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Results for “Translational repression”

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PubMed1997

Structure and function of the iron-responsive element from human ferritin L chain mRNA.

We report the cloning and functional characterization of the iron responsive element (IRE) of human ferritin light (L) chain mRNA from a cDNA library of primary human T lymphocytes. Comparison of this palindromic cDNA element to the IRE predicted from the reported genomic sequence revealed significant differences, resulting in a stem-loop structure with lower stability than the IRE of the heavy (H) chain mRNA. Nevertheless, the L subunit IRE mediated efficient binding of the iron regulatory protein (IRP) in a manner comparable to that of human ferritin H chain mRNA in vitro. In accordance with previous observations on H form transcripts, the cis-acting regulatory IRE motif of human ferritin L chain mRNA was capable of repressing translation under iron deprivation but permitted mobilization of the transcripts into polysomes following iron repletion in vivo.

Base Sequence↗

PubMed1999

The timing of lin-4 RNA accumulation controls the timing of postembryonic developmental events in Caenorhabditis elegans.

The lin-4 gene encodes a small RNA that is required to translationally repress lin-14 toward the end of the first larval stage of Caenorhabditis elegans development. To determine if the timing of LIN-14 protein down-regulation depends on the temporal profile of lin-4 RNA level, we analyzed the stage-specificity of lin-4 RNA expression during wild-type development and examined the phenotypes of transgenic worms that overexpress lin-4 RNA during the first larval stage. We found that lin-4 RNA first becomes detectable at approximately 12 h of wild-type larval development and rapidly accumulates to nearly maximum levels by 16 h. This profile of lin-4 RNA accumulation corresponded to the timing of LIN-14 protein down-regulation. Transgenic strains that express elevated levels of lin-4 RNA prior to 12 h of development display reduced levels of LIN-14 protein and precocious phenotypes consistent with abnormally early loss of lin-14 activity. These results indicate that the temporal profile of lin-4 RNA accumulation specifies the timing of LIN-14 down-regulation and thereby controls the timing of postembryonic developmental events.

Animals↗

PubMed2001

CPEB degradation during Xenopus oocyte maturation requires a PEST domain and the 26S proteasome.

Cytoplasmic poly(A) elongation is widely utilized during the early development of many organisms as a mechanism for translational activation. Targeting of mRNAs for this mechanism requires the presence of a U-rich element, the cytoplasmic polyadenylation element (CPE), and its binding protein, CPEB. Blocking cytoplasmic polyadenylation by interfering with the CPE or CPEB prevents the translational activation of mRNAs that are crucial for oocyte maturation. The CPE sequence and CPEB are also important for translational repression of mRNAs stored in the Xenopus oocyte during oogenesis. To understand the contribution of protein metabolism to these two roles for CPEB, we have examined the mechanisms influencing the expression of CPEB during oogenesis and oocyte maturation. Through a comparison of CPEB mRNA levels, protein synthesis, and accumulation, we find that CPEB is synthesized during oogenesis and stockpiled in the oocyte. Minimal synthesis of CPEB, <3.6%, occurs during oocyte maturation. In late oocyte maturation, 75% of CPEB is degraded coincident with germinal vesicle breakdown. Using proteasome and ubiquitination inhibitors, we demonstrate that CPEB degradation occurs via the proteasome pathway, most likely through ubiquitin-conjugated intermediates. In addition, we demonstrate that degradation requires a 14 amino acid PEST domain.

Amino Acid Sequence↗

PubMed2002

Three-dimensional ultrastructural analysis of RNA distribution within germinal granules of Xenopus.

