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Results for “Translational repression”

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PubMed1998

Mapping of a minimal AU-rich sequence required for lipopolysaccharide-induced binding of a 55-kDa protein on tumor necrosis factor-alpha mRNA.

In monocyte/macrophage cells, the translation of tumor necrosis factor-alpha (TNF-alpha) mRNA is tightly controlled. In unstimulated cells, TNF-alpha mRNA is translationally repressed. However, upon stimulation of the cells with various agents (e.g. lipopolysaccharides (LPS) and viruses), this repression is overcome and translation occurs. The key element in this regulation is the AU-rich sequence present in the 3'-untranslated region of TNF-alpha mRNA. Several groups have described the binding of proteins on AU-rich elements (AREs). We have previously reported the binding of two cytosolic protein complexes (1 and 2) to the TNF-alpha mRNA ARE, one of which (complex 2) is observed only following induction of TNF-alpha production by LPS. In this report, we have demonstrated that complex 1 involves a long fragment of the ARE, whereas the formation of the LPS-inducible complex 2 requires a minimal sequence which corresponds to the nonanucleotide UUAUUUAUU. Furthermore, we show that the RNA-binding protein involved in complex 2 has an apparent molecular mass of 55 kDa. Finally, we tested other AREs for their ability to form complex 2. We observed that the ARE derived from granulocyte/macrophage colony-stimulating factor mRNA, which does contain the nonanucleotide, is able to sustain the LPS-induced binding of the 55-kDa protein. However, c-myc mRNA, which does not contain the nonanucleotide, is unable to promote the formation of any LPS-induced complex.

Animals↗

PubMed2002

Identification of critical amino acid residues on human dihydrofolate reductase protein that mediate RNA recognition.

Previous studies have shown that human dihydrofolate reductase (DHFR) acts as an RNA-binding protein, in which it binds to its own mRNA and, in so doing, results in translational repression. In this study, we used RNA gel mobility shift and nitrocellulose filter-binding assays to further investigate the specificity of the interaction between human DHFR protein and human DHFR mRNA. Site-directed mutagenesis was used to identify the critical amino acid residues on DHFR protein required for RNA recognition. Human His-Tag DHFR protein specifically binds to human DHFR mRNA, while unrelated proteins including thymidylate synthase, p53 and glutathione-S-transferase were unable to form a ribonucleoprotein complex with DHFR mRNA. The Cys6 residue is essential for RNA recognition, as mutation at this amino acid with either an alanine (C6A) or serine (C6S) residue almost completely abrogated RNA-binding activity. Neither one of the cysteine mutant proteins was able to repress the in vitro translation of human DHFR mRNA. Mutations at amino acids Ile7, Arg28 and Phe34, significantly reduced RNA-binding activity. An RNA footprinting analysis identified three different RNA sequences, bound to DHFR protein, ranging in size from 16 to 45 nt, while a UV cross-linking analysis isolated an approximately 16 nt RNA sequence bound to DHFR. These studies begin to identify the critical amino acid residues on human DHFR that mediate RNA binding either through forming direct contact points with RNA or through maintaining the protein in an optimal structure that allows for the critical RNA-binding domain to be accessible.

Amino Acids↗

PubMed2006

Insulin stimulates muscle protein synthesis in neonates during endotoxemia despite repression of translation initiation.

