AU-rich elements (AREs) present in the 3′ untranslated regions of many protooncogene, cytokine, and lymphokine messages target them for rapid degradation. HuR, a ubiquitously expressed member of the ELAV (embryonic lethal abnormal vision) family of RNA binding proteins, selectively binds AREs and stabilizes ARE-containing mRNAs in transiently transfected cells. Here, we identify four mammalian proteins that bind regions of HuR known to be essential for its ability to shuttle between the nucleus and the cytoplasm and to stabilize mRNA: SETα, SETβ, pp32, and acidic protein rich in leucine (APRIL). Three have been reported to be protein phosphatase 2A inhibitors. All four ligands contain long, acidic COOH-terminal tails, while pp32 and APRIL share a second motif: rev-like leucine-rich repeats in their NH2-terminal regions. We show that pp32 and APRIL are nucleocytoplasmic shuttling proteins that interact with the nuclear export factor CRM1 (chromosomal region maintenance protein 1). The inhibition of CRM1 by leptomycin B leads to the nuclear retention of pp32 and APRIL, their increased association with HuR, and an increase in HuR's association with nuclear poly(A)+ RNA. Furthermore, transcripts from the ARE-containing c-fos gene are selectively retained in the nucleus, while the cytoplasmic distribution of total poly(A)+ RNA is not altered. These data provide evidence that interaction of its ligands with HuR modulate HuR's ability to bind its target mRNAs in vivo and suggest that CRM1 is instrumental in the export of at least some cellular mRNAs under certain conditions. We discuss the possible role of these ligands upstream of HuR in pathways that govern the stability of ARE-containing mRNAs.

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2␣ phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2␣ phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2␣ to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2␣ phosphorylation-dependent and -independent pathways that target translation initiation.

AU-rich elements (AREs) located in the 3′ untranslated region target the mRNAs encoding many protooncoproteins, cytokines, and lymphokines for rapid degradation. HuR, a ubiquitously expressed member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, binds ARE sequences and selectively stabilizes ARE-containing reporter mRNAs when overexpressed in transiently transfected cells. HuR appears predominantly nucleoplasmic but has been shown to shuttle between the nucleus and cytoplasm via a novel shuttling sequence HNS. We report generation of a mouse monoclonal antibody 3A2 that both immunoblots and immunoprecipitates HuR protein; it recognizes an epitope located in the first of HuR's three RNA recognition motifs. This antibody was used to probe HuR interactions with mRNA before and after heat shock, a condition that has been reported to stabilize ARE-containing mRNAs. At 37°C, approximately one-third of the cytoplasmic HuR appears polysome associated, and in vivo UV crosslinking reveals that HuR interactions with poly(A) + RNA are predominantly cytoplasmic rather than nuclear. This comprises evidence that HuR directly interacts with mRNA in vivo . After heat shock, 12–15% of HuR accumulates in discrete foci in the cytoplasm, but surprisingly the majority of HuR crosslinks instead to nuclear poly(A) + RNA, whose levels are dramatically increased in the stressed cells. This behavior of HuR differs from that of another ARE-binding protein, hnRNP D, which has been implicated as an effector of mRNA decay rather than mRNA stabilization and of the general pre-RNA-binding protein hnRNP A1. We interpret these differences to mean that the temporal association of HuR with ARE-containing mRNAs is different from that of these other two proteins.

A potential p120 GTPase-activating protein (RasGAP) effector, G3BP (RasGAP Src homology 3 [SH3] binding protein), was previously identified based on its ability to bind the SH3 domain of RasGAP. Here we show that G3BP colocalizes and physically interacts with RasGAP at the plasma membrane of serumstimulated but not quiescent Chinese hamster lung fibroblasts. In quiescent cells, G3BP was hyperphosphorylated on serine residues, and this modification was essential for its activity. Indeed, G3BP harbors a phosphorylation-dependent RNase activity which specifically cleaves the 3-untranslated region of human c-myc mRNA. The endoribonuclease activity of G3BP can initiate mRNA degradation and therefore represents a link between a RasGAP-mediated signaling pathway and RNA turnover.The Ras protein belongs to a family of low-molecular-weight GTPases which are essential components of multiple receptormediated signal transduction pathways controlling cell proliferation, differentiation, and cytoskeletal organization (23). Activated Ras is bound to GTP, while the GDP-bound form of Ras is inactive (27). Extracellular stimuli induce the exchange of GDP for GTP on Ras through a series of protein-protein interactions involving activated receptors, adaptor proteins (such as Grb2 or Shc), and Ras guanine nucleotide exchange factors (5,9,33,38). Mutations in the Ras gene which lock Ras in the GTP-bound form lead to cell growth in the absence of mitogenic signals and are associated with an oncogenic phenotype (17). Physiological inactivation of Ras involves interaction with GTPase-activating proteins (GAPs) (40), such as p120 (RasGAP) (41,43) or the product of the NF1 gene (neurofibromin) (26,44), which accelerate the hydrolysis of Ras-associated GTP, thereby converting Ras from an active to an inactive form. Disruption of either the RasGAP or the NF1 gene in mice results in an embryonic lethal phenotype (3, 14), indicating that Ras inactivation is a key process in normal cell signaling and development.In addition to being a negative regulator of Ras, RasGAP may also represent a downstream target of Ras (35). RasGAP is a widely expressed modular protein which comprises several structural features that likely enable it to function in the transduction cascade (29). While the carboxyl-terminal domain of RasGAP constitutes a catalytic domain (25), the N-terminal region is believed to mediate interactions with other signaling proteins (20). The N-terminal region is characterized by a Src homology 3 (SH3) domain flanked by two SH2 domains, as well as pleckstrin homology (PH) and calcium-dependent lipid binding domains (4, 34). Upon activation of many growth factor receptors, RasGAP becomes phosphorylated and associates with cytosolic proteins as well as with the autophosphorylated tyrosine kinase receptors (19). RasGAP has been shown to form a complex with G3BP (RasGAP SH3 binding protein) in a Ras-GTP-dependent manner (32). G3BP is composed of 466 amino acid and has a predicted molecular mass of 52 kDa; the carboxyl-terminal region contai...