The germ plasm is a specialized region of oocyte cytoplasm that contains determinants of germ cell fate. In Xenopus oocytes, the germ plasm is a part of the METRO region of mitochondrial cloud. It contains the germinal granules and a variety of coding and noncoding RNAs that include Xcat2, Xlsirts, Xdazl, DEADSouth, Xpat, Xwnt11, fatVg, B7/Fingers, C10/XFACS, and mitochondrial large and small rRNA. We analyzed the distribution of these 11 different RNAs within the various compartments of germ plasm during Xenopus oogenesis and development by using whole-mount electron microscopy in situ hybridization. Serial EM sections were used to reconstruct a three-dimensional image of germinal granule distribution within the METRO region of the cloud and the distribution of RNAs on the granules in oocytes and embryos. We found that, in the oocytes, the majority of RNAs were associated either with the precursor of germinal granules or with the germ plasm matrix. Only Xcat2, Xpat, and DEADSouth RNAs were associated with the mature germinal granules in oocytes, while only Xcat2 and Xpat were associated with germinal granules in embryos. However, Xcat2 was the only RNA that was consistently sequestered inside the germinal granules, while the others were located on the periphery. Xdazl, which functions in germ cell migration/formation, was detected on the matrix between granules. Later in development, Xcat2 mRNA was released from the germinal granules. This coincides with the timing of its translational derepression. These results demonstrate that there is a dynamic three-dimensional architecture to the germinal granules that changes during oogenesis and development. They also indicate that association of specific RNAs with the germinal granules is not a prerequisite for their serving a germ cell function; however, it may be related to their state of translational repression.

Animals↗

PubMed2000

The RGG domain in hnRNP A2 affects subcellular localization.

The heterogeneous nuclear ribonucleoproteins (hnRNP) associate with pre-mRNA in the nucleus and play an important role in RNA processing and splice site selection. In addition, hnRNP A proteins function in the export of mRNA to the cytoplasm. Although the hnRNP A proteins are predominantly nuclear, hnRNP A1 shuttles rapidly between the nucleus and the cytoplasm. HnRNP A2, whose cytoplasmic overexpression has been identified as an early biomarker of lung cancer, has been less well studied. Cytosolic hnRNP A2 overexpression has also been noted in brain tumors, in which it has been correlated with translational repression of Glucose Transporter-1 expression. We now examine the role of arginine methylation on the nucleocytoplasmic localization of hnRNP A2 in the HEK-293 and NIH-3T3 mammalian cell lines. Treatment of either cell line with the methyltransferase inhibitor adenosine dialdehyde dramatically shifts hnRNP A2 localization from the nuclear to the cytoplasmic compartment, as shown both by immunoblotting and by immunocytochemistry. In vitro radiolabeling with [(3)H]AdoMet of GST-tagged hnRNP A2 RGG mutants, using recombinant protein arginine methyltransferase (PRMT1), shows (i) that hnRNP A2 is a substrate for PRMT1 and (ii) that methylated residues are found only in the RGG domain. Deletion of the RGG domain (R191-G253) of hnRNP A2 results in a cytoplasmic localization phenotype, detected both by immunoblotting and by immunocytochemistry. These studies indicate that the RGG domain of hnRNP A2 contains sequences critical for cellular localization of the protein. The data suggest that hnRNP A2 may contain a novel nuclear localization sequence, regulated by arginine methylation, that lies in the R191-G253 region and may function independently of the M9 transportin-1-binding region in hnRNP A2.

3T3 Cells↗

PubMed1995

Citrate utilization gene cluster of the Lactococcus lactis biovar diacetylactis: organization and regulation of expression.

The transport of citrate in Lactococcus lactis biovar diacetylactis is mediated by the citrate permease P. This polypeptide is encoded by the citP gene carried by plasmid pCIT264. In this report, we characterize the citP transcript, identify a cluster of two genes cotranscribed with citP and describe their post-transcriptional regulation. The transcriptional promoter is located 1500 nucleotides upstream of the citP gene and the transcriptional terminator is positioned next to the 3'-end of this gene. The DNA sequence was determined of the region upstream of the citP gene, including the promoter. Two partially overlapping open reading frames, citQ and citR were identified, which could encode polypeptides of 3.9 and 13 kDa respectively. These two genes, together with citP, constitute the cit cluster. Moreover, an IS-like element located between the cit promoter and the citQ open reading frame was identified. This element includes an open reading frame ORF1, which could encode a 33 kDa polypeptide. A translational fusion between the citP and a cat reporter gene showed that translation of citR and citP is coupled, and regulated by CitR. The cit mRNA was subjected to specific cleavage after addition of rifampicin to the bacterial cultures. We propose that expression of the cit cluster is controlled at the post-transcriptional level by mRNA processing at a putative complex secondary structure and by translational repression mediated by CitR.