Skeletal muscle protein synthesis is reduced in neonatal pigs in response to endotoxemia. To examine the role of insulin in this response, neonatal pigs were infused with endotoxin (LPS, 0 and 10 mug.kg(-1).h(-1)), whereas glucose and amino acids were maintained at fasting levels and insulin was clamped at fasting or fed (2 or 10 muU/ml) levels. Fractional rates of protein synthesis and translational control mechanisms were examined in longissimus dorsi muscle and liver. In the presence of fasting insulin, LPS reduced muscle protein synthesis (-29%), and increasing insulin to fed levels accelerated muscle protein synthesis in both groups (controls, +44%; LPS, +64%). LPS, but not insulin, increased liver protein synthesis by +28%. In muscle of fasting neonatal pigs, LPS reduced 4E-BP1 phosphorylation and eIF4E to eIF4G binding. In muscle of controls, but not LPS pigs, raising insulin to fed levels increased 4E-BP1 and S6K1 phosphorylation and eIF4E to eIF4G binding. In muscle and liver, neither LPS nor insulin altered eIF2B activity. eEF2 phosphorylation decreased in response to insulin in both LPS and control animals. The results suggest that, in endotoxemic neonatal animals, the response of protein synthesis to insulin is maintained despite suppression of mTOR-dependent translation initiation and eIF4E availability for eIF4F assembly. Maintenance of an anabolic response to the feeding-induced rise in insulin likely exerts a protective effect for the neonate to the catabolic processes induced by sepsis.

Animals↗

PubMed1992

Translation of poly(A)-binding protein mRNA is regulated by growth conditions.

Translational efficiency of a minor group of mRNAs is regulated by serum levels in 3T6 fibroblasts. Included within this group is the poly(A)-binding protein (PABP) mRNA. We analyzed the distribution of PABP mRNA in polysome profiles and found a large percentage of this mRNA to be translationally repressed in both actively growing (approximately 60%) and resting cells (approximately 70%). Elevated serum levels induced a distinct bimodal distribution of this mRNA between actively translated and repressed fractions. Similarly, treatment of cells with low doses of cycloheximide also generated a partial shift of repressed PABP mRNA into the actively translated fraction. In an attempt to characterize the factors which regulate PABP mRNA translation we have identified the proteins which bind to this mRNA in vitro. Sequences within the 5' untranslated region were found to be sufficient for binding of all proteins to this mRNA. We suggest that this region and the proteins associated with it may be essential for translation control of PABP mRNA.

Blood Physiological Phenomena↗

PubMed2005

MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function.

MicroRNAs (miRNAs) repress translation of target mRNAs by interaction with partially mismatched sequences in their 3' UTR. The mechanism by which they act on translation has remained largely obscure. We examined the translation of mRNAs containing four partially mismatched miRNA-binding sites in the 3' UTR in HeLa cells cotransfected with a cognate miRNA. The mRNAs were prepared by in vitro transcription and were engineered to employ different modes of translation initiation. We find that the 5' cap structure and the 3' poly(A) tail are each necessary but not sufficient for full miRNA-mediated repression of mRNA translation. Replacing the cap structure with an internal ribosome entry site from either the cricket paralysis virus or the encephalomyocarditis virus impairs miRNA-mediated repression. Collectively, these results demonstrate that miRNAs interfere with the initiation step of translation and implicate the cap-binding protein eukaryotic initiation factor 4E as a molecular target.

Eukaryotic Initiation Factor-4E↗

PubMed2004

CiYB1 is a major component of storage mRNPs in ascidian oocytes: implications in translational regulation of localized mRNAs.

In ascidian eggs, the existence of several localized maternal cytoplasmic determinants has been proposed and the importance of localized mRNAs for tissue differentiation has been demonstrated. We previously identified the ascidian Y-box proteins (CiYB1, 2 and 3), homologues of which are known to be involved in the storage of maternal mRNA in oocytes of other organisms. In this study, we found that CiYB1 protein is abundant in the gonad, egg, and embryo. Purification of messenger ribonucleoprotein (mRNP) particles from the gonad revealed that CiYB1 was one of their major components. A significant change in the distribution of CiYB1 protein from stored mRNP particles in the gonad to the ribosome fraction in eggs and embryos was observed. This change correlates most likely with the shift of stored maternal mRNAs to polyribosomes. Moreover, we found that CiYB1 colocalized with Cipem and Ci-macho1 mRNAs, which are localized at the posterior end of the embryo at the cleavage stage. Cipem and Ci-macho1 mRNAs were co-immunoprecipitated with CiYB1 in the oocyte and embryo lysates. The formation of a complex between Cipem mRNA and CiYB1 protein resulted in translational repression in the in vitro translation system. Our results indicate that associating with CiYB1 protein contributes to the translational control of the localized mRNA in eggs and embryos.