Mitogen activation of mRNA decay pathways likely involves specific endoribonucleases, such as G3BP, a phosphorylation-dependent endoribonuclease that associates with RasGAP in dividing but not quiescent cells. G3BP exclusively cleaves between cytosine and adenine (CA) after a specific interaction with RNA through the carboxyl-terminal RRM-type RNA binding motif. Accordingly, G3BP is tightly associated with a subset of poly(A)؉ mRNAs containing its high-affinity binding sequence, such as the c-myc mRNA in mouse embryonic fibroblasts. Interestingly, c-myc mRNA decay is delayed in RasGAP-deficient fibroblasts, which contain a defective isoform of G3BP that is not phosphorylated at serine 149. A G3BP mutant in which this serine is changed to alanine remains exclusively cytoplasmic, whereas a glutamate for serine substitution that mimics the charge of a phosphorylated serine is translocated to the nucleus. Thus, a growth factor-induced change in mRNA decay may be modulated by the nuclear localization of a site-specific endoribonuclease such as G3BP.

The inhibition of the ubiquitin-dependent proteasome system (UPS) via specific drugs is one type of approach used to combat cancer. Although it has been suggested that UPS inhibition prevents the rapid decay of AU-rich element (ARE)-containing messages, very little is known about the cellular mechanisms leading to this effect. Here we establish a link between the inhibition of UPS activity, the formation of cytoplasmic stress granules (SGs), and mRNA metabolism. The assembly of the SGs requires the phosphorylation of the translation initiation factor eIF2alpha by a mechanism involving the stress kinase GCN2. On prolonged UPS inhibition and despite the maintenance of eIF2alpha phosphorylation, SGs disassemble and translation recovers in an Hsp72 protein-dependent manner. The formation of these SGs coincides with the disassembly of processing bodies (PBs), known as mRNA decay entities. As soon as the SGs assemble, they recruit ARE-containing messages such as p21(cip1) mRNA, which are stabilized under these conditions. Hence, our findings suggest that SGs could be considered as one of the players that mediate the early response of the cell to proteasome inhibitors by interfering temporarily with mRNA decay pathways.

The transport of messenger RNAs (mRNAs) from the nucleus to the cytoplasm involves adapter proteins that bind the mRNA as well as receptor proteins that interact with the nuclear pore complex. We demonstrate the utility of cell-permeable peptides designed to interfere with interactions between potential adapter and receptor proteins to define the pathways accessed by particular mRNAs. We show that HuR, a protein implicated in the stabilization of short-lived mRNAs containing AU-rich elements (AREs), serves as an adapter for c-fos mRNA export through two pathways. One involves the HuR shuttling domain, HNS, which exhibits a heat shock-sensitive interaction with transportin 2 (Trn2); the other involves two protein ligands of HuR-pp32 and APRIL-which contain leucine-rich nuclear export signals (NES) recognized by the export receptor CRM1. Heterokaryon and in situ hybridization experiments reveal that the peptides selectively block the nucleocytoplasmic shuttling of their respective adapter proteins without perturbing the overall cellular distribution of polyadenylated mRNAs.

The formation of muscle fibers involves the sequential expression of many proteins that regulate key steps during myoblast-to-myotube transition. MyoD, myogenin, and the cyclin-dependent kinase inhibitor p21 cip1 are major players in the initiation and maintenance of the differentiated state of mouse embryonic muscle cells (C2C12). The messenger RNAs encoding these three proteins contain typical AU-rich elements (AREs) in their 3-untranslated regions (3-UTRs), which are known to affect the half-life of many short-lived mRNAs. HuR, an RNA-binding protein that regulates both the stability and cellular movement of ARE-containing mRNAs, interacts and stabilizes the p21 cip1 message under UV stress in human RKO colorectal carcinoma cells. Here, by the use of gel shift experiments and immunoprecipitation followed by reverse transcription-PCR analysis, we show that HuR interacts with MyoD, myogenin, and p21 cip1 mRNAs through specific sequences in their 3-UTRs. To demonstrate the implication of endogenous HuR in myogenesis, we knocked down its expression in myoblasts using RNA interference and observed a significant reduction of HuR expression, associated with complete inhibition of myogenesis. Moreover, the expression of MyoD and myogenin mRNAs, as well as proteins, is significantly reduced in the HuR knockdown C2C12 cells. We were able to completely re-establish the myogenic process of these defective cells by introducing back HuR protein conjugated to a cell-permeable peptide. Finally, HuR accumulates in the cytoplasm during myogenesis. Thus, our results clearly demonstrated that endogenous HuR plays a crucial role in muscle differentiation by regulating the expression and/or the nuclear export of ARE-containing mRNAs that are essential for this process.