Binding of the La autoantigen to the 5' untranslated region of a chimeric human translation elongation factor 1A reporter mRNA inhibits translation in vitro.

Human translation elongation factor 1A (EF1A) is a member of a large class of mRNAs, including ribosomal proteins and other translation elongation factors, which are coordinately translationally regulated under various conditions. Each of these mRNAs contains a terminal oligopyrimidine tract (TOP) that is required for translational control. A human growth hormone (hGH) expression construct containing the promoter region and 5' untranslated region (UTR) of EF1A linked to the hGH coding region (EF1A/hGH) was translationally repressed following rapamycin treatment in similar fashion to endogenous EF1A in human B lymphocytes. Mutation of two nucleotides in the TOP motif abolished the translational regulation. Gel mobility shift assays showed that both La protein from human B lymphocyte cytoplasmic extracts as well as purified recombinant La protein specifically bind to an in vitro-synthesized RNA containing the 5' UTR of EF1A mRNA. Moreover, extracts prepared from rapamycin-treated cells showed increased binding activity to the EF1A 5' UTR RNA, which correlates with TOP mRNA translational repression. In an in vitro translation system, recombinant La dramatically decreased the expression of EF1A/hGH construct mRNA, but not mRNAs lacking an intact TOP element. These results indicate that TOP mRNA translation may be modulated through La binding to the TOP element.     

Differential translation of TOP mRNAs in rapamycin-treated human B lymphocytes

TOP mRNAs (contain a 5' terminal oligopyrimidine tract) are differentially translated in rapamycin-treated human B lymphocytes. Following rapamycin treatment, ribosomal protein (rp) and translation elongation factor TOP mRNAs were translationally repressed, whereas hnRNP A1 TOP mRNA was not. Poly(A)-binding protein (Pabp1) TOP mRNA was translationally repressed under all conditions tested. To investigate the mechanism involved, chimeric mRNAs containing the hnRNP A1 5' untranslated region (UTR) linked to the human growth hormone (hGH) reporter were analyzed. Wild-type hnRNP A1 construct mRNA behaved similarly to endogenous hnRNP A1, whereas a single mutation (guanosine to cytidine) within the TOP element resulted in increased translational regulation. These results suggest that TOP mRNA translation can be modulated and that all TOP mRNAs are not translated with equal efficiency.     

Amino Acid-Induced Translation of TOP mRNAs Is Fully Dependent on Phosphatidylinositol 3-Kinase-Mediated Signaling, Is Partially Inhibited by Rapamycin, and Is Independent of S6K1 and rpS6 Phosphorylation

Vertebrate TOP mRNAs contain an oligopyrimidine tract at their 5' termini (5'TOP) and encode components of the translational machinery. Previously it has been shown that they are subject to selective translational repression upon growth arrest and that their translational behavior correlates with the activity of S6K1. We now show that the translation of TOP mRNAs is rapidly repressed by amino acid withdrawal and that this nutritional control depends strictly on the integrity of the 5'TOP motif. However, neither phosphorylation of ribosomal protein (rp) S6 nor activation of S6K1 per se is sufficient to relieve the translational repression of TOP mRNAs in amino acid-starved cells. Likewise, inhibition of S6K1 activity and rpS6 phosphorylation by overexpression of dominant-negative S6K1 mutants failed to suppress the translational activation of TOP mRNAs in amino acid-refed cells. Furthermore, TOP mRNAs were translationally regulated by amino acid sufficiency in embryonic stem cells lacking both alleles of the S6K1 gene. Inhibition of mTOR by rapamycin led to fast and complete repression of S6K1, as judged by rpS6 phosphorylation, but to only partial and delayed repression of translational activation of TOP mRNAs. In contrast, interference in the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway by chemical or genetic manipulations blocked rapidly and completely the translational activation of TOP mRNAs. It appears, therefore, that translational regulation of TOP mRNAs, at least by amino acids, (i) is fully dependent on PI3-kinase, (ii) is partially sensitive to rapamycin, and (iii) requires neither S6K1 activity nor rpS6 phosphorylation.     