Amino Acid Sequence↗

PubMed2006

The 5' UTR and 3' UTR of the sperm mitochondria-associated cysteine-rich protein mRNA regulate translation in spermatids by multiple mechanisms in transgenic mice.

The Smcp mRNA encoding the sperm mitochondria-associated cysteine-rich protein is translationally repressed in round spermatids and translationally active in elongated spermatids. The patterns of transcription and translation of fusions of the Smcp promoter, the green fluorescent protein coding region (Gfp) and various combination of the Smcp and Gfp 5' UTR and 3' UTR have been studied in transgenic mice. 518 nt of Smcp 5' flanking region and 8 nt of 5' UTR drive transcription of mRNAs containing the Gfp coding region in early round spermatids at the same transcription start site as the natural Smcp gene. Transcripts containing both the Gfp 5' and 3' UTRs are translationally active in step 2 spermatids as detected by GFP fluorescence in squashes of living seminiferous tubules from adult testes, and the presence of polysomal mRNAs in sucrose gradient analyses of testes from 21-day-old prepubertal mice, which contain early round spermatids and lack elongated spermatids. By comparison, expression of GFP is delayed until steps 5 and 10, respectively, in transcripts containing the Smcp 5' UTR and Gfp 3' UTR and the Gfp 5' UTR and the Smcp 3' UTR. Sucrose gradient analysis of 21-day-old testes demonstrates that transcripts containing the Smcp 3' UTR exhibit a bimodal distribution in free-mRNPs and polysomes, indicating that the 3' UTR blocks the expression of GFP after the transcripts have entered the elongation phase, a mechanism that may involve microRNAs. The Smcp 5' UTR reduces the levels and size of polysomes in adult testis. In addition, the natural Smcp mRNA contains a positive control element that counteracts the inhibition of translation by the Smcp 5' UTR in adult testis, and the Smcp 3' UTR strongly localizes GFP fluorescence in step 10 spermatids.

3' Untranslated Regions↗

PubMed2004

Cytoprotection by pre-emptive conditional phosphorylation of translation initiation factor 2.

Transient phosphorylation of the alpha-subunit of translation initiation factor 2 (eIF2alpha) represses translation and activates select gene expression under diverse stressful conditions. Defects in the eIF2alpha phosphorylation-dependent integrated stress response impair resistance to accumulation of malfolded proteins in the endoplasmic reticulum (ER stress), to oxidative stress and to nutrient deprivations. To study the hypothesized protective role of eIF2alpha phosphorylation in isolation of parallel stress signaling pathways, we fused the kinase domain of pancreatic endoplasmic reticulum kinase (PERK), an ER stress-inducible eIF2alpha kinase that is normally activated by dimerization, to a protein module that binds a small dimerizer molecule. The activity of this artificial eIF2alpha kinase, Fv2E-PERK, is subordinate to the dimerizer and is uncoupled from upstream stress signaling. Fv2E-PERK activation enhanced the expression of numerous stress-induced genes and protected cells from the lethal effects of oxidants, peroxynitrite donors and ER stress. Our findings indicate that eIF2alpha phosphorylation can initiate signaling in a cytoprotective gene expression pathway independently of other parallel stress-induced signals and that activation of this pathway can single-handedly promote a stress-resistant preconditioned state.

Activating Transcription Factor 4↗

PubMed2004

Translational control at the synapse.

