The degradation of some proto-oncogene and lymphokine mRNAs is controlled in part by an AU-rich element (ARE) in the 3' untranslated region. It was shown previously (G. Brewer, Mol. Cell. Biol. 11:2460-2466) that two polypeptides (37 and 40 kDa) copurified with fractions of a 130,000 x g postribosomal supernatant (S130) from K562 cells that selectively accelerated degradation of c-myc mRNA in a cell-free decay system. These polypeptides bound specifically to the c-myc and granulocyte-macrophage colony-stimulating factor 3' UTRs, suggesting they are in part responsible for selective mRNA degradation. In the present work, we have purified the RNA-binding component of this mRNA degradation activity, which we refer to as AUFl. Using antisera specific for these polypeptides, we demonstrate that the 37-and 40-kDa polypeptides are immunologically cross-reactive and that both polypeptides are phosphorylated and can be found in a complex(s) with other polypeptides. Immunologically related polypeptides are found in both the nucleus and the cytoplasm. The antibodies were also used to clone a cDNA for the 37-kDa polypeptide. This cDNA contains an open reading frame predicted to produce a protein with several features, including two RNA recognition motifs and domains that potentially mediate protein-protein interactions. These results provide further support for a role of this protein in mediating ARE-directed mRNA degradation.The c-myc gene is important for the control of cellular growth, differentiation, and transformation (reviewed in references 17 and 41). It belongs to the class of immediateearly genes whose expression is required to drive cells from Go to G1 following stimulation of quiescent cells by growth factors. However, the c-myc gene is not unique in terms of having an essential role in cellular growth processes. It has been known for decades that specific and timely changes in the expression of multiple genes are required for proper embryonic development and cell maturation (20). Expression of genes such as c-myc seems to be regulated not only at the levels of transcription, attenuation, nuclear processing, and translation but also at the level of mRNA turnover (reviewed in reference 41). Indeed, direct half-life measurements indicated that c-myc mRNA has a half-life of 15 to 40 min (19). These and other studies (41) demonstrated that the control of c-myc mRNA turnover might be an important means of regulating both the level and the timing of c-myc expression.Many proto-oncogene mRNAs are very unstable. The rapid turnover of c-myc mRNA is controlled by sequences in the 3' untranslated region (3'UTR) or by coding region sequences (reviewed in references 33 and 60). A common feature of many labile mRNAs, such as those for c-myc, c-fos, and granulocyte-macrophage colony-stimulating factor (GM-CSF), is the presence of an AU-rich element (ARE) in the 3'UTR which is one cis-acting element responsible for their rapid degradation (reviewed in reference 3, 48, and 60). It AUUUA (70). This might be important because...

Long-lasting synaptic plasticity and memory requires mRNA translation, yet little is known as to how this process is regulated. To explore the role that the translation repressor 4E-BP2 plays in hippocampal long-term potentiation (LTP) and learning and memory, we examined 4E-BP2 knock-out mice. Interestingly, genetic elimination of 4E-BP2 converted early-phase LTP to late-phase LTP (L-LTP) in the Schaffer collateral pathway, likely as a result of increased eIF4F complex formation and translation initiation. A critical limit for activityinduced translation was revealed in the 4E-BP2 knock-out mice because L-LTP elicited by traditional stimulation paradigms was obstructed. Moreover, the 4E-BP2 knock-out mice also exhibited impaired spatial learning and memory and conditioned fear-associative memory deficits. These results suggest a crucial role for proper regulation of the eIF4F complex by 4E-BP2 during LTP and learning and memory in the mouse hippocampus.

All nuclear-encoded mRNAs contain a 5' cap structure (m7GpppN, where N is any nucleotide), which is recognized by the eukaryotic translation initiation factor 4E (eIF4E) subunit of the eIF4F complex. The eIF4E-binding proteins constitute a family of three polypeptides that reversibly repress cap-dependent translation by binding to eIF4E, thus preventing the formation of the eIF4F complex. We investigated the biological function of 4E-BP1 by disrupting its gene (Eif4ebp1) in the mouse. Eif4ebp1-/- mice manifest markedly smaller white fat pads than wild-type animals, and knockout males display an increase in metabolic rate. The males' white adipose tissue contains cells that exhibit the distinctive multilocular appearance of brown adipocytes, and expresses the uncoupling protein 1 (UCP1), a specific marker of brown fat. Consistent with these observations, translation of the peroxisome proliferator-activated receptor-gamma co-activator 1 (PGC1), a transcriptional co-activator implicated in mitochondrial biogenesis and adaptive thermogenesis, is increased in white adipose tissue of Eif4ebp1-/- mice. These findings demonstrate that 4E-BP1 is a novel regulator of adipogenesis and metabolism in mammals.

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The eukaryotic mRNA 3' poly(A) tail acts synergistically with the 5' cap structure to enhance translation. This effect is mediated by a bridging complex, composed of the poly(A) binding protein (PABP), eIF4G, and the cap binding protein, eIF4E. PABP-interacting protein 1 (Paip1) is another factor that interacts with PABP to coactivate translation. Here, we describe a novel human PABP-interacting protein (Paip2), which acts as a repressor of translation both in vitro and in vivo. Paip2 preferentially inhibits translation of a poly(A)-containing mRNA, but has no effect on the translation of hepatitis C virus mRNA, which is cap- and eIF4G-independent. Paip2 decreases the affinity of PABP for polyadenylate RNA, and disrupts the repeating structure of poly(A) ribonucleoprotein. Furthermore, Paip2 competes with Paip1 for PABP binding. Thus, Paip2 inhibits translation by interdicting PABP function.

