Translation Of Globin mRNA Research Articles

Phosphorylation of ribosomal protein S6 by cAMP-dependent protein kinase and mitogen-stimulated S6 kinase differentially alters translation of globin mRNA.

The effects of phosphorylation of ribosomal protein S6 by two different protein kinases, the cAMP-dependent protein kinase and the mitogen-stimulated S6 kinase, or translation of globin mRNA in a reconstituted system and on binding of globin mRNA to 40 S ribosomal subunits were examined. The cAMP-dependent protein kinase incorporated 1.5 mol of phosphate/mol of 40 S ribosomal subunits. Phosphorylation of S6 by the cAMP-dependent protein kinase had no effect on binding of 3' terminus-labeled globin mRNA to 40 S ribosomal subunits. [3H]Leucine incorporation with 40 S ribosomal subunits phosphorylated by the cAMP-dependent protein kinase was identical to that observed with nonphosphorylated 40 S ribosomal subunits, although on occasion, a slight inhibition (less than 10%) was observed; there was no effect on the rate of synthesis of either the alpha or beta chains of globin. Phosphorylation with the mitogen-stimulated S6 kinase (2.5 mol/mol) did not alter binding of globin mRNA to 40 S ribosomal subunits; however, translation of globin mRNA in the reconstituted protein-synthesizing system was stimulated up to 4-fold over that observed with nonphosphorylated subunits. Synthesis of both the alpha and beta chains of globin was enhanced by phosphorylation as shown by electrophoretic analysis. Since the sites phosphorylated by the mitogen-stimulated S6 kinase are identical to those observed in vivo in response to insulin and growth-promoting compounds, the data support the hypothesis that enhanced synthesis of specific proteins may be due to phosphorylation of S6 and that differential phosphorylation of S6 can alter translation of natural mRNA.

Inhibition of rabbit globin mRNA translation by sequence-specific oligodeoxyribonucleotides.

Oligodeoxyribonucleotides 8-12 nucleotides in length whose sequences are complementary to the 5' end, the initiation codon regions, or the coding regions of rabbit globin mRNA were tested for their ability to inhibit translation in a rabbit reticulocyte lysate and in a wheat germ extract. The oligomers interact specifically with their target mRNAs as shown by their ability to serve as primers with reverse transcriptase. In the reticulocyte lysate, oligomers complementary to the 5' end or the initiation codon regions inhibit translation of both alpha- and beta-globin mRNA, whereas oligomers complementary to the coding regions have little or no effect. This suggests that reticulocyte ribosomes are able to displace the oligomers from the mRNA during the elongation but not the initiation step of translation. In the wheat germ system, translation was effectively inhibited by all oligomers regardless of their binding site on the message. In contrast to their behavior in the reticulocyte system, the oligomers inhibited alpha- and beta-globin synthesis in a specific manner. This observation suggests that control of alpha- and beta-globin mRNA translation is coordinated in the reticulocyte lysate system but not in the wheat germ extract. The results of our studies indicate that oligodeoxyribonucleotides may be useful probes for studying control of mRNA translation in cell-free systems.

Human rhinovirus 14 infection of HeLa cells results in the proteolytic cleavage of the p220 cap-binding complex subunit and inactivates globin mRNA translation in vitro.

One of the characteristics of picornavirus infection of cells in tissue culture is a specific inhibition of utilization of host cell mRNA for protein synthesis. In this study we show that human rhinovirus 14 is similar to poliovirus in that the inhibition of host cell translation that occurs during infection correlates with the proteolytic cleavage of an Mr 220,000 subunit of the cap-binding protein complex.

Effects of trichothecenes (T-2 toxin) on protein synthesis in vitro by brain polysomes and messenger RNA

1. The effects of T-2 toxin on protein synthesis were tested in two reticulocyte lysate In vitro systems pretreated with micrococcal nuclease. One of the test systems contained purified globin mRNA and was initiation dependent. The other contained rat brain polysomes and incorporated amino acids by an elongation dependent process. 2. T-2 toxin inhibited the translation of globin mRNA at all concentrations tested, from 10 −8M to 10 −4M. Rat brain polysomes were much less sensitive to T-2 toxin than globin mRNA. While high concentrations of the toxin (10 −4 M ) led to partial inhibition of protein synthesis by polysomes, low concentrations (10 −8 M and 10 −6 M) stimulated protein synthesis. 3. Comparison of the above results with those obtained by other workers suggests that the T-2 toxin may inhibit not only the initiation step of translation, but also elongation and termination, depending upon the concentration of the toxin and the nature of the translation system. A similar mechanism may operate for all the trichothecene toxins that exert their effect through binding to ribosomal peptidyl transferase.