The strength of synaptic connections can undergo long-lasting changes, and such long-term plasticity is thought to underlie higher brain functions such as learning and memory. De novo synthesis of proteins is required for such plastic changes. This model is now supported by several lines of experimental data. Components of translational machinery have been identified in dendrites, including ribosomes, translation-al factors, numerous RNAs, and components of posttranslational secretory pathways. Various RNAs have been shown to be actively and rapidly transported to dendrites. Dendritic RNAs typically contain transport-specifying elements (dendritic targeting elements). Such dendritic targeting elements associate with trans-acting factors to form transport-competent ribonucleoprotein particles. It is assumed that molecular motors mediate transport of such particles along dendritic cytoskeletal elements. Once an mRNA has arrived at its dendritic destination site, appropriate spatiotemporal control of its translation, for example, in response to transsynaptic activity, becomes vital. Such local translational control, recent evidence indicates, is implemented at different levels and through various pathways. In the default state, translation is assumed to be repressed, and several mechanisms, some including small untranslated RNAs, have been proposed to contribute to such repression. Translational control at the synapse thus provides a molecular basis for the long-term, input-specific modulation of synaptic strength.

Animals↗

PubMed2025

Degradation of ribosomal RNA during Plasmodium falciparum gametocytogenesis.

The life cycle of Plasmodium falciparum is characterized by complex regulatory changes that allow adaptation of the parasites to different environmental conditions, which are especially pronounced during transmission between the mammalian host and the insect vector. Previous studies have shown that P. falciparum uses three types of ribosomal RNAs (rRNA A, S1, and S2 types) at different stages of its life cycle. We used Oxford Nanopore Technologies direct RNA sequencing to investigate the dynamics of rRNA usage throughout the parasite's intraerythrocytic development, as well as in salivary gland sporozoites. Our study revealed a preponderance of A-type rRNAs during the intraerythrocytic cycle and gametocytogenesis, while S-type rRNAs slowly increase in abundance in mosquito stages starting 3 days post-infection. Salivary gland sporozoites showed an even proportion of all rRNA types. By examining the length distributions of rRNA molecules, we detected an extensive and specific degradation of rRNAs during gametocytogenesis, starting in stage II gametocytes and continuing until the final stages of gametocyte development. We hypothesize that rRNA degradation may be linked to the global translational repression and metabolic quiescence described in stage V gametocytes, similar to mechanisms observed in bacterial and eukaryotic stress responses.IMPORTANCEOur study uses Oxford Nanopore direct RNA sequencing of tightly synchronized blood-stage Plasmodium falciparum parasites to investigate the expression of ribosomal RNAs during asexual and sexual development. P. falciparum utilizes distinct types of rRNA during its development. However, due to the challenges of differentiating these highly similar molecules, their regulation and the mechanism underlying the switch between rRNA types remain unclear. We observe significant rRNA degradation in mature gametocytes, leading us to propose that this potentially leads to a reduced number of functional ribosomes when parasites become quiescent and translation is repressed.

Plasmodium falciparum↗

PubMed1988

[An inhibitor of translation in cellular mRNA containing the 100S structure from Krebs II ascites carcinoma cells infected with encephalomyocarditis virus].

As shown previously, the bulk of cellular mRNA in Krebs II ascite carcinoma cells infected with encephalomyocarditis virus during active virus-specific synthesis is bound to ribosomes within the 100S structure which is inactive in protein synthesis. In order to elucidate the reasons for the translation repression of cellular mRNA within the 100S structure, a fraction of loosely bound proteins which are liberated by treatment of the 100S structure with 0.5 M KCl an which contain sum translation factors, was obtained. This fraction was shown to contain an inhibitor which non-specifically represses the translation of endogenous viral and cellular mRNA within the composition of polyribosomes and of exogenous poly(A)-containing cellular mRNAs from ascite carcinoma cells.

Animals↗

PubMed1996

Isolation and characterization of the 102-kilodalton RNA-binding protein that binds to the 5' and 3' translational enhancers of tobacco mosaic virus RNA.

