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Results for “Translational repression”

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PubMed2005

STAR proteins quaking-6 and GLD-1 regulate translation of the homologues GLI1 and tra-1 through a conserved RNA 3'UTR-based mechanism.

The binding of the STAR protein GLD-1 to an element in the tra-2 3' untranslated region (3'UTR), called the TGE (tra GLI element), represses tra-2 translation, allowing for hermaphrodite spermatogenesis in Caenorhabditis elegans. GLD-1 is a member of the STAR family that includes the mammalian quaking (Qk) proteins. Here, we show that the 3'UTR of the nematode homologue of GLI1, called tra-1, also contains a TGE, through which translation is regulated by GLD-1. We find that GLD-1 activity is required for proper TRA-1 protein expression in hermaphrodites. RNA gel shift assays show that GLD-1 binds the predicted sites. Using reporter transgenes, we show that the human GLI1 (hGLI1) 3'UTR controls translation in the mouse embryo. We demonstrate that the addition of the mouse QK isoform-6 (QKI-6) protein to a mammalian cell line that lacks QKI-6 can confer regulation on reporter and GLI1 mRNAs in a TGE-specific manner, and reduction of QKI-6 activity with siRNA disrupts translational control. Further, siRNA knockdown of QKI-6 increases the activity of a reporter transgene that monitors the transcriptional activity of mouse Gli1 (mGli1) and increases mouse Gli1 protein. We show by immunoprecipitation that QKI-6 antibody specifically co-purifies TGE-containing mRNAs in ribonucleoproteins. Thus, we find that the mouse QKI-6 protein acts through the mGli1 and hGLI1 RNAs to repress translation. Our results suggest that STAR family-dependent translational control of GLI mRNAs is ancient, and that it existed before the division of nematodes and mammals.

3' Untranslated Regions↗

PubMed1987

Identification of the proteins in direct contact with duck globin mRNA.

Proteins in direct contact with translationally active and repressed duck globin mRNA were determined by irradiating blood or lysates with ultraviolet light. Cross-linked proteins from polyribosomes and free mRNP particles were 14C-labeled by reductive methylation and identified on SDS-polyacrylamide gels upon autoradiography. Results indicate that ten cross-linked proteins are common to both polysomal and free mRNP, however, a 44 kDa protein appears to be specific for repressed mRNP particles. Furthermore, the notable lack of cross-linked proteins in the 20-30 kDa range in free mRNP supports the view that the characteristic low molecular mass 'prosomal' proteins, previously found associated with translationally repressed duck globin free mRNP [(1984) EMBO J. 3, 29-34], do not interact directly with the mRNA molecule.

Animals↗

PubMed2004

The STAR/Maxi-KH domain protein GLD-1 mediates a developmental switch in the translational control of C. elegans PAL-1.

Translational control is an essential mechanism of gene control utilized throughout development, yet the molecular mechanisms underlying translational activation and repression are poorly understood. We have investigated the translational control of the C. elegans caudal homolog, pal-1, and found that GLD-1, a member of the evolutionarily conserved STAR/Maxi-KH domain family, acts through a minimal pal-1 3' UTR element to repress pal-1 translation in the distal germline. We also provide data suggesting that GLD-1 may repress pal-1 translation after initiation. Finally, we show that GLD-1 represses the distal germline expression of the KH domain protein MEX-3, which was previously shown to repress PAL-1 expression in the proximal germline and which appears specialized to control PAL-1 expression patterns in the embryo. Hence, GLD-1 mediates a developmental switch in the control of PAL-1 repression, allowing MEX-3 to accumulate and take over the task of PAL-1 repression in the proximal germline, where GLD-1 protein levels decline.

3' Untranslated Regions↗

PubMedDate not supplied

[The ratio of coat protein to bacteriophage f2 RNA in the translational repressor complex].

