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An AU-rich element (ARE) consisting of repeated canonical AUUUA motifs confers rapid degradation to many cytokine mRNAs when present in the 3 untranslated region. Destabilization of mRNAs with AREs (AREmRNAs) is consistent with the interaction of ARE-binding proteins such as tristetraprolin and the four AUF1 isoforms. However, the association of the AUF1-mRNA interaction with decreased ARE-mRNA stability is correlative and has not been directly tested. We therefore determined whether overexpression of AUF1 isoforms promotes ARE-mRNA destabilization and whether AUF1 isoforms are limiting components for ARE-mRNA decay. We show that the p37 AUF1 isoform and, to a lesser extent, the p40 isoform possess ARE-mRNAdestabilizing activity when overexpressed. Surprisingly, overexpressed p37 AUF1 also destabilized reporter mRNAs containing a noncanonical but AU-rich 3 untranslated region. Since overexpressed p37 AUF1 could interact in vivo with the AU-rich reporter mRNA, AUF1 may be involved in rapid turnover of mRNAs that lack canonical AREs. Moreover, overexpression of p37 AUF1 restored the ability of cells to rapidly degrade AREmRNAs when that ability was saturated and inhibited by overexpression of ARE-mRNAs. Finally, activation of ARE-mRNA decay often involves a translation-dependent step, which was eliminated by overexpression of p37 AUF1. These data indicate that the p37 AUF1 isoform and, to some extent, the p40 isoform are limiting factors that facilitate rapid decay of AU-rich mRNAs.Most cytokine and many proto-oncogene mRNAs display short half-lives, which are conferred by an AU-rich element (ARE) in the 3Ј untranslated region (UTR) that functions as a cytoplasmic destabilizing motif (reviewed in reference 20). As many as 8% of other human mRNA 3Ј UTRs also contain AREs (2). The granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE is prototypical; it consists of eight AUUUA pentamers, five of which are contiguous, which are sufficient to confer a short half-life to reporter mRNAs (40).The mechanism for ARE-mediated mRNA decay is poorly understood. The ARE promotes mRNA deadenylation in vitro (16) and in vivo (42) and may stimulate mRNA-decapping activity (19). A number of proteins can bind various ARE sequences in vitro, including hnRNP proteins A1, A2, C, and L (21-23), but a role in controlling mRNA stability is unlikely (36). To date, three ARE-binding proteins have been shown to be involved in regulating rapid mRNA decay in vivo: HuR (33); tristetraprolin (TTP) (10); and the ARE-and poly(U)-binding and degradation factor, AUF1 (5), also known as hnRNP D (48). HuR stabilizes ARE-containing reporter mRNAs when ectopically overexpressed (15, 35), and antisense RNA knockdown of endogenous HuR expression increases the half-lives of certain ARE-containing mRNAs (ARE-mRNAs) (31,38,49). TTP is important for the destabilization of tumor necrosis factor and GM-CSF mRNAs, as shown in knockout mice (10, 45) and in tissue culture by ectopic-overexpression studies (27).AUF1 consists of four isoforms of 37, 40, 42, ...

