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PubMed2000

Developmental and light-regulated post-translational control of 3-hydroxy-3-methylglutaryl-CoA reductase levels in potato.

In plants, the first committed step in the cytosolic pathway for biosynthesis of isoprenoids is catalysed by 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). We have added an eight amino-acid-residue epitope tag to a potato (Solanum tuberosum L.) HMGR isoform and expressed this novel protein (HMGR-FLAG) in transgenic plants. Despite high levels of transcript accumulation in all leaf stages of transgenic plants, high levels of HMGR-FLAG protein were found only in apical meristematic tissue, suggesting post-translational regulation of potato HMGR affected by plant development. Protein immunoblots, and determination of enzymatic activity and transcript accumulation in the HMGR-FLAG transgenic and the non-transgenic parental plant lines, show that HMGR levels decrease dramatically in the dark. Again, the mechanism of this control occurs at a post-translational level. After 2.5 h in darkness, levels of HMGR-FLAG are approximately one-half of those in plants in the light; protein levels recover rapidly when dark-treated plants are returned to the light. In non-transgenic plants, hmg transcript levels are reduced in the dark, whereas dark treatments do not affect transgene hmg transcripts expressed under the control of a constitutive promoter. Furthermore, transcripts for HMGR-FLAG remain associated with polyribosomes in dark-treated tissues. Addition of inhibitors of cysteine proteases during microsomal protein extraction is required for recovery of immunoreactive HMGR-FLAG. The epitope-tagged isozyme has been used to show for the first time that a regulated decrease in plant HMGR activity correlates closely with a loss of the HMGR protein. We have used whole plants to demonstrate that developmental and light-regulated control of HMGR occurs post-translationally in vivo.

Blotting, Northern↗

PubMed1995

Deregulation of translational control of the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A leads to multinucleated cells.

Efficient translation of the mRNA encoding the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A (PP2A) is prevented by an out of frame upstream AUG and a stable stem-loop structure (delta G = -55.9 kcal/mol) in the 5'-untranslated region (5'-UTR). Deletion of the 5'-UTR allows efficient translation of the PR65 alpha message in vitro and overexpression in COS-1 cells. Insertion of the 5'-UTR into the beta-galactosidase leader sequence dramatically inhibits translation of the beta-galactosidase message in vitro and in vivo, confirming that this sequence functions as a potent translation regulatory sequence. Cells transfected or microinjected with a PR65 alpha expression vector lacking the 5'-UTR, express high levels of PR65 alpha, accumulating in both nucleus and cytoplasm. PR65 alpha overexpressing rat embryo fibroblasts (REF-52 cells) become multinucleated. These data and previous results (Mayer-Jaekel, R. E., Ohkura, H., Gomes, R., Sunkel, C. E., Baumgartner, S., Hemmings, B. A., and Glover, D. M. (1993) Cell 72, 621-633) suggest that PP2A participates in the regulation of both mitosis and cytokinesis.

Animals↗

PubMed1999

Translational control of specific genes during differentiation of HL-60 cells.

Eukaryotic gene expression can be regulated through selective translation of specific mRNA species. Nevertheless, the limited number of known examples hampers the identification of common mechanisms that regulate translation of specific groups of genes in mammalian cells. We developed a method to identify translationally regulated genes. This method was used to examine the regulation of protein synthesis in HL-60 cells undergoing monocytic differentiation. A partial screening of cellular mRNAs identified five mRNAs whose translation was specifically inhibited and five others that were activated as was indicated by their mobilization onto polysomes. The specifically inhibited mRNAs encoded ribosomal proteins, identified as members of the 5'-terminal oligopyrimidine tract mRNA family. Most of the activated transcripts represented uncharacterized genes. The most actively mobilized transcript (termed TA-40) was an untranslated 1.3-kilobase polyadenylated RNA with unusual structural features, including two Alu-like elements. Following differentiation, a significant change in the cytoplasmic distribution of Alu-containing mRNAs was observed, namely, the enhancement of Alu-containing mRNAs in the polysomes. Our findings support the notion that protein synthesis is regulated during differentiation of HL-60 cells by both global and gene-specific mechanisms and that Alu-like sequences within cytoplasmic mRNAs are involved in such specific regulation.

Alu Elements↗

PubMed1988

The first and fourth upstream open reading frames in GCN4 mRNA have similar initiation efficiencies but respond differently in translational control to change in length and sequence.

