Unknown,Transcriptomics,Genomics,Proteomics

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Rip-Chip profiling of HnRNP A1 nuclear complexes in HuR WT and HuR KO cells

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Olga Papadodima

PROVIDER: E-GEOD-49225 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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Rip-Chip profiling of HnRNP A1 nuclear complexes in HuR WT and HuR KO cells

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