Correlation between immunosuppressive activity and translation regulatory activity

An immunoglobulin negative material from the eluate of an anti-idiotype immunosorbent column [1] exhibited potent immunosuppressive activity. This material also inhibited the translation of globin mRNA in a cell-free reticulocyte lysate system. The translation inhibitory activity of this material was not attributable to nucleases which were separable by a blue-dextran agarose column. Further correlation between immunosuppressive activity and translation inhibitory activity was observed when GTP or GTP analogue was included in experimental systems. These results suggest that the immunosuppressive factor (or factors) may contain a translation inhibitory factor. The biochemical mechanism of immunosuppression is discussed.

Preparation of a cell-free translation system from a wild-type strain of Neurospora crassa and translation of pyruvate kinase messenger RNA.

A cell-free in vitro translation system exhibiting high activity has been developed from wild-type Neurospora crassa mycelium. The isolation is simple and fast, and the homogenization does not appear to affect the activity of mycelial proteases and nucleases. This system is capable of supporting efficient translation of exogenously added homologous RNA as demonstrated by the experiments with PK-specific mRNA. In addition, it translates heterologous RNA efficiently, shown by the translation of globin mRNA. We did not examine the Neurospora lysate for post-translational modification activity. The procedure used for the preparation of Neurospora cell-free extracts should be readily applicable to the other filamentous fungi.

Preferential stimulation of rabbit α globin mRNA translation by a cap-binding protein complex

A cap-binding protein complex (Edery et al. (1983) J. Biol. Chem. 258, 11398–11403) is shown here to stimulate preferentially the translation of endogenous α versus β globin mRNA in a rabbit reticulocyte lysate. Several initiation factors (eIF-2, eIF-3, eIF-4A, eIF-4B, eIF-4C, eIF-4E and eIF-5) and elongation factor 1 were found to have no such discriminatory effect. These results are in contrast to several previous reports and demonstrate that the only factor capable of relieving translational competition between α and β globin mRNAs is the cap-binding protein complex.

The cytoplasmic 4S translation inhibitory RNA species of chick embryonic muscle: fractionation of biologically active subspecies by high performance liquid chromatography.

A 4S RNA species (iRNA) isolated from chick embryonic muscle which is a potent inhibitor of mRNA translation in vitro shows heterogeneity in the 70-100 nucleotide size range (Sarkar, S., Mukherjee, A.K., and Guha, C. (1981) J. Biol. Chem., 256, 5077-5086). The iRNA was fractionated by HPLC on different size exclusion columns using a variety of elution conditions. Chromatography of iRNA on a TSK 4000 SW column and elution with a low ionic strength buffer gave three components, one of which contained a pure subspecies of about 90-100 nucleotides size, as shown by a single band on PAGE analysis in 99% formamide. The biological activity of this purified subspecies showed that this is a more potent inhibitor of globin mRNA translation than unfractionated iRNA (Sarkar, S., Mukherjee, A.K., and Guha, C., (1981) J. Biol. Chem. 256, 5077-5086). Partial resolution of three additional low molecular weight iRNA subspecies in the 70-80 nucleotide size range in biologically active form was obtained on chromatography of unfractionated iRNA on TSK 4000 SW column in the presence of 0.5 M NaCl or on TSK 3000 SW column in the presence of low salt. The fractionation of iRNA by HPLC appears to be primarily based on size. These results strongly suggest that HPLC may also be useful for the fractionation of a variety of low molecular weight eukaryotic nuclear and cytoplasmic RNAs with retention of biological activity.

Messenger RNA—ribonucleoprotein interaction during the initiation of protein synthesis

The interaction of globin mRNA with proteins during translation has been investigated in order to establish whether and to what extent messenger-ribonucleoprotein complexes are involved in protein synthesis. We present evidence for the functional importance of two minor messenger RNA-associated proteins (55 kDa and 60 kDa) during the initiation of globin mRNA translation in reticulocyte lysates. The formation of an mRNA complex containing the major 78 kDa and 52 kDa messenger-ribonucleoproteins was not detected.