Alternate exon insertion controls selective ubiquitination and degradation of different AUF1 protein isoforms

The A+U-rich element (ARE) in the 3′ non-coding region (3′ NCR) of short-lived cytokine mRNAs binds several regulatory proteins, including hnRNP D/AUF1, which comprises four isoforms of 37, 40, 42 and 45 kDa. ARE-mRNA degradation involves ubiquitin–proteasome activity, and one or more AUF1 proteins are thought to be ubiquitinated. Here we have characterized the mechanism for differential ubiquitination and degradation of the different AUF1 protein isoforms. We demonstrate in an in vitro ubiquitination system that the p37, followed by the p40 protein, are strongly ubiquitinated, whereas the p42 and p45 forms are not. Over expression in cells of enzymes that control the ubiquitin cycle were found to control p37 and p40 AUF1 protein levels through ubiquitination and proteasome activity, but not p42 and p45 forms. The p42 and p45 AUF1 proteins share a C-terminal exon 7 that is not found in the p37/p40 isoforms. Our studies show that exon 7 blocks ubiquitination and rapid degradation of AUF1 proteins, whereas its deletion permits ubiquitination to occur and promotes rapid turnover of AUF1 proteins. Thus, the stabilities of AUF1 isoforms are differentially controlled by insertion of an alternate exon that regulates ubiquitin targeting activity.     

The TSC-mTOR Pathway Mediates Translational Activation of TOP mRNAs by Insulin Largely in a Raptor- or Rictor-Independent Manner

The stimulatory effect of insulin on protein synthesis is due to its ability to activate various translation factors. We now show that insulin can increase protein synthesis capacity also by translational activation of TOP mRNAs encoding various components of the translation machinery. This translational activation involves the tuberous sclerosis complex (TSC), as the knockout of TSC1 or TSC2 rescues TOP mRNAs from translational repression in mitotically arrested cells. Similar results were obtained upon overexpression of Rheb, an immediate TSC1-TSC2 target. The role of mTOR, a downstream effector of Rheb, in translational control of TOP mRNAs has been extensively studied, albeit with conflicting results. Even though rapamycin fully blocks mTOR complex 1 (mTORC1) kinase activity, the response of TOP mRNAs to this drug varies from complete resistance to high sensitivity. Here we show that mTOR knockdown blunts the translation efficiency of TOP mRNAs in insulin-treated cells, thus unequivocally establishing a role for mTOR in this mode of regulation. However, knockout of the raptor or rictor gene has only a slight effect on the translation efficiency of these mRNAs, implying that mTOR exerts its effect on TOP mRNAs through a novel pathway with a minor, if any, contribution of the canonical mTOR complexes mTORC1 and mTORC2. This conclusion is further supported by the observation that raptor knockout renders the translation of TOP mRNAs rapamycin hypersensitive.     

Forskolin induction of late-LTP and up-regulation of 5' TOP mRNAs translation via mTOR, ERK, and PI3K in hippocampal pyramidal cells

The late phase of long-term potentiation (LTP) requires activation of the mammalian target of rapamycin (mTOR) pathway and synthesis of new proteins. mTOR regulates protein synthesis via phosphorylation of 4E-binding proteins (4E-BPs) and S6K, and via selective up-regulation of 5' terminal oligopyrimidine (5' TOP) mRNAs that encode components of the translational machinery. In this study, we explored the regulation of 5' TOP mRNAs during late-LTP (L-LTP). Synaptic plasticity was studied at Schaffer collateral – CA1 pyramidal cell synapses in rat organotypic hippocampal slices. Forskolin, an adenylate cyclase activator, induced L-LTP in organotypic slices that was mTOR-dependent. To determine if 5' TOP mRNAs are specifically up-regulated during L-LTP, we generated a 5' TOP-myr-dYFP reporter to selectively monitor 5' TOP translation. Confocal imaging experiments in cultured slices revealed an increase in somatic and dendritic fluorescence after forskolin treatment. This up-regulation was dependent on an intact TOP sequence and was mTOR, extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)-dependent. Our findings indicate that forskolin induces L-LTP in hippocampal neurons and up-regulates 5' TOP mRNAs translation via mTOR, suggesting that up-regulation of the translational machinery is a candidate mechanism for the stabilization of LTP.     

