Relief of microRNA-mediated translational repression in human cells subjected to stress

In metazoans, most microRNAs imperfectly base-pair with the 3' untranslated region (3'UTR) of target mRNAs and prevent protein accumulation by either repressing translation or inducing mRNA degradation. Examples of specific mRNAs undergoing microRNA-mediated repression are numerous, but whether the repression is a reversible process remains largely unknown. Here we show that cationic amino acid transporter 1 (CAT-1) mRNA and reporters bearing its 3'UTR can be relieved from the microRNA miR-122-induced inhibition in human hepatocarcinoma cells subjected to different stress conditions. The derepression of CAT-1 mRNA is accompanied by its release from cytoplasmic processing bodies and its recruitment to polysomes. The derepression requires binding of HuR, an AU-rich-element binding protein, to the 3'UTR of CAT-1 mRNA. We propose that proteins interacting with the 3'UTR will generally act as modifiers altering the potential of miRNAs to repress gene expression.     

The 3' untranslated region of bovine follicle-stimulating hormone beta messenger RNA downregulates reporter expression: involvement of AU-rich elements and transfactors

FSHbeta mRNA has a unique 3' untranslated region (3'UTR) that is highly conserved across the species. Sequence analyses of the mouse, rat, human, bovine, and ovine 3'UTRs revealed the presence of elements implicated in mRNA instability and translational control such as AU-Rich Element (ARE) and lipoxygenase differentiation control elements. Bovine FSHbeta 3'UTR down-regulated reporter expression in alphaT3-1 and NIH3T3 cells, but not in HEK 293 cells, suggesting the involvement of a cell-specific factor or mechanism. The presence of a 3'UTR did not influence reporter mRNA stability, but it did decrease its association with polysomes, indicating that the downregulatory effect may be exerted at the translational level. The segment spanning 601-800 bases (U4) of the bovine FSHbeta 3'UTR was found to be the most effective downregulating segment, its effect being equal to that of the full-length 3'UTR. RNA electrophoretic mobility shift assay with U4 showed the presence of specific transfactors in the cytosolic preparations of bovine pituitary and the cell lines. U4 contained an ARE that appeared to be functional, because the mutated U4 ARE was ineffective in downregulating the reporter expression and inhibiting [(32)P]-labeled U4-transfactor complex formation. Downregulation of reporter activity by the full-length 3'UTR and U4 could be overcome by overexpression of HuR, a protein known to stabilize ARE-containing mRNAs in NIH3T3 cells, but not in the alphaT3-1 cells, once again indicating that factors other than HuR may also be involved in the regulation of FSHbeta in the pituitary.     

Post-transcriptional regulation of RNase-L expression is mediated by the 3'-untranslated region of its mRNA

RNase-L mediates critical cellular functions including antiviral, pro-apoptotic, and tumor suppressive activities; accordingly, its expression must be tightly regulated. Little is known about the control of RNASEL expression; therefore, we examined the potential regulatory role of a conserved 3'-untranslated region (3'-UTR) in its mRNA. The 3'-UTR mediated a potent decrease in the stability of RNase-L mRNA, and of a chimeric beta-globin-3'-UTR reporter mRNA. AU-rich elements (AREs) are cis-acting regulatory regions that modulate mRNA stability. Eight AREs were identified in the RNase-L 3'-UTR, and deletion analysis identified positive and negative regulatory regions associated with distinct AREs. In particular, AREs 7 and 8 served a strong positive regulatory function. HuR is an ARE-binding protein that stabilizes ARE-containing mRNAs, and a predicted HuR binding site was identified in the region comprising AREs 7 and 8. Co-transfection of HuR and RNase-L enhanced RNase-L expression and mRNA stability in a manner that was dependent on this 3'-UTR region. Immunoprecipitation demonstrated that RNase-L mRNA associates with a HuR containing complex in intact cells. Activation of endogenous HuR by cell stress, or during myoblast differentiation, increased RNase-L expression, suggesting that RNase-L mRNA is a physiologic target for HuR. HuR-dependent regulation of RNase-L enhanced its antiviral activity demonstrating the functional significance of this regulation. These findings identify a novel mechanism of RNase-L regulation mediated by its 3'-UTR.     

