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PubMed1991

Ferritin gene transcription is regulated by iron in soybean cell cultures.

Iron-regulated ferritin synthesis in animals is dominated by translational control of stored mRNA; iron-induced transcription of ferritin genes, when it occurs, changes the subunit composition of ferritin mRNA and protein and is coupled to translational control. Ferritins in plants and animals have evolved from a common progenitor, based on the similarity of protein sequence; however, sequence divergence occurs in the C termini; structure prediction suggests that plant ferritin has the E-helix, which, in horse ferritin, forms a large channel at the tetrameric interface. In contemporary plants, a transit peptide is encoded by ferritin mRNA to target the protein to plastids. Iron-regulated synthesis of ferritin in plants and animals appears to be very different since the 50- to 60-fold increases of ferritin protein, previously observed to be induced by iron in cultured soybean cells, is accompanied by an equivalent accumulation of hybridizable ferritin mRNA and by increased transcription of ferritin genes. Ferritin mRNA from iron-induced cells and the constitutive ferritin mRNA from soybean hypocotyls are identical. The iron-induced protein is translocated normally to plastids. Differences in animal ferritin structure coincide with the various iron storage functions (reserve for iron proteins and detoxification). In contrast, the constancy of structure of soybean ferritin, iron-induced and constitutive, coupled with the potential for vacuolar storage of excess iron in plants suggest that rapid synthesis of ferritin from a stored ferritin mRNA may not be needed in plants for detoxification of iron.

Amino Acid Sequence↗
PubMed2026

Design and optimization of a kinase-controlled allosteric switch.

Post-translational control enables rapid and precise regulation of cell behavior. Despite these advantages, general strategies to build phosphorylation-based synthetic circuits are limited. Here we reasoned that engineered allostery, a technique that has been applied to design light- and chemically gated protein switches, could also be used to engineer phosphorylation-controlled protein switches (phospho-switches). Using an allosterically controllable Gal4 transcription factor as a scaffold, we show that a classic kinase Förster resonance energy transfer biosensor architecture can be used as a starting point for phospho-switch design. We optimize all features of the phospho-switch to develop an ERK-controlled transcription factor with a 20-fold phosphorylation-dependent change in transcriptional output. The resulting synthetic ERK-responsive transcription factor responds with comparable sensitivity to the c-fos promoter and reveals spatial ERK signaling patterns in mammalian developmental organoids. We further show that our switch architecture can be generalized to other input kinases and allosterically controlled targets. This work provides a general platform for a new generation of kinase-responsive tools for biosensing and synthetic biology applications.

Allosteric Regulation↗
PubMed2004

Insights into the receptor transcription and signaling: implications in opioid tolerance and dependence.

Drug addiction has great social and economical implications. In order to resolve this problem, the molecular and cellular basis for drug addiction must be elucidated. For the past three decades, our research has focused on elucidating the molecular mechanisms behind morphine tolerance and dependence. Although there are many working hypotheses, it is our premise that cellular modulation of the receptor signaling, either via transcriptional or post-translational control of the receptor, is the basis for morphine tolerance and dependence. Thus, in the current review, we will summarize our recent work on the transcriptional and post-translational control of the opioid receptor, with special emphasis on the mu-opioid receptor, which is demonstrated to mediate the in vivo functions of morphine.

Animals↗
PubMed1990

A fractionated reticulocyte lysate retains high efficiency for protein synthesis.

The rabbit reticulocyte lysate is a highly efficient system for protein synthesis in vitro, and therefore has been used frequently in studies of translational control. However, when the lysate is fractionated into ribosomes and soluble proteins by centrifugation, there is a severe loss of activity, thereby making the system less suitable for identifying components involved in translational control. We have devised a new method of cell fractionation which employs rapid (20-min) centrifugation to pellet ribosomes. The post-ribosomal supernatant (S-100), high salt-washed ribosomes, and the high salt wash are all required in the reconstituted protein synthesis assay, and greater than 70% of the activity of the unfractionated lysate is attained. Proteins in the high salt wash have been further fractionated by ammonium sulfate precipitation, and the assay system has been used to measure the activities of eIF-4B and eIF-4F. This highly active fractionated lysate system should be useful in measuring the specific activities of translational components and may be appropriate for detecting changes due to covalent modifications such as phosphorylation.

Animals↗
PubMed2001

Translational silencing of ceruloplasmin requires the essential elements of mRNA circularization: poly(A) tail, poly(A)-binding protein, and eukaryotic translation initiation factor 4G.

