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R.Winzen and M.Kracht contributed equally to this workStabilization of mRNAs contributes to the strong and rapid induction of genes in the inflammatory response. The signaling mechanisms involved were investigated using a tetracycline-controlled expression system to determine the half-lives of interleukin (IL)-6 and IL-8 mRNAs. Transcript stability was low in untreated HeLa cells, but increased in cells expressing a constitutively active form of the MAP kinase kinase kinase MEKK1. Destabilization and signal-induced stabilization was transferred to the stable β-globin mRNA by a 161-nucleotide fragment of IL-8 mRNA which contains an AU-rich region, as well as by defined AU-rich elements (AREs) of the c-fos and GM-CSF mRNAs. Of the different MEKK1-activated signaling pathways, no significant effects on mRNA degradation were observed for the SAPK/JNK, extracellular regulated kinase and NF-κB pathways. Selective activation of the p38 MAP kinase (⍧SAPK2) pathway by MAP kinase kinase 6 induced mRNA stabilization. A dominant-negative mutant of p38 MAP kinase interfered with MEKK1 and also IL-1-induced stabilization. Furthermore, an active form of the p38 MAP kinase-activated protein kinase (MAPKAP K2 or MK2) induced mRNA stabilization, whereas a negative interfering MK2 mutant interfered with MAP kinase kinase 6-induced stabilization. These findings indicate that the p38 MAP kinase pathway contributes to cytokine/stress-induced gene expression by stabilizing mRNAs through an MK2-dependent, ARE-targeted mechanism.

SUMMARY MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3′UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.

AU-rich RNA destabilizing elements (AREs) are found in mRNAs encoding proteins with diversified functions and synthesized under a vast variety of physiological conditions (Chen and Shyu 1995), suggesting that AREs are a key player in controlling gene expression post-transcriptionally. The potent RNA-destabilizing ability of AREs coupled with transient transcription of the corresponding genes is a prerequisite for achieving a tight temporal and spatial regulation of a transient expression of mRNA (Treisman 1985;Schiavi et al. 1992;Ross 1995). Whereas AREs are found in many different labile mRNAs, they are most commonly found in the cytokine mRNAs whose half-lives change in cells undergoing a stress response, an immune response, and responding to tissue repair (Caput et al. 1986;Shaw and Kamen 1986;Greenberg and Belasco 1993;Chen and Shyu 1995). It is now clear that the regulation of cytoplasmic mRNA turnover plays a critical role in determining the duration and level of expression of many cytokines. Recently, much has been learned concerning the key sequence features of AREs that are necessary for exerting their destabilizing function (Chen and Shyu 1995;Xu et al. 1997). Increasing reports have also been made on how alterations of certain signaling transduction pathways in lymphoid or myeloid cell lines lead to changes of the stability of cytokine mRNAs via mechanisms that require AREs, for example, IL-2, IL-3, and IL-8 mRNAs (Sirenko et al. 1997;Chen et al. 1998;Ming et al. 1998

Regulation of cytoplasmic deadenylation has a direct impact on the fate of mRNA and, consequently, its expression in the cytoplasm. AU-rich elements (AREs) found in the 3 untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. AREs direct accelerated deadenylation as the first step in mRNA turnover. Recently we have proposed that AREs can be divided into three different classes. mRNAs bearing either the class I AUUUA-containing ARE or the class III non-AUUUA ARE display synchronous poly(A) shortening, whereas class II ARE-containing mRNAs are deadenylated asynchronously, with the formation of poly(A) ؊ intermediates. In this study, we have systematically characterized the deadenylation kinetics displayed by various AREs and their mutant derivatives. We find that a cluster of five or six copies of AUUUA motifs in close proximity forming various degrees of reiteration is the key feature that dictates the choice between processive versus distributive deadenylation. An AU-rich region 20 to 30 nucleotides long immediately 5 to this cluster of AUUUA motifs can greatly enhance the destabilizing ability of the AUUUA cluster and is, therefore, an integral part of the class I and class II AREs. These two features are the defining characteristics of class II AREs. Our results are consistent with the interpretation that the pentanucleotide AUUUA, rather than the nonamer UUAUUUA(U/A)(U/A), is both an essential and the minimal sequence motif of AREs. Our study provides the groundwork for future characterization of AREbinding proteins identified by in vitro gel shift assays in order to stringently define their potential role in the ARE-mediated decay pathway. Moreover, transformation of deadenylation kinetics from one type to the other by mutations of AREs implies the existence of cross talk between the ARE and 3 poly(A) tail, which dictates the decay kinetics.