Simplified in vitro system for study of eukaryotic mRNA translation by measuring di- and tripeptide formation.

An in vitro system for measurement of rabbit globin mRNA translation has been developed based on the formation of the NH2-terminal dipeptide, fMet-Val. The basic components include a partially purified initiation factor preparation from rabbit reticulocytes supplemented with eukaryotic initiation factor 4A, purified and formylated yeast Met-tRNAi, and rabbit liver or Escherichia coli Val-tRNA1Val. Picomole quantities of fMet-Val are synthesized, dependent on mRNA, and the dipeptide is readily assayed by a simple extraction procedure. In the presence of Leu-tRNA or His-tRNA, the tripeptides fMet-Val-Leu and fMet-Val-His are synthesized, corresponding to the NH2-terminal sequence of alpha- and beta-globin, respectively. Therefore, tripeptide synthesis provides a simple means to distinguish between the expression of the alpha- and beta-globin mRNA species.

Desferrioxamine inhibits induced erythroid differentiation of human leukemic K-562 cells

The iron chelator desferrioxamine reversibly inhibits heme accumulation and globin synthesis in human leukemic K-562 cells induced to express erythroid genes by butyric acid. These results suggest that iron metabolism can modulate globin gene expression. In addition we describe experimental conditions (6.25 μg/ml desferrioxamine) which do not suppress transcription of globin genes and translation of globin mRNA but prevent heme synthesis. Therefore expression of globin genes in butyric acid induced K-562 cells does not require accumulation of heme molecules. Human leukemic K-562 cells cultured with different inducers and treated with desferrioxamine should be used as a useful model system to further analyse the relationship between expression of erythroid genes, iron metabolism and heme accumulation.

New initiation factor activity required for globin mRNA translation.

A reconstituted reticulocyte translation system originally designed to be deficient in eukaryotic initiation factor 4B (eIF-4B) was used to identify a new activity required for maximal synthesis of rabbit globin. This new activity purifies as a stable, high molecular weight complex by a variety of chromatographic procedures and is termed eIF-4F. The purified globin stimulatory activity also restores translation of capped mRNAs in extracts of poliovirus-infected HeLa cells. Like restoring activity that was obtained as a protein complex by different procedures (Tahara, S. M., Morgan, M. A. and Shatkin, A. J. (1981) J. Biol. Chem. 256, 791-794), eIF-4F includes the 24,000-dalton cap binding protein and major polypeptides of Mr approximately 200,000 and approximately 46,000. The latter component comigrates with eIF-4A by two-dimensional gel electrophoresis and, like eIF-4A, chemically cross-links to the 5'-end of capped mRNA by an ATP-dependent, m7GDP-sensitive reaction. Unlike eIF-4F, cap binding protein of Mr approximately 24,000 isolated by affinity chromatography on m7GDP-Sepharose does not stimulate globin synthesis in the reconstituted system.

The effects of lectin transformation on cytoplasmic polyadenylated RNA from human lymphocytes.

The translational activity of cytoplasmic poly(A)+ RNA from resting human lymphocytes was approximately 20% of that from phytohemagglutinin-transformed lymphocytes in a rabbit reticulocyte lysate assay. Translation assays in the presence of cap analogues suggested that the mRNA from resting cells was relatively deficient in functional 5'-terminal cap structures. Neither mRNA fraction inhibited the translation of globin mRNA in the cell-free assay, and both preparations were essentially pure as shown by hybridisation with [3H]poly(U). The size distribution and poly(A) tail length of poly(A)+ RNA was similar in the resting and transformed cell and both preparations directed the synthesis of peptides of molecular weight 15 000 to 90 000. Two dimensional gels of total proteins from resting and transformed lymphocytes showed predominantly quantitative changes. However cross-hydridising cDNA and mRNA from resting and transformed cells after the common sequences have been removed by hydroxylapatite chromatography showed that about 4% of the cytoplasmic poly(A)+ RNA from transformed lymphocytes was not present in resting cells. This difference may result from transformation-specific gene expression.

Stimulation of Xenopus oocyte protein synthesis by microinjected adenovirus RNA.