Ceruloplasmin (Cp) is a glycoprotein secreted by the liver and monocytic cells and probably plays roles in inflammation and iron metabolism. We showed previously that gamma interferon (IFN-gamma) induced Cp synthesis by human U937 monocytic cells but that the synthesis was subsequently halted by a transcript-specific translational silencing mechanism involving the binding of a cytosolic factor(s) to the Cp mRNA 3' untranslated region (UTR). To investigate how protein interactions at the Cp 3'-UTR inhibit translation initiation at the distant 5' end, we considered the "closed-loop" model of mRNA translation. In this model, the transcript termini are brought together by interactions of poly(A)-binding protein (PABP) with both the poly(A) tail and initiation factor eIF4G. The effect of these elements on Cp translational control was tested using chimeric reporter transcripts in rabbit reticulocyte lysates. The requirement for poly(A) was shown since the cytosolic inhibitor from IFN-gamma-treated cells minimally inhibited the translation of a luciferase reporter upstream of the Cp 3'-UTR but almost completely blocked the translation of a transcript containing a poly(A) tail. Likewise, a requirement for poly(A) was shown for silencing of endogenous Cp mRNA. We considered the possibility that the cytosolic inhibitor blocked the interaction of PABP with the poly(A) tail or with eIF4G. We found that neither of these interactions were inhibited, as shown by immunoprecipitation of PABP followed by quantitation of the poly(A) tail by reverse transcription-PCR and of eIF4G by immunoblot analysis. We considered the alternate possibility that these interactions were required for translational silencing. When PABP was depleted from the reticulocyte lysate with anti-human PABP antibody, the cytosolic factor did not inhibit translation of the chimeric reporter, thus showing the requirement for PABP. Similarly, in lysates treated with anti-human eIF4G antibody, the cytosolic extract did not inhibit the translation of the chimeric reporter, thereby showing a requirement for eIF4G. These data show that translational silencing of Cp requires interactions of three essential elements of mRNA circularization, poly(A), PABP, and eIF4G. We suggest that Cp mRNA circularization brings the cytosolic Cp 3'-UTR-binding factor into the proximity of the translation initiation site, where it silences translation by an undetermined mechanism. These results suggest that in addition to its important function in increasing the efficiency of translation, transcript circularization may serve as an essential structural determinant for transcript-specific translational control.

3' Untranslated Regions↗
PubMed2001

EDEN-dependent translational repression of maternal mRNAs is conserved between Xenopus and Drosophila.

Translational control is a key level in regulating gene expression in oocytes and eggs because many mRNAs are synthesized and stored during oogenesis for latter use at various stages of oocyte maturation and embryonic development. Understanding the molecular mechanisms that underlie this translational control is therefore crucial. Another important issue is the evolutionary conservation of these mechanisms--in other words the determination of their universal and specific aspects. We report here a comparative analysis of a translational repression mechanism that depends on the EDEN (embryo deadenylation element) element. This small cis-acting element, localized in the 3' untranslated region of c-mos and Eg mRNAs, was shown to be involved in a deadenylation process. We demonstrate here that in Xenopus embryos, mRNAs that contain an EDEN are translationally repressed. Next, transgenic flies were used to study the effect of the EDEN motif on translation in Drosophila oocytes. We show that this element also causes the translational repression of a reporter gene in Drosophila demonstrating that the EDEN-dependent translational repression is functionally conserved between Xenopus and Drosophila.

3' Untranslated Regions↗
PubMed1988

Regulation of ornithine decarboxylase mRNA translation by polyamines. Studies using a cell-free system and a cell line with an amplified ornithine decarboxylase gene.

