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PubMed2005

A dual upstream open reading frame-based autoregulatory circuit controlling polyamine-responsive translation.

A novel form of translational regulation is described for the key polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC). Plant AdoMetDC mRNA 5' leaders contain two highly conserved overlapping upstream open reading frames (uORFs): the 5' tiny and 3' small uORFs. We demonstrate that the small uORF-encoded peptide is responsible for constitutively repressing downstream translation of the AdoMetDC proenzyme ORF in the absence of increased polyamine levels. This first example of a sequence-dependent uORF to be described in plants is also functional in Saccharomyces cerevisiae. The tiny uORF is required for normal polyamine-responsive AdoMetDC mRNA translation, and we propose that this is achieved by control of ribosomal recognition of the occluded small uORF, either by ribosomal leaky scanning or by programmed -1 frameshifting. In vitro expression demonstrated that both the tiny and the small uORFs are translated. This tiny/small uORF configuration is highly conserved from moss to Arabidopsis thaliana, and a more diverged tiny/small uORF arrangement is found in the AdoMetDC mRNA 5' leader of the single-celled green alga Chlamydomonas reinhardtii, indicating an ancient origin for the uORFs.

Adenosylmethionine Decarboxylase↗

PubMed1995

Translational repressor activity is equivalent and is quantitatively predicted by in vitro RNA binding for two iron-responsive element-binding proteins, IRP1 and IRP2.

Iron regulatory proteins (IRPs) bind to specific RNA stem-loop structures known as iron-responsive elements (IREs) which mediate the post-transcriptional regulation of many genes of iron metabolism. Most studies have focused on the role of IRP1, which has previously been shown to bind with high affinity to IREs and mediate repression of in vitro translation of ferritin mRNAs. More recently, a second IRP has been identified that is expressed in all tissues and that binds IREs (Rouault, T. A., Haile, D. H., Downey, W. E., Philpott, C. C., Tang, C., Samaniego, F., Chin, J., Paul, I., Orloff, D., Harford, J. B., and Klausner, R. D. (1992) BioMetals 5, 131-140; Henderson, B. R., Seiser, C., and Kuhn, L. C. (1993) J. Biol. Chem. 268, 27327-27334; Guo, B., Yu, Y., and Leibold, E. A. (1994) J. Biol. Chem. 269, 24252-24260; Samaniego, F., Chin, J., Iwai, K., Rouault, T. A., and Klausner, R. D. (1994) J. Biol. Chem. 269, 30904-30910). Here we report that purified recombinant IRP2 inhibits translation of ferritin mRNAs with a molar efficacy equal to that of recombinant IRP1. There is a quantitative correlation between binding to isolated RNA target motifs, as judged by gel retardation assays, and translational repressor function as assayed in an in vitro translation system. In contrast to IRP1, IRP2 is not inactivated for RNA binding by alkylation with N-ethylmaleimide or phenylmaleimide, and as we would therefore predict, IRP2 treated with N-ethylmaleimide remains an effective repressor of ferritin translation. As IRP1 and IRP2 clearly have equal capability of mediating translational repression in vitro, the contributions of both IRPs to overall regulation must be considered in describing the pathways of iron regulated gene expression in individual cells.

Alkylation↗

PubMed2002

[RNA interference for mammalian cells].

Knock-out of mammalian genes is technically troublesome and time-consuming compared to those of lower animals. RNA interference (RNAi) is a strategy of sequence-specific post-translational gene silencing, and it has been successfully applied for disruption of gene transcripts in C. elegans. Gene silencing by RNAi had not been accomplished in mammalian cells until recently, because bystander activation of DNA-dependent protein kinase (PKR) coincides to disturb gene silencing by long double-stranded RNA (dsRNA), resulting in non-specific repression of translation of many proteins. A breakthrough has come recently with a report suggesting that 20- or 21-bp duplex RNA with 2 bp 3' overhang are made by DICER protein that cleaves long dsRNA during RNAi reactions in vivo and the resultant short dsRNAs mediate RNAi. Those short dsRNAs, namely, small interference RNA (siRNA), barely activate PKR. Using synthesized 21 bp siRNA, T. Tuschl's group has challenged to establish an artificial RNAi method suitable for mammalian cells. Their report was favorable in that siRNA specifically suppressed targeted gene translation in mammalian cells during culture without activation of PKR. Recently K. Taira's group developed the vector-based siRNA expression system by which RNAi is feasible in mammalian cells. Almost all genes can be targeted by RNAi. RNAi methods require minimal time and labor; therefore, mammalian gene knockdown by RNAi will become popular in the near future.

