Unknown,Transcriptomics,Genomics,Proteomics

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Chemokine Transcripts as Targets of the RNA-Binding Protein HuR in Human Airway Epithelium

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Jinshui Fan

PROVIDER: E-GEOD-25264 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Posttranscriptional regulation is emerging as a key factor in glucocorticoid (GC)-mediated gene regulation. We investigated the role of the human glucocorticoid receptor (GR) as an RNA-binding protein and its effect on mRNA turnover in human airway epithelial cells. Cell treatment with the potent GC budesonide accelerated the decay of CCL2 mRNA (t1/2=8±1 min vs. 62±17 min in DMSO-treated cells) and CCL7 mRNA (t1/2=15±4 min vs. 114±37 min), but not that of CCL5 mRNA (t1/2=231±8 min vs. 266±5 min) in the BEAS-2B cell line. This effect was inhibited by pre-incubation with an anti-GR antibody, indicating that GR itself plays a role in the turnover of these transcripts. Co-immunoprecipitation and biotin pulldown experiments showed that GR associates with CCL2 and CCL7 mRNAs, but not CCL5 mRNA. These methods confirmed CCL2 mRNA targeting by GR in human primary airway epithelial cells. Association of the GR was localized to the 5’UTR of CCL2 mRNA, and further mapped to nucleotides 44-60. The collection of transcripts associated with GR, identified by immunoprecipitation of GR-mRNA complexes followed by microarray analysis, revealed 479 transcripts that associated with GR. Computational analysis of the primary sequence and secondary structures of these transcripts yielded a GC-rich motif, which was shown to bind to GR in vitro. This motif was used to predict binding of GR to an additional 7889 transcripts. These results indicate that cytoplasmic GR interacts with a subset of mRNA through specific sequences and can regulate turnover rates, suggesting a novel posttranscriptional role for GR as an RNA-binding protein. The BEAS-2B cell line is derived from human tracheal epithelium transformed by an adenovirus 12-SV40 hybrid virus. For isolation of glucocorticoid receptor-associated transcripts, cytoplasmic lysates obtained from 1.6x107 cells/condition, 4 replicates per condition, were used to generate a collection of transcripts associated with GR by immunoprecipitation of the GR-mRNA complexes. The RNA from these IP's were extracted and labeled with the Illumina TotalPrep RNA Amplification Kit which generates biotin-labeled cRNA. This was hybridized to Illumina's Sentrix HumanRef-8,v3 Expression BeadChips. The data from 3 replicates of GR-IP and 4 replicates of Control-IP were used for global analysis of GR-mRNA interactions.

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Chemokine Transcripts as Targets of the RNA-Binding Protein HuR in Human Airway Epithelium

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