One of the mechanisms underlying the regulation of the bacteriophage f2 RNA translation is the repression of the phage RNA-replicase formation by coat protein. This repression is due to the formation of a complex between f2 RNA and coat protein (complex I). In this work the mechanism of complex I formation as well as the effect of this complex on the f2 RNA-replicase formation was followed by inhibition of alanine incorporation into RNA-replicase polypeptide which was separated by polyacrylamide gel electrophoresis. The molar ratios of protein to f2 RNA in complex I were analyzed by sucrose gradient sedimentation. It was been found that complex I consists of six molecules of coat protein bound per one molecule of RNA. Ribonuclease digestion of the glutaraldehyde-fixed complex resulted in a mixture of products in which the hexamers of coat protein molecules were predominant. This indicates that the six molecules of coat protein bound to f2 RNA are neighbouring. It has been also shown that under conditions required for phage protein synthesis, coat protein occurs in solution is dimer. The results show that the translational repression of the RNA-replicase cistron is due to the cooperative attachment of three dimers of coat protein to phage template, forming a hexameric cluster on the RNA strand. The proposed mechanism of the complex I formation seems to be in good agreement with the sequence of events in the phage F2 life cycle. It is known that shortly after infection of the host cell the coat protein and phage RNA-replicase begin to be synthesised. According to our findings, the first portions of coat protein do not affect the translation of the RNA-replicase gene since at low concentration the coat protein occure in the form of monomers. At a later period of phage development, when the concentration of coat protein is sufficiently high to promote the formation of protein dimers, the translational repressor complex is formed and the RNA-replicase gene becomes inoperative.

Coliphages↗

PubMed2002

Structural basis of translational control by Escherichia coli threonyl tRNA synthetase.

Escherichia coli threonyl-tRNA synthetase (ThrRS) represses the translation of its own messenger RNA by binding to an operator located upstream of the initiation codon. The crystal structure of the complex between the core of ThrRS and the essential domain of the operator shows that the mRNA uses the recognition mode of the tRNA anticodon loop to initiate binding. The final positioning of the operator, upon which the control mechanism is based, relies on a characteristic RNA motif adapted to the enzyme surface. The finding of other thrS operators that have this conserved motif leads to a generalization of this regulatory mechanism to a subset of Gram-negative bacteria.

Anticodon↗

PubMed1996

The expression of E.coli threonyl-tRNA synthetase is regulated at the translational level by symmetrical operator-repressor interactions.

Threonyl-tRNA synthetase from Escherichia coli represses the translation of its own mRNA by binding to the operator region located upstream from the ribosome binding site. The operator contains two stemloop structures which interact specifically with the homodimeric enzyme. Here, we provide in vitro and in vivo evidence that these two stem-loop structures are recognized by the enzyme in an analogous way and mimic the anticodon arm of E.coli tRNA(Thr). Determination of the stoichiometry of the different RNA-threonyl-tRNA synthetase complexes reveals that two tRNA(Thr) molecules bind to the enzyme whereas only one thrS operator interacts with the homodimeric enzyme. A model is presented in which the two anticodon-like domains of the operator bind symmetrically to the two tRNA(Thr) anticodon recognition sites (one per subunit) of the dimeric threonyl-tRNA synthetase. Although symmetrical operator-repressor interactions in transcriptional control are widespread, this report stresses the importance of such interactions in translational regulation of gene expression.

Anticodon↗

PubMed1981

Processing of mRNA by ribonuclease III regulates expression of gene 1.2 of bacteriophage T7.

A bacteriophage T7 mutation, HS9, is phenotypically defective in gene 1.2, although it maps outside the gene. The single nucleotide change responsible for the HS9 mutation lies within the RNAase III recognition site immediately following gene 1.2. This RNAase III recognition site, responsible for the processing of the mRNA encoding genes 1.1 and 1.2, contains two cleavage sites, separated by 29 bases. The HS9 mutation prevents cutting by RNAase III at one site in vitro, yielding a mRNA containing an additional 29 bases at its 3' end. The ten second-site reversion mutations of HS9 are all located in the RNAase III recognition site and either restore or eliminate cutting at both sites. RNAase III mutants of Escherichia coli phenotypically suppress the HS9 mutation. We propose that the extra 29 bases at the 3' end of the mRNA hybridize to the ribosome-binding site of gene 1.1; gene 1.1 immediately precedes gene 1.2 on the same mRNA molecule. Such hybridization prevents the initiation of translation of this mRNA containing gene 1.1. A strong polar effect represses the translation of gene 1.2.

Base Sequence↗

PubMed2006

Implication of nanos2-3'UTR in the expression and function of nanos2.