The third and fourth AUG codons in GCN4 mRNA efficiently repress translation of the GCN4-coding sequences under normal growth conditions. The first AUG codon is approximately 30-fold less inhibitory and is required under amino acid starvation conditions to override the repressing effects of AUG codons 3 and 4. lacZ fusions constructed to functional, elongated versions of the first and fourth upstream open reading frames (URFs) were used to show that AUG codons 1 and 4 function similarly as efficient translational start sites in vivo, raising the possibility that steps following initiation distinguish the regulatory properties of URFs 1 and 4. In accord with this idea, we observed different consequences of changing the length and termination site of URF1 versus changing those of URFs 3 and 4. The latter were lengthened considerably, with little or no effect on regulation. In fact, the function of URFs 3 and 4 was partially reconstituted with a completely heterologous URF. By contrast, certain mutations that lengthen URF1 impaired its positive regulatory function nearly as much as removing its AUG codon did. The same mutations also made URF1 a much more inhibitory element when it was present alone in the mRNA leader. These results strongly suggest that URFs 1 and 4 both function in regulation as translated coding sequences. To account for the phenotypes of the URF1 mutations, we suggest the most ribosomes normally translate URF1 and that the mutations reduce the number of ribosomes that are able to complete URF1 translation and resume scanning downstream. This effect would impair URF1 positive regulatory function if ribosomes must first translate URF1 in order to overcome the strong translational block at the 3'-proximal URFs. Because URF1-lacZ fusions were translated at the same rate under repressing and derepressing conditions, it appears that modulating initiation at URF1 is not the means that is used to restrict the regulatory consequences of URF1 translation to starvation conditions.

Base Sequence↗

PubMedDate not supplied

[Translational control of the picornavirus phenotype].

Picornaviruses are small animal viruses with positive-stranded genomic RNA, which is translated using cap-independent internal translation initiation. The key role in this is played by cis elements of the 5'-untranslated region (5'-UTR) and, in particular, by the internal ribosome entry site (IRES). The function of translational cis elements requires both canonical translation initiation factors (eIFs) and additional IRES trans-acting factors (ITAFs). All known ITAFs are cell RNA-binding proteins which play a variety of functions in noninfected cells. Specific features of translational cis elements substantially affect the phenotype and, in particular, tissue tropism and pathogenic properties of picornaviruses. It is clear that, in some cases, the molecular mechanism of this is a change in interactions between viral cis elements and ITAFs. The properties and tissue distribution of ITAFs may determine the biological properties of other viruses that also use the IRES-dependent translation initiation. Since this mechanism is also involved in translation of several cell mRNAs, ITAF may contribute to the regulation of the most important aspects of the living activity in noninfected cells.

Picornaviridae↗

PubMed2001

Translational control of protein synthesis: implications for understanding changes in skeletal muscle mass.

Gain or loss of skeletal muscle mass is due largely to the establishment of an imbalance between rates of protein synthesis and degradation. A key determinant of the rate of protein synthesis is translation initiation, a process regulated in part through binding of initiator methionyl-tRNA (met-tRNAi) and messenger RNA (mRNA) to a 40S ribosomal subunit. Either the met-tRNAi or mRNA binding step can become limiting for protein synthesis. Furthermore, the mRNA binding step can modulate translation of specific mRNAs with or without changes in the overall rate of protein synthesis. This report highlights molecular mechanisms involved in mediating control of the mRNA binding step in translation initiation. Particular attention is given to the effect of exercise on this step and to how the branched-chain amino acid leucine stimulates muscle protein synthesis after exercise. Potential mechanisms for exercise-induced increase in muscle mass are discussed.

Animals↗

PubMed1991

A quantitative model for translational control of the GCN4 gene of Saccharomyces cerevisiae.

Expression of the GCN4 gene of Saccharomyces cerevisiae is regulated at the translational level by short open reading frames (uORFs) present in the leader sequence of its mRNA. Under conditions of amino acid sufficiency, these sequences restrict the flow of initiating ribosomes to the GCN4 AUG start codon. Mutational analysis of GCN4 has led to a model in which ribosomes must translate the 5'-proximal uORF1 and reassemble an initiation complex in order to translate GCN4. This reassembly process is thought to be rapid when amino acids are abundant, such that reinitiation occurs at uORF2, uORF3, or uORF4. Reinitiation at these sites prevents translation of GCN4, presumably because ribosomes dissociate from the mRNA following termination at uORFs 2 to 4. Because of reduced initiation factor activity under starvation conditions, a substantial fraction of ribosomal subunits scanning downstream from uORF1 are not ready to reinitiate when they reach uORFs 2 to 4, but become competent to do so while scanning the additional sequences between uORF4 and GCN4. Examination of the effects of point mutations in the ATG codons of the different uORFs suggests a quantitative model for this control mechanism that describes the probability of reinitiation as a function of the distance scanned downstream from uORF1. This model accounts for the phenotypes of a number of deletion and insertion mutations that alter the intercistronic spacing between the uORFs and GCN4. The correspondence between observed and predicted results implies that the differential rates of reinitiation at GCN4 versus uORFs 2 to 4 are determined largely by the different scanning times required to reach each of these start sites following translation of uORF1. In addition, it supports the notion that an increased scanning-time requirement for reinitiation in amino acid-starved cells forms the basis for translational derepression of GCN4 expression.

