNA targets of HuR. 2) HuR knockdown to identify mRNAs whose expression are dependent on HuR. All 12 samples were normalized with PLIER using Affymetrix power tools. To identify RNA targets of HuR, HuR RIP samples were compared to Mock RIP samples. To identify RNA regulated by HuR, HuR knockdown samples were compared to mock knockdown samples.
2011-08-18 | E-GEOD-29778 | biostudies-arrayexpress
Project description:RNA-binding proteins coordinate the fates of multiple RNAs, but the principles underlying these global interactions remain poorly understood. We elucidated regulatory mechanisms of the RNA-binding protein HuR, by integrating data from diverse high-throughput targeting technologies, specifically PAR-CLIP, RIP-chip, and whole-transcript expression profiling. The number of binding sites per transcript, degree of HuR-association, and degree of HuR-dependent RNA stabilization were positively correlated. Pre-mRNA and mature mRNA containing both intronic and 3' UTR binding sites were more highly stabilized than transcripts with only 3' UTR or only intronic binding sites, suggesting that HuR couples pre-mRNA processing with mature mRNA stability. We also observed HuR-dependent splicing changes and substantial binding of HuR in poly-pyrimidine tracts of pre-mRNAs. Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression. We conclude that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing. HuR (ELAVL1) PAR-CLIP
2011-08-19 | E-GEOD-29779 | biostudies-arrayexpress
Project description:The purpose of the study was to identify mRNA bound to HuR in the presence of doxorubicin in MCF7 cells. We collected cytoplasmic RNA from untreated and treated cells and detected differentially expressed genes (DEGs). We also coimmunoprecipitated HuR and IgG (as control) from doxorubicin treated cells. Comparison between HuR RIP and IgG RIP signals was used to discriminate specific mRNA bound to HuR. HuR coimmmunoprecipitated material was hybridized together with cytoplasmic mRNA of doxorubicin treated cells, enabling the fold enrichment calculation and the selection of mRNAs bound to HuR. Keywords: RIP-Chip, HuR, doxorubicin, MCF7, HuR consensus binding, post-transcriptional regulation.
2011-12-31 | GSE33055 | GEO
Project description:Elavl1/HuR is a ubiquitous and conserved RNA-binding protein that binds to a U-rich RNA motif that shuttles between nucleus and cytoplasm. In epithelia, the elevated expression of HuR assumingly promotes degeneration and cancer suggesting that its generic suppression may provide clinical benefits. In this study we focused on biological and clinical functions of HuR in intestinal epithelial cells and we presented evidence that changes in HuR levels induce polarized distortions in these cells to support different pathologic outcomes. In this experiment we investigate Elavl1 targets via Elavl1 Immunopercipitation (RIP-chip) by arrays and compare relative to background. Control and TgATF-HuR mice were treated with Dimethylhydrazine (DMH)/Dextran Sodium Sulphate (DSS) for 60 days and tumors where dissected from large intestines; those with sizes between 10-15mm2 were pooled to generate samples with 4 tumors/sample and snap frozen. Three samples per genotype were used either for RIP analyses or total RNA extraction. Isolated RNA was used for microarray or qRT-PCR analyses.
2018-01-01 | E-MTAB-4018 | biostudies-arrayexpress
Project description:The purpose of the study was to identify mRNA bound to HuR in the presence of doxorubicin in MCF7 cells. We collected cytoplasmic RNA from untreated and treated cells and detected differentially expressed genes (DEGs). We also coimmunoprecipitated HuR and IgG (as control) from doxorubicin treated cells. Comparison between HuR RIP and IgG RIP signals was used to discriminate specific mRNA bound to HuR. HuR coimmmunoprecipitated material was hybridized together with cytoplasmic mRNA of doxorubicin treated cells, enabling the fold enrichment calculation and the selection of mRNAs bound to HuR. Keywords: RIP-Chip, HuR, doxorubicin, MCF7, HuR consensus binding, post-transcriptional regulation. We subjected MCF7 cells to starvation for 24h and then we added doxorubicin at final concentration of 10 uM, profiling before and after 4 hours of treatment in biological quadruplicate (only on cytoplasmic mRNAs, as HuR was found in the cytoplasm). Differentially expressed genes, altered during the treatment, were identified. Data derived from HuR RIP-Chip and IgG RIP-Chip (in biological quadruplicate) allowed the identification of specific mRNAs bound to HuR. The comparison between HuR RIP-Chip and cytoplasmic extracts from doxorubicin treated cells (in biological triplicate) identified those genes that were more strictly bound to HuR independently from their expression levels.