The heterogeneous nuclear ribonucleoprotein D family of proteins also known as AUF1 consists of four isoforms implicated in both nuclear and cytoplasmic functions. The AUF1 proteins are largely nuclear but also are found in the cytoplasm and are thought to undergo nucleocytoplasmic shuttling. The nucleocytoplasmic distribution and potential shuttling activity of the individual AUF1 isoforms have not been previously studied in detail. Therefore, we characterized the nucleocytoplasmic transport of each of the heterogeneous nuclear ribonucleoprotein D/AUF1 isoforms. All four AUF1 proteins were found to undergo rapid nucleocytoplasmic shuttling in a manner that is transcription-independent, carrier-mediated, and energy-requiring. Nucleocytoplasmic shuttling of the AUF1 proteins is shown to utilize a novel arrangement of nuclear import and export signals. Mutagenesis of the AUF1 proteins and fusion of polypeptides to a green fluorescent protein reporter demonstrated that a nuclear import signal is located in the C-terminal domain of the protein and is found only in the two smaller isoforms. Further mapping demonstrated that nuclear export is facilitated by sequences in AUF1 exon 7 found in the C-terminal domain of the two larger AUF1 isoforms. A subset of AUF1 proteins are shown to directly interact in vitro using purified recombinant proteins and in vivo in the absence of RNA. These results suggest that nuclear import of AUF1 is facilitated by sequences found only in the two smaller isoforms and that nuclear export is facilitated by sequences (exon 7 and the C-terminal domain) found only in the two larger isoforms. This novel arrangement of signals might represent a mechanism to assure co-shuttling of a subset of AUF1 proteins that interact in a heterocomplex.Transport of proteins across the nuclear pore complex involves the recognition of specific signal sequences by transport receptors and adapter proteins (reviewed in Ref. 1). Import receptors (importins) and export receptors (exportins) shuttle back and forth across the nuclear pore complex with cargo proteins and other macromolecules. The export receptor exportin 1/CRM1 1 interacts directly with a leucine-rich nuclear export sequence in the cargo protein or with adapter molecules (1). The best characterized nuclear import receptors are members of the importin-␤ superfamily of RanGTP-binding proteins. Facilitated nuclear import involves nuclear localization sequences (NLSs) within cargo proteins containing any of several canonical basic amino acid motifs (2). Some cargo proteins utilize non-canonical NLSs for transport such as the M9 domain, which consists of a glycine-rich amino acid sequence that promotes both protein import and export, and therefore functions as a shuttling sequence (3-5). The M9 domain was originally identified in heterogeneous nuclear ribonucleoproteins (hnRNP) A1 and binds to the import receptor, transportin (5).Apart from hnRNPs C1/C2 and U, all of the other hnRNPs appear to undergo nucleocytoplasmic shuttling (1, 2). hnRNP A1 represents ...

The cationic amino acid transporter, Cat-1, is a high affinity transporter of the essential amino acids, arginine and lysine. Expression of the cat-1 gene increases during nutritional stress as part of the adaptive response to starvation. Amino acid limitation induces coordinate increases in stability and translation of the cat-1 mRNA, at a time when global protein synthesis decreases. It is shown here that increased cat-1 mRNA stability requires an 11 nucleotide AU-rich element within the distal 217 bases of the 3-untranslated region. When this 217-nucleotide nutrient sensor AU-rich element (NS-ARE) is present in a chimeric mRNA it confers mRNA stabilization during amino acid starvation. HuR is a member of the ELAV family of RNA-binding proteins that has been implicated in regulating the stability of ARE-containing mRNAs. We show here that the cytoplasmic concentration of HuR increases during amino acid starvation, at a time when total cellular HuR levels decrease. In addition, RNA gel shift experiments in vitro demonstrated that HuR binds to the NS-ARE and binding was dependent on the 11 residue AU-rich element. Moreover, HuR binding to the NS-ARE in extracts from amino acid-starved cells increased in parallel with the accumulation of cytoplasmic HuR. It is proposed that an adaptive response of cells to nutritional stress results in increased mRNA stability mediated by HuR binding to the NS-ARE.

An AU rich element (ARE) in the 3 noncoding region promotes the rapid degradation of mammalian cytokine and proto-oncogene mRNAs, such as tumor necrosis factor-␣, granulocyte-macrophage colony-stimulating factor (GM-CSF) and c-fos. Destabilization of ARE-mRNAs involves the association of ARE-binding proteins tristetraprolin or AUF1 and proteasome activity, of which the latter has not been characterized. Here, we show that the stability of a model short-lived mRNA containing the GM-CSF ARE was regulated by the level of ubiquitin-conjugating activity in the cell, which links ARE-mRNA decay to proteasome activity. Increased expression of a cytokine-inducible deubiquitinating protein (DUB) that impairs addition of ubiquitin to proteins fully blocked ARE-mRNA decay, whereas increased expression of a DUB that promotes ubiquitin addition to proteins strongly accelerated ARE-mRNA decay. AREmRNA turnover was found to be activated by the ubiquitinaddition reaction and blocked by the ubiquitin-removal reaction. Saturation of the ARE-mRNA decay machinery by high levels of ARE-mRNA, which is well established but not understood, was found to be relieved by increased expression of a DUB that promotes ubiquitin addition to proteins. Finally, inhibition of proteasome activity also blocked accelerated ARE-mRNA decay that is mediated by increased ubiquitin recycling. These results demonstrate that both ubiquitinating activity and proteasome activity are essential for rapid turnover of a model cytokine ARE-mRNA containing the GM-CSF ARE.