Amino Acids↗

PubMed1979

Translation of partially purified poly(A)+ protamine messenger RNA components in wheat germ and rabbit reticulocyte cell-free systems. Evidence for translational control mechanisms.

The coding properties of individual poly(A)+ protamine mRNA subcomponents have been explored by analysis of their translation products in two different cell-free protein synthesis systems, the rabbit reticulocyte lysate and the wheat germ S-30, both of which can translate total protamine mRNA. The products synthesized in the reticulocyte lysate in the presence of total poly(A)+ PmRNA consisted mainly of protamine components CII and CIII with component CI only a minor product. However, in the wheat germ S-30, the same mRNA preparation supported the synthesis of all three protamine components, in approximately equal amounts. In addition a new polypeptide, a putative fourth protamine component, labelled CO, was also synthesized. The translation products of subcomponents of poly(A)+ PmRNA separated as individual bands on polyacrylamide gels were similarly analyzed and it was shown that each of the isolated poly(A)+ PmRNA species could stimulate the incorporation of [3H]arginine into protamines in both translational systems. Although each mRNA band stimulated the synthesis of one particular protamine polypeptide predominantly in a given cell-free system, the same RNA preparation was found to direct preferentially the synthesis of a different protamine component in the second cell-free system. The products synthesized in the rabbit reticulocyte lysate in the presence of the individual mRNA species still showed component CI present as a minor product.

Animals↗

PubMed1986

Translational control of gene expression in a normal fibroblast. Characterization of a subclass of mRNAs with unusual kinetic properties.

The translation of a small number of mRNAs in mouse SC-1 fibroblasts can be stimulated by cycloheximide, under conditions where the synthesis of most proteins is inhibited. These mRNAs are ordinarily present in small polyribosomes or messenger ribonucleoprotein particles, although the addition of cycloheximide drives them into large (greater than or equal to 5) polysomes. These mRNAs cannot be translated in vitro unless they are extracted with phenol. With such treatment, however, they are translated with normal competitive efficiencies. In iron-poor media, the mRNA for ferritin exhibits several of the distinctive kinetic properties of this class of mRNAs. With iron supplementation, however, ferritin translation appears normal. These observations are consistent with the existence of translational induction/repression systems in eukaryotes. Several types of evidence suggest that repressors may act by interfering with the interaction between mRNAs and limiting translational initiation components.

Animals↗

PubMed1999

Cytoplasmic proteins interact with a translational control element in the protein-coding region of proopiomelanocortin mRNA.

Previous studies have indicated that proopiomelanocortin (POMC) is translationally regulated. We proposed that the regulatory mechanism involves an interaction between trans-acting protein factors and a cis-acting stem-loop structure in the coding region of POMC mRNA. Functional interactions were tested by examining the translation of mouse POMC mRNA in a rabbit reticulocyte system. Specific binding was demonstrated with ultraviolet-crosslinking and RNA gel mobility shift assays. The evidence presented supports our hypothesis that the translational regulation of POMC gene expression involves recognition of the stem-loop by RNA-binding proteins. Furthermore, POMC stem-loop RNA-binding proteins specifically recognized a predicted stem-loop found in the coding region of corticotropin-releasing hormone, suggesting a novel mechanism of gene regulation that may extend to other neuropeptides as well.

Animals↗

PubMed1998

The 5' untranslated region, signal peptide, and the coding sequence of the carboxyl terminus of IL-15 participate in its multifaceted translational control.

