The 3′ Untranslated Region of Tumor Necrosis Factor Alpha mRNA Is a Target of the mRNA-Stabilizing Factor HuR

Abstract

Posttranscriptional regulation is important for tumor necrosis factor alpha (TNF-␣) expression in monocytes and macrophages, and an AU-rich element (ARE) in the 3 untranslated region (UTR) of TNF-␣ mRNA is implicated in control of its translation and mRNA stability. Regulation of mRNA turnover is thought to be mediated by trans-acting proteins, which bind the ARE and stabilize or destabilize the transcript. However, with the exception of the destabilizing factor tristetraprolin, the identity and function of the proteins binding the TNF-␣ mRNA ARE have not been established. To identify other proteins involved in the posttranscriptional control of TNF-␣, the subcellular location of TNF-␣ mRNA was determined in the macrophage-like cell line RAW 264.7. TNF-␣ mRNA was located in the pellet following centrifugation of cytoplasm at 100,000 ؋ g (P100 fraction). This fraction also contained proteins which formed two distinct ARE-specific complexes with the TNF-␣ mRNA 3 UTR in electrophoretic mobility shift assays (EMSAs). A protein present in these two complexes was purified and identified by peptide mass mapping and tandem mass spectrometry as HuR. In EMSAs both complexes were supershifted by an anti-HuR antibody, while Western blotting also demonstrated the presence of HuR in the P100 extract. A HeLa cell tetracycline-regulated reporter system was used to determine the effect of HuR on mRNA stability. In this system, overexpression of HuR resulted in stabilization of an otherwise unstable reporter-mRNA containing the TNF-␣ ARE. These results demonstrate that the TNF-␣ ARE is a target of the mRNA-stabilizing factor HuR.

Distinct Domains of AU-Rich Elements Exert Different Functions in mRNA Destabilization and Stabilization by p38 Mitogen-Activated Protein Kinase or HuR

“…HuR has been found to interact with several AREs in vitro and in cells, and its overexpression induced stabilization of mRNAs for vascular endothelial growth factor (23), nitric oxide synthase II (35), eotaxin (1), and different ARE-containing reporter RNAs (14,15,33). Furthermore, suppression of HuR amounts by antisense RNA or small interfering RNA approaches inhibited stabilization induced by hypoxia (23), cytokines (35), or UV light (43) or during cell cycle progression (42).…”

The Role of Human Antigen R, an RNA-binding Protein, in Mediating the Stabilization of Toll-Like Receptor 4 mRNA Induced by Endotoxin

“…Activation of HuR results from the activation of p38 MAPK. MAPKAPK-2, which is phosphorylated by p38 MAPK, regulates the stability of mRNAs for TNF-␣, 26 through their AREs. Importantly, the activation of MAPKAPK-2, which increases the cytoplasmic accumulation of HuR 27 ; dominant negative mutants of MAPKAPK-2 changes the cytoplasmic HuR level 27 and blocks cytokine-induced COX-2 28 mRNA degradation.…”

p38 Mitogen-activated Protein Kinase Stabilizes mRNAs That Contain Cyclooxygenase-2 and Tumor Necrosis Factor AU-rich Elements by Inhibiting Deadenylation

“…In Vitro Transcription of Riboprobes-␣-32 P-Labeled riboprobes were synthesized by in vitro transcription from linearized DNA templates using T7 RNA polymerase, 12 M cold UTP, and 50 Ci of [␣-32 P]UTP (800 Ci mmol Ϫ1 ) as described previously (31).…”

“…It is an in vitro substrate of MAPKAPK-2 (30); however, TTP is not necessarily involved in the basic stabilization mechanism because it is not expressed in HeLa cells in which regulation of reporter mRNAs by p38 MAPK has been studied. 2 HuR, which is expressed in HeLa cells (31), also binds the p38 MAPK-responsive COX-2 and TNF AREs (31)(32)(33)(34). HuR is thought to stabilize mRNA by inhibiting the decay of hypo-adenylated intermediates (35,36).…”