Unknown,Transcriptomics,Genomics,Proteomics

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Intestinal epithelial HuR balances counteracting RNA networks to protect against intestinal inflammation and cancer. PART 3 RIP chip by array

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Dimitris Kontoyiannis

PROVIDER: E-MTAB-4018 | 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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Intestinal epithelial HuR balances counteracting RNA networks to protect against intestinal inflammation and cancer. PART 3 RIP chip by array

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