The translational control of ornithine decarboxylase (ODCase) by polyamines has been studied using a cellular as well as a cell-free system. A mutant L1210 cell line, in which ODCase represents 4-5% of all soluble protein synthesized, was isolated by stepwise selection for resistance to the ODCase inhibitor 2-difluoromethylornithine (DFMO). The exceptionally high expression of ODCase in these cells was due to amplification of the ODCase gene. When the cells were grown in the absence of DFMO, dramatic increases in cellular putrescine and spermidine levels occurred. These increases were accompanied by a rapid decrease in ODCase synthesis. The change in ODCase synthesis was not associated with an alteration in the amount of ODCase mRNA, demonstrating a translational control in these cells. The effects of polyamines on ODCase mRNA translation were also studied in rabbit reticulocyte lysates using mRNA isolated from the DFMO-resistant cells. Low concentrations of spermidine stimulated synthesis of ODCase and that of total protein, when added to gel-filtered lysates. Notably, optimal stimulation of ODCase synthesis was achieved at a spermidine concentration lower than that required for an optimal rate of total protein synthesis. Higher concentrations of spermidine were inhibitory, and their effects of ODCase synthesis were stronger than on protein synthesis in general, resulting in a decrease in the fraction of protein synthesis accounted for by ODCase. The present results demonstrate that at least part of the feedback regulation of ODCase exerted by the polyamines is due to direct inhibition of ODCase mRNA translation.

Animals↗
PubMed1975

Growth hormone, cyclic nucleotides and the rapid control of translation in heart muscle.

Perfused rat heart incorporated L-[14C]tyrosine into protein at a constant rate for up to 75 min. A purified bovine growth-hormone preparation (1 mug/ml) stimulated the incorporation to a new constant rate that was more than three times the control rate by 10 min after hormone addition to perfusate. The hormone, however, did not alter the intracellular tracer amino acid pool, and the relationship of this to the aminoacyl-tRNA precursor pool is discussed. It is concluded that the increased incorporation largely reflected a rapid increase in protein synthesis at the ribosomes. Measurements of cyclic nucleotide contents during the perfusion showed that these appeared to vary in a systematic way during the perfusion. This strands in contrast with the constant values given by several other parameters measured in this preparation. Futher, the cyclic nucleotide variation seems to be independent of external effectors. The steady-state performance of the heart correlates more closely the [cyclic AMP]/[cyclic GMP] ratio than with the content of the individual cyclic nucleotides. At 10 min after the addition of growth hormone a slight decrese in cyclic AMP content and a large decrease in cyclic GMP were found, suggesting that the hormone's effect in stimulating protein synthesis may be mediated by a decrease in cyclic nucleotide concentrations or an increase in the [cyclic AMP]/[cyclic |p] ratio. The findings are also consistent with an intracellularly directed role for these nucleotides, and the possibility that the cyclic nucleotide changes are an indirect result of growth-hormone action is discussed.

Amino Acids↗
PubMed1992

Molecular control of dentinogenesis: a reaction.

This paper represents an invited reaction to three papers presented at the International Conference on Pathobiology of the Dentin/Pulp Complex, June, 1991. Repair dentinogenesis following transplantation into normal and germs free animals are correlated with results elucidating the expression of dentin phosphoproteins, collagen, and osteocalcin. The importance of transcription and translation controls of dentin matrix components are discussed and reviewed. In addition, possible implications of a molecular chaperone protein, Hsp47, in controlling dentinogenesis is introduced. Future research directions are developed and include: (a) identification of odontoblast precursors; (b) delineation of markers for odontoblasts at varying degrees of differentiation; (c) characterization of environmental conditions leading to odontoblast differentiation; (d) determination of the nature of repair and regenerated tissues; (e) elucidation of transcription and translation control factors, and (f) mapping the human genome for dentin matrix constituents.

Collagen↗
PubMed1997

In vivo antisense oligodeoxynucleotide mapping reveals masked regulatory elements in an mRNA dormant in mouse oocytes.

In mouse oocytes, tissue-type plasminogen activator (tPA) mRNA is under translational control. The newly transcribed mRNA undergoes deadenylation and translational silencing in growing oocytes, while readenylation and translation occur during meiotic maturation. To localize regulatory elements controlling tPA mRNA expression, we identified regions of the endogenous transcript protected from hybridization with injected antisense oligodeoxynucleotides. Most of the targeted sequences in either the 5' untranslated region (5'UTR), coding region, or 3'UTR were accessible to hybridization, as revealed by inhibition of tPA synthesis and by RNase protection. Two protected regions were identified in the 3'UTR of tPA mRNA in primary oocytes: the adenylation control element (ACE) and the AAUAAA polyadenylation signal. These sequences were previously shown to be involved in the translational control of injected reporter transcripts. During the first hour of meiotic maturation, part of the ACE and the AAUAAA hexanucleotide became accessible to hybridization, suggesting a partial unmasking of the 3'UTR of this mRNA before it becomes translationally competent. Our results demonstrate that in vivo antisense oligodeoxynucleotide mapping can reveal the dynamics of regulatory features of a native mRNA in the context of the intact cell. They suggest that specific regions in the 3'UTR of tPA mRNA function as cis-acting masking determinants involved in the silencing of tPA mRNA in primary oocytes.