Amino Acid Sequence↗

PubMed1992

An iron-sulfur cluster plays a novel regulatory role in the iron-responsive element binding protein.

Post-transcriptional regulation of genes important in iron metabolism, ferritin and the transferrin receptor (TfR), is achieved through regulated binding of a cytosolic protein, the iron-responsive element binding protein (IRE-BP), to RNA stem-loop motifs known as iron-responsive elements (IREs). Binding of the IRE-BP represses ferritin translation and represses degradation of the TfR mRNA. The IRE-BP senses iron levels and accordingly modifies binding to IREs through a novel sensing mechanism. An iron-sulfur cluster of the IRE-BP reversibly binds iron; when cytosolic iron levels are depleted, the cluster becomes depleted of iron and the IRE-BP acquires the capacity to bind IREs. When cytosolic iron levels are replete, the IRE-BP loses RNA binding capacity, but acquires enzymatic activity as a functional aconitase. RNA binding and aconitase activity are mutually exclusive activities of the IRE-BP, and the state of the iron-sulfur cluster determines how the IRE-BP will function.

Aconitate Hydratase↗

PubMed1988

Translational control in E. coli: the case of threonyl-tRNA synthetase.

Genetic studies have shown that expression of the E. coli threonyl-tRNA synthetase (thrS) gene is negatively auto-regulated at the translational level. A region called the operator, located 110 nucleotides downstream of the 5' end of the mRNA and between 10 and 50 bp upstream of the translational initiation codon in the thrS gene, is directly involved in that control. The conformation of an in vitro RNA fragment extending over the thrS regulatory region has been investigated with chemical and enzymatic probes. The operator locus displays structural similarities to the anti-codon arm of threonyl tRNA. The conformation of 3 constituent mutants containing single base changes in the operator region shows that replacement of a base in the anti-codon-like loop does not induce any conformational change, suggesting that the residue concerned is directly involved in regulation. However mutation in or close to the anti-codon-like stem results in a partial or complete rearrangement of the structure of the operator region. Further experiments indicate that there is a clear correlation between the way the synthetase recognises each operator, causing translational repression, and threonyl-tRNA.

Amino Acyl-tRNA Synthetases↗

PubMed2000

Functional analysis of the leader peptide of the yeast gene CPA1 and heterologous regulation by other fungal peptides.

The 25-amino-acid leader peptide present at the 5' end of yeast CPA1 mRNA is responsible for the translational repression of that gene by arginine. We show here that the active domain of the yeast peptide is highly specific and extends over amino acids 6-23. The region between amino acids 6-21 is well conserved between similar peptides present upstream of CPA1-homologous genes in other fungi. The Neurospora crassa arg-2 peptide represses the expression of CPA1, whereas the peptide from Aspergillus nidulans has only a weak regulatory effect. Such results suggest that the N- and C-terminal amino acids of the peptide may influence its regulatory activity. We also show that the transcription start sites of CPA1 are not modified when the cells are grown in the presence of arginine, nor in a strain carrying an inactive peptide.

Amino Acid Sequence↗

PubMed2007

Conservation of large foci formation in arrested oocytes of Caenorhabditis nematodes.

