Level Of Translation Initiation Research Articles

Tuning Riboswitch Regulation through Conformational Selection

The SMK box riboswitch, which represents one of three known classes of S-adenosylmethionine (SAM)-responsive riboswitches, regulates gene expression in bacteria at the level of translation initiation. In contrast to most riboswitches, which contain separate domains responsible for ligand recognition and gene regulation, the ligand-binding and regulatory domains of the SMK box riboswitch are coincident. This property was exploited to allow the first atomic-level characterization of a functionally intact riboswitch in both the ligand-bound state and the ligand-free state. NMR spectroscopy revealed distinct mutually exclusive RNA conformations that are differentially populated in the presence or in the absence of the effector metabolite. Isothermal titration calorimetry and in vivo reporter assay results revealed the thermodynamic and functional consequences of this conformational equilibrium. We present a comprehensive model of the structural, thermodynamic, and functional properties of this compact RNA regulatory element.

The SAM‐responsive SMK box is a reversible riboswitch

The S(MK) (SAM-III) box is an S-adenosylmethionine (SAM)-responsive riboswitch found in the 5' untranslated region of metK genes, encoding SAM synthetase, in many members of the Lactobacillales. SAM binding causes a structural rearrangement in the RNA that sequesters the Shine-Dalgarno (SD) sequence by pairing with a complementary anti-SD (ASD) sequence; sequestration of the SD sequence inhibits binding of the 30S ribosomal subunit and prevents translation initiation. We observed a slight increase in the half-life of the metK transcript in vivo when Enterococcus faecalis cells were depleted for SAM, but no significant change in overall transcript abundance, consistent with the model that this riboswitch regulates at the level of translation initiation. The half-life of the SAM-S(MK) box RNA complex in vitro is shorter than that of the metK transcript in vivo, raising the possibility of reversible binding of SAM. We used a fluorescence assay to directly visualize reversible switching between the SAM-free and SAM-bound conformations. We propose that the S(MK) box riboswitch can make multiple SAM-dependent regulatory decisions during the lifetime of the transcript in vivo, acting as a reversible switch that allows the cell to respond rapidly to fluctuations in SAM pools by modulating expression of the SAM synthetase gene.

Transcription regulation of the Escherichia coli pcnB gene coding for poly(A) polymerase I: roles of ppGpp, DksA and sigma factors

Poly(A) polymerase I (PAP I), encoded by the pcnB gene, is a major enzyme responsible for RNA polyadenylation in Escherichia coli, a process involved in the global control of gene expression in this bacterium through influencing the rate of transcript degradation. Recent studies have suggested a complicated regulation of pcnB expression, including a complex promoter region, a control at the level of translation initiation and dependence on bacterial growth rate. In this report, studies on transcription regulation of the pcnB gene are described. Results of in vivo and in vitro experiments indicated that (a) there are three σ70-dependent (p1, pB, and p2) and two σS-dependent (pS1 and pS2) promoters of the pcnB gene, (b) guanosine tetraphosphate (ppGpp) and DksA directly inhibit transcription from pB, pS1 and pS2, and (c) pB activity is drastically impaired at the stationary phase of growth. These results indicate that regulation of the pcnB gene transcription is a complex process, which involves several factors acting to ensure precise control of PAP I production. Moreover, inhibition of activities of pS1 and pS2 by ppGpp and DksA suggests that regulation of transcription from promoters requiring alternative σ factors by these effectors of the stringent response might occur according to both passive and active models.

Blocking Variant Surface Glycoprotein Synthesis in Trypanosoma brucei Triggers a General Arrest in Translation Initiation

BackgroundThe African trypanosome Trypanosoma brucei is covered with a dense layer of Variant Surface Glycoprotein (VSG), which protects it from lysis by host complement via the alternative pathway in the mammalian bloodstream. Blocking VSG synthesis by the induction of VSG RNAi triggers an unusually precise precytokinesis cell-cycle arrest.Methodology/Principal FindingsHere, we characterise the cells arrested after the induction of VSG RNAi. We were able to rescue the VSG221 RNAi induced cell-cycle arrest through expression of a second different VSG (VSG117 which is not recognised by the VSG221 RNAi) from the VSG221 expression site. Metabolic labeling of the arrested cells showed that blocking VSG synthesis triggered a global translation arrest, with total protein synthesis reduced to less than 1–4% normal levels within 24 hours of induction of VSG RNAi. Analysis by electron microscopy showed that the translation arrest was coupled with rapid disassociation of ribosomes from the endoplasmic reticulum. Polysome analysis showed a drastic decrease in polysomes in the arrested cells. No major changes were found in levels of transcription, total RNA transcript levels or global amino acid concentrations in the arrested cells.ConclusionsThe cell-cycle arrest phenotype triggered by the induction of VSG221 RNAi is not caused by siRNA toxicity, as this arrest can be alleviated if a second different VSG is inserted downstream of the active VSG221 expression site promoter. Analysis of polysomes in the stalled cells showed that the translation arrest is mediated at the level of translation initiation rather than elongation. The cell-cycle arrest induced in the presence of a VSG synthesis block is reversible, suggesting that VSG synthesis and/or trafficking to the cell surface could be monitored during the cell-cycle as part of a specific cell-cycle checkpoint.