Animals↗
PubMed2001

A novel bacterial gene-finding system with improved accuracy in locating start codons.

Although a number of bacterial gene-finding programs have been developed, there is still room for improvement especially in the area of correctly detecting translation start sites. We developed a novel bacterial gene-finding program named GeneHacker Plus. Like many others, it is based on a hidden Markov model (HMM) with duration. However, it is a 'local' model in the sense that the model starts from the translation control region and ends at the stop codon of a coding region. Multiple coding regions are identified as partial paths, like local alignments in the Smith-Waterman algorithm, regardless of how they overlap. Moreover, our semiautomatic procedure for constructing the model of the translation control region allows the inclusion of an additional conserved element as well as the ribosome-binding site. We confirmed that GeneHacker Plus is one of the most accurate programs in terms of both finding potential coding regions and precisely locating translation start sites. GeneHacker Plus is also equipped with an option where the results from database homology searches are directly embedded in the HMM. Although this option does not raise the overall predictability, labeled similarity information can be of practical use. GeneHacker Plus can be accessed freely at http://elmo.ims.u-tokyo.ac.jp/GH/.

Algorithms↗
PubMed1992

Complementary DNA synthesis in situ: methods and applications.

In situ transcription is the synthesis of cDNA within cells. This chapter has illustrated some of the application of IST to the study of gene expression in complex cell environments. While the importance of transcription in modulating cellular activity has been long appreciated, the role of translational control mechanisms in regulating central nervous system functioning is just beginning to be recognized. Previous limitations in the availability of tissue have made it difficult to construct cDNA libraries from defined cell populations, to examine translational control, and to quantitate differences in the amount of mRNA for many distinct mRNAs in the same sample. In situ transcription facilitates all of these procedures, making it possible to characterize aspects of gene regulation that were previously difficult. Indeed, taken to its furthest extreme it is now possible to characterize gene expression in single live cells. This level of analysis allows basic questions, such as How different morphologically identical cells are at the level of gene expression, and How synaptic connectivity and glial interactions influence gene expression in single cells, to be experimentally approached. The ability to characterize gene expression in small amounts of tissue and single cells is critical to gaining an understanding of the contribution of specific cell types to the physiology of the central nervous system.

Animals↗
PubMed1993

Synthesis of two bacteriophage lambda S proteins in an in vivo system.

Bacteriophage lambda has two genes which are essential for lysis: R, a gene encoding a 158-amino-acid (aa) transglycosylase that attacks the peptidoglycan, and S, a gene encoding two inner-membrane-associating proteins, designated S105 and S107 for their predicted lengths in aa residues. S105 and S107 are thought to have opposing roles in lysis, with the former acting as the lethal lysis effector and the latter as a lysis inhibitor. Here, we used a T7-polymerase-mediated expression system to show that S105 and S107 are synthesized at a constant ratio of about 2.5:1 throughout the period leading up to lysis, indicating that lysis scheduling does not require a translationally controlled switch from inhibitor (S107) to effector (S105) synthesis. However, evidence is presented that the mRNA sequences immediately 5' to the ribosome-binding site (RBS) of the S gene are required for the rather limited translation, but not the stability, of the S mRNA. No difference could be found in the pattern of ternary complex formation over the two S start codons in in vitro toe-printing assays with the wild-type mRNA and with mRNA deleted of the upstream sequences. Nevertheless, these results may suggest a role for translational control in S gene expression, if not in its temporal regulation or in the partition between S105 and S107 production.

Amino Acid Sequence↗
PubMedDate not supplied

Control of the hypoxic response through regulation of mRNA translation.