Within the rhabditid phylogeny of nematodes, the great majority of species are gonochoristic, having evolved as obligate male/female species. In contrast, the well-studied nematode model system, Caenorhabditis elegans, is androdioecious, utilizing a hermaphroditic/male reproductive system. We have previously determined that in the arrested oocytes of old-aged C. elegans hermaphrodites with depleted sperm, large cytoplasmic ribonucleoprotein foci form. The formation of these foci is reversible, as they dissociate within 3 h after a male mates with the hermaphrodite, resupplying it with sperm. The functional significance of these oocyte foci is not known and previously has not been clear for a hermaphroditic species in which oocytes of young adults wait only approximately 23 min to be fertilized. One hypothesis is that the foci function to maintain maternal mRNAs in oocytes while fertilization is delayed. In this paper, we examine four gonochoristic rhabditid species: Caenorhabditis remanei, Caenorhabditis sp. CB5161, Caenorhabditis sp. PS1010, and Rhabditella axei DF5006. We demonstrate that in three of these four species, ovulation arrests in unmated females until mating occurs and large cytoplasmic foci develop in arrested oocytes. The oocyte foci contain nuclear pore proteins and, in C. remanei at least, the RNA-binding protein MEX-3 as well as RNA. We speculate that these foci maintain the integrity of ooctyes, possibly maintaining the stability or translational repression of maternal mRNAs in unmated females. We further speculate that their presence in oocytes of old-aged C. elegans hermaphrodites is due to conservation from an ancestral gonochoristic state.

Animals↗

PubMed2001

Complex control of the developmental regulatory locus brlA in Aspergillus nidulans.

brlA is a primary regulator of asexual development in Aspergillus nidulans. Activation of brlA is necessary and sufficient for conidiophore development. It is known that brlA produces two overlapping transcripts, designated brlAalpha and brlAbeta. We found that expression of brlA is subject to complex regulation, in that activation of the two brlA transcripts is regulated at different levels. While brlAalpha is regulated at the transcriptional level, brlAbeta is regulated at both the transcriptional and translational levels. brlAalpha expression requires both abaA and brlA, but overexpression of brlAbeta can induce brlAalpha in an abaA mutant. brlAbetamuORF, a short ORF located upstream of the brlA initiator codon, regulates expression of brlA by damping translation of the brlAbeta ORF, and translational repression of brlA expression prevents premature development in A. nidulans. Transcriptional control of brlAbeta is apparently independent of BrlA. In order to understand better the transcriptional control of brlAalpha and brlAbeta, we have made 5' deletions in the essential approximately 2-kb upstream control sequences that regulate brlAbeta transcription and fused them to the E. coli lacZ reporter gene. Various deletions in this region resulted in only minor changes in the regulation of beta-galactosidase expression. The results of the deletion experiments indicate that there are probably several cis-acting control sequences involved in the regulation of brlAbeta. As a complementary approach, we fused various fragments of the 2034-bp brlAbeta and 754-bp brlAalpha control sequences to an otherwise inactive amdS::lacZ fusion, in order to search for regions that are sufficient to place the reporter under developmental control. We identified two approximately 600-bp brlAbeta fragments extending from -2901 to -2293 and -967 to -414, respectively, and a approximately 150-bp brlAalpha segment from -271 to -127, that confer activity on the inactive amdS promoter. brlA is overexpressed in an abaA null mutant and one site for abaA-dependent repression is apparently located in the -742 to -414 brlAbeta fragment. This indicates that abaA-mediated repression of brlA expression occurs through control of brlAbeta, but apparently involves a mechanism that does not require AbaA binding to brlA(p) sequences, because there are no AbaA binding sites in this region.

Aspergillus nidulans↗

PubMedDate not supplied

Diversity of endogenous small non-coding RNAs in Oryza sativa.

Small non-coding RNAs play important roles in regulating cell functions by controlling mRNA turnover and translational repression in eukaryotic cells. Here we isolated 162 endogenous small RNA molecules from Oryza sativa, which ranged from 16 to 35 nt in length. Further analysis indicated that they represented a diversity of small RNA molecules, including 17 microRNAs (miRNAs), 30 tiny non-coding RNAs (tncRNAs) and 20 repeat-associated small interfering RNAs (rasiRNAs). Among 17 miRNAs, 13 were novel miRNA candidates and their potential targets were important regulatory genes in the rice genome. We also found that a cluster of small RNAs, including many rasiRNAs, matched to a nuclear DNA fragment that evolutionarily derived from chloroplast. These results demonstrate clearly the existence of distinct types of small RNAs in rice and further suggest that small RNAs may control gene regulation through diverse mechanisms.