Translational control of gene expression is an important component of the regulation of cellular differentiation and development. To elucidate the function of the 3'untranslated region (UTR) of the nanos2 gene in mice, we compared the phenotypes of lacZ knock-in mice with or without a native nanos2 3'UTR and found that this region of the nanos2 gene has a potential role during translational regulation in germ cells. The nanos2-3'UTR functions to repress the translation of mRNA in oocytes, but enhances the production of protein in the male gonads. To further understand the significance of the nanos2 3'UTR in vivo, we generated the mouse line nanos2pA/pA, which lacks this region endogenously. In nanos2(-/pA) mice, the number of germ cell-depleted seminiferous tubules was increased when compared with that of nanos2pA/pA mice, indicating a dose-dependent defect in spermatogenesis. These results suggest that the level of nanos2 protein is critical for normal spermatogenesis, and that this pathway may be regulated through the nanos2-3'UTR. We found that the defects in nanos2pA/pA and nanos2(-/pA) mice were caused by apoptosis of gonocytes in the embryonic gonads and gonocyte/spermatogonia in neonatal testes. In addition, it was noted that the nanos2 expression was restricted to a particular subset of spermatogonia after birth, which indicates that nanos2 plays a role in the maintenance and differentiation of gonocytes/spermatogonia in neonatal testes.

3' Untranslated Regions↗

PubMed1996

A testis cytoplasmic RNA-binding protein that has the properties of a translational repressor.

Translation of the mouse protamine 1 (Prm-1) mRNA is repressed for several days during male germ cell differentiation. With the hope of cloning genes that regulate the translational repression of Prm-1, we screened male germ cell cDNA expression libraries with the 3' untranslated region of the Prm-1 RNA. From this screen we obtained two independent clones that encode Prbp, a Prm-1 RNA-binding protein. Prbp contains two copies of a double-stranded-RNA-binding domain. In vitro, the protein binds to a portion of the Prm-1 3' untranslated region previously shown to be sufficient for translational repression in transgenic mice, as well as to poly(I). poly(C). Prbp protein is present in multiple forms in cytoplasmic extracts prepared from wild-type mouse testes and is absent from testes of germ cell-deficient mouse mutants, suggesting that Prbp is restricted to the germ cells of the testis. Immunocytochemical localization confirmed that Prbp is present in the cytoplasmic compartment of late-stage meiotic cells and haploid round spermatids. Recombinant Prbp protein inhibits the translation of multiple mRNAs in a wheat germ lysate, suggesting that Prbp acts to repress translation in round spermatids. While this protein lacks complete specificity for Prm-1-containing RNAs in vitro, the properties of Prbp are consistent with it acting as a general repressor of translation.

Amino Acid Sequence↗

PubMedDate not supplied

[Translational regulation of potato virus X RNA-coat protein complexes: the key role of a coat protein N-terminal peptide].

The efficiency of in vitro translation of potato virus X (PVX) RNA within vRNP complexes assembled from genomic RNA and viral CP was examined. The vRNP particles contain the 5'-proximal RNA segments encapsidated by helically arranged CP head-like portions heterogeneous in length and the CP-free RNA tail. Translation of RNA is completely repressed upon incubation with PVX CP and is accompanied by vRNP particles production. By contrast, translation is activated in vRNPs in vitro assembled using two CP forms, differing in the principals of their N-terminal peptides modification. The N-terminal peptide of PVX CP represents the major phosphorylation site(s) for Thr/Ser-specific protein kinases. It was shown that: (i) CP phosphorylation results in a translational activation of vRNP; (ii) removal of N-terminal peptide from CP abolished activation and CP retains the translation repressing ability. It was suggested that substitution of Ser/Thr residues by non-phosphorylated Ala/Gly in N-terminal peptide of the mutant CP will led to a complete inhibition of vRNP translation. However, opposite results were obtained in our experiments: (i) RNA of such mutant virus (PVX-ST) was efficiently translated within the virions; (ii) RNA of a wild-type (wt) PVX also efficiently translated in mixedly assembled vRNP "wt PVX RNA + PVX-ST CP"; (iii) opposite result (repression of translation) was obtained with "mixed" vRNP (PVX-ST RNA + wtPVX CP). Therefore, the N-terminal peptide located at the surface of the particle and of the particles plays a key role in translation activation of the RNA encapsidated in vRNP and native virions.

Capsid Proteins↗

PubMed2002

Dendritic BC1 RNA: functional role in regulation of translation initiation.