Assembly of Functional Ribonucleoprotein Complexes by AU-rich Element RNA-binding Protein 1 (AUF1) Requires Base-dependent and -independent RNA Contacts

AU-rich element RNA-binding protein 1 (AUF1) regulates the stability and/or translational efficiency of diverse mRNA targets, including many encoding products controlling the cell cycle, apoptosis, and inflammation by associating with AU-rich elements residing in their 3′-untranslated regions. Previous biochemical studies showed that optimal AUF1 binding requires 33–34 nucleotides with a strong preference for U-rich RNA despite observations that few AUF1-associated cellular mRNAs contain such extended U-rich domains. Using the smallest AUF1 isoform (p37^AUF1) as a model, we employed fluorescence anisotropy-based approaches to define thermodynamic parameters describing AUF1 ribonucleoprotein (RNP) complex formation across a panel of RNA substrates. These data demonstrated that 15 nucleotides of AU-rich sequence were sufficient to nucleate high affinity p37^AUF1 RNP complexes within a larger RNA context. In particular, p37^AUF1 binding to short AU-rich RNA targets was significantly stabilized by interactions with a 3′-purine residue and largely base-independent but non-ionic contacts 5′ of the AU-rich site. RNP stabilization by the upstream RNA domain was associated with an enhanced negative change in heat capacity consistent with conformational changes in protein and/or RNA components, and fluorescence resonance energy transfer-based assays demonstrated that these contacts were required for p37^AUF1 to remodel local RNA structure. Finally, reporter mRNAs containing minimal high affinity p37^AUF1 target sequences associated with AUF1 and were destabilized in a p37^AUF1-dependent manner in cells. These findings provide a mechanistic explanation for the diverse population of AUF1 target mRNAs but also suggest how AUF1 binding could regulate protein and/or microRNA binding events at adjacent sites.     

Transduction of growth or mitogenic signals into translational activation of TOP mRNAs is fully reliant on the phosphatidylinositol 3-kinase-mediated pathway but requires neither S6K1 nor rpS6 phosphorylation

Translation of terminal oligopyrimidine tract (TOP) mRNAs, which encode multiple components of the protein synthesis machinery, is known to be controlled by mitogenic stimuli. We now show that the ability of cells to progress through the cell cycle is not a prerequisite for this mode of regulation. TOP mRNAs can be translationally activated when PC12 or embryonic stem (ES) cells are induced to grow (increase their size) by nerve growth factor and retinoic acid, respectively, while remaining mitotically arrested. However, both growth and mitogenic signals converge via the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway and are transduced to efficiently translate TOP mRNAs. Translational activation of TOP mRNAs can be abolished by LY294002, a PI3-kinase inhibitor, or by overexpression of PTEN as well as by dominant-negative mutants of PI3-kinase or its effectors, PDK1 and protein kinase Balpha (PKBalpha). Likewise, overexpression of constitutively active PI3-kinase or PKBalpha can relieve the translational repression of TOP mRNAs in quiescent cells. Both mitogenic and growth signals lead to phosphorylation of ribosomal protein S6 (rpS6), which precedes the translational activation of TOP mRNAs. Nevertheless, neither rpS6 phosphorylation nor its kinase, S6K1, is essential for the translational response of these mRNAs. Thus, TOP mRNAs can be translationally activated by growth or mitogenic stimuli of ES cells, whose rpS6 is constitutively unphosphorylated due to the disruption of both alleles of S6K1. Similarly, complete inhibition of mammalian target of rapamycin (mTOR) and its effector S6K by rapamycin in various cell lines has only a mild repressive effect on the translation of TOP mRNAs. It therefore appears that translation of TOP mRNAs is primarily regulated by growth and mitogenic cues through the PI3-kinase pathway, with a minor role, if any, for the mTOR pathway.     

Translational discrimination of ribosomal protein mRNAs in the early Drosophila embryo

Most Drosophila mRNAs are actively translated in the early embryo, with the exception of the poorly translated ribosomal protein (r-protein) mRNAs. Two possible mechanisms for this translational discrimination were tested: (1) Translation of r-protein mRNAs is discriminated against by the limited activity of translational initiation factors in the early embryo and (2) translation of r-protein mRNAs is repressed by trans-acting factors that reversibly bind these mRNAs. Exogenously provided initiation factors promoted partial recruitment of r-protein mRNAs into polysomes, suggesting that modulation of initiation factor activity may play a role in the translational discrimination of r-protein mRNAs during embryogenesis. No evidence for involvement of reversibly binding trans-acting factors was obtained, although there are limitations in the interpretation of the latter experiments.     