Base Sequence↗

PubMed1987

Putative nuclease-sensitive control element in unfertilized eggs of the sea urchin Lytechinus pictus.

Very limited nuclease-treatment of actively translating lysates from unfertilized eggs of the sea urchin Lytechinus pictus results in activation of the lysate. This activation is reflected by the ability of the nuclease-treated (or 'nuclease-activated') lysates to more efficiently utilize exogenous added RNAs. The activation may be reversed by adding small aliquots of untreated lysate to the nuclease-activated lysates. These results suggest that a nuclease-sensitive negative control element may be involved in the translational repression of unfertilized eggs, and that a deactivation of this control element is partially responsible for the dramatic activation of protein synthesis following fertilization in sea urchin eggs.

Ovum↗

PubMed1987

S4-alpha mRNA translation regulation complex. II. Secondary structures of the RNA regulatory site in the presence and absence of S4.

The secondary structure of the Escherichia coli alpha mRNA leader sequence has been determined using nucleases specific for single- or double-stranded RNA. Three different length alpha RNA fragments were studied at 0 degrees C and 37 degrees C. A very stable eight base-pair helix forms upstream from the ribosome initiation site, defining a 29 base loop. There is evidence for base-pairing between nucleotides within this loop and for a "pseudo-knot" interaction of some loop bases with nucleotides just 3' to the initiation codon, forming a region of complex structure. A weak helix also pairs sequences near the 5' terminus of the alpha mRNA with bases near the Shine-Dalgarno sequence. Affinity constants for the translational repressor S4 binding different length alpha mRNA fragments indicate that most of the S4 recognition features must be contained within the main helix and hairpin regions. Binding of S4 to the alpha mRNA alters the structure of the 29 base hairpin region, and probably melts the weak pairing between the 5' and 3' termini of the leader. The pseudo-knot structure and the conformational changes associated with it provide a link between the structures of the S4 binding site and the ribosome binding site. The alpha mRNA may therefore play an active role in mediating translational repression.

Autoradiography↗

PubMed1989

S4-16 S ribosomal RNA complex. Binding constant measurements and specific recognition of a 460-nucleotide region.

The region of the Escherichia coli 16 S ribosomal RNA recognized by the ribosomal protein S4 has been defined by assaying a set of 13 16 S rRNA fragments for S4 binding. The fragments were prepared by transcription in vitro, and binding constants were measured in three ways: retention of labeled RNA fragments on nitrocellulose filters by S4; co-sedimentation of labeled S4 with RNA fragments in sucrose gradients; and the distribution of labeled S4 between two RNAs of different sizes in a sucrose gradient. All three methods gave similar relative binding strengths for a variety of 16 S rRNA and non-specific (23 S rRNA) sequences, with the exception of two of the largest 16 S rRNA fragments; these gave smaller association constants in the filter retention assay than in the other methods. We found that specific complexes of S4 with these larger RNAs do not bind well to filters, leaving non-specific complexes to dominate the assay. Specific complexes with RNAs less than or equal to 891 nucleotides were retained efficiently by S4 on filters, and gave reliable binding constants. All 16 S rRNA fragments containing nucleotides 39 to 500 bound S4 with the same affinity as intact 16 S rRNA, while all fragments with endpoints within 39 to 500 bound at least tenfold more weakly. This sequence must be able to fold independently of the rest of the rRNA. Comparison of this minimal 462-nucleotide S4 binding site with S4 footprinting results suggests that S4 binding might alter the conformations of RNA neighboring the 39 to 500 region in the intact 16 S rRNA. Specific S4-rRNA binding is not sensitive to KCl concentration, but a more normal salt dependence is seen in K2SO4 (delta logK/delta log[K+] approximately -3.3). This duplicates the behavior of the specific S4-alpha mRNA translational repression complex, arguing that S4 recognizes both the mRNA and rRNA substrates by the same mechanism. Mg2+ is not required to form the specific rRNA complex, at least under conditions which stabilize RNA structure (0.35 M-KCl, 5 degrees C).