Tobacco mosaic virus (TMV) is a positive-sense, single-stranded RNA virus the genome of which acts as a mRNA in the cytoplasm. On infection, TMV mRNA is efficiently and selectively translated by the host translation machinery despite the lack of a poly(A) tail, which is normally required for efficient translation. Both the 68-base 5' leader (Omega) and the 205-base 3' untranslated region of TMV promote efficient translation. A 25-base poly(CAA) region within Omega and the upstream pseudoknot domain, a 72-base region composed of three RNA pseudoknots, are responsible for the translational regulation. We have identified, purified, and characterized a 102-kDa RNA-binding protein (p102) from wheat that binds specifically to the poly(CAA) region within Omega and the upstream pseudoknot domain within the TMV 3' untranslated region. Polyclonal antibodies raised against wheat p102 were used to demonstrate that p102 is widely conserved in plant species. Moreover, specific RNA binding activity was detected in all plant species tested. Addition of anti-p102 antibodies to an in vitro translation lysate derived from wheat germ repressed translation, which was subsequently reversed by supplementing the lysate with p102. These findings suggest that this protein may play an important role in determining translational efficiency in plants.

Antibodies↗

PubMed1998

Localization-dependent translation requires a functional interaction between the 5' and 3' ends of oskar mRNA.

The precise restriction of proteins to specific domains within a cell plays an important role in early development and differentiation. An efficient way to localize and concentrate proteins is by localization of mRNA in a translationally repressed state, followed by activation of translation when the mRNA reaches its destination. A central issue is how localized mRNAs are derepressed. In this study we demonstrate that, when oskar mRNA reaches the posterior pole of the Drosophila oocyte, its translation is derepressed by an active process that requires a specific element in the 5' region of the mRNA. We demonstrate that this novel type of element is a translational derepressor element, whose functional interaction with the previously identified repressor region in the oskar 3' UTR is required for activation of oskar mRNA translation at the posterior pole. The derepressor element only functions at the posterior pole, suggesting that a locally restricted interaction between trans-acting factors and the derepressor element may be the link between mRNA localization and translational activation. We also show specific interaction of two proteins with the oskar mRNA 5' region; one of these also recognizes the 3' repressor element. We discuss the possible involvement of these factors as well as known genes in the process of localization-dependent translation.

Animals↗

PubMed2002

Control of PERK eIF2alpha kinase activity by the endoplasmic reticulum stress-induced molecular chaperone P58IPK.

P58(IPK) is an Hsp40 family member known to inhibit the interferon (IFN)-induced, double-stranded RNA-activated, eukaryotic initiation factor 2alpha (eIF2alpha) protein kinase R (PKR) by binding to its kinase domain. We find that the stress of unfolded proteins in the endoplasmic reticulum (ER) activates P58(IPK) gene transcription through an ER stress-response element in its promoter region. P58(IPK) interacts with and inhibits the PKR-like ER-localized eIF2alpha kinase PERK, which is normally activated during the ER-stress response to protect cells from ER stress by attenuating protein synthesis and reducing ER client protein load. Levels of phosphorylated eIF2alpha were lower in ER-stressed P58(IPK)-overexpressing cells and were enhanced in P58(IPK) mutant cells. In the ER-stress response, PKR-like ER kinase (PERK)-mediated translational repression is transient and is followed by translational recovery and enhanced expression of genes that increase the capacity of the ER to process client proteins. The absence of P58(IPK) resulted in increased expression levels of two ER stress-inducible genes, BiP and Chop, consistent with the enhanced eIF2alpha phosphorylation in the P58(IPK) deletion cells. Our studies suggest that P58(IPK) induction during the ER-stress response represses PERK activity and plays a functional role in the expression of downstream markers of PERK activity in the later phase of the ER-stress response.

Animals↗

PubMed2001

Carbon- and nitrogen-quality signaling to translation are mediated by distinct GATA-type transcription factors.

The target of rapamycin (Tor) proteins sense nutrients and control transcription and translation relevant to cell growth. Treating cells with the immunosuppressant rapamycin leads to the intracellular formation of an Fpr1p-rapamycin-Tor ternary complex that in turn leads to translational down-regulation. A more rapid effect is a rich transcriptional response resembling that when cells are shifted from high- to low-quality carbon or nitrogen sources. This transcriptional response is partly mediated by the nutrient-sensitive transcription factors GLN3 and NIL1 (also named GAT1). Here, we show that these GATA-type transcription factors control transcriptional responses that mediate translation by several means. Four observations highlight upstream roles of GATA-type transcription factors in translation. In their absence, processes caused by rapamycin or poor nutrients are diminished: translation repression, eIF4G protein loss, transcriptional down-regulation of proteins involved in translation, and RNA polymerase I/III activity repression. The Tor proteins preferentially use Gln3p or Nil1p to down-regulate translation in response to low-quality nitrogen or carbon, respectively. Functional consideration of the genes regulated by Gln3p or Nil1p reveals the logic of this differential regulation. Besides integrating control of transcription and translation, these transcription factors constitute branches downstream of the multichannel Tor proteins that can be selectively modulated in response to distinct (carbon- and nitrogen-based) nutrient signals from the environment.