Gene Silencing↗

PubMed2005

Nanos downregulates transcription and modulates CTD phosphorylation in the soma of early Drosophila embryos.

nanos (nos) specifies posterior development in the Drosophila embryo by repressing the translation of maternal hb mRNA. In addition to this somatic function, nos is required in the germline progenitors, the pole cells, to establish transcriptional quiescence. We have previously reported that nos is required to keep the Sex-lethal establishment promoter, Sxl-Pe, off in the germline of both sexes. We show here that nos also functions to repress Sxl-Pe activity in the surrounding soma. Sxl-Pe is inappropriately activated in the soma of male embryos from nos mothers, while Sxl-Pe can be repressed in female embryos by ectopic Nos protein. nos appears to play a global role in repressing transcription in the soma as the effects of nos on promoter activity are correlated with changes in the phosphorylation status of the carboxy terminal domain (CTD) repeats of the large RNA polymerase II subunit. Finally, we present evidence indicating that the suppression of transcription in the soma by Nos protein is important for normal embryonic development.

3' Untranslated Regions↗

PubMed2003

Adenosine-rich elements present in the 5'-untranslated region of PABP mRNA can selectively reduce the abundance and translation of CAT mRNAs in vivo.

The poly(A)-binding protein (PABP) is a highly conserved eukaryotic protein whose synthesis is regulated at the post-transcriptional level. The binding of PABP to the poly(A)-rich element found in the 5'-untranslated region (5'UTR) of PABP mRNA specifically inhibits its own translation. In this report, we show that similar adenosine-rich elements in the 5'UTR of the chloramphenicol acetyl-transferase (CAT) gene can significantly reduce the reporter mRNA abundance and translation in human 293 cells. The reduction in mRNA level, but not CAT expression, is dependent on the size of the 5'UTR poly(A) element. Furthermore, one 5'UTR-tethered PABP molecule is enough to inhibit CAT expression without affecting its mRNA level. We propose that the control of PABP synthesis may involve mRNA decay and the repression of translation.

5' Untranslated Regions↗

PubMed1992

Alteration of translation and stability of mRNA for the poly(A)-binding protein during myogenesis.

The regulation of synthesis of various factors involved in mRNA translation during differentiation of muscle cells was examined. The steady-state levels of mRNAs coding for eukaryotic initiation factor (eIF) 2 alpha, 2 beta and elongation factor (eEF)-1 alpha were measured in both proliferating rat L6 myoblast and differentiated myotubes. The steady-state levels of these mRNAs were not altered during myogenesis. Furthermore, the distribution of these mRNAs between repressed and translated populations remained unchanged. Recent studies suggest a role for poly(A)-binding protein (PABP) in translation initiation. Therefore, we also examined the expression of PABP mRNA during myogenesis. The PABP mRNA was less abundant in myotubes compared to myoblasts. However, the synthesis of PABP remained unchanged. In myoblasts, approximately 50-60% of the total mRNA was associated with polyribosomes, whereas in myotubes more than 80% of the mRNA was associated with polyribosomes. These results, therefore, suggest that the PABP mRNA was more efficiently translated in differentiated myotubes than in the proliferating myoblasts. Measurement of the stability and transcription of PABP mRNA showed that, while transcription was not affected during myogenesis, the stability of the mRNA was reduced in differentiated cells. The t1/2 of PABP mRNA in myoblasts was 13 h compared to 7.5 h in myotubes. This observation suggests that the reduced steady-state level of PABP mRNA in myotube were largely due to the change in stability of this mRNA during myogenesis.

Animals↗

PubMed2006

CPEB3 and CPEB4 in neurons: analysis of RNA-binding specificity and translational control of AMPA receptor GluR2 mRNA.

CPEB is a sequence-specific RNA-binding protein that promotes polyadenylation-induced translation in oocytes and neurons. Vertebrates contain three additional genes that encode CPEB-like proteins, all of which are expressed in the brain. Here, we use SELEX, RNA structure probing, and RNA footprinting to show that CPEB and the CPEB-like proteins interact with different RNA sequences and thus constitute different classes of RNA-binding proteins. In transfected neurons, CPEB3 represses the translation of a reporter RNA in tethered function assays; in response to NMDA receptor activation, translation is stimulated. In contrast to CPEB, CPEB3-mediated translation is unlikely to involve cytoplasmic polyadenylation, as it requires neither the cis-acting AAUAAA nor the trans-acting cleavage and polyadenylation specificity factor, both of which are necessary for CPEB-induced polyadenylation. One target of CPEB3-mediated translation is GluR2 mRNA; not only does CPEB3 bind this RNA in vitro and in vivo, but an RNAi knockdown of CPEB3 in neurons results in elevated levels of GluR2 protein. These results indicate that CPEB3 is a sequence-specific translational regulatory protein.

