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PubMed2004

Transcriptional and translational control of ornithine decarboxylase during Ras transformation.

ODC (ornithine decarboxylase) activity is induced following ras activation. However, the Ras effector pathways responsible are unknown. These experiments used NIH-3T3 cells expressing partial-loss-of-function Ras mutants to activate selectively pathways downstream of Ras and examined the contribution of each pathway to ODC induction. Overexpression of Ras12V, a constitutively active mutant, resulted in ODC activities up to 20-fold higher than controls. Stable transfections of Ras partial-loss-of-function mutants and constitutively active forms of MEK (MAPK kinase) and Akt indicated that activation of more than one Ras effector pathway is necessary for the complete induction of ODC activity. The increase in ODC activity in Ras12V-transformed cells is not owing to a substantial change in ODC protein half-life, which increased by <2-fold. Northern-blot analysis and reporter assays suggested that the mechanism of ODC induction involves both a modest increase in the transcription of ODC mRNA and a much more considerable increase in the translation of mRNA into protein. ODC transcription was controlled through a pathway dependent on Raf/MEK/ERK (where ERK stands for extracellular-signal-regulated kinase) activation, whereas activation of the phosphoinositide 3-kinase and the Raf/MEK/ERK pathways were necessary for translational regulation of ODC. The increase in ODC synthesis was accompanied by changes in phosphorylation of eukaryotic initiation factor 4E and its binding protein 4E-BP1. Results show that the phosphoinositide 3-kinase pathway regulates phosphorylation of both proteins, whereas the Raf/MEK/ERK pathway affects only the eukaryotic initiation factor 4E phosphorylation.

Animals↗
PubMed2006

Translational control of cytochrome c by RNA-binding proteins TIA-1 and HuR.

Stresses affecting the endoplasmic reticulum (ER) globally modulate gene expression patterns by altering posttranscriptional processes such as translation. Here, we use tunicamycin (Tn) to investigate ER stress-triggered changes in the translation of cytochrome c, a pivotal regulator of apoptosis. We identified two RNA-binding proteins that associate with its approximately 900-bp-long, adenine- and uridine-rich 3' untranslated region (UTR): HuR, which displayed affinity for several regions of the cytochrome c 3'UTR, and T-cell-restricted intracellular antigen 1 (TIA-1), which preferentially bound the segment proximal to the coding region. HuR did not appear to influence the cytochrome c mRNA levels but instead promoted cytochrome c translation, as HuR silencing greatly diminished the levels of nascent cytochrome c protein. By contrast, TIA-1 functioned as a translational repressor of cytochrome c, with interventions to silence TIA-1 dramatically increasing cytochrome c translation. Following treatment with Tn, HuR binding to cytochrome c mRNA decreased, and both the presence of cytochrome c mRNA within actively translating polysomes and the rate of cytochrome c translation declined. Taken together, our data suggest that the translation rate of cytochrome c is determined by the opposing influences of HuR and TIA-1 upon the cytochrome c mRNA. Under unstressed conditions, cytochrome c mRNA is actively translated, but in response to ER stress agents, both HuR and TIA-1 contribute to lowering its biosynthesis rate. We propose that HuR and TIA-1 function coordinately to maintain precise levels of cytochrome c production under unstimulated conditions and to modify cytochrome c translation when damaged cells are faced with molecular decisions to follow a prosurvival or a prodeath path.

3' Untranslated Regions↗
PubMed1976

Novel mechanism for translational control in regulation of ferritin synthesis by iron.

Poly(A)-containing RNA was isolated from the polyribosomal and post-ribosomal fractions of the livers of normal and iron-treated rats. These RNA fractions were then translated in a wheat germ system to provide a measure of the amount of ferritin mRNA present in each fraction. Following iron administration, there was a 2-fold increase in the amount of ferritin mRNA in the polyribosomal fraction. This increase was not inhibited by prior treatment of the rats with actinomycin D or cordycepin, suggesting a cytoplasmic control mechanism. In normal rats, the post-ribosomal fraction contained an amount of ferritin mRNA equal to that in the polyribosomes. When iron was administered, this untranslated ferritin mRNA became reduced to negligible quantities, thus accounting for the doubling of the ferritin mRNA content of the polyribosomal fraction. A scheme is proposed in which translation of the ferritin mRNA in the post-ribosomal fraction is prevented by adhering ferritin subunits. Iron administration removes this inhibition of the translation of ferritin mRNA by promoting aggregation of these subunits into ferritin.

Animals↗
PubMed1992

Analysis of mRNAs under translational control during Xenopus embryogenesis: isolation of new ribosomal protein clones.