We previously reported that the AUG-burdened 5' untranslated region (UTR) of IL-15 mRNA impedes its translation. Here we demonstrate that the nucleotide or protein sequences of the IL-15 signal peptide and carboxyl terminus also contribute to the poor translation of IL-15 transcripts. In particular, the exchange of the IL-15 signal peptide coding sequence with that of IL-2 increased cellular and secreted levels of IL-15 protein 15- to 20-fold in COS cells, while IL-2 transcripts with the IL-15 signal peptide generated 30- to 50-fold less IL-2 protein than wild-type IL-2. Furthermore, the addition of an artificial epitope tag to the 3' coding sequence of IL-15 increased its protein production 5- to 10-fold. Combining these two IL-15 message modifications, in addition to removing the 5' UTR, increased IL-15 synthesis 250-fold compared with a wild-type construct with an intact 5' UTR. These data suggest that IL-15 mRNA, unlike IL-2 mRNA, may exist in translationally inactive pools. By storing translationally quiescent IL-15 mRNA, cells might respond to intracellular infections or other stimuli by rapidly transforming IL-15 message into one that can be efficiently translated.

Animals↗

PubMed1991

Translational control of discoidin lectin expression in drsA suppressor mutants of Dictyostelium discoideum.

Genetic analysis in Dictyostelium discoideum has identified regulatory genes which control the developmental expression of the discoidin lectin multigene family. Among these, the drsA mutation is a dominant second-site suppressor of another mutation, disB, which has the discoidinless phenotype. We now demonstrate a novel mechanism by which the drsA allele exerts its suppressive effect on the disB mutation. Interestingly, drsA does not merely bypass the disB mutation and restore the wild-type pattern of lectin expression. Rather, drsA mutant cells have high levels of discoidin lectin synthesis during growth but do not express lectins during aggregation. In contrast, wild-type cells only express lectin protein during the aggregation period of development. Phenocopies of the drsA mutation show a pattern of discoidin expression similar to that seen in the bona fide mutant. These data suggest that there may be a mechanism of negative feedback, resulting from the high levels of discoidin lectin made during growth, which inhibits further discoidin lectin expression during development. Northern (RNA) analysis of developing drsA mutant cells shows that these cells contain high levels of discoidin mRNA, although no discoidin lectin protein is being translated from these messages. Therefore, expression of the discoidin gene family can be controlled at the level of translation as well as transcription.

Blotting, Northern↗

PubMed1996

Translational control during recovery from heat shock in the absence of heat shock proteins.

Heat shock results in a reduction in translational efficiency and an increase in mRNA stability in plants that is proportional to the severity of the stress. To determine whether the absence of heat shock proteins may affect the observed changes during translation or mRNA turnover, the effect that inhibiting transcription during a heat shock has on the subsequent translation of a reporter mRNA was examined. The presence of actinomycin D repressed heat shock protein synthesis in response to the application of a heat shock by more than 80%. The translational efficiency of the reporter mRNA was subject to a greater degree of repression following a heat shock when transcription was inhibited than it was in heat-shocked cells in which transcription was not inhibited. In contrast, inhibiting transcription during a heat shock did not prevent the heat-mediated increase in mRNA stability. These data suggest that ongoing transcription is needed during a heat shock to support a basal level of translational activity in the subsequent recovery from the stress but does not appear to be required for the heat-mediated increase in mRNA stability.

Animals↗

PubMed1986

Multiple upstream AUG codons mediate translational control of GCN4.

GCN4 encodes a transcriptional activator of amino acid biosynthetic genes in yeast that is regulated at the translational level. The 5' leader of GCN4 mRNA contains four small open-reading-frames. By constructing point mutations in the initiation codons of these sequences, we show that they are essential for translational repression of GCN4. Each upstream AUG codon can repress translation; however, the two 3' proximal AUG codons are much more inhibitory than the 5' proximal AUG codons. Unexpectedly, the first AUG codon is required for efficient GCN4 expression under starvation conditions. This positive function appears to involve antagonism of the inhibitory effect of the 3' proximal AUG codons since it is dispensable in the absence of these sequences. The interaction between the upstream AUG codons is modulated by the trans-acting factors GCN2 and GCD1 in response to amino acid availability.

Amino Acids↗

PubMed1991

Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control.