Binding Sites↗

PubMed1996

Anti-interferon activity of adenovirus-2-encoded VAI and VAII RNAs in translation in cultured human cells.

The mode of anti-interferon action of VAI and VAII RNAs of adenovirus type 2 (Ad2) was studied by transfecting interferon-alpha (IFN-alpha)-treated KB cells in culture with a plasmid construct containing the VAI or VAII RNA gene and an SV40 promoter-chloramphenicol acetyltransferase (CAT) gene construct as reporter (pSV2-CAT). The longer the treatment of KB cells with IFN-alpha (2,000 IU/ml) lasted, the higher was the inhibition of CAT expression. A maximum of 76% inhibition was attained without pronounced cytotoxicity during 48 h of treatment. The earlier the VAI RNA gene was transfected, the higher was the enhancement of CAT expression. CAT activity increased from 113 to 157% in normal cells and 200-400% in IFN-alpha treated cells, as compared with the corresponding controls without VAI RNA transfection. The level of CAT mRNA was neither appreciably decreased by IFN-alpha treatment, nor detectably increased by VAI or VAII RNA. The effect of VA RNA thus appeared to be on translation rather than on transcription. The relative constancy of the level of CAT mRNA indicated that IFN-alpha inhibition of CAT expression was not due to the activation of RNase L, but due mainly to translational repression. The level of VAII RNA expressed was only 9-13% of that of VAI RNA. Nevertheless, VAII RNA gene transfection stimulated CAT activity to 112% of the control in non-IFN-alpha-treated cells, and 126-182% in IFN-alpha-inhibited cells. When IFN-alpha treatment was started late after VAI RNA cotransfection, CAT expression increased to 169% which was higher than the expression in cotransfected control cells without IFN-alpha treatment. The enhanced level of CAT activity was in remarkable contrast to the IFN-alpha inhibited level of 25% without VA RNA co-transfection when IFN-alpha was added upon seeding. The enhanced CAT activity in cells treated late with IFN-alpha could be ascribed to higher levels of VA RNAs.

Adenoviruses, Human↗

PubMed1989

Modeling loop structures in proteins and nucleic acids: an RNA stem-loop.

We have used a novel modeling technique, based on combining information from several preexisting structures, to generate a three-dimensional (3D) model for the RNA stem-loop responsible for translational repression of the MS2 RNA bacteriophage replicase. Specific features of the model have been tested experimentally by chemical and enzymatic structural probes; results from these experiments have been used to "improve" the model by fixing initial assumptions. The new model and chemical modification data are in part consistent, and further predictions are being tested. The modeling algorithm has a wide range of potential applications, particularly to loop regions in proteins and nucleic acids.

Algorithms↗

PubMed1991

The maternal gene product D7 is not required for early Xenopus development.

The Xenopus D7 gene codes for a novel protein whose expression is restricted to early development. D7 protein is synthesized for the first time during oocyte maturation (1988, Genes Dev. 2, 1296-1306). Injection of D7 RNA into the full-grown oocyte and its subsequent translation into D7 protein neither induced oocyte maturation nor affected the kinetics of hormone-induced maturation. Overexpression of D7 protein by 20-fold in the early Xenopus embryo by injection of D7 RNA into fertilized eggs did not affect subsequent development. Oocytes specifically lacking D7 mRNA were generated by oligodeoxynucleotide-mediated RNA destruction within the oocyte. Unfertilized eggs generated from such oocytes lacked detectable D7 protein, but nevertheless could be activated and fertilized. Embryos generated from such eggs, estimated to contain less than 5% of wildtype levels of D7 protein, developed normally up to the tailbud stage. Thus the D7 protein, the product of a maternal mRNA that is under strict translational repression in oocytes, appears not to be required for oocyte maturation, activation, fertilization or early embryonic development in Xenopus.

Animals↗