We have analyzed several randomly selected mRNAs, of the relatively abundant category, on the basis of maternal or zygotic origin and translational efficiency at different developmental stages. For this purpose, clones from a Xenopus embryo cDNA library were hybridized with cDNA probes prepared with poly(A)+RNA from polysomes and from mRNPs of embryos at different stages. The results obtained indicate that the majority of the relatively abundant mRNAs (38 out of 61) is subject to some kind of translational regulation during embryogenesis. Moreover, 30 clones have been selected as corresponding to mRNAs that behave, from the point of view of transcriptional and translational regulation, similarly to previously studied ribosomal protein (r-protein) mRNAs. Sequence analysis of 20 of these selected cDNAs has shown that half of them are in fact homologous to already sequenced r-protein mRNAs. Unexpectedly we have found that also the mRNA for alpha-cardiac actin and another mRNA homologous to creatine kinase M mRNA have a similar translational regulation during embryogenesis.

Actins↗
PubMed1999

Role of two upstream open reading frames in the translational control of oncogene mdm2.

Overexpression of oncoprotein MDM2 has been found in a significant number of human soft tissue tumors. In a subset of these tumors, overexpression is a result of enhanced translation of mdm2 mRNA. There are two transcripts from the mdm2 gene that differ only in their 5' leaders: a long form (L-mdm2) and a short form (S-mdm2) that arise from the use of different promoters. L-mdm2 mRNA contains two upstream open reading frames (uORFs) and this mRNA was loaded with ribosomes inefficiently in comparison with S-mdm2. The 5' leader of L-mdm2 was sufficient to transfer translational repression to a reporter gene and the two uORFs acted synergistically to achieve full suppression. In contrast, the 5' leader of S-mdm2 allowed efficient translation of an attached reporter gene in the tumor cells. These results are consistent with a model in which overexpression of MDM2 in certain tumors results from a change in mRNA structure due to a switch in promoter usage.

5' Untranslated Regions↗
PubMed1993

Translational control by a long range RNA-RNA interaction; a basepair substitution analysis.

One of the two mechanisms that regulate expression of the replicase cistron in the single stranded RNA coliphages is translational coupling. This mechanism prevents ribosomes from binding at the start of the replicase cistron unless the upstream coat cistron is being translated. Genetic analysis had identified a maximal region of 132 nucleotides in the coat gene over which ribosomes should pass to activate the replicase start. Subsequent deletion studies in our laboratory had further narrowed down the regulatory region to 12 nucleotides. Here, we identify a long-distance RNA-RNA interaction of 6 base pairs as the basis of the translational polarity. The 3' side of the complementarity region is located in the coat-replicase intercistronic region, some 20 nucleotides before the start codon of the replicase. The 5' side encodes amino acids 31 and 32 of the coat protein. Mutations that disrupt the long-range interaction abolish the translational coupling. Repair of basepairing by second site base substitutions restores translational coupling.

Base Composition↗
PubMed1997

Thermally regulated translational control of FRQ mediates aspects of temperature responses in the neurospora circadian clock.

Two forms of FRQ, a central component of the Neurospora circadian clock, arise through alternative in-frame initiation of translation. Either form alone suffices for a functional clock at some temperatures, but both are always necessary for robust rhythmicity. Temperature regulates the ratio of FRQ forms by favoring different initiation codons at different temperatures; when either initiation codon is eliminated, the temperature range permissive for rhythmicity is demonstrably reduced. This temperature-influenced choice of translation-initiation site represents a novel adaptive mechanism that extends the physiological temperature range over which clocks function. Additionally, a temperature-dependent threshold level of FRQ is required to establish the feedback loop comprising the oscillator. These data may explain how temperature limits permissive for rhythmicity are established, thus providing a molecular understanding for a basic characteristic of circadian clocks.

Biological Clocks↗
PubMed1997

Cytoplasmic protein mRNA interaction mediates cGMP-modulated translational control of the asialoglycoprotein receptor.

Expression of the asialoglycoprotein receptor by the human hepatocellular carcinoma cell line HuH-7 in response to intracellular cGMP concentrations was previously shown to be regulated at the translational level. In a cell-free system, initiation of asialoglycoprotein receptor mRNA translation was dependent on the presence of the 7-methylguanylate cap site and was independent of 8-bromo-cGMP levels in which the cells were grown prior to RNA isolation. Stable transfection of COS-7 cells with deletion constructs of the asialoglycoprotein receptor H2b subunit localized the cGMP-responsive cis-acting element to the mRNA 5'-untranslated region (UTR). Addition of biotin (an activator of guanylate cyclase) induced the expression of beta-galactosidase present as a chimeric plasmid containing the H2b 187-nucleotide 5'-UTR. An RNA gel retardation assay identified a 37-nucleotide cognate sequence within this 187-nucleotide region. Titration of the 5'-UTR with a cytosolic fraction isolated from HuH-7 grown in the presence or absence of 8-bromo-cGMP or biotin provided direct evidence for an RNA-binding protein responsive to intracellular levels of cGMP. Based on these findings, it seems reasonable to propose that reduction of intracellular levels of cGMP by biotin deprivation results in a negative trans-acting factor associating with the 5'-UTR of asialoglycoprotein receptor mRNAs, thereby inhibiting translation.

