RNA Immunoprecipitation-sequencing from mouse liver using HuR antibody
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ABSTRACT:
PROVIDER: PRJNA601341 | ENA |
REPOSITORIES: ENA
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Project description:We report the HuR-RNA interactions in the liver by performing RNA-immunoprecipitation sequencing (RIP-seq). RIP-seq was performed in healthy livers of wildtype (WT) mice using a HuR antibody. We found that 1380 cytoplasmic-target mRNAs bound to HuR, as assessed by the comparison between the HuR-specific antibody and the IgG control
2021-09-15 | GSE143703 | GEO
Project description:HuR shRNA adenoviruses were delivered into WT or humanized mice intravenously at 2 × 109 pfu/mouse for both control virus and HuR shRNA virus . After seven days, liver tissue samples were harvested after a four hours fasting and stored immediately in liquid nitrogen till further analysis.The frozen liver tissue samples were homogenized in Trizol reagent (Invitrogen) using TissueLyser LT system (Qiagen). The isolated RNA was purified by MagMAX RNA Extraction Kit (ThermoFisher) and the construction of strand specific sequencing libraries using TruSeq Stranded Total RNA Prep kit (Illumina) and the sequencing was performed at NHLBI DNA Sequencing and Genomics Core using Illumina HiSeq 3000 paired-end sequencing platform.
2021-01-04 | GSE161462 | 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:Sequencing of liver RNA from HuR WT and HuR KO mice
| PRJNA600219 | ENA
Project description:RNA was isolated from material that had been immunoprecipitated (IP) from Hela cells using antibodies recognizing RNA-binding proteins HuR or AUF1, as well as using a control IgG1 antibody. RNA was reverse-transcribed in the presence of [alpha-33P]dCTP and the radiolabeled product used to hybridize human cDNA arrays. The experiment was repeated using three independent sample sets. The samples were numbered HuR-1, HuR-2, HuR-3, AUF1-1, AUF1-2, AUF1-3, IgG1-1, IgG1-2, IgG1-3. HuR represents RNA from IP reactions using an anti-HuR antibody, AUF1 represents RNA from IP reactions using an anti-AUF1 antibody and, IgG1 represents RNA from IP reactions using an anti-IgG1 antibody. The numbers 1, 2 and 3 correspond to the three independent experimental datasets. Keywords = RNa-binding protein Keywords = mRNA stability Keywords = exosome Keywords = polysome Keywords = RNA motif Keywords: ordered
2004-05-04 | GSE1361 | GEO
Project description:Analysis of RNA immunoprecipitation of HuR, a RNA binding protein (RBP), in breast cancer cell lines. This approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich (ARE) regions of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR associated mRNAs found individually or in both cell types. Two novel HuR targets, CD-9 and CALM-2, were identified and validated by quantitative RT-PCR and biotin pulldown analysis. Our findings reveal that the differential regulation of these two cancer-related genes by HuR was contingent upon the cellular environment. RNA immunoprecipitation of the HuR RNA binding protein by 3A2 antibody and IgG (control) from two human breast cancer cell lines, MCF-7 and MDA-MB-231 .
2010-06-24 | E-GEOD-17820 | biostudies-arrayexpress
Project description:RNA was isolated from material that had been immunoprecipitated (IP) from Hela cells using antibodies recognizing RNA-binding proteins HuR or AUF1, as well as using a control IgG1 antibody. RNA was reverse-transcribed in the presence of [alpha-33P]dCTP and the radiolabeled product used to hybridize human cDNA arrays. The experiment was repeated using three independent sample sets. The samples were numbered HuR-1, HuR-2, HuR-3, AUF1-1, AUF1-2, AUF1-3, IgG1-1, IgG1-2, IgG1-3. HuR represents RNA from IP reactions using an anti-HuR antibody, AUF1 represents RNA from IP reactions using an anti-AUF1 antibody and, IgG1 represents RNA from IP reactions using an anti-IgG1 antibody. The numbers 1, 2 and 3 correspond to the three independent experimental datasets. Keywords = RNa-binding protein Keywords = mRNA stability Keywords = exosome Keywords = polysome Keywords = RNA motif Keywords: ordered
2010-06-09 | E-GEOD-1361 | biostudies-arrayexpress
Project description:The purpose of this study is to generate RNA binding profile of RNA-binding protein HuR in human breast cancer cell line MDA-MB-231 cells by ribonucleoprotein immunoprecipitation sequencing (RIP-deq). RNA immunoprecipitated by mouse anti-HuR antibody or mouse IgG was collected for library prepareation and deep sequencing, in duplicate, using Illumina Hiseq 2500 system. About 30 million sequence reads per sample were mapped to the human genome (build hg38), the corresponding peaks were then detected and annotated. Finally, the RIP peaks that correspond to significant transcript abundance change were identified and compiled.
2019-04-05 | GSE129361 | GEO
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
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-49856 | biostudies-arrayexpress