Identification and characterization of proteins that selectively interact with isoforms of the mRNA binding protein AUF1 (hnRNP D)

The mRNAs that encode certain cytokines and proto-oncogenes frequently contain a typical AU-rich motif that is located in their 3'-untranslated region. The protein AUF1 is the first factor identified that binds to AU-rich regions and mediates the fast degradation of the target mRNAs. AUF1 exists as four different isoforms (p37, p40, p42 and p45) that are generated by alternative splicing. The fact that AUF1 does not degrade mRNA itself had led to the suggestion that other AUF1 interacting proteins might be involved in the process of selective mRNA degradation. Here we used the yeast two-hybrid system in order to identify proteins that bind to AUF1. We detected AUF1 itself, as well as the ubiquitin-conjugating enzyme E2I and three RNA binding proteins: NSEP-1, NSAP-1 and IMP-2, as AUF1 interacting proteins. We confirmed all interactions in vitro and mapped the protein domains that are involved in the interaction with AUF1. Gel-shift assays with the recombinant purified proteins suggest that the interacting proteins and AUF1 can bind simultaneously to an AU-rich RNA oligonucleotide. Most interestingly, the AUF1 interacting protein NSEP-1 showed an endoribonuclease activity in vitro. These data suggest the possibility that the identified AUF1 interacting proteins might be involved in the regulation of mRNA stability mediated by AUF1.     

The mTOR signaling pathway mediates control of ribosomal protein mRNA translation in rat liver

Previous studies have shown that oral administration of leucine to fasted rats results in a preferential increase in liver in the translation of mRNAs containing an oligopyrimidine sequence at the 5'-end of the message (i.e. a TOP sequence). TOP mRNAs include those encoding the ribosomal proteins (rp) and translation elongation factors. In cells in culture, the preponderance of evidence suggests that translation of TOP mRNAs is regulated by the mammalian target of rapamycin (mTOR), a protein kinase that signals through ribosomal protein S6 kinase (S6K1) to rpS6. However, the results of previous studies were recently challenged by several reports suggesting that translation of TOP mRNAs is independent of mTOR, S6K1, and S6 phosphorylation. The purpose of the present study was to evaluate the role of mTOR in the stimulation of TOP mRNA translation by leucine in vivo. Fasted rats were treated with the mTOR inhibitor, rapamycin, prior to oral administration of leucine. It was found that rapamycin severely attenuated leucine-induced signaling through mTOR in liver. In addition, rapamycin prevented the enhanced translation of TOP mRNAs in rats administered leucine, as assessed by a decrease in the proportion of TOP mRNAs associated with polysomes (i.e. those mRNAs being actively translated). Instead, in rapamycin-treated rats, ribosomal protein mRNAs accumulated in the fraction containing monosomes (mRNA bound to one ribosome). The results suggest that in liver in vivo, mTOR-dependent signaling is critical for maximal stimulation of TOP mRNA translation.     

Translational regulation of terminal oligopyrimidine mRNAs induced by serum and amino acids involves distinct signaling events

Various mitogenic or growth inhibitory stimuli induce a rapid change in the association of terminal oligopyrimidine (TOP) mRNAs with polysomes. It is generally believed that such translational control hinges on the mammalian target of rapamycin (mTOR)-S6 kinase pathway. Amino acid availability affects the translation of TOP mRNAs, although the signaling pathway involved in this regulation is less well characterized. To investigate both serum- and amino acid-dependent control of TOP mRNA translation and the signaling pathways involved, HeLa cells were subjected to serum and/or amino acid deprivation and stimulation. Our results indicate the following. 1). Serum and amino acid deprivation had additive effects on TOP mRNA translation. 2). The serum content of the medium specifically affected TOP mRNA translation, whereas amino acid availability affected both TOP and non-TOP mRNAs. 3). Serum signaling to TOP mRNAs involved only a rapamycin-sensitive pathway, whereas amino acid signaling depended on both rapamycin-sensitive and rapamycin-insensitive but wortmannin-sensitive events. 4). Eukaryotic initiation factor-2alpha phosphorylation increased during amino acid deprivation, but not following serum deprivation. Interestingly, rapamycin treatment suggests a novel connection between the mTOR pathway and eukaryotic initiation factor-2alpha phosphorylation in mammalian cells, which may not, however, be involved in TOP mRNA translational regulation.     

Selective degradation of AU-rich mRNAs promoted by the p37 AUF1 protein isoform

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 (ARE-mRNAs) 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-mRNA-destabilizing 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 ARE-mRNAs 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.