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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Concurrent binding and modifications of AUF1 and HuR mediate the pH-responsive stabilization of phosphoenolpyruvate carboxykinase mRNA in kidney cells.

Onset of metabolic acidosis leads to a pronounced increase in renal expression of phosphoenolpyruvate carboxykinase (PEPCK). This response, which is mediated in part by stabilization of PEPCK mRNA, is effectively modeled by treating LLC-PK(1)-F(+)-9C cells with an acidic medium. siRNA knockdown of HuR prevented the pH-responsive increase in PEPCK mRNA half-life suggesting that HuR is necessary for this response. A recruitment assay, using a reporter mRNA in which the pH response elements of the PEPCK 3'-UTR were replaced with six MS2 stem-loop sequences, was developed to test this hypothesis. The individual recruitment of a chimeric protein containing the MS2 coat protein and either HuR or p40AUF1 failed to produce a pH-responsive stabilization. However, the concurrent expression of both chimeric proteins was sufficient to produce a pH-responsive increase in the half-life of the reporter mRNA. siRNA knockdown of AUF1 produced slight increases in basal levels of PEPCK mRNA and protein, but partially inhibited the pH-responsive increases. Complete inhibition of the latter response was achieved by knockdown of both RNA-binding proteins. The results suggest that binding of HuR and AUF1 has opposite effects on basal expression, but may interact to mediate the pH-responsive increase in PEPCK mRNA. Two-dimensional gel electrophoresis indicated that treatment with acidic medium caused a decrease in phosphorylation of HuR, but may increase phosphorylation of the multiple AUF1 isoforms. Thus, the pH-responsive stabilization of PEPCK mRNA requires the concurrent binding of HuR and AUF1 and may be mediated by changes in their extent of covalent modification.[1]


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