Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

The RNA-binding protein HuR coordinates global changes in gene expression during patterning of early peripheral nerves

ABSTRACT: Identify mRNA targets of the RNA-binding protein HuR in vivo during Schwann cell development using RIP-chip analysis. IP protocol of endogenous mRNA-transfected HuR complexes was performed as described in Keene et al. (2006). In brief, 500 mg of whole-cell lysate obtained from a pool of NB or P5 sciatic nerves from C57BL6J mice were incubated with a suspension of Protein Sepharose beads (Sigma-Aldrich), pre-coated with 15 mg of either IgG1 (BD Pharmingen) or anti-HuR (Santa Cruz Biotechnology) antibodies. mRNAs were isolated using the phenol-chloroform method.

ORGANISM(S): Mus musculus

SUBMITTER: ashwin woodhoo

PROVIDER: E-MEXP-3519 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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The RNA-binding protein human antigen R controls global changes in gene expression during Schwann cell development.

Iruarrizaga-Lejarreta Marta M Varela-Rey Marta M Lozano Juan José JJ Fernández-Ramos David D Rodríguez-Ezpeleta Naiara N Embade Nieves N Lu Shelly C SC van der Kraan Peter M PM Blaney Davidson Esmeralda N EN Gorospe Myriam M Mirsky Rhona R Jessen Kristján R KR Aransay Ana María AM Mato José M JM Martínez-Chantar María L ML Woodhoo Ashwin A

The Journal of neuroscience : the official journal of the Society for Neuroscience 20120401 14

An important prerequisite to myelination in peripheral nerves is the establishment of one-to-one relationships between axons and Schwann cells. This patterning event depends on immature Schwann cell proliferation, apoptosis, and morphogenesis, which are governed by coordinated changes in gene expression. Here, we found that the RNA-binding protein human antigen R (HuR) was highly expressed in immature Schwann cells, where genome-wide identification of its target mRNAs in vivo in mouse sciatic ne ...[more]

PMID: 22492050

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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.

Project description:IL-20 cytokines are involved in the establishment of psoriasis, a common chronic skin inflammation epidemiologically associated with metabolic syndrome, but molecular mechanisms underlying their over-expression remain to be elucidated. We find that keratinocytes (KCs) expressed IL-20 and lymphocytes expressed IL-22 cytokines up-regulation occurs at post-transcriptional level with stabilization of their RNA messengers. Looking at psoriatic epidermis, we observe that the p38/MK2 pathway is not activated but that the RNA-binding protein (RBP) HuR re-localizes in keratinocytes cytoplasm, suggesting post-transcriptional regulation of numerous mRNAs. HuR ribonucleoprotein immunoprecipitations analyzed by high-throughput sequencing (RIP-Seq) identify potential pre-mature and mature RNA targets for uninvolved and involved skin and confirms that HuR activity is displaced from the nucleus to the cytoplasm. Numerous psoriasis up-regulated transcripts are HuR targets and HuR knockdown reduces expression of transcripts like beta-defensin-2, CXCL-10 or IL-2, suggesting an implication of HuR in pathophysiological processes such as morphological, immune and metabolic inflammatory responses. Finally, metabolic disorders affecting psoriatic keratinocytes are responsible for HuR cytoplasmic localization since a decreased activity of the cellular metabolic sensor AMPK, that is observed in human psoriatic epidermis, is sufficient to promote HuR cytosolic localization as well as IL-20 over-production both in human keratinocytes and in vivo in mouse epidermis where it then initiates psoriasis-like histological changes. These results may provide insights into molecular links between metabolism and post-transcriptional networks during chronic inflammation, as illustrated in psoriasis by mechanisms connecting AMPK, HuR and IL-20. Analysis of HuR-binding RNA in uninvolved versus involved psoriatic samples by RIP-Seq. Samples from five different patients were used for both uninvolved and involved skin. RIP-Seq was also made using a control IgG.

Dataset Information

The RNA-binding protein HuR coordinates global changes in gene expression during patterning of early peripheral nerves

Publications

The RNA-binding protein human antigen R controls global changes in gene expression during Schwann cell development.

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