C-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation

The Myc/Max/Mad family of transcription factors and the eukaryotic initiation factor 4F (eIF4F) complex play fundamental roles in regulating cell growth, proliferation, differentiation, and oncogenic transformation. eIF4F is involved in the recruitment of ribosomes to mRNAs and is thought to generally be the rate-limiting phase of translation. Here, we show that c-Myc directly activates transcription of the three subunits of eIF4F (eIF4E, eIF4AI, and eIF4GI). These transcriptional effects are mediated through canonical E-boxes (5'CACGTG3') present in the promoters of these genes. In addition, the c-Myc antagonist Mad1 down-regulates the expression of eIF4F subunits. We also show that MycER activation stimulates protein synthesis at the level of translation initiation. Increased eIF4F levels result in stimulation of c-Myc mRNA translation specifically, as assessed by quantitative reverse transcription-PCR. We use a murine model of lymphomagenesis to show the expression of eIF4F subunits is also up-regulated by c-Myc in vivo. Our results suggest the presence of a feedforward loop involving c-Myc and eIF4F that serves to link transcription and translation and that could contribute to the effects of c-Myc on cell proliferation and neoplastic growth.

ERK and mTOR Signaling Couple β-Adrenergic Receptors to Translation Initiation Machinery to Gate Induction of Protein Synthesis-dependent Long-term Potentiation

beta-Adrenergic receptors critically modulate long-lasting synaptic plasticity and long-term memory in the mammalian hippocampus. Persistent long-term potentiation of synaptic strength requires protein synthesis and has been correlated with some forms of hippocampal long-term memory. However, the intracellular processes that initiate protein synthesis downstream of the beta-adrenergic receptor are unidentified. Here we report that activation of beta-adrenergic receptors recruits ERK and mammalian target of rapamycin signaling to facilitate long-term potentiation maintenance at the level of translation initiation. Treatment of mouse hippocampal slices with a beta-adrenergic receptor agonist results in activation of eukaryotic initiation factor 4E and the eukaryotic initiation factor 4E kinase Mnk1, along with inhibition of the translation repressor 4E-BP. This coordinated activation of translation machinery requires concomitant ERK and mammalian target of rapamycin signaling. Taken together, our data identify distinct signaling pathways that converge to regulate beta-adrenergic receptor-dependent protein synthesis during long-term synaptic potentiation in the hippocampus. We suggest that beta-adrenergic receptors play a crucial role in gating the induction of long-lasting synaptic plasticity at the level of translation initiation, a mechanism that may underlie the ability of these receptors to influence the formation of long-lasting memories.

Alternatively spliced isoforms of the human elk-1 mRNA within the 5′ UTR: implications for ELK-1 expression

The expression of cellular proteins that play central roles in the regulation of cell growth and differentiation is frequently tightly controlled at the level of translation initiation. In this article, we provide evidence that the ETS domain transcription factor ELK-1 forms part of this class of genes. Its mRNA 5′ UTR is composed of a complexed mosaic of elements, including uAUGs, uORFs and RNA structure, that interplay to modulate ribosomal access to the ELK-1 AUG start codon. Superimposed upon this is the generation of two different 5′ UTRs via alternative splicing. The two spliced isoforms show altered cellular and tissue distributions and behave differently in polysomal recruitment assays in the presence of the drug rapamycin. We propose that repression is therefore the sum of a series of interplaying negative elements within the 5′ UTRs, a situation which may reflect the need for tight translational control of ELK-1 in different tissues and under changing physiological conditions.