In neurons, local protein synthesis in synaptodendritic microdomains has been implicated in the growth and plasticity of synapses. Prerequisites for local translation are the targeted transport of RNAs to distal sites of synthesis in dendrites and translational control mechanisms to limit synthesis to times of demand. Here we identify dendritic BC1 RNA as a specific repressor of translation. Experimental use of internal ribosome entry mechanisms and sucrose density gradient centrifugation showed that BC1-mediated repression targets translation at the level of initiation. Specifically, BC1 RNA inhibited formation of the 48S preinitiation complex, i.e., recruitment of the small ribosomal subunit to the messenger RNA (mRNA). However, 48S complex formation that is independent of the eukaryotic initiation factor 4 (eIF4) family of initiation factors was found to be refractory to inhibition by BC1 RNA, a result that implicates at least one of these factors in the BC1 repression pathway. Biochemical experiments indicated a specific interaction of BC1 RNA with eIF4A, an RNA unwinding factor, and with poly(A)-binding protein. Both proteins were found enriched in synaptodendritic microdomains. Significantly, BC1-mediated repression was shown to be effective not only in cap-dependent translation initiation but also in eIF4-dependent internal initiation. The results suggest a functional role of BC1 RNA as a mediator of translational control in local protein synthesis in nerve cells.

Animals↗

PubMed1995

Evidence that XR family interspersed RNA may regulate translation in Xenopus oocytes.

It has been shown that about two thirds of Xenopus oocyte or sea urchin egg cytoplasmic poly(A)+ RNA contains interspersed repetitive sequences. The functional significance of this interspersed RNA has remained unknown. Here the function of a subfamily of interspersed RNA (XR family; McGrew and Richter, 1989: Dev Biol 134:267-270) in Xenopus oocytes was studied. We found that the elimination of T7 XR (one of the two complementary strands of the XR repeat) interspersed RNA by complementary oligodeoxynucleotides significantly inhibited protein synthesis. On the other hand, the injection of in vitro synthesized T7 XR RNA stimulated translation. Moreover, the insertion of the T7 XR RNA sequence into globin mRNA repressed the translation of the globin mRNA. In order to explain these results, we analyzed interactions between the XR interspersed RNA and oocyte proteins. We found that the major XR RNA binding proteins were p56 and p60, which could be the known mRNA "masking" proteins that bind mRNA and inhibit translation. Further, a 42 kD protein has been identified that appears to bind T7 XR RNA relatively specifically, although it interacts with mRNA with a lower affinity. Based on all of these data, we have proposed that interspersed RNA may be involved in regulating translation by competing with mRNA to interact with certain proteins that can regulate translation.

Animals↗

PubMed1993

Symmetrical interactions between the translational operator of the thrS gene and dimeric threonyl transfer RNA synthetase.

Threonyl-tRNA synthetase from Escherichia coli represses the translation of its coding gene, thrS, by binding an operator located in the leader region of its messenger RNA. Published data on the structure of this leader region and on its interaction with threonyl-tRNA synthetase support a model in which each of two stem-and-loop structures mimics the anticodon arm of tRNA(Thr) and binds a different subunit of one synthetase dimer.

Anticodon↗

PubMed2004

Translational control by internal ribosome entry site in Saccharomyces cerevisiae.

To confirm the active involvement of the internal ribosome entry site (IRES)-dependent translation in living eukaryotes, Saccharomyces cerevisiae and HAP4 IRES were used for in vitro and in vivo experiments. Since HAP4 protein might be required for activating mRNAs transcription in yeast cells when they are released from catabolite repression, the translational efficiency of HAP4 mRNA is presumed to increase under such a condition. The in vitro experiment showed clearly that the translational mechanism was shifted from the cap-dependent to the completely IRES-dependent translation when the yeast cells were derepressed from catabolite repression. From in vivo experiment, it was confirmed that the IRES-dependent translational efficiency was in a low level at the beginning of the stationary growth phase, and was enhanced at the glucose-exhausted phase. These results indicate that yeast cells on the catabolite derepressed condition could get a large amount of HAP4 protein for the completely IRES-dependent translation, while the IRES-dependent translational efficiency is increased. It has been proven that IRES functions directing the initiation of translation in living yeast cells.

Gene Expression Regulation, Fungal↗

PubMed1992

Translational control by poly(A) elongation during Xenopus development: differential repression and enhancement by a novel cytoplasmic polyadenylation element.

