HuR Binding Sites Research Articles

MRNA openers and closers: modulating AU-rich element-controlled mRNA stability by a molecular switch in mRNA secondary structure.

Approximately 3 000 genes are regulated in a time-, tissue-, and stimulus-dependent manner by degradation or stabilization of their mRNAs. The process is mediated by interaction of AU-rich elements (AREs) in the mRNA's 3'-untranslated regions with trans-acting factors. AU-rich element-controlled genes of fundamentally different functional relevance depend for their activation on one positive regulator, HuR. Here we present a methodology to exploit this central regulatory process for specific manipulation of AU-rich element-controlled gene expression at the mRNA level. With a combination of single-molecule spectroscopy, computational biology, and molecular and cellular biochemistry, we show that mRNA recognition by HuR is dependent on the presentation of the sequence motif NNUUNNUUU in single-stranded conformation. The presentation of the HuR binding site in the mRNA secondary structure appears to act analogously to a regulatory on/off switch that specifically controls HuR access to mRNAs in cis. Based on this finding we present a methodology for manipulating ARE mRNA levels by actuating this conformational switch specifically in a target mRNA. Computationally designed oligonucleotides (openers) enhance the NNUUNNUUU accessibility by rearranging the mRNA conformation. Thereby they increase in vitro and endogenous HuR-mRNA complex formation which leads to specific mRNA stabilization (as demonstrated for TNFalpha and IL-2, respectively). Induced HuR binding both inside and outside the AU-rich element promotes functional IL-2 mRNA stabilization. This opener-induced mRNA stabilization mimics the endogenous IL-2 response to CD28 stimulation in human primary T-cells. We therefore propose that controlled modulation of the AU-rich element conformation by mRNA openers or closers allows message stabilization or destabilization in cis to be specifically triggered. The described methodology might provide a means for studying distinct pathways in a complex cellular network at the node of mRNA stability control. It allows ARE gene expression to be potentially silenced or boosted. This will be of particular value for drug-target validation, allowing the diseased phenotype to ameliorate or deteriorate. Finally, the mRNA openers provide a rational starting point for target-specific mRNA stability assays to screen for low-molecular-weight compounds acting as inhibitors or activators of an mRNA structure rearrangement.

The RNA-binding Protein HuR Regulates the Expression of Cyclooxygenase-2

The cyclooxygenase-2 (COX-2) gene encodes the inducible prostaglandin synthase enzyme implicated in inflammation, cell growth, and tumorigenesis. Regulation of the COX-2 gene expression at the post-transcriptional level is poorly understood. For example, protein factors that regulate the post-transcriptional mRNA metabolism of COX-2 have not been fully characterized. In this study, we demonstrate that the RNA-binding protein HuR binds to COX-2 mRNA and regulates its expression. We show that there are three binding sites for HuR in the 3'-untranslated region of human COX-2. These sites are located at the following positions in the COX-2 3'-untranslated region: 39-84 nucleotides (nt), 1155-1187 nt, and 1244-1256 nt (hereinafter referred to as Sites I, II and III, respectively). Although all three sites are present in the 4.6-kb COX-2 mRNA, only site I is present in the shorter 2.8-kb isoform. HuR in MDA-MB-231 cell extracts associated with COX-2 mRNA at the identified sites. Further, HuR location in the cytoplasm was induced by serum withdrawal, a stimulus known to induce COX-2 mRNA. Down-regulation of HuR by two independent methods, namely RNA interference as well as antisense RNA expression, significantly attenuated serum withdrawal-induced increase in COX-2 mRNA (both the 4.6- and 2.8-kb isoforms) and protein levels. These data suggest that HuR binding to COX-2 is critical for its post-transcriptional mRNA stabilization.

A 40-bp RNA Element That Mediates Stabilization of Vascular Endothelial Growth Factor mRNA by HuR

VEGF is a critical mediator of hypoxia-induced angiogenesis in numerous physiological and pathophysiological conditions. The hypoxic induction of VEGF is due in large part to an increase in the stability of its mRNA. We recently demonstrated that the stabilization of VEGF mRNA by hypoxia is dependent upon the RNA-binding protein HuR. This report describes the identification of a 40-bp functional HuR binding site in the VEGF mRNA 3'-untranslated region. This element can confer HuR-mediated stabilization of a heterologous gene in vitro and in vivo. Furthermore, the element is sufficient to confer an increase in the hypoxic induction of a heterologous gene. Deletion of the HuR binding site within this 40-bp element as mapped by RNase T1 and lead footprinting uncouples a stabilizing sequence from a destabilizing sequence, thus providing a novel RNA-protein regulatory model that might be exploited to manipulate VEGF expression and hypoxia-induced angiogenesis.

The identification of an endonuclease that cleaves within an HuR binding site in mRNA.

Messenger RNAs (mRNAs) that contain U-rich elements are targeted for rapid decay. Selective inhibition of this decay results in a rapid increase in steady state level. Thus, this is an important regulatory step in gene expression. Previously, we have found that these mRNAs are selectively stabilized by a specific mRNA binding protein called HuR. The mechanism of action of HuR is not well understood. It has been postulated that HuR stabilizes mRNA by the displacement or inhibition of factors that specifically cleave or deadenyl-ate these mRNAs. In this paper, we report the identification and characterization of a novel endo-nuclease that cleaves within an HuR binding site in p27kip1 mRNA. The specificity of this endonuclease and its inhibition by HuR argue for it playing a role in the postranscriptional regulation of gene expression.

HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation.

Colorectal carcinoma RKO cells expressing reduced levels of the RNA-binding protein HuR (ASHuR) displayed markedly reduced growth. In synchronous RKO populations, HuR was almost exclusively nuclear during early G(1), increasing in the cytoplasm during late G(1), S and G(2). The expression and half-life of mRNAs encoding cyclins A and B1 similarly increased during S and G(2), then declined, indicating that mRNA stabilization contributed to their cell cycle-regulated expression. In gel-shift assays using radiolabeled cyclin RNA transcripts and RKO protein extracts, only those transcripts corresponding to the 3'-untranslated regions of cyclins A and B1 formed RNA-protein complexes in a cell cycle-dependent fashion. HuR directly bound mRNAs encoding cyclins A and B1, as anti-HuR antibodies supershifted such RNA-protein complexes. Importantly, the expression and half-life of mRNAs encoding cyclins A and B1 were reduced in ASHuR RKO cells. Our results indicate that HuR may play a critical role in cell proliferation, at least in part by mediating cell cycle-dependent stabilization of mRNAs encoding cyclins A and B1.