3' Untranslated Regions↗

PubMed2001

Cloning of cDNA for cathepsin B mRNA 3'-untranslated-region-binding protein (CBBP), and characterization of recombinant CBBP.

Previously, we purified the cathepsin B mRNA 3'-untranslated-region-binding protein (CBBP) from Sarcophaga and suggested its participation in the translational regulation of cathepsin B mRNA in this insect. In this study, we isolated a full length cDNA for CBBP. CBBP was an RNA-binding protein that contained four RGG boxes and four zinc finger motifs required for RNA binding. CBBP was shown to be localized in both the nuclei and cytoplasm of Sarcophaga hemocytes. Recombinant CBBP bound to the entire region of cathepsin B mRNA and repressed its translation in vitro.

3' Untranslated Regions↗

PubMed1999

Translational regulation of ornithine decarboxylase and other enzymes of the polyamine pathway.

It has long been known that polyamines play an essential role in the proliferation of mammalian cells, and the polyamine biosynthetic pathway may provide an important target for the development of agents that inhibit carcinogenesis and tumor growth. The rate-limiting enzymes of the polyamine pathway, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are highly regulated in the cell, and much of this regulation occurs at the level of translation. Although the 5' leader sequences of ODC and AdoMetDC are both highly structured and contain small internal open reading frames (ORFs), the regulation of their translation appears to be quite different. The translational regulation of ODC is more dependent on secondary structure, and therefore responds to the intracellular availability of active eIF-4E, the cap-binding subunit of the eIF-4F complex, which mediates translation initiations. Cell-specific translation of AdoMetDC appears to be regulated exclusively through the internal ORF, which causes ribosome stalling that is independent of eIF-4E levels and decreases the efficiency with which the downstream ORF encoding AdoMetDC protein is translated. The translation of both ODC and AdoMetDC is negatively regulated by intracellular changes in the polyamines spermidine and spermine. Thus, when polyamine levels are low, the synthesis of both ODC and AdoMetDC is increased, and an increase in polyamine content causes a corresponding decrease in protein synthesis. However, an increase in active eIF-4E may allow for the synthesis of ODC even in the presence of polyamine levels that repress ODC translation in cells with lower levels of the initiation factor. In contrast, the amino acid sequence that is encoded by the upstream ORF is critical for polyamine regulation of AdoMetDC synthesis and polyamines may affect synthesis by interaction with the putative peptide, MAGDIS.

Adenosylmethionine Decarboxylase↗

PubMed1993

Translational regulation via iron-responsive elements by the nitric oxide/NO-synthase pathway.

Nitric oxide (NO) produced from L-arginine by NO synthases (NOS) is a transmitter known to be involved in diverse biological processes, including immunomodulation, neurotransmission and blood vessel dilatation. We describe a novel role of NO as a signaling molecule in post-transcriptional gene regulation. We demonstrate that induction of NOS in macrophage and non-macrophage cell lines activates RNA binding by iron regulatory factor (IRFs), the central trans regulator of mRNAs involved in cellular iron metabolism. NO-induced binding of IRF to iron-responsive elements (IRE) specifically represses the translation of transfected IRE-containing indicator mRNAs as well as the biosynthesis of the cellular iron storage protein ferritin. These findings define a new biological function of NO and identify a regulatory connection between the NO/NOS pathway and cellular iron metabolism.

Amino Acid Oxidoreductases↗

PubMed1990

The translational regulation of threonyl-tRNA synthetase. Functional relationship between the enzyme, the cognate tRNA and the ribosome.

The E. coli threonyl-tRNA synthetase gene is negatively autoregulated at the translational level by a direct binding of the enzyme to the leader region of the thrS mRNA. This region folds in four well-defined domains. The enzyme binds to the leader at two major sites: the first is a stem-loop structure located in domain II upstream of the translational initiation site (domain I) which shares structural analogies with the anticodon arm of several tRNA(Thr) isoacceptors. The second site corresponds to a stable stem-loop structure located in domain IV. Both sites are separated by a large unpaired region (domain III). In vivo and in vitro experiments show that the structural integrity of both sites is required for the regulatory process. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. tRNA(Thr) suppresses this inhibitory effect by displacing the mRNA from the enzyme at both the upstream stem-loop structure and the tRNA-like anticodon arm.