Amino Acids↗

PubMed2003

Cell cycle regulation of hepatitis C and encephalomyocarditis virus internal ribosome entry site-mediated translation in human embryonic kidney 293 cells.

We have established stably transformed human embryonic kidney cell lines (HEK293) containing bicistronic constructs to study regulation of viral internal ribosome entry site (IRES)-mediated translation in vivo. These cells produce Renilla luciferase (Rluc) in a cap-dependent manner, while Firefly luciferase (Luc) synthesis is mediated by IRES elements. Using these cell lines, we demonstrate here that IRES-mediated translation directed by both hepatitis C (HCV) and encephalomyocarditis (EMCV) virus varies with the cell cycle. Experiments involving arrest of the cell lines at different phases of the cell cycle, release of synchronized cells from cell cycle arrest, as well as direct sorting of the cells based on position in the cell cycle have shown that the activity of the HCV and EMCV IRES elements is lowest during the G2/M phase in HEK293 cells. These results suggest that cellular trans-acting factors either stimulate viral IRES-mediated translation during G1 and S phases or repress translation during the G2/M phase in HEK293 cells.

5' Untranslated Regions↗

PubMed1990

Ferritin is a translationally regulated heat shock protein of avian reticulocytes.

Heat-shock avian reticulocytes exhibit enhanced synthesis of a greater than 450-kDa protein. Biochemical, immunochemical, and visual criteria were used to identify this protein as the iron storage protein ferritin. The 21-kDa ferritin subunits synthesized during heat shock are similar in size and pI to the subunits that are constitutively synthesized. The 2-6-fold heat shock-induced increase in ferritin synthesis appears to be regulated at the translational level as it is insensitive to actinomycin D. Northern and dot-blot hybridization analyses of cytoplasmic RNAs with avian H-ferritin cDNA fragments support the contention that the heat shock stimulation of ferritin synthesis is translationally regulated. These latter studies demonstrate that the heat shock-induced synthesis of ferritin does not involve a change in the amount of total cytoplasmic ferritin mRNAs, but rather appears to entail a translocation of cytoplasmic H-ferritin mRNAs from a polyribosome-free, translationally repressed state to a polyribosome-associated, translationally active state. These results suggest that thermally stressed avian reticulocytes have a critical and functional need for the synthesis of additional ferritin and that its enhanced synthesis, unlike the new and/or enhanced synthesis of the well-established avian heat shock proteins, is regulated wholly at the translational level.

Animals↗

PubMed1984

Nucleotide sequence of the rpoA-rplQ DNA of Escherichia coli: a second regulatory binding site for protein S4?

The "alpha-operon" of E.coli is a unit of regulation comprising the following known genes, mostly encoding ribosomal proteins (in order of transcription, and with their products named in brackets): rpsM (S13), rpsK (S11), rpsD (S4), rpoA (alpha-subunit of RNA polymerase), rplQ (L17). There is evidence that S4 tightly regulates all of these genes, except rpoA, by repressing translation of the polycistronic mRNA. Binding of S4 to the S13 start-site is thought to regulate the first three genes. We have extended the 'rpsD-rpoA' sequences previously determined by others, to include all of rpoA and rplQ. The rpoA-rplQ intercistronic region shows strong primary, and potential secondary structural homologies with the S4-binding sites on 16S rRNA and S13 mRNA. We suggest that S4 represses L17 translation directly.

Bacterial Proteins↗