The ability of injected heterologous mRNAs to compete with endogenous mRNAs in Xenopus oocytes was assayed. In confirmation of previous reports, globin mRNA translation in oocytes results in a concomitant decrease in endogenous protein synthesis. In contrast, injection of adenovirus 5 mRNA into the oocyte results in a stimulation of endogenous protein synthesis. The stimulation is dose dependent and does not require nuclear transcription in the oocyte. Preliminary mapping data suggest that the stimulatory RNA is a product of one of the viral immediate early genes.

Effects of ethionine treatment on protein-synthesizing apparatus of rat liver 80 S ribosomes and 40 S ribosomal subunits

The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete cell-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsCl isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [ 14C]- or [ 3H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 S subunits by SDS-polyacrylamide slab gel electrophoresis revealed that eIF-3 subunits and two unidentified protein fractions of molecular weight of 2.3·10 4 and 2.1·10 4 were decreased in ethionine-treated rat liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with N-[ 3 H]ethylmaleimide or N-[ 14 C]ethylmaleimide , and the 3H to 14C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially eIF-3, as the results of conformational changes of 40 S subunits.

A polypeptide which reverses cap analogue inhibition of cell-free protein synthesis. Purification and binding to capped oligonucleotides.

An assay was developed to detect the component which recognizes the methylated 5'-terminus of messenger RNA (cap) during initiation of translation. Globin mRNA translation in a reticulocyte cell-free system was partially inhibited with cap analogues, and protein fractions were added to the system in an attempt to reverse inhibition. Such an activity was detected in the 500 mM KCl extract of rabbit reticulocyte ribosomes. The activity (cap analogue inhibition reversal) was purified 800-fold by precipitation with ammonium sulfate saturation, batchwise chromatography on DEAE-cellulose, centrifugation on sucrose gradients containing 100 and 500 mM KCl, and column chromatography on DEAE-cellulose and phosphocellulose. At some stages multiple peaks of activity were detected. Electrophoretic analysis of the final preparation revealed a single polypeptide of 24,000 daltons, making it likely that it is the same as the cap-binding protein detected by Sonenberg et al. (Sonenberg, N., Morgan, M. A., Merrick, W. C., and Shatkin, A. J. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4843-4847), using a cross-linking assay. Direct binding of purified fractions of cap analog inhibition reversal factor to capped oligonucleotides of the form m7Gppp(Np)6-8G[32P]Cp could be demonstrated by both gel filtration on Sephadex G-50 and nitrocellulose membrane filtration. Binding was stimulated by MgCl2 and nucleoside triphosphates. An approximate association constant between oligonucleotide and protein of 3 X 10(8) M-1 was obtained.

Translational control by messenger RNA competition for eukaryotic initiation factor 2.

Translation of globin mRNA in a micrococcal nuclease-treated reticulocyte lysate was studied in the presence of increasing amounts of Mengovirus RNA, under conditions in which the number of translation initiation events remains constant as judged by the transfer of label from N-formyl[35S]methionyl-tRNAf into protein. The translation of globin mRNA is progressively inhibited by low concentrations of Mengovirus RNA, free of detectable traces of double-stranded RNA, concomitant with the increasing synthesis of Mengovirus RNA-directed products. On a molar basis, Mengovirus RNA apparently competes about 35 times more effectively than globin mRNA for a critical component in translation. The competition is relieved by the addition of highly purified eukaryotic initiation factor 2 (eIF-2). Addition of eIF-2 does not stimulate overall protein synthesis, but shifts it in favor of globin synthesis. No stimulation of globin mRNA translation by eIF-2 is seen when Mengovirus RNA is absent. These experiments show that Mengovirus RNA competes, directly or indirectly, with globin mRNA for eIF-2. In direct binding experiments using isolated mRNA and eIF-2, Mengovirus RNA is shown to compete with globin mRNA for eIF-2 and to exhibit a 30-fold higher affinity for this factor. The binding of Mengovirus RNA to eIF-2 is much more resistant to increasing salt concentrations than is the binding of globin mRNA, again reflecting its high affinity. These results reveal a direct correlation between the ability of these mRNA species to compete in translation and their ability to bind to initiation factor eIF-2. They suggest that the affinity of a given mRNA species for eIF-2 is essential in determining its translation, relative to that of other mRNA species. Messenger RNA competition for eIF-2 may contribute significantly to the selective translation of viral RNA in infected cells.

Regulation of protein synthesis: translational control by procollagen-derived fragments.