Identification of AUF1 as a rapamycin-responsive binding protein to the 5′-terminal oligopyrimidine element of mRNAs

In vertebrates, mRNAs containing a 5′-terminal oligopyrimidine (TOP) motif are coordinately post-transcriptionally regulated. Binding of specific proteins to this element has been proposed to downregulate expression of TOP mRNAs at the level of translational initiation. We previously reported that rapamycin induces binding activity to the TOP element of ribosomal protein (r-protein) L32 mRNA. In this study, we adapt DEAE-cellulose/oligo dT-cellulose tandem column chromatography to purify TOP element-binding proteins from bovine submaxillary lymph nodes (SLN). We also show by northwestern blot analysis that two proteins of molecular weight 47 kDa (47BP) and 43 kDa (43BP) specifically bind to a 32P-labeled riboprobe containing TOP regulatory element of the r-protein L32. Microsequencing of the purified 47BP revealed an internal sequence of 15 amino acids identical to the consensus sequence of the 2× RBD-Gly family. Western blot analysis of the cytoplasm fractions using an AUF1 antibody revealed that these two proteins are p45 AUF1 and p42 AUF1. Increases of the four isoforms of AUF1 protein were observed in 100,000 g supernatant fractions of rapamycin-administered rat SLN. Furthermore, decreases of p45 AUF1 and p42 and/or p40 AUF1 were observed in the polysomal fractions of BJAB cells in which translation of TOP mRNAs was selectively suppressed by rapamycin treatment. Taken together, these results suggest that AUF1 is a TOP mRNA-binding protein that may participate in the translational suppression of TOP mRNAs resulting from rapamycin treatment.

The Embryonic Pregnancy Signal Oestradiol Influences Gene Expression at the Level of Translational Initiation in Porcine Endometrial Cells

In the pig, conceptus-derived oestrogens (days 11 and 12 of pregnancy) seem to be a critical component of the signalling mechanism for maternal recognition of pregnancy. Embryonic oestrogens can mediate effects on endometrial function by interactions with epithelial and stromal oestrogen receptors (ER). Recent data demonstrate that cell membrane ER interacts with the phosphatidylinositol 3-kinase/Akt pathway in several types of cells. The protein kinase Akt is involved in the control of cell growth, survival and proliferation. One distinct function of the Akt signalling cascade is its ability to phosphorylate the eukaryotic initiation factor-4E (eIF-4E)-binding protein 1 (4E-BP1). This phosphorylation suppresses the inhibitory effect of 4E-BP1 on the translation initiation factor eIF4E and in such a way potentially stimulates gene expression at the level of translational initiation. The aim of the present study was to examine if embryonic oestradiol (E(2)) transmits its effect by such a mechanism. Endometrial cells of cyclic gilts (day 13 of the oestrous cycle, n = 4) were cultured and supplemented with vehicle (control), E(2) (50 and 100 pm/l) or with the selective ER modulator raloxifen (10 and 1000 nm/l), and incubated for 24 h. The cell viability was detected by MTT assay, the abundance and phosphorylation of Akt, 4E-BP1 and ERalpha was analysed by Western blotting. Incubation with E(2) or raloxifen did not alter endometrial cell viability. The phosphorylation of Akt at Ser(473) seems to be increased by E(2) (p < 0.05) and decreased by raloxifen (p > 0.05). Raloxifen (1000 nm/l) induced a band shift in 4E-BP1 to the highest electrophoretic mobility which reflects a decrease in phosphorylation (p < 0.05), whereas an influence of E(2) on 4E-BP1 phosphorylation could not be detected. The decrease (p < 0.05) of the abundance of the 80 kDa ERalpha form both by E(2) and raloxifen indicates that the E(2)-stimulated Akt phosphorylation and the inhibition of 4E-BP1 phosphorylation by raloxifen is an E(2) ER-transmitted process. Therefore, embryonic oestrogens can potentially transmit their effect by influencing signalling cascades which modulate gene expression at the level of translational initiation.

Small-Molecule Inhibition of the Interaction between the Translation Initiation Factors eIF4E and eIF4G

Assembly of the eIF4E/eIF4G complex has a central role in the regulation of gene expression at the level of translation initiation. This complex is regulated by the 4E-BPs, which compete with eIF4G for binding to eIF4E and which have tumor-suppressor activity. To pharmacologically mimic 4E-BP function we developed a high-throughput screening assay for identifying small-molecule inhibitors of the eIF4E/eIF4G interaction. The most potent compound identified, 4EGI-1, binds eIF4E, disrupts eIF4E/eIF4G association, and inhibits cap-dependent translation but not initiation factor-independent translation. While 4EGI-1 displaces eIF4G from eIF4E, it effectively enhances 4E-BP1 association both in vitro and in cells. 4EGI-1 inhibits cellular expression of oncogenic proteins encoded by weak mRNAs, exhibits activity against multiple cancer cell lines, and appears to have a preferential effect on transformed versus nontransformed cells. The identification of this compound provides a new tool for studying translational control and establishes a possible new strategy for cancer therapy.

