Transcriptome-wide identification and validation of interactions between the miRNA machinery and HuR on mRNA targets
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA397421 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR5906480.fastq.gz | Fastqsanger.gz | |||
| SRR5906490.fastq.gz | Fastqsanger.gz | |||
| SRR5906500.fastq.gz | Fastqsanger.gz | |||
| SRR5906481.fastq.gz | Fastqsanger.gz | |||
| SRR5906491.fastq.gz | Fastqsanger.gz |
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Project description:The 3’ UTR of messenger RNAs serves as the regulatory region that mediates post-transcriptional control by microRNAs and RNA-binding proteins (RBPs). Aside from individual sequence-specific binding and regulation, examples of interaction between these factors at particular 3’ UTR sites have emerged in recent studies. However, the whole picture of such higher-order regulatory modules across the transcriptome is lacking. Here, we investigate the interactions between HuR, a ubiquitous RBP, and Ago2, a core effector of the miRNA pathway, at the transcriptome-wide level. Using HITS-CLIP, we map HuR and miRNA binding sites on human 3’UTRs and assess their co-occurrence. Additionally, we demonstrate global effects of HuR knockdown on Ago2 occupancy, suggesting a co-regulatory relationship. Focusing on sites of Ago2-HuR overlap, 13 candidates were screened in luciferase reporter assays, compared to miRNA site mutant controls. Eleven of the sites showed a repressive activity, which displayed significant de-repression upon subsequent testing of the reporters in Dicer-null cells, substantiating miRNA dependence. To experimentally test for HuR’s role in co-regulation, we tested the reporters in CRISPR-generated HuR KO cells. Three of the miRNA sites demonstrated altered activities, indicating that HuR has an effect on miRNA repression at those sites. Our study presents an efficient search and validation system for studying miRNA-HuR interactions, which expands our understanding of the combinatorial post-transcriptional control of gene expression at the 3’ UTR.
2018-01-19 | GSE102320 | GEO
Project description:The 3’ UTR of messenger RNAs serves as the regulatory region that mediates post-transcriptional control by microRNAs and RNA-binding proteins (RBPs). Aside from individual sequence-specific binding and regulation, examples of interaction between these factors at particular 3’ UTR sites have emerged in recent studies. However, the whole picture of such higher-order regulatory modules across the transcriptome is lacking. Here, we investigate the interactions between HuR, a ubiquitous RBP, and Ago2, a core effector of the miRNA pathway, at the transcriptome-wide level. Using HITS-CLIP, we map HuR and miRNA binding sites on human 3’UTRs and assess their co-occurrence. Additionally, we demonstrate global effects of HuR knockdown on Ago2 occupancy, suggesting a co-regulatory relationship. Focusing on sites of Ago2-HuR overlap, 13 candidates were screened in luciferase reporter assays, compared to miRNA site mutant controls. Eleven of the sites showed a repressive activity, which displayed significant de-repression upon subsequent testing of the reporters in Dicer-null cells, substantiating miRNA dependence. To experimentally test for HuR’s role in co-regulation, we tested the reporters in CRISPR-generated HuR KO cells. Three of the miRNA sites demonstrated altered activities, indicating that HuR has an effect on miRNA repression at those sites. Our study presents an efficient search and validation system for studying miRNA-HuR interactions, which expands our understanding of the combinatorial post-transcriptional control of gene expression at the 3’ UTR.
2018-01-19 | GSE102319 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2018-01-19 | GSE102321 | GEO
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:HuR is a regulator of mRNA turnover or translation of inflammatory genes through binding to adenylate-uridylate-rich elements (ARE) and related motifs present in the 3’untranslated region (UTR) of mRNAs. We aimed to identify HuR targets in the human airway epithelial cell line BEAS-2B challenged with TNFa plus IFNg, a strong stimulus for inflammatory epithelial responses. Ribonucleoprotein (RNP) complexes from resting and cytokine-treated cells were immunoprecipitated (IP) using anti-HuR and isotype-control antibody, and eluted mRNAs were reverse-transcribed and hybridized to an inflammatory-focused gene array. The chemokines CCL2, CCL8, CXCL1 and CXCL2 ranked highest among 27 signaling and inflammatory genes significantly enriched in the HuR RNP-IP from stimulated cells over the control IP. Among these, 20 displayed published HuR binding motifs. Association of HuR with the four endogenous chemokine mRNAs was validated by single-gene RNP-IP, and shown to be 3’UTR-dependent by biotin pull-down assay. Cytokine treatment increased mRNA stability only for CCL2 and CCL8, and transient silencing and overexpression of HuR affected only CCL2 and CCL8 expression in primary and transformed epithelial cells. Cytokine-induced CCL2 mRNA was predominantly cytoplasmic; conversely, CXCL1 mRNA remained mostly nuclear and unaffected, as CXCL2, by changes in HuR levels. Increase in cytoplasmic HuR and HuR target expression partially relied on the inhibition of AMP-dependent kinase, a negative regulator of HuR nucleocytoplasmic shuttling. We postulate that HuR critically regulates the epithelial response, by associating with multiple adenylate-uridylate-rich elements (ARE)-bearing, functionally related inflammatory transcripts. On the basis of genome-wide studies probing the relationship between RNA-binding proteins and the functional profile of their associated transcripts, we combined the specific HuR immunoprecipitation of RNPs and the genome-scale microarray to profile the target mRNAs of HuR in the human airway epithelial cell line BEAS-2B challenged with very strong inflammatory stimulation, TNFa plus IFNG. The array platform we used is the Human Autoimmune and Inflammatory Response Gene Array (SuperArray Bioscience, Frederick, MD). To validate the association of HuR and its target mRNAs, we planned to apply biotin pull-down assay. HuR overexpression and knockdown assays were also planned to further verify the association of HuR and its target mRNAs. Overall, we did three biological replicates for the IP array experiments, which include both IgG1 control IP and HuR IP arrays.
