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Identification of a target RNA motif for RNA-binding protein HuR.

ABSTRACT: HuR, a protein that binds to specific mRNA subsets, is increasingly recognized as a pivotal posttranscriptional regulator of gene expression. Here, HuR was immunoprecipitated under conditions that preserved HuR-RNA interactions, and HuR-bound target mRNAs were identified by cDNA array hybridization. Analysis of primary sequences and secondary structures shared among HuR targets led to the identification of a 17- to 20-base-long RNA motif rich in uracils. This HuR motif was found in almost all mRNAs previously reported to be HuR targets, was located preferentially within 3' untranslated regions of all unigene transcripts examined, and was conserved in >50% of human and mouse homologous genes. Importantly, the HuR motif allowed the successful prediction and subsequent validation of novel HuR targets from gene databases. This study describes an HuR target RNA motif and presents a general strategy for identifying target motifs for RNA-binding proteins.

SUBMITTER: Lopez de Silanes I

PROVIDER: S-EPMC365732 | biostudies-literature | 2004 Mar

REPOSITORIES: biostudies-literature

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Identification of a target RNA motif for RNA-binding protein HuR.

López de Silanes Isabel I Zhan Ming M Lal Ashish A Yang Xiaoling X Gorospe Myriam M

Proceedings of the National Academy of Sciences of the United States of America 20040223 9

HuR, a protein that binds to specific mRNA subsets, is increasingly recognized as a pivotal posttranscriptional regulator of gene expression. Here, HuR was immunoprecipitated under conditions that preserved HuR-RNA interactions, and HuR-bound target mRNAs were identified by cDNA array hybridization. Analysis of primary sequences and secondary structures shared among HuR targets led to the identification of a 17- to 20-base-long RNA motif rich in uracils. This HuR motif was found in almost all mR ...[more]

PMID: 14981256

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Project description:ObjectiveGlomerular injury is a prominent pathological feature of diabetic kidney disease (DKD). Constitutively active NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species that mediates hyperglycemia-induced mesangial cell (MC) fibrotic injury. However, the mechanism that Nox4 utilizes to achieve its biological outcome remains elusive, and the signaling pathways that regulate this isoform oxidase are not well understood. Here, our goal is to study the detailed mechanism by which NAPDH oxidase 4 (Nox4) is post-transcriptionally regulated in MC during diabetic pathology.MethodsWe studied the protein expression of HuR, Nox4 and matrix proteins by western blotting, while we assessed the mRNA stability of Nox4 by RT-PCR and polysomal assay, examined in vitro cultured glomerular mesangial cells treated by high glucose (HG) and diabetic animal induced by STZ. The binding assay between HuR and the Nox4 promoter was done by immuno-precipiating with HuR antibody and detecting the presence of Nox4 mRNA, or by pull-down by using biotinlyated labeled Nox4 promoter RNA and detecting the presence of the HuR protein. The binding was also confirmed in MCs where Nox4 promoter-containing luciferage constructs were transfected. ROS levels were measured with DHE/DCF dyes in cells, or lucigenin chemiluminescence for Nox enzymatic levels, or HPLC assay for superoxide. HuR protein was inhibited by antisense oligo that utilized osmotic pumps for continuous delivery in animal models. The H1bAc1 ratio was measured by an ELISA kit for mice.ResultsWe demonstrate that in MCs, high glucose (HG) elicits a rapid upregulation of Nox4 protein via translational mechanisms. Nox4 mRNA 3' untranslated region (3'-UTR) contains numerous AU-rich elements (AREs) that are potential binding sites for the RNA-binding protein human antigen R (HuR). We show that HG promotes HuR activation/expression and that HuR is required for HG-induced Nox4 protein expression/mRNA translation, ROS generation, and subsequent MC fibrotic injury. Through a series of invitro RNA-binding assays, we demonstrate that HuR acts via binding to AREs in Nox4 3'-UTR in response to HG. The invivo relevance of these observations is confirmed by the findings that increased Nox4 is accompanied by the binding of HuR to Nox4 mRNA in kidneys from type 1 diabetic animals, and further suppressing HuR expression showed a reno-protective role in a type 1 diabetic mouse model via reducing MC injury, along with the improvement of hyperglycemia and renal function.ConclusionsWe established for the first time that HuR-mediated translational regulation of Nox4 contributes to the pathogenesis of fibrosis of the glomerular microvascular bed. Thus therapeutic interventions affecting the interplay between Nox4 and HuR could be exploited as valuable tools in designing treatments for DKD.

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Identification of a target RNA motif for RNA-binding protein HuR.

Publications

Identification of a target RNA motif for RNA-binding protein HuR.

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