The DAZL family proteins are PABP-binding proteins that regulate translation in germ cells

DAZL proteins are germ-cell-specific RNA-binding proteins essential for gametogenesis. The precise molecular role of these proteins in germ-cell development remains enigmatic; however, they appear to function in the cytoplasm. In order to directly address the function of vertebrate DAZL proteins, we have used Xenopus laevis oocytes as a model system. Here we demonstrate that members of this family, including Xdazl, mouse Dazl, human DAZL, human DAZ and human BOULE, have the ability to stimulate translation and function at the level of translation initiation. We show that DAZL proteins interact with poly(A)-binding proteins (PABPs), which are critical for the initiation of translation. Mapping and tethered function experiments suggest that these interactions are physiologically important. This leads to an attractive hypothesis whereby DAZL proteins activate translationally silent mRNAs during germ cell development through the direct recruitment of PABPs.

Interleukins 2 and 15 Regulate Ets1 Expression via ERK1/2 and MNK1 in Human Natural Killer Cells

Interleukins (IL)-2 and IL-15 regulate natural killer (NK) cell proliferation, survival, and cytolytic activity. Ets1 is a transcription factor expressed early in NK cell differentiation. Because IL-2Rbeta, IL-2Rgamma, IL-15, and Ets1 knock-out mice similarly lack NK cells, we explored a molecular connection between IL-2R signaling and Ets1. Here we report the post-transcriptional regulation of Ets1 by IL-2R signaling in human NK cells. IL-2 and IL-15 stimulation leads to increased Ets1 protein levels with no significant change in mRNA levels. Pulse and pulse-chase experiments show that IL-2 stimulation results in both a marked increase in the nascent translation of Ets1 and an increased protein half-life. Pharmacological inhibition of MEK specifically blocks IL-2- and IL-15-induced translation, whereas p38, phosphatidylinositol 3-kinase, and mTOR inhibitors had no effect on Ets1 levels. Fli1, an Ets family member, exhibited a different mechanism of regulation, illustrating the specificity of IL-2R beta and gamma subunit signaling on the regulation of Ets1 expression. Expression of a dominant negative form of MNK1, a regulator of the translation initiation factor eIF4E, blocks the expression of Ets1 as do the dominant negative forms of the common IL-2R beta and gamma chains. Expression of Ets1 is regulated similarly in normal peripheral human NK cells. Taken together, our findings provide a direct link between IL-2R subunit signaling and Ets1 expression and helps to explain the interdependence of the IL-2R subunits and Ets1 for NK cell development and function.

Leucine acutely reverses burn-induced alterations in translation initiation in heart.

Myocardial dysfunction is a common manifestation of thermal injury, the etiology of which appears to be multifactorial. We have previously demonstrated that burn injury impairs cardiac protein synthesis at the level of translation initiation. The purpose of the present study was to determine whether oral administration of leucine, which is known to stimulate translation initiation in skeletal muscle, can ameliorate burn-induced changes in signal transduction pathways known to regulate protein synthesis in cardiac muscle. To address this aim, thermal injury was produced by a 40% total body surface area full-thickness scald burn in anesthetized rats, and the animals were studied in the fasted condition 24 h later; appropriate time-matched nonburned control rats were also included. Separate groups of control and burn rats also received an oral gavage of leucine. To identify potential mechanisms responsible for regulating mRNA translation in cardiac muscle, several eukaryotic initiation factors (eIFs) were examined using immunoprecipitation and immunoblotting techniques. Hearts from burned rats demonstrated a redistribution of eIF4E as evidenced by the increased binding of the translational repressor 4E-BP1 with eIF4E, a decreased amount of eIF4E bound with eIF4G, and a decreased amount of the hyperphosphorylated gamma-isoform of 4E-BP1. Furthermore, constitutive phosphorylation of mTOR, the ribosomal protein S6, and eIF4G was also decreased in hearts from burned rats. In control rats, leucine failed to alter eIF4E distribution but did increase the phosphorylation of S6K1 and S6. However, in hearts from burn rats, leucine acutely reversed the alterations in eIF4E distribution as well as the changes in S6, eIF4G, and mTOR phosphorylation. These data suggest that oral administration of leucine can acutely reverse multiple defects in cardiac translation initiation produced by thermal injury.