Anticodon↗

PubMed1999

Degradation of FinP antisense RNA from F-like plasmids: the RNA-binding protein, FinO, protects FinP from ribonuclease E.

Transfer of F-like plasmids is regulated by the FinOP system, which controls the expression of traJ, a positive regulator of the transfer operon. F FinP is a 79 base antisense RNA, composed of two stem-loops, complementary to the 5' untranslated leader of traJ mRNA. Binding of FinP to the traJ leader sequesters the traJ ribosome binding site, preventing its translation and repressing plasmid transfer. The FinO protein binds stem-loop II of FinP and traJ mRNA and promotes duplex formation in vitro. FinO stabilizes FinP, increasing its effective concentration in vivo. To determine how FinO protects FinP from decay, the degradation of FinP was examined in a series of ribonuclease-deficient strains. Using Northern blot analysis, full-length FinP was found to be stabilized sevenfold in an RNase E-deficient strain. The major site of RNase E cleavage was mapped on synthetic FinP, to the single-stranded region between stem-loops I and II. A secondary site near the 5' end ( approximately 10 bases) was also observed. A GST-FinO fusion protein protected FinP from RNase E cleavage at both sites in vitro. Two duplexes between FinP and traJ mRNA were detected in an RNase III-deficient strain. The larger duplex resulted from extension of the FinP transcript at its 3' end, suggesting readthrough at the terminator that corresponds to FinP stem-loop II. A point mutant of finP (finP305; C30U) that is unable to repress traJ in the presence of FinO was also characterized. The pattern of RNase E digestion of finP305 RNA differed from FinP, and GST-FinO did not protect finP305 RNA from cleavage in vitro. The half-life of finP305 RNA decreased more than tenfold in vivo, such that the steady-state levels of finP305 RNA, in the presence of FinO, were insufficient to significantly reduce the level of traJ mRNA available for translation, allowing derepressed levels of transfer.

Bacterial Outer Membrane Proteins↗

PubMed2004

The fragile X protein controls microtubule-associated protein 1B translation and microtubule stability in brain neuron development.

The fragile X mental retardation protein (FMRP) is a selective RNA-binding protein implicated in regulating translation of its mRNA ligands. The absence of FMRP results in fragile X syndrome, one of the leading causes of inherited mental retardation. Delayed dendritic spine maturation was found in fragile X mental retardation patients as well as in Fmr1 knockout (KO) mice, indicating the functional requirement of FMRP in synaptic development. However, the biochemical link between FMRP deficiency and the neuronal impairment during brain development has not been defined. How FMRP governs normal synapse development in the brain remains elusive. We report here that the developmentally programmed FMRP expression represses the translation of microtubule associated protein 1B (MAP1B) and is required for the accelerated decline of MAP1B during active synaptogenesis in neonatal brain development. The lack of FMRP results in misregulated MAP1B translation and delayed MAP1B decline in the Fmr1 KO brain. Furthermore, the aberrantly elevated MAP1B protein expression leads to abnormally increased microtubule stability in Fmr1 KO neurons. Together, these results indicate that FMRP plays critical roles in controlling cytoskeleton organization during neuronal development, and the abnormal microtubule dynamics is a conceivable underlying factor for the pathogenesis of fragile X mental retardation.

Animals↗

PubMed1992

Selection and characterization of randomly produced mutants of gene V protein of bacteriophage M13.

Gene V protein of bacteriophage Ff (M13, f1, fd) is a master regulator of phage DNA replication and phage mRNA translation. It exerts these two functions by binding to single-stranded viral DNA or to specific sequences in the 5' ends of its target mRNAs, respectively. To study the structure/function relationship of gene V protein, M13 gene V was inserted in a phagemid expression vector and a library of missense and nonsense mutants was constructed by random chemical mutagenesis. Phagemids encoding gene V proteins with decreased biological activities were selected and the nucleotide sequences of their gene V fragments were determined. Furthermore, the mutant proteins were characterized both with respect to their ability to inhibit the production of phagemid DNA transducing particles and their ability to repress the translation of a chimeric lacZ reporter gene whose expression is controlled by the promoter and translational initiation signals of M13 gene II. From the data obtained, it can be deduced that the mechanism by which gene V protein binds to single-stranded DNA differs from the mechanism by which it binds to its target sequence in the gene II mRNA.

Bacteriophages↗

PubMed2005

Involvement of Hu and heterogeneous nuclear ribonucleoprotein K in neuronal differentiation through p21 mRNA post-transcriptional regulation.