热激蛋白Hsp72促进热休克下HuR对p21CIP1的调控作用

RNA结合蛋白HuR可以结合并调控靶标mRNA稳定性与翻译,但影响HuR 结合活性的因素有待探讨。本研究从蛋白质-蛋白质相互作用角度对影响HuR 与RNA结合活性的因素做了探讨。结果发现,热激蛋白Hsp72在细胞浆与HuR相互作用并促进HuR与p21 (KIP1) 3′UTR（3′非翻译区）的结合; 热休克下Hsp72总蛋白质及细胞浆蛋白质水平上调、但HuR总蛋白质及细胞浆蛋白质水平不变|热休克下HuR与p21 3′UTR的相互作用加强、p21蛋白及mRNA水平上调。上述结果提示,Hsp72可通过与HuR相互作用促进后者与p21 mRNA的结合,进而加强热休克下HuR对p21的表达的促进作用。这些结果为进一步解析HuR的生物学作用机制提供了实验依据。     

Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs.

The messenger RNAs of many proto-oncogenes, cytokines and lymphokines are targeted for rapid degradation through AU-rich elements (AREs) located in their 3'' untranslated regions (UTRs). HuR, a ubiquitously expressed member of the Elav family of RNA binding proteins, exhibits specific affinities for ARE-containing RNA sequences in vitro which correlate with their in vivo decay rates, thereby implicating HuR in the ARE-mediated degradation pathway. We have transiently transfected HuR into mouse L929 cells and observed that overexpression of HuR enhances the stability of beta-globin reporter mRNAs containing either class I or class II AREs. The increase in mRNA stability parallels the level of HuR overexpression, establishing an in vivo role for HuR in mRNA decay. Furthermore, overexpression of HuR deletion mutants lacking RNA recognition motif 3 (RRM 3) does not exert a stabilizing effect, indicating that RRM 3 is important for HuR function. We have also developed polyclonal anti-HuR antibodies. Immunofluorescent staining of HeLa and L929 cells using affinity-purified anti-HuR antibody shows that both endogenous and overexpressed HuR proteins are localized in the nucleus. By forming HeLa-L929 cell heterokaryons, we demonstrate that HuR shuttles between the nucleus and cytoplasm. Thus, HuR may initially bind to ARE-containing mRNAs in the nucleus and provide protection during and after their export to the cytoplasmic compartment.     

Phosphorylation of HuR by Chk2 regulates SIRT1 expression

The RNA binding protein HuR regulates the stability of many target mRNAs. Here, we report that HuR associated with the 3' untranslated region of the mRNA encoding the longevity and stress-response protein SIRT1, stabilized the SIRT1 mRNA, and increased SIRT1 expression levels. Unexpectedly, oxidative stress triggered the dissociation of the [HuR-SIRT1 mRNA] complex, in turn promoting SIRT1 mRNA decay, reducing SIRT1 abundance, and lowering cell survival. The cell cycle checkpoint kinase Chk2 was activated by H(2)O(2), interacted with HuR, and was predicted to phosphorylate HuR at residues S88, S100, and T118. Mutation of these residues revealed a complex pattern of HuR binding, with S100 appearing to be important for [HuR-SIRT1 mRNA] dissociation after H(2)O(2). Our findings demonstrate that HuR regulates SIRT1 expression, underscore functional links between the two stress-response proteins, and implicate Chk2 in these processes.     

HuR binds a cyclic nucleotide-dependent, stabilizing domain in the 3' untranslated region of Na(+)/glucose cotransporter (SGLT1) mRNA.

Differentiation-dependent expression of the Na(+)/glucose cotransporter (SGLT1) is accompanied by a large, cAMP-dependent increase in stability of its mRNA. Stabilization is mediated by protein binding to a critical uridine-rich element (URE) in its 3' untranslated region. In the present study, we demonstrate that HuR, an RNA binding protein of the embryonic lethal abnormal vision family, binds the SGLT1 URE. HuR binding was increased after elevation of intracellular cAMP levels and was dependent on protein phosphorylation. This interaction was prevented by a substitution mutation previously shown to block cAMP-dependent reporter message stabilization. These results implicate HuR as a key mediator of cAMP-dependent SGLT1 mRNA stabilization.