Ribosome Loading onto the mRNA Cap Is Driven by Conformational Coupling between eIF4G and eIF4E

The eukaryotic initiation factor 4G (eIF4G) is the core of a multicomponent switch controlling gene expression at the level of translation initiation. It interacts with the small ribosomal subunit interacting protein, eIF3, and the eIF4E/cap-mRNA complex in order to load the ribosome onto mRNA during cap-dependent translation. We describe the solution structure of the complex between yeast eIF4E/cap and eIF4G (393–490). Binding triggers a coupled folding transition of eIF4G (393–490) and the eIF4E N terminus resulting in a molecular bracelet whereby eIF4G (393–490) forms a right-handed helical ring that wraps around the N terminus of eIF4E. Cofolding allosterically enhances association of eIF4E with the cap and is required for maintenance of optimal growth and polysome distributions in vivo. Our data explain how mRNA, eIF4E, and eIF4G exists as a stable mRNP that may facilitate multiple rounds of ribosomal loading during translation initiation, a key determinant in the overall rate of protein synthesis.

The L box regulon: lysine sensing by leader RNAs of bacterial lysine biosynthesis genes.

Expression of amino acid biosynthesis genes in bacteria is often repressed when abundant supplies of the cognate amino acid are available. Repression of the Bacillus subtilis lysC gene by lysine was previously shown to occur at the level of premature termination of transcription. In this study we show that lysine directly promotes transcription termination during in vitro transcription with B. subtilis RNA polymerase and causes a structural shift in the lysC leader RNA. We find that B. subtilis lysC is a member of a large family of bacterial lysine biosynthesis genes that contain similar leader RNA elements. By analogy with related regulatory systems, we designate this leader RNA pattern the "L box." Genes in the L box family from Gram-negative bacteria appear to be regulated at the level of translation initiation rather than transcription termination. Mutations of B. subtilis lysC that disrupt conserved leader features result in loss of lysine repression in vivo and loss of lysine-dependent transcription termination in vitro. The identification of the L box pattern also provides an explanation for previously described mutations in both B. subtilis and Escherichia coli lysC that result in lysC overexpression and resistance to the lysine analog aminoethylcysteine. The L box regulatory system represents an example of gene regulation using an RNA element that directly senses the intracellular concentration of a small molecule.

Regulation of riboflavin biosynthesis and transport genes in bacteria by transcriptional and translational attenuation.

The riboflavin biosynthesis in bacteria was analyzed using comparative analysis of genes, operons and regulatory elements. A model for regulation based on formation of alternative RNA structures involving the RFN elements is suggested. In Gram-positive bacteria including actinomycetes, Thermotoga, Thermus and Deinococcus, the riboflavin metabolism and transport genes are predicted to be regulated by transcriptional attenuation, whereas in most Gram-negative bacteria, the riboflavin biosynthesis genes seem to be regulated on the level of translation initiation. Several new candidate riboflavin transporters were identified (impX in Desulfitobacterium halfniense and Fusobacterium nucleatum; pnuX in several actinomycetes, including some Corynebacterium species and Strepto myces coelicolor; rfnT in Rhizobiaceae). Traces of a number of likely horizontal transfer events were found: the complete riboflavin operon with the upstream regulatory element was transferred to Haemophilus influenzae and Actinobacillus pleuropneumoniae from some Gram-positive bacterium; non-regulated riboflavin operon in Pyrococcus furiousus was likely transferred from Thermotoga; and the RFN element was inserted into the riboflavin operon of Pseudomonas aeruginosa from some other Pseudomonas species, where it had regulated the ribH2 gene.

Translation initiation and its deregulation during tumorigenesis.

Regulation of protein synthesis at the level of translation initiation is fundamentally important for the control of cell proliferation under normal physiological conditions. Conversely, misregulation of protein synthesis is emerging as a major contributory factor in cancer development. Most bulk protein synthesis is initiated via recognition of the mRNA 5′ cap and subsequent recognition of the initiator AUG codon by a directional scanning mechanism. However, several key regulators of tumour development are translated by a cap-independent pathway. Here we review eukaryotic translation initiation, its regulation and the ways in which this regulation can break down during tumorigenesis.British Journal of Cancer (2002) 86, 1023–1027. DOI: 10.1038/sj/bjc/6600222 www.bjcancer.com© 2002 Cancer Research UK

Developmental changes in the feeding-induced stimulation of translation initiation in muscle of neonatal pigs.