2010-11-10 | E-GEOD-25264 | biostudies-arrayexpress
Project description:HuR is a regulator of mRNA turnover or translation of inflammatory genes through binding to adenylate-uridylate-rich elements (ARE) and related motifs present in the 3’untranslated region (UTR) of mRNAs. We aimed to identify HuR targets in the human airway epithelial cell line BEAS-2B challenged with TNFa plus IFNg, a strong stimulus for inflammatory epithelial responses. Ribonucleoprotein (RNP) complexes from resting and cytokine-treated cells were immunoprecipitated (IP) using anti-HuR and isotype-control antibody, and eluted mRNAs were reverse-transcribed and hybridized to an inflammatory-focused gene array. The chemokines CCL2, CCL8, CXCL1 and CXCL2 ranked highest among 27 signaling and inflammatory genes significantly enriched in the HuR RNP-IP from stimulated cells over the control IP. Among these, 20 displayed published HuR binding motifs. Association of HuR with the four endogenous chemokine mRNAs was validated by single-gene RNP-IP, and shown to be 3’UTR-dependent by biotin pull-down assay. Cytokine treatment increased mRNA stability only for CCL2 and CCL8, and transient silencing and overexpression of HuR affected only CCL2 and CCL8 expression in primary and transformed epithelial cells. Cytokine-induced CCL2 mRNA was predominantly cytoplasmic; conversely, CXCL1 mRNA remained mostly nuclear and unaffected, as CXCL2, by changes in HuR levels. Increase in cytoplasmic HuR and HuR target expression partially relied on the inhibition of AMP-dependent kinase, a negative regulator of HuR nucleocytoplasmic shuttling.
2010-11-10 | GSE25264 | GEO
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.
2011-08-19 | GSE29778 | GEO
Project description:Transcriptome-wide identification and validation of interactions between the miRNA machinery and HuR on mRNA targets [HuR]
| PRJNA397429 | ENA
Project description:BackgroundHuR/ELAV1, a ubiquitous RNA-binding protein, belongs to the RNA-binding protein family and is crucial for stabilizing and regulating the translation of various mRNA targets, influencing gene expression. Elevated HuR levels are associated with multiple disorders, including cancer and neurodegenerative diseases. Despite the identification of small molecule inhibitors targeting HuR, their detailed characterization remains limited. Recently, Eltrombopag, an FDA-approved drug for immune thrombocytopenic purpura and chemotherapy-induced thrombocytopenia, emerged as a potential HuR inhibitor. However, the specific molecular pathways influenced by both HuR and Eltrombopag are not fully understood. ResultsOur study demonstrates that Eltrombopag operates via HuR inhibition, affecting gene expression regulation at the posttranscriptional level. We show that both HuR knockout and Eltrombopag treatment modulate iron metabolism by decreasing ferritin heavy chain (FTH1) and light chain (FTL) synthesis while increasing the expression of Iron-regulatory protein 2 (IRP2), a key regulator of ferritin translation. Additionally, HuR inhibition reduces the levels of Glycoprotein Hormones, Alpha Polypeptide (CGA), a marker associated with hormone-induced tumors, suggesting a potential use of Eltrombopag in treatment of cancers overexpressing CGA. We observed that the main of control is manifested at the level of translation inhibition, with proteosome-mediated regulation also playing an important role.ConclusionsThese findings uncover novel posttranscriptional mechanisms governed by HuR and its inhibitor, elucidating pathways relevant to HuR-mediated regulation and molecular therapies aimed at targeting this protein.
2024-12-20 | PXD050698 | Pride
Project description:Upon muscle injury the high mobility group box 1 (HMGB1) protein is up-regulated and secreted to initiate reparative responses. Here we show that HMGB1 controls myogenesis both in vitro and in vivo, during development and after adult muscle injury. HMGB1 expression in muscle cells is regulated at the translational level: the miRNA miR-1192 inhibits HMGB1 translation and the RNA-binding protein HuR promotes it. HuR binds to a cis-element, HuRBS, located in the 3'UTR of the HMGB1 transcript, and at the same time miR-1192 is recruited to an adjacent seed element. The binding of HuR to the HuRBS prevents the recruitment of Argonaute 2 (Ago2), overriding miR-1192-mediated translation inhibition. Depleting HuR reduces myoblast fusion and silencing miR-1192 re-establishes the fusion potential of HuR-depleted cells. We propose that HuR promotes the commitment of myoblasts to myogenesis by enhancing the translation of HMGB1 and suppressing the translation inhibition mediated by miR-1192. RNA content was extracted following immunoprecipitation of HuR using a monoclonal antibody (3A2) and the levels of mRNA were compared to an IgG control in order to determine which transcripts were enriched in the HuR ribonucleoprotein complex.
2013-08-14 | E-GEOD-49855 | biostudies-arrayexpress