GCN4 encodes a transcriptional activator of amino acid-biosynthetic genes in Saccharomyces cerevisiae that is regulated at the translational level by upstream open reading frames (uORFs) in its mRNA leader. uORF4 (counting from the 5' end) is sufficient to repress GCN4 under nonstarvation conditions; uORF1 is required to overcome the inhibitory effect of uORF4 and stimulate GCN4 translation in amino acid-starved cells. Insertions of sequences with the potential to form secondary structure around uORF4 abolish derepression, indicating that ribosomes reach GCN4 by traversing uORF4 sequences rather than by binding internally to the GCN4 start site. By showing that wild-type regulation occurred even when uORF4 was elongated to overlap GCN4 by 130 nucleotides, we provide strong evidence that those ribosomes which translate GCN4 do so by ignoring the uORF4 AUG start codon. This conclusion is in accord with the fact that translation of a uORF4-lacZ fusion was lower in a derepressed gcd1 mutant than in a nonderepressible gcn2 strain. We also show that increasing the distance between uORF1 and uORF4 to the wild-type spacing that separates uORF1 from GCN4 specifically impaired the ability of uORF1 to derepress GCN4 translation. As expected, this alteration led to increased uORF4-lacZ translation in gcd1 cells. Our results suggest that under starvation conditions, a substantial fraction of ribosomes that translate uORF1 fail to reassemble the factors needed for reinitiation by the time they scan to uORF4, but become competent to reinitiate after scanning the additional sequences to GCN4. Under nonstarvation conditions, ribosomes would recover more rapidly from uORF1 translation, causing them all to reinitiate at uORF4 rather than at GCN4.

Amino Acids↗

PubMedDate not supplied

Patient safety and inpatient glycemic control: translating concepts into action.

OBJECTIVE: (1) To summarize current knowledge regarding patient safety issues and their impact on inpatient glycemic control and (2) to provide a systematic and practical approach to improving patient safety in this area. METHODS: Insulin delivery in inpatient settings is examined, as are the barriers to safe insulin therapy. A distinction is made between safety strategies of proven value and those that are less useful. Characteristics of safer insulin algorithms are discussed. RESULTS: Errors in insulin administration remain a common cause of injuries. Despite strong evidence of the dangers of sliding-scale insulin regimens in inpatient settings, sliding-scale insulin regimens remain a widely used but ineffective (and occasionally dangerous) method. To correctly analyze inpatient processes of care, a systems approach is needed, and the relationship between quality of care, latent medical errors, injurious patient errors, and patient safety must be understood. In complex systems such as hospitals, catastrophic error has complex roots involving many individuals, and the design of the system often has latent flaws that make human error likely, even predictable. The concept of the scope of awareness is useful in understanding why it is ineffective to focus only on those at the point of care, and how improvement is more apt to occur with a team approach and the evaluation of systemic factors, at the "blunt end" of care. Error-prone organizations are often very unsafe, and certain practices tend to be common among them. Data are now available showing the negative impact of poorly supervised trainees and the extent of the problem worldwide on patient safety. CONCLUSION: Redesign of patient care provided in hospitals regarding glucose control is long overdue. Practical strategies are given that focus on improving patient safety for the patient needing inpatient glycemic control. Such techniques now have a sufficient evidentiary base. Therefore, hospitals should move rapidly to make euglycemia a goal for all inpatients, and to make patient safety in glycemic control a reality, not just for a few, but for all patients in our care.

Humans↗

PubMed1986

Accumulation of nonamidated preprogastrin and preprocholecystokinin products in porcine pituitary corticotrophs. Evidence of post-translational control of cell differentiation.

Using gel and ion-exchange chromatography monitored by radioimmunoassays specific for sequences essential in the processing of preprogastrin and preprocholecystokinin, the products were characterized in extracts of porcine pituitary lobes before and after incubation with trypsin, carboxypeptidase B, and arylsulfatase. The intermediate and neural lobes contained only fully activated (i.e. alpha-amidated) preprogastrin products (component I, gastrin-34, and gastrin-17). In contrast, the anterior lobe contained, in addition to traces of alpha-amidated gastrin (2 pmol/g), hundredfold higher amounts of a nonamidated progastrin (189 pmol/g; Mr approximately 7000) and two nonamidated procholecystokinin fragments (75 pmol/g; Mr approximately 7000 and 5000). These results show that hormone genes, in spite of translation of their mRNA, are not necessarily expressed in functional peptides in cells outside the principal production regions. Hence, the study indicates that differentiation of endocrine cells may be controlled at the post-translational level.

Amino Acid Sequence↗

PubMed2001

Translational control by the ER transmembrane kinase/ribonuclease IRE1 under ER stress.

Under conditions of endoplasmic reticulum (ER) stress, mammalian cells induce both translational repression and the unfolded protein response that transcriptionally activates genes encoding ER-resident molecular chaperones. To date, the only known pathway for translational repression in response to ER stress has been the phosphorylation of eIF-2alpha by the double-stranded RNA-activated protein kinase (PKR) or the transmembrane PKR-like ER kinase (PERK). Here we report another pathway in which the ER transmembrane kinase/ribonuclease IRE1beta induces translational repression through 28S ribosomal RNA cleavage in response to ER stress. The evidence suggests that both pathways are important for efficient translational repression during the ER stress response.

Amino Acid Sequence↗