Animals↗
PubMed1992

The specificity of translational control switched with transfer RNA identity rules.

The interaction of Escherichia coli threonyl-transfer RNA (tRNA) synthetase with the leader sequence of its own messenger RNA inhibits ribosome binding, resulting in negative translational feedback regulation. The leader sequence resembles the substrate (tRNA(Thr)) of the enzyme, and the nucleotides that mediate the correct recognition of the leader and the tRNA may be the same. A mutation suggested by tRNA identity rules that switches the resemblance of the leader sequence from tRNA(Thr) to tRNA(Met) causes the translation of the threonyl-tRNA synthetase messenger RNA to become regulated by methionyl-tRNA synthetase. This identity swap in the leader messenger RNA indicates that tRNA identity rules may be extended to interactions of synthetases with other RNAs.

Bacterial Proteins↗
PubMed1986

Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control.

Avian retroviral mRNAs contain a long 5' untranslated leader of approximately 380 nucleotides. The leader includes sequences required for viral replication and three AUG codons which precede the AUG codon used for translational initiation of the gag and env genes. We have used sensitive, quantitative assays of viral gene transcription and translation to analyze the role of this mRNA leader in viral gene expression. By substituting segments from related viruses, we had previously shown that the endogenous avian provirus ev-1 contained a defective leader segment (B. R. Cullen, A. M. Skalka, and G. Ju, Proc. Natl. Acad. Sci. USA 80:2946-2950, 1983). The sequence analysis presented here, followed by comparison with the nondefective ev-2 endogenous provirus segment, identified the critical changes at nucleotides 4 and 7 upstream of the initiator AUG. These differences do not alter the most conserved nucleotides within the consensus sequence which precedes eucaryotic initiation codons, but lie within a nine-nucleotide region that is otherwise highly conserved among avian retrovirus strains. Analysis of a series of deletion mutants indicated that other sequences within the leader are also required for efficient expression. Characterization of the altered transcripts demonstrated that the presence of the defective ev-1 segment or the deletion of a ca. 200-nucleotide leader segment did not affect the steady-state level or splicing efficiency of these mRNAs. Thus, we conclude that the reduced expression of these mRNAs is due to a translational deficiency. These results indicate that specific leader sequences, other than the previously identified consensus nucleotides which precede eucaryotic AUG initiator codons, can influence eucaryotic gene translation.

Animals↗
PubMed1979

A kinetic model of protein synthesis. Application to hemoglobin synthesis and translational control.

We present a kinetic model of protein synthesis which encompasses initiation, elongation, and termination parameters. We have investigated the dependence of the total rate of protein synthesis and the size of the translating polysomes on each of these parameters and in particular on the level of active 40 S ribosomes and initiation factors. This model qualitatively fits experimental data for the ratio of alpha- to beta-globin synthesis in reticulocytes, both under normal conditions and in the presence of inhibitors of chain initiation. This model has also been used to examine the effect that limiting amounts of certain tRNAs might have on the total rate of protein synthesis. In addition, the role of initiation factor discrimination and mRNA length are examined with respect to the differential translation of mRNAs.

Animals↗
PubMed1998

Translational control elements in the major human transforming growth factor-beta 1 mRNA.

Polysome analysis indicates that the major 2.4 kb transforming growth factor-beta 1 (TGF-beta 1) transcript is poorly translated, both in cultured cells, and in vivo in mouse liver. In contrast, the TGF-beta 2 transcripts are efficiently translated. The contribution of the 5'- and 3'-untranslated regions (UTRs) to the translational inhibition of the full-length TGF-beta 1 transcript was studied by deletion analysis. Despite their high G + C content, both UTRs stimulated translation in vitro. However, polysome analysis of synthetic TGF-beta 1 mRNAs transfected into MCF-7 cells suggests that the cell contains a limited pool of trans-acting factors that interact with the 5'UTR to make it inhibitory in vivo. Further deletion analysis in vitro revealed multiple stimulatory and inhibitory regions in the 5'UTR. This has important implications for the translatability of the naturally occurring shorter TGF-beta 1 transcripts and provides a framework for higher resolution mapping studies. Overall, the poor translational efficiency of the major TGF-beta 1 mRNA in vivo appears to be due to a combination of poor initiation sequence context, and inhibitory interactions of limiting transacting factors with cis-inhibitory elements embedded in an otherwise stimulatory 5'UTR.