2011-12-31 | E-GEOD-33055 | biostudies-arrayexpress
Project description:Elavl1/HuR is a ubiquitous and conserved RNA-binding protein that binds to a U-rich RNA motif that shuttles between nucleus and cytoplasm. In epithelia, the elevated expression of HuR assumingly promotes degeneration and cancer suggesting that its generic suppression may provide clinical benefits. In this study we focused on biological and clinical functions of HuR in intestinal epithelial cells and we presented evidence that changes in HuR levels induce polarized distortions in these cells to support different pathologic outcomes. Here we study the differentiality in mRNA abundance between Control mice and mice overexpressing Elavl1 (TgATF-HuR). Control and TgATF-HuR mice were treated with Dimethylhydrazine (DMH)/Dextran Sodium Sulphate (DSS) for 60 days and tumors where dissected from large intestines; those with sizes between 10-15mm2 were pooled to generate samples with 4 tumors/sample and snap frozen. Three samples per genotype were used either for RIP analyses or total RNA extraction. Isolated RNA was used for microarray or qRT-PCR analyses.
2020-07-01 | E-MTAB-4017 | biostudies-arrayexpress
Project description:RNA-binding proteins coordinate the fates of multiple RNAs, but the principles underlying these global interactions remain poorly understood. We elucidated regulatory mechanisms of the RNA-binding protein HuR, by integrating data from diverse high-throughput targeting technologies, specifically PAR-CLIP, RIP-chip, and whole-transcript expression profiling. The number of binding sites per transcript, degree of HuR-association, and degree of HuR-dependent RNA stabilization were positively correlated. Pre-mRNA and mature mRNA containing both intronic and 3' UTR binding sites were more highly stabilized than transcripts with only 3' UTR or only intronic binding sites, suggesting that HuR couples pre-mRNA processing with mature mRNA stability. We also observed HuR-dependent splicing changes and substantial binding of HuR in poly-pyrimidine tracts of pre-mRNAs. Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression. We conclude that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing.
2011-08-19 | GSE29779 | GEO
Project description:Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic fibroblast (MEF) cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicative of their concerted function in mRNA metabolism. Ribonucleoprotein Immunoprecipitation (RIP) using hnRNP A1 specific antibody was performed in nuclear extracts from HuR WT and HuR KO Mouse Embryonic Fibroblasts (MEFs). RNA isolated from these IPs together with nuclear RNA from the two cell types, was subjected to microarray analysis. Three biological replicates, representing three independent experiments, are available for each condition except in the case of nuclear RNA isolated from HuR WT MEFs that one replicate didn’t pass the quality control.
2013-11-01 | E-GEOD-49225 | biostudies-arrayexpress
Project description:Post-transcriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. We used PAR-CLIP, a method based on RNA-protein crosslinking, to identify transcriptome wide ~26,000 HuR binding sites. These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. Surprisingly, many sites were intronic, implicating HuR in splicing. Upon HuR knock down, mRNA levels and protein synthesis of thousands of target genes was down regulated, validating functionality. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Additionally, HuR knock down triggered strong and specific up regulation of miR-7. In summary, we identified thousands of direct and functional HuR targets, found a human miRNA controlled by HuR, and propose a role for HuR in splicing.
2011-06-30 | GSE29943 | GEO
Project description:Post-transcriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. We used PAR-CLIP, a method based on RNA-protein crosslinking, to identify transcriptome wide ~26,000 HuR binding sites. These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. Surprisingly, many sites were intronic, implicating HuR in splicing. Upon HuR knock down, mRNA levels and protein synthesis of thousands of target genes was down regulated, validating functionality. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Additionally, HuR knock down triggered strong and specific up regulation of miR-7. In summary, we identified thousands of direct and functional HuR targets, found a human miRNA controlled by HuR, and propose a role for HuR in splicing. PolyA mRNA was extracted from anti HuR siRNA treated and mock transfected HeLa cells to identify changes in mRNA expression and splicing. 2x100 paired end sequencing was performed according to the protocol on the Illumina HiSeq. PARCLIP was performed as in Hafner et. Al 2010 but with an antibody against endogenous HuR (3A2, Santa Cruz, sc-5261) in unstressed HeLa cells. We used, independently, 4-thiouridine (4SU) and 6-thioguanosine (6SG) to assess a possible nucleotide bias. As our proteomics measurements required labeling of cells in a special medium we also performed PAR-CLIP on cells grown in SILAC medium. Altogether we performed three PAR-CLIP experiments: 4SU labeling in standard DMEM medium, 4SU labeling in SILAC medium (as a replicate) and 6SG labeling in SILAC medium. Small RNA was extracted from anti HuR siRNA treated and mock transfected HeLa cells to identify changes in mRNA expression. Sequencing was performed on Illumina GAII using the standard sRNA 36cycle protocol.
2011-06-29 | E-GEOD-29943 | biostudies-arrayexpress