Rapid degradation of many labile mRNAs is regulated in part by an A؉U-rich element (ARE) in their 3-untranslated regions. Extensive mutational analyses of various AREs have identified important components of the ARE, such as the nonamer motif UUAUUUAUU, two copies of which serve as a potent mRNA destabilizer. To investigate the roles of trans-acting factors in ARE-directed mRNA degradation, we previously purified and molecularly cloned the RNA-binding protein AUF1 and demonstrated that both cellular and recombinant AUF1 bind specifically to AREs as shown by UV cross-linking assays in vitro. In the present work, we have examined the in vitro RNA-binding properties of AUF1 using gel mobility shift assays with purified recombinant His 6 -AUF1 fusion protein. We find that ARE binding affinities of AUF1 correlate with the potency of an ARE to direct degradation of a heterologous mRNA. These results support a role for AUF1 in ARE-directed mRNA decay that is based upon its affinity for different AREs.Control of mRNA stability is an important component of eukaryotic gene expression and involves cis-acting elements that can be found in the coding region and/or UTRs 1 of mRNAs (reviewed in Refs. 1-6). One type of cis-acting instability element is comprised of the AREs found in the 3Ј-UTRs of many unstable mRNAs (reviewed in Ref. 7). Many ARE-containing mRNAs are degraded by a sequential pathway involving removal of the poly(A) tract followed by degradation of the mRNA body (8 -10). In most cases the poly(A) tract is thought to protect the mRNA from ribonuclease attack so that its removal permits degradation of the mRNA body (reviewed in Ref. 11). While it has been known for almost a decade that AREs are important for mRNA instability (12-15), the mechanism(s) by which they mediate mRNA turnover is still unknown.Despite the presence of AREs in many different mRNAs, there is no single evolutionarily conserved AϩU-rich instability sequence. Typically AREs contain multiple copies of the pentanucleotide AUUUA, often in conjunction with one or more U-rich regions (14). In addition, transfection studies indicate that as the number of tandemly repeated AUUU motifs is increased in a reporter mRNA, its instability increases. Likewise, two copies of the nonameric motif UUAUUUAUU act as a more potent destabilizer than a single nonameric motif (16,17). Together, these analyses suggest that potent destabilizing AREs are high affinity binding sites for a mRNA decay factor(s).In order to investigate how AREs function in mRNA turnover, we utilized a cell-free mRNA decay system to identify proteins that may be relevant to ARE-directed mRNA decay (8,18,19). To this end, we previously reported the purification, molecular cloning, and characterization of the ARE-binding protein AUF1 (20). Cellular AUF1 purified from cytoplasmic extracts of K562 human erythroid leukemia cells consists of a 37-and a 40-kDa isoform. Cloning of the 37-kDa isoform, p37 AUF1 , revealed two nonidentical RNA recognition motifs (21) and a short glutamine-rich region i...

mRNA turnover is an important regulatory component of gene expression and is significantly influenced by ribonucleoprotein (RNP) complexes which form on the mRNA. Studies of human ␣-globin mRNA stability have identified a specific RNP complex (␣-complex) which forms on the 3 untranslated region (3UTR) of the mRNA and appears to regulate the erythrocyte-specific accumulation of ␣-globin mRNA. One of the protein activities in this multiprotein complex is a poly(C)-binding activity which consists of two proteins, ␣CP1 and ␣CP2. Neither of these proteins, individually or as a pair, can bind the ␣-globin 3UTR unless they are complexed with the remaining non-poly(C) binding proteins of the ␣-complex. With the yeast two-hybrid screen, a second ␣-complex protein was identified. This protein is a member of the previously identified A؉U-rich (ARE) binding/degradation factor (AUF1) family of proteins, which are also known as the heterogeneous nuclear RNP (hnRNP) D proteins. We refer to these proteins as AUF1/hnRNP-D. Thus, a protein implicated in ARE-mediated mRNA decay is also an integral component of the mRNA stabilizing ␣-complex. The interaction of AUF1/hnRNP-D is more efficient with ␣CP1 relative to ␣CP2 both in vitro and in vivo, suggesting that the ␣-complex might be dynamic rather than a fixed complex. AUF1/hnRNP-D could, therefore, be a general mRNA turnover factor involved in both stabilization and decay of mRNA.Regulation of mRNA stability is an important control mechanism that dictates gene expression. The spectrum of mRNA decay rates can range from several minutes, in the case of some proto-oncogene and cytokine mRNAs, to days, as in the case of the globin mRNAs. Irregularities in mRNA stability could have profound consequences that can be manifested in different clinical phenotypes.The turnover rate of a given mRNA is determined by both cis elements and trans factors (47). Most RNA polymerase II transcripts contain an m 7 G cap structure at their 5Ј termini and a poly(A) tract at their 3Ј termini. Both of these structures contribute to the stability and translatability of an mRNA (29,50). The unique 5Ј-5Ј phosphodiester bond of the m 7

In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors ( beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreased (approximately 40%) beta2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to beta1-AR 3'-untranslated region mRNA.