3' Untranslated Regions↗
PubMed1991

Translational control of human cytomegalovirus gp48 expression.

Posttranscriptional controls modulate the expression of several human cytomegalovirus genes. Previous studies have shown that one cytomegalovirus gene transcript leader contains AUG codons which inhibit translation of a downstream reading frame. However, two other cytomegalovirus gene transcript leaders of similar structure do not inhibit translation. We have extended these studies to the analysis of the structural glycoprotein gp48, whose predominant transcript contains three upstream AUG codons. The 5' leader of this transcript strongly inhibits downstream translation in fibroblasts. Analyses of deletions and point mutations identify the second upstream AUG codon as an essential component of the inhibitory signal. Other leader sequences, but neither the first nor the third AUG codon, are also required. Intriguingly, the inhibitory signal appears also to depend on the amino acid coding information of the short reading frame associated with the second AUG codon. Insights derived from these studies are germane to understanding the translational regulation of other viral and cellular genes of similar structure.

Base Sequence↗
PubMed2005

The utrophin A 5'-untranslated region confers internal ribosome entry site-mediated translational control during regeneration of skeletal muscle fibers.

Utrophin up-regulation in muscle fibers of Duchenne muscular dystrophy patients represents a potential therapeutic strategy. It is thus important to delineate the regulatory events presiding over utrophin in muscle in attempts to develop pharmacological interventions aimed at increasing utrophin expression. A number of studies have now shown that under several experimental conditions, the abundance of utrophin is increased without a corresponding elevation in its mRNA. Here, we examine whether utrophin expression is regulated at the translational level in regenerating muscle fibers. Treatment of mouse tibialis anterior muscles with cardiotoxin to induce muscle degeneration/regeneration led to a large (approximately 14-fold) increase in the levels of utrophin A with a modest change in expression of its transcript (40%). Isolation of the mouse utrophin A 5'-untranslated region (UTR) revealed that it is relatively long with a predicted high degree of secondary structure. In control muscles, the 5'-UTR of utrophin A caused an inhibition upon translation of a reporter protein. Strikingly, this inhibition was removed during regeneration, indicating that expression of utrophin A in regenerating muscles is translationally regulated via its 5'-UTR. Using bicistronic reporter vectors, we observed that this translational effect involves an internal ribosome entry site in the utrophin A 5'-UTR. Thus, internal ribosome entry site-mediated translation of utrophin A can, at least partially, account for the discordant expression of utrophin A protein and transcript in regenerating muscle. These findings provide a novel target for up-regulating levels of utrophin A in Duchenne muscular dystrophy muscle fibers via pharmacological interventions.

5' Untranslated Regions↗
PubMed1990

Maturation-specific polyadenylation and translational control: diversity of cytoplasmic polyadenylation elements, influence of poly(A) tail size, and formation of stable polyadenylation complexes.

Early embryonic development in Xenopus laevis is programmed in part by maternally derived mRNAs, many of which are translated at the completion of meiosis (oocyte maturation). Polysomal recruitment of at least one of these mRNAs, G10, is regulated by cytoplasmic poly(A) elongation which, in turn, is dependent upon the cytoplasmic polyadenylation element (CPE) UUUUUUAUAAAG and the hexanucleotide AAUAAA (L. L. McGrew, E. Dworkin-Rastl, M. B. Dworkin, and J. D. Richter, Genes Dev. 3:803-815, 1989). We have investigated whether sequences similar to the G10 RNA CPE that are present in other RNAs could also be responsible for maturation-specific polyadenylation. B4 RNA, which encodes a histone H1-like protein, requires a CPE of the sequence UUUUUAAU as well as the polyadenylation hexanucleotide. The 3' untranslated regions of Xenopus c-mos RNA and mouse HPRT RNA also contain U-rich CPEs since they confer maturation-specific polyadenylation when fused to Xenopus B-globin RNA. Polyadenylation of B4 RNA, which occurs very early during maturation, is limited to 150 residues, and it is this number that is required for polysomal recruitment. To investigate the possible diversity of factors and/or affinities that might control polyadenylation, egg extracts that faithfully adenylate exogenously added RNA were used in competition experiments. At least one factor is shared by B4 and G10 RNAs, although it has a much greater affinity for B4 RNA. Additional experiments demonstrate that an intact CPE and hexanucleotide are both required to compete for the polyadenylation apparatus. Gel mobility shift assays show that two polyadenylation complexes are formed on B4 RNA. Optimal complex formation requires an intact CPE and hexanucleotide but not ongoing adenylation. These data, plus additional RNA competition studies, suggest that stable complex formation is enhanced by an interaction of the trans-acting factors that bind the CPE and polyadenylation hexanucleotide.

Animals↗