The Hu family is a group of neuronal RNA-binding proteins required for neuronal differentiation in the developing nervous system. Previously, Hu proteins have been shown to enhance the stabilization and/or translation of target mRNAs, such as p21 (CIP1), by binding to AU-rich elements in untranslated regions (UTRs). In this study, we show that Hu induces p21 expression, cell cycle arrest, and neuronal differentiation in mouse neuroblastoma N1E-115 cells. p21 expression is also up-regulated during Me2SO-induced differentiation in N1E-115 cells and is controlled by post-transcriptional mechanisms through its 3'-UTR. To investigate the molecular mechanisms of Hu functions, we used a proteomics strategy to isolate Hu-interacting proteins and identified heterogeneous nuclear ribonucleoprotein (hnRNP) K. hnRNP K also specifically binds to CU-rich sequences in p21 mRNA 3'-UTR and represses its translation in both nonneuronal and neuronal cells. Further, using RNA interference experiments, we show that the Hu-p21 pathway contributes to the regulation of neurite outgrowth and proliferation in N1E-115 cells, and this pathway is antagonized by hnRNP K. Our results suggest a model in which the mutually antagonistic action of two RNA-binding proteins, Hu and hnRNP K, control the timing of the switch from proliferation to neuronal differentiation through the post-transcriptional regulation of p21 mRNA.

3' Untranslated Regions↗

PubMed1987

Differentiation apparently repressed by the nucleus. Rapidly-induced pigmentation of enucleated melanoma cells.

There is evidence for cytoplasmic control over gene expression in cell differentiation, but still very little is known of the intracellular mechanism, nuclear, cytoplasmic, or both, which actively initiates the differentiation of one cell type into another. Here the role of the cytoplasm was examined in the induction of differentiation of cultured mouse melanoma cells by melanocyte-stimulating hormone and alkaline medium. Intact cells were compared with cytoplasts, cells enucleated by centrifugation in the presence of cytochalasin D (CD). Surprisingly, early inductions of pigment (melanin) synthesis and of the principal melanin-synthesizing enzyme activity, tyrosinase, could be achieved in cytoplasts. Indeed these early changes were slower in nucleated cells and were accelerated by the inhibitor of protein synthesis, cycloheximide. Thus the initial activation of tyrosinase and melanin synthesis--although not necessarily any other or later aspects of melanoma cell differentiation--is apparently controlled through a labile, transcription- and translation-dependent repression. To our knowledge this is a novel mechanism for the initiation of differentiation; its generality remains to be tested.

Animals↗

PubMed1982

Translational control of bacteriophage f1 gene II and gene X proteins by gene V protein.

The gene II region of bacteriophage f1 DNA codes for two proteins, the 46 kd gene II protein and the 13 kd gene X protein, which results from an in-phase start at codon 300 of gene II. Using antigene II protein IgG, we show that the intracellular concentration of both proteins is controlled by the phage gene V protein. In wild-type f1-infected cells, the amount of gene II protein reaches a plateau of about 1500 molecules per cell at 20 min after infection, as measured by blot immunoassay. Similarly, the amount of gene X protein reaches a peak of about 500 molecules per cell around 10 min after infection. In contrast, when the gene V protein is inactive, both gene II and gene X proteins continue to accumulate at a high rate for at least 40 min after infection. This difference is caused by decreased synthesis of gene II and gene X proteins in the presence of gene V protein, which represses the translation of these two proteins.

Antibodies, Viral↗

PubMed2003

The function of circadian RNA-binding proteins and their cis-acting elements in microalgae.

An endogenous clock regulates the temporal expression of genes/mRNAs that are involved in the circadian output pathway. In the bioluminescent dinoflagellate Gonyaulax polyedra circadian expression of the luciferin-binding protein (LBP) is controlled at the translational level. Thereby, a clock-controlled RNA-binding protein, called circadian controlled translational regulator (CCTR), interacts specifically with an UG-repeat, which is situated in the lbp 3' UTR. Its binding activity correlates negatively with the amount of LBP during a circadian cycle. In the green alga Chlamydomonas reinhardtii, a clock-controlled RNA-binding protein (CHLAMY 1) was identified, which represents an analog of the CCTR from the phylogenetically diverse alga G. polyedra. CHLAMY 1 binds specifically to the 3' UTRs of several mRNAs and recognizes them all via a common cis-acting element, composed of at least seven UG-repeats. The binding strength of CHLAMY 1 is strongest to mRNAs, whose products are key components of nitrogen metabolism resulting in arginine biosynthesis as well as of CO2 metabolism. Since temporal activities of processes involved in nitrogen metabolism have an opposite phase than CHLAMY 1 binding activity, the protein might repress the translation of the cognate mRNAs.

Animals↗