Hypoxia is a common feature of most solid tumors which negatively impacts their treatment response. This is due in part to the biological changes that result from a coordinated cellular response to hypoxia. A large part of this response is driven by a transcriptional program initiated via stabilization of HIF, promoting both angiogenesis and cell survival. However, hypoxia also results in a rapid inhibition of protein synthesis which occurs through the repression of the initiation step of mRNA translation. This inhibition is fully reversible and occurs in all cell lines tested to date. Inhibition of translation is mediated by two distinct mechanisms during hypoxia. The first is through phosphorylation and inhibition of an essential eukaryotic initiation factor, eIF2alpha. Phosphorylation of this factor occurs through activation of the PERK kinase as part of a coordinated ER stress response program known as the UPR. Activation of this program promotes cell survival during hypoxia and facilitates tumor growth. Translation during hypoxia can also be inhibited through the inactivation of a second eukaryotic initiation complex, eIF4F. At least part of this inhibition is mediated through a REDD1 and TSC1/TSC2 dependent inhibition of the mTOR kinase. Inhibition of mRNA translation is hypothesized to affect the cellular tolerance to hypoxia in part by promoting energy homeostasis. However, regulation of translation also results in a specific increase in the synthesis of a subset of hypoxia induced proteins. Consequently, both arms of translational control during hypoxia influence hypoxia induced gene expression and the hypoxic phenotype.

Animals↗
PubMed2000

Regulated translation initiation controls stress-induced gene expression in mammalian cells.

Protein kinases that phosphorylate the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) are activated in stressed cells and negatively regulate protein synthesis. Phenotypic analysis of targeted mutations in murine cells reveals a novel role for eIF2alpha kinases in regulating gene expression in the unfolded protein response (UPR) and in amino acid starved cells. When activated by their cognate upstream stress signals, the mammalian eIF2 kinases PERK and GCN2 repress translation of most mRNAs but selectively increase translation of Activating Transcription Factor 4 (ATF4), resulting in the induction of the downstream gene CHOP (GADD153). This is the first example of a mammalian signaling pathway homologous to the well studied yeast general control response in which eIF2alpha phosphorylation activates genes involved in amino acid biosynthesis. Mammalian cells thus utilize an ancient pathway to regulate gene expression in response to diverse stress signals.

Activating Transcription Factor 4↗
PubMed1988

Translational activation of the lck proto-oncogene.

The lck proto-oncogene, a member of the src gene family, encodes a lymphocyte-specific protein tyrosine kinase (p56lck) that is implicated in the pathogenesis of lymphoid neoplasia. We report here that 5' lck sequence elements, containing AUG codons, significantly reduce the in vivo efficiency of p56lck translation from the normal messenger RNA. This result provides a quantitative explanation for the overexpression of p56lck in two retrovirally-induced murine lymphomas that express abnormal lck transcripts from which the physiological 5' translational control region has been deleted and non AUG-containing viral sequences substituted. In contrast to other mammalian genes, most proto-oncogenes contain AUG codons 5' to the authentic initiation codon, suggesting that cells can regulate translational start sites in these mRNAs. Abrogation of translational control may be a general mechanism of proto-oncogene activation in malignancy.

Animals↗
PubMedDate not supplied

Regulation of the isoforms of metallothionein.

The objective of this research was to study the regulation of hepatic MT-I and MT-II in rats. Induction of MTs following Cd, Zn, and glucocorticoid treatments was studied in adult rats and the ontogeny of MTs was evaluated in developing rats. The isoforms were quantitated by anion-exchange HPLC and their respective mRNAs were evaluated by Northern blot hybridization with mouse cRNA probes. In untreated adult rat liver, MT-II was more abundant than MT-I. Cd treatment increased MT-I and MT-II to similar concentrations. Low dosages of Zn increased MT-II levels three times higher than MT-I levels, whereas higher dosages increased the isoforms to similar levels. Differences in the degradation of Zn-induced MTs were observed: apparent half-lives for MT-I and MT-II were 12.2 and 21.9 h, respectively. MT-II was the predominant glucocorticoid-inducible isoform. MT-I and MT-II mRNAs increased coordinately after glucocorticoid treatment, but MT-II mRNA was more abundant than MT-I mRNA. In immature rats, MT-I and MT-II were first detected in fetal liver on gestation day 18 and increased to maximum hepatic concentrations during the first week postpartum. Thereafter, MT levels decreased, reaching adult levels by 35 d. In contrast, MT mRNAs reached maximal levels during late gestation that were maintained throughout the first three weeks postpartum. Discoordination between MT mRNA and protein levels is suggestive of translational control of MTs in developing rats. In summary, MT-II is more abundant than MT-I in untreated rats and in rats treated with glucocorticoids or low dosages of zinc. In addition to transcriptional regulations of MTs, translational control of MTs may be important in regulating hepatic MT concentrations, particularly in developing rats, and differences in the degradation of MT-I and MT-II may influence tissue concentrations of the isoforms.

Animals↗