The rapid gain in skeletal muscle mass in the neonate is associated with a marked elevation in skeletal muscle protein synthesis in response to feeding. The feeding-induced response decreases with development. To determine whether the response to feeding is regulated at the level of translation initiation, the expression, phosphorylation, and function of a number of eukaryotic initiation factors (eIF) were examined. Pigs at 7 and 26 days of age were either fasted overnight or fed porcine milk after an overnight fast. In muscle of 7-day-old pigs, the hyperphosphorylated form of the eIF4E repressor protein, 4E-binding protein 1 (4E-BP1), was undetectable in the fasting state but rose to 60% of total 4E-BP1 after feeding; eIF4E phosphorylation was unaffected by feeding status. The amount of eIF4E in the inactive 4E-BP1. eIF4E complex was reduced by 80%, and the amount of eIF4E in the active eIF4E. eIF4G complex was increased 14-fold in muscle of 7-day-old pigs after feeding. The amount of 70-kDa ribosomal protein S6 (p70(S6)) kinase in the hyperphosphorylated form rose 2.5-fold in muscle of 7-day-old pigs after feeding. Each of these feeding-induced responses was blunted in muscle of 26-day-old pigs. eIF2B activity in muscle was unaffected by feeding status but decreased with development. Feeding produced similar changes in eIF characteristics in liver and muscle; however, the developmental changes in liver were not as apparent as in skeletal muscle. Thus the results demonstrate that the developmental change in the acute stimulation of skeletal muscle protein synthesis by feeding is regulated by the availability of eIF4E for 48S ribosomal complex formation. The results further suggest that the overall developmental decline in skeletal muscle protein synthesis involves regulation by eIF2B.

The 3′ UTR of Human MnSOD mRNA Hybridizes to a Small Cytoplasmic RNA and Inhibits Gene Expression

Human MnSOD localizes to the mitochondria and plays a key protective role by detoxifying oxygen free radicals. The MnSOD mRNA 3′ UTR contains a 280-bp region (Alu-like element or Alu-E) that shows high homology to human Alu and 7SL sequences. MnSOD 3′ UTR probes hybridize to a specific cytoplasmic RNA species of ∼300 nucleotides. This antisense RNA is most likely 7SL RNA based on its size, ubiquitousness, high levels, and lack of inducibility. Hybridization of this small RNA to the MnSOD 3′ UTR may modulate posttranscriptional MnSOD gene expression. This regulation could occur by several means including inhibition of translation and mRNA destabilization. Regulation at the level of translational initiation does not seem to occur as MnSOD mRNA containing the Alu-E is efficiently bound by ribosomes. To test the role of the MnSOD 3′ UTR, and in particular the Alu-E in gene expression, luciferase reporter gene constructs were made containing various regions of the MnSOD 3′ UTR including the Alu-E. These constructs were transfected into human A549 lung carcinoma cells and luciferase activity was measured. Reporter constructs containing the MnSOD 3′ UTR and the Alu-E repress luciferase activity. Taken together, these results suggest that naturally occurring antisense RNA may bind MnSOD mRNA and repress its expression. These results also suggest that other mRNAs containing Alu elements may be similarly repressed.

Regulation of SOCS-1 Expression by Translational Repression

Accumulating evidence demonstrates that cytokine receptor signaling is negatively regulated by a family of Src homology 2 domain-containing adaptor molecules termed SOCS (suppressor of cytokine signaling). Previous studies have indicated that the expression of SOCS-related molecules is tightly controlled at the level of transcription. Furthermore, it has been reported that SOCS polypeptides are relatively unstable in cells, unless they are associated with elongins B and C. Herein, we document the existence of a third mechanism of regulation of SOCS function. Our data showed that expression of SOCS-1, a member of the SOCS family, is strongly repressed at the level of translation initiation. Structure-function analyses indicated that this effect is mediated by the 5' untranslated region of socs-1 and that it relates to the presence of two upstream AUGs in this region. Further studies revealed that socs-1 translation is cap-dependent and that it is modulated by eIF4E-binding proteins. In combination, these results uncover a novel level of regulation of SOCS-related molecules. Moreover, coupled with previous findings, they suggest that SOCS expression is tightly regulated through multiple mechanisms, in order to avoid inappropriate interference with cytokine-mediated effects.