RNA-binding proteins HuR and AUF1 bind to many common AU-rich target mRNAs and exert opposing influence on target mRNA stability, but the functional interactions between HuR and AUF1 have not been systematically studied. Here, using common target RNAs encoding p21 and cyclin D1, we provide evidence that HuR and AUF1 can bind target transcripts on both distinct, nonoverlapping sites, and on common sites in a competitive fashion. In the nucleus, both proteins were found together within stable ribonucleoprotein complexes; in the cytoplasm, HuR and AUF1 were found to bind to target mRNAs individually, HuR colocalizing with the translational apparatus and AUF1 with the exosome. Our results indicate that the composition and fate (stability, translation) of HuR- and/or AUF1-containing ribonucleoprotein complexes depend on the target mRNA of interest, RNA-binding protein abundance, stress condition, and subcellular compartment

In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.

We report the antiapoptotic effect of RNA‐binding protein HuR, a critical regulator of the post‐transcriptional fate of target transcripts. Among the most prominent mRNAs complexing with HuR is that encoding prothymosin α (ProTα), an inhibitor of the apoptosome. In HeLa cells, treatment with the apoptotic stimulus ultraviolet light (UVC) triggered the mobilization of ProTα mRNA to the cytoplasm and onto heavier polysomes, where its association with HuR increased dramatically. Analysis of a chimeric ProTα mRNA directly implicated HuR in regulating ProTα production: ProTα translation and cytoplasmic concentration increased in HuR‐overexpressing cells and declined in cells in which HuR levels were lowered by RNA interference. Importantly, the antiapoptotic influence engendered by HuR was vitally dependent on ProTα expression, since use of oligomers that blocked ProTα translation abrogated the protective effect of HuR. Together, our data support a regulatory scheme whereby HuR binds the ProTα mRNA, elevates its cytoplasmic abundance and translation, and thereby elicits an antiapoptotic program.

Stresses affecting the endoplasmic reticulum (ER) globally modulate gene expression patterns by altering posttranscriptional processes such as translation. Here, we use tunicamycin (Tn) to investigate ER stress-triggered changes in the translation of cytochrome c, a pivotal regulator of apoptosis. We identified two RNA-binding proteins that associate with its ∼900-bp-long, adenine- and uridine-rich 3′ untranslated region (UTR): HuR, which displayed affinity for several regions of the cytochrome c 3′UTR, and T-cell-restricted intracellular antigen 1 (TIA-1), which preferentially bound the segment proximal to the coding region. HuR did not appear to influence the cytochrome c mRNA levels but instead promoted cytochrome c translation, as HuR silencing greatly diminished the levels of nascent cytochrome c protein. By contrast, TIA-1 functioned as a translational repressor of cytochrome c, with interventions to silence TIA-1 dramatically increasing cytochrome c translation. Following treatment with Tn, HuR binding to cytochrome c mRNA decreased, and both the presence of cytochrome c mRNA within actively translating polysomes and the rate of cytochrome c translation declined. Taken together, our data suggest that the translation rate of cytochrome c is determined by the opposing influences of HuR and TIA-1 upon the cytochrome c mRNA. Under unstressed conditions, cytochrome c mRNA is actively translated, but in response to ER stress agents, both HuR and TIA-1 contribute to lowering its biosynthesis rate. We propose that HuR and TIA-1 function coordinately to maintain precise levels of cytochrome c production under unstimulated conditions and to modify cytochrome c translation when damaged cells are faced with molecular decisions to follow a prosurvival or a prodeath path.

The RNA-binding protein TIAR has been proposed to inhibit protein synthesis transiently by promoting the formation of translationally silent stress granules. Here, we report the selective binding of TIAR to several mRNAs encoding translation factors such as eukaryotic initiation factor 4A (eIF4A) and eIF4E (translation initiation factors), eEF1B (a translation elongation factor), and c-Myc (which transcriptionally controls the expression of numerous translation regulatory proteins). TIAR bound the 3-untranslated regions of these mRNAs and potently suppressed their translation, particularly in response to low levels of short-wavelength UV (UVC) irradiation. The UVC-imposed global inhibition of the cellular translation machinery was significantly relieved after silencing of TIAR expression. We propose that the TIAR-mediated inhibition of translation factor expression elicits a sustained repression of protein biosynthesis in cells responding to stress.

Nuclear import of HuR, a shuttling RNA-binding protein, is associated with reduced stability of its target mRNAs. Increased function of the AMP-activated protein kinase (AMPK), an enzyme involved in responding to metabolic stress, was recently shown to reduce the cytoplasmic levels of HuR. Here, we provide evidence that importin ␣1, an adaptor protein involved in nuclear import, contributes to the nuclear import of HuR through two AMPK-modulated mechanisms. First, AMPK triggered the acetylation of importin ␣1 on Lys 22 , a process dependent on the acetylase activity of p300. Second, AMPK phosphorylated importin ␣1 on Ser 105 . Accordingly, expression of importin ␣1 proteins bearing K22R or S105A mutations failed to mediate the nuclear import of HuR in intact cells. Our results point to importin ␣1 as a critical downstream target of AMPK and key mediator of AMPK-triggered HuR nuclear import.

The growth arrest- and DNA damage-inducible gene GADD45alpha is potently upregulated in response to stress stimuli. Here, two RNA binding proteins, the mRNA decay-promoting AUF1 and the translational suppressor TIAR, were found to interact specifically with the 3' untranslated region (UTR) of the GADD45alpha mRNA in HeLa cells. These associations were prominent in unstimulated cells, decreasing dramatically after treatment with the genotoxin methyl methanesulfonate (MMS). Analysis of both endogenous and chimeric GADD45alpha mRNA revealed that in untreated cells AUF1 strongly reduced GADD45alpha mRNA stability, whereas TIAR potently inhibited GADD45alpha translation. After genotoxic stress, AUF1 and TIAR dissociated from the GADD45alpha mRNA, thereby allowing coordinated elevations in both GADD45alpha mRNA half-life and translation rate, respectively. We propose that the posttranscriptional derepression of GADD45alpha critically contributes to its potent upregulation after DNA damage.

The stability of mRNAs undergoing translation has long been a controversial question. Here, we systematically investigate links between mRNA turnover and translation during the endoplasmic reticulum (ER) stress response, a process during which protein synthesis is potently regulated. cDNA array-based approaches to assess the stability and translational status of each mRNA were devised. First, ER stress-triggered changes in mRNA stability were studied by comparing differences in steady-state mRNA levels with differences in gene transcription. Second, changes in translational status were monitored by studying ER stress-induced shifts in the relative distribution of each mRNA along sucrose gradients. Together, the array-derived data reveal complex links between mRNA stability and translation, with all regulatory groups represented: both stabilized and destabilized mRNAs were found among translationally induced as well as translationally suppressed mRNA collections. Remarkably, however, the subset of stabilized mRNAs was prominently enriched in translationally suppressed transcripts, suggesting that ER stress was capable of causing the stabilization of mRNAs associated with a global reduction in protein synthesis. The cDNA array-based approach described here can be applied to global analyses of mRNA turnover and translation and can serve to investigate subsets of mRNAs subject to joint posttranscriptional control.