One characteristic of oocyte maturation and embryogenesis in Xenopus laevis is the activation of translationally repressed (masked) maternal mRNAs by cytoplasmic poly(A) elongation. At maturation, poly(A) elongation is controlled by two cis-acting elements in the 3'-untranslated regions (UTRs) of responsive mRNAs, the hexanucleotide AAUAAA and the cytoplasmic polyadenylation element (CPE), consisting of UUUUUAU or other similar sequences. To investigate poly(A) elongation and translational activation during embryogenesis, we have focused on Cl2 RNA, a representative transcript that undergoes these processes. By injecting radiolabeled Cl2 RNA into fertilized eggs and allowing development to proceed, we found that maximal polyadenylation of this RNA is reached by the 4000-cell blastula stage and that it requires two cis-acting elements, the hexanucleotide AAUAAA and a novel CPE, which is dodecauridine. Interestingly, a shortening of the distance between the two elements changes the timing of maximal polyadenylation to the four-cell stage. The injection of a chloramphenicol acetyl transferase (CAT)-Cl2 chimeric RNA into fertilized eggs not only results in embryonic polyadenylation of the transcript but also 5- to 15-fold more CAT activity compared with eggs injected with CAT RNA or CAT-Cl2 chimeric RNA that is prevented from undergoing poly(A) elongation by a mutation in the polyadenylation hexanucleotide. However, eggs injected with a CAT-Cl2 chimeric RNA that is preadenylated but that cannot undergo further poly(A) elongation contain no more CAT activity than eggs injected with the same RNA without a poly(A) tail, suggesting that the process of poly(A) elongation, and not poly(A) tail length, is important for translation. Finally, we show that precocious poly(A) elongation of Cl2 RNA during oocyte maturation is prevented by a large "masking" element that includes the dodecauridine CPE. The dual role of the CPE in both repression and activation of poly(A) elongation is discussed.

Animals↗

PubMed2002

A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins.

Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 expression. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. In Drosophila, the fly homolog of the FMR1 protein (dFMR1) binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. We have isolated a dFMR1-associated complex that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The dFMR1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. We show that Dmp68 is also required for efficient RNAi. We further show that dFMR1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that dFMR1 is part of the RNAi-related apparatus. Our findings suggest a model in which the RNAi and dFMR1-mediated translational control pathways intersect in Drosophila. Our findings also raise the possibility that defects in an RNAi-related machinery may cause human disease.

Amino Acid Sequence↗

PubMed1995

Binding of pumilio to maternal hunchback mRNA is required for posterior patterning in Drosophila embryos.

Posterior patterning in Drosophila embryos is governed by nanos (nos), which acts by repressing the translation of maternal transcripts of the hunchback (hb) gene. Sites in hb mRNA that mediate this repression, named nanos response elements (NREs), have been identified. However, we know of no evidence of a direct interaction between nos, or any other protein, and the NRE. Here, we show that two proteins present in embryonic extracts, neither one nos, bind specifically to the NRE in vitro. Furthermore, we show that binding in vitro correlates with NRE function in vivo. One of the NRE-binding factors is encoded by pumilio (pum), a gene that, like nos, is essential for abdominal segmentation. These and other observations suggest that pum acts by recognizing the NRE and then recruiting nos. Presumably, the resulting complex inhibits some component of the translation machinery.

Animals↗

PubMed1989

Thymidine kinase synthesis is repressed in nonreplicating muscle cells by a translational mechanism that does not affect the polysomal distribution of thymidine kinase mRNA.

The molecular basis for replication-dependent expression of thymidine kinase (TK) activity (EC 2.7.1.21) was investigated in mouse skeletal muscle cells transformed with multiple copies of the chicken TK gene. When shifted to mitogen-depleted medium, proliferating myoblasts irreversibly withdraw from the cell cycle and commit to terminal differentiation. Early after commitment, postreplicative myocytes maintain nearly proliferative levels of TK mRNA but have greatly reduced levels of TK activity. Metabolic labeling studies with [35S]methionine indicated that the decrease in TK activity was associated with a 10-fold reduction in the rate of TK protein synthesis. Commitment had little effect on the stability or catalytic efficiency of TK protein. The decrease in TK synthetic rate in the continued presence of TK mRNA indicated that translation of TK mRNA was repressed in committed cells. The distribution of TK mRNA between ribonucleoprotein particles and polysomes was determined. In both proliferative cells and committed cells, TK mRNA levels were maximal in polysomes containing five to seven ribosomes. Thus, the synthesis of TK protein in nonreplicating muscle cells was inhibited by a translational mechanism that did not alter the average number of ribosomes engaged by TK mRNA.

Animals↗