Previous studies have shown that a type I procollagen-derived peptide, called pN alpha 1(I)-Col 1, selectively inhibits collagen synthesis by fibroblasts in culture and the translation of procollagen mRNA in a rabbit reticulocyte lysate system. We prepared the 10,700-dalton peptide from dermatosparactic calf skin, which contained high levels of incompletely processed type I procollagen, by collagenase digestion. Time-course and dose-response studies showed that the peptide specifically inhibited the translation of procollagen mRNA in preparations of human fibroblast RNA while not affecting the translation of globin mRNA or of other messenger RNAs in fibroblast RNA. After reduction and alkylation, the peptide lost its specificity but became a nonspecific inhibitor of translation. Enzymatic cleavage enabled us to localize the nonspecific activity to a short sequence -Pro-Thr-Asp-Glu, an assignment confirmed by peptide synthesis. Using pactamycin, a specific inhibitor of translational initiation, we showed that the intact peptide acts on procollagen mRNA translation by inhibition of polypeptide chain elongation or termination, or both, whereas the nonspecific inhibitory activity of the unfolded peptide and its derivatives can be attributed to an inhibition of chain initiation. Although the native peptide may function in feedback regulation of collagen synthesis, the physiological role of the lower molecular weight fragments, if any, is uncertain.

Molecular characteristics of a non-deletion alpha-thalassaemia of the Po River Delta.

The form of alpha-thalassaemia of the Po river delta presents haematological and globin biosynthetic characteristics similar to alpha-thalassaemia-1 but never gives rise to HB H disease nor to hydrops foetalis. In alpha-thalassaemic subjects originally from this region globin mRNA translation and alpha-globin gene arrangement have been investigated. The data obtained indicate that alpha-globin synthesis and reticulocyte alpha-globin mRNA are reduced by one fourth; in addition, since the defect in alpha-globin synthesis does not change with cell ageing, a possible instability of alpha-globin mRNA is excluded. Restriction enzyme analysis of the DNA shows a normal alpha-globin gene organization. This form of alpha-thalassaemia is therefore of the non-deletion type; its molecular lesion is either at the level of alpha-globin mRNA transcription or processing. The fact that this, as well as other forms of non-deletion alpha-thalassaemia, have a phenotypic expression similar to alpha-thalassaemia 1 is discussed.

Selective inhibition of Escherichia coli protein synthesis and growth by nonionic oligonucleotides complementary to the 3' end of 16S rRNA.

A series of nonionic oligonucleotide analogues, the deoxyribooligonucleoside methylphosphonates, were synthesized. The base sequences of these compounds, d(ApGpGp), d(ApGpGp)(2), and d[(ApGpGp)(2)T], are complementary to the Shine-Dalgarno sequence (-A-C-C-U-C-C-U-) found at the 3' end of bacterial 16S rRNA. These nonionic oligonucleotide analogues were tested for their ability to inhibit the in vitro translation of mRNAs in cell-free systems of Escherichia coli and rabbit reticulocyte. In the E. coli system, both d(ApGpGp)(2) and d[(ApGpGp)(2)T] effectively inhibited MS-2 RNA-directed protein synthesis but they had much less effect on either poly(U)- or poly(A)-directed polypeptide synthesis. In the reticulocyte system, these compounds had no significant effect on the translation of globin mRNA. The observation that d[(ApGpGp)(2)[(3)H]T)] binds to 70S ribosomes (association constant, 2.0 x 10(4) M(-1), 37 degrees C) together with the specificity of the inhibitory action of these compounds on protein synthesis strongly suggests that inhibition of translation is a consequence of analogue binding to Shine-Dalgarno sequence of 16S rRNA. The oligonucleoside methylphosphonates inhibited both protein synthesis (without concurrent inhibition of RNA synthesis) and colony formation by E. coli ML 308-225 (a permeable mutant) whose cell wall contains negligible quantities of lipopolysaccharide but had no effect on wild-type E. coli B. Our preliminary results on the uptake of oligodeoxyribonucleoside methylphosphonates by E. coli B show that these cells are not permeable to oligomers longer than 4 nucleotidyl units. Although oligodeoxyribonucleoside methylphosphonates are taken up by mammalian cells in culture, this series of analogues had negligible inhibitory effects on colony formation by transformed human cells. This study indicates that this class of nonionic oligonucleotide analogues can be used to probe and regulate the function and structure of nucleic acids of defined sequence within living cells.