The translation of globin messenger RNA with use of muscle initiation factors.

The ability of embryonic chicken muscle initiation factors to translate rabbit globin messenger RNA in an efficient, fractionated cell-free system has been examined. Although muscle factors stimulate leucine incorporation to only 15--35% the levels achieved with rabbit reticulocyte initiation factors, they synthesize more than one globin chain per mRNA molecule and both alpha and beta globin are produced. Increasing the ribosome concentration and adding the polyamine spermidine to the system produce stimulatory effects which are quantitatively and qualitatively similar for both factor preparations. The lower efficiency of synthesis of muscle factors relative to reticulocyte factors is also apparent when mRNA from encephalomyocarditis virus or embryonic chicken muscle polysomes are used in the cell-free system. These results do not support a specific restriction in the capacity of muscle factors to translate globin mRNA. Furthermore, the similarity of the effects of presumed non-specific components on the activity of muscle and reticulocyte factors suggests that globin synthesis in the cell-free system may be controlled in a similar fashion for both preparations.     

Identification of Mouse Haemoglobin Messenger RNA

RETICULOCYTE polyribosomes contain 9S RNA with many of the properties expected for the haemoglobin messenger RNA (mRNA)^1–12. Proof that this RNA is the haemoglobin (Hb) mRNA, however, can be obtained only by showing that it directs the synthesis of globin chains. Laycock and Hunt¹³ added an RNA isolated from rabbit reticulocytes to an E. coli cell-free preparation and observed the synthesis of material, with the properties of globin in the presence of N-acetylvalyl tRNA. We added the mouse reticulocyte 9S RNA to a rabbit reticulocyte cell-free system and have shown that material is synthesized which co-chromatographs with mouse globin β-chains¹⁴. We now present evidence that the material synthesized under the direction of the mouse 9S RNA is indeed mouse haemoglobin β-chains.     

Translation of Globin Messenger RNA in a Heterologous Cell-free System

MESSENGER-SPECIFIC initiation factors, capable of discriminating between classes of messenger RNAs (mRNAs) or different cistrons in viral RNA, have been implicated in the regulation of protein synthesis in bacteria^1–5. Comparable but less detailed observations have also been made in eukaryotic systems^6–10. For example, RNA extracted from a mammalian virus (encephalomyocarditis virus, EMC) cannot be translated in a reticulocyte cell-free system unless the system is fortified with an extract from responsive cells—in this case, Krebs II ascites cells⁶. Such results imply the existence of tissue-specific factors and lead to questions whether this incompatibility is reciprocated by an inability of the Krebs II ascites cell system to respond to the mRNA for globin.     

Polyribosome binding of rabbit globin messenger RNA and messenger ribonucleoprotein labelled with bacteriophage-T4 RNA ligase and 5''-[32P] phosphocytidine 3''-phosphate

Rabbit polyribosomal globin messenger RNA (mRNA) and messenger ribonucleoprotein (mRNP) were labelled at the 3′ poly(A) tail to high specific activity with T4 RNA ligase and [5′-³²P]pCp without consequent loss of functional activity. Labelled message was translated in both micrococcal nuclease treated and untreated rabbit reticulocyte lysates, as shown by the formation of labelled polyribosomes. The utilisation of labelled messenger was abolished by T2 toxin or sodium fluoride which are known to inhibit protein synthesis.     

Induction of globin mRNA accumulation by hemin in cultured erythroleukemic cells

The role of heme in erythroid development is investigated in erythroleukemic (Friend) cells. Exogenous hemin induces the accumulation of globin mRNA and globin protein in T3-Cl2 erythroleukemia cells to levels comparable to those induced by polar solvents, such as dimethylsulfoxide (DMSO). The hemin concentration required for maximal induction (10^?4 M) is the same as that which stimulates globin message translation in reticulocytes or cell-free reticulocyte lysates. Hemin and DMSO together cause T3-Cl2 cells to accumulate 8–9 fold more globin mRNA than either inducer individually. The kinetics of globin mRNA induction in hemin as compared to DMSO are very different: globin message accumulation begins 4 hr after hemin addition, but not until 30–40 hr after DMSO addition. Biliverdin induces 20–40 fold less hemoglobin than hemin; delta-aminolevulinic acid and porphobilinogen do not induce.     

Inhibitors of cytoplasmic protein synthesis purified from rat liver mitochondria

Summary Purified mitochondria from rat liver were found to contain protein synthesis inhibitors, that could be extracted by disruption of mitochondrial membranes and fractionated by gel filtration into two fractions of low and high molecular weight. Small size inhibitors were also released from the latter peak by high ionic strength followed by gel filtration. Both types of factors inhibit incorporation of radioactive amino acids into protein by liver cytoplasmic polysomes programmed with endogenous mRNA or poly U, and by rabbit reticulocyte lysates programmed with added globin mRNA and by incubations of Walker carcinoma cells. They decrease to the same level the cytoplasmic synthesis of proteins for the mitochondrial and extra-mitochondrial compartments in intact cells, but do not appear to inhibit substantially endogenous mitochondrial protein synthesis. Inhibitors were purified by paper chromatography and reverse phase high performance liquid chromatography into fractions which block with the same kinetics the incorporation of [¹⁴]leucine and [³⁵]methionine into protein in systems able to initiate protein synthesis, such as reticulocyte lysates or intact cells, but differ in this respect in incubations of liver ribosomes where re-binding of mRNA is a limiting step. Some of these factors behave as oligopeptides that are assumed to inhibit in vitro primarily the initiation stage but whose function in vivo is still undetermined.     

Structure of methemerythrin at 5 A resolution.

Rabbit globin messenger RNA was labelled in vitro with ¹²⁵I to specific activities in the range 20 to 200 × 10⁶ cts/min per μg. This ¹²⁵I-labelled mRNA bound to rabbit reticulocyte ribosomes with the kinetics and sensitivity to inhibitors expected from its participation in the normal process of the initiation of protein synthesis. Furthermore, when modified in 25% of its cytidine residues with unlabelled iodide, the mRNA coded for the same series of initiation peptides as did the unmodified mRNA. Using the techniques of RNA fingerprinting, the binding reaction was shown to select against contaminants and against “globin mRNA” molecules which lack a particular oligonucleotide implicated in the initiation process. When the ¹²⁵I-labelled mRNA was bound to ribosomes, both the initiating 40 S subunits and the 80 S ribosomes protected a fraction of the mRNA from digestion by pancreatic ribonuclease. Fingerprint analysis showed that highly specific regions of the mRNA were protected by the 40 S subunits and 80 S ribosomes and that these two protected regions were not identical.     

Protein synthesis in oocytes of Xenopus laevis is not regulated by the supply of messenger RNA

The control of protein synthesis in oocytes of Xenopus laevis has been investigated by injecting oocytes with mRNA and polysomes followed by labeling with ¹⁴C-amino acid mixtures. Contrary to previous reports in which injected oocytes were labeled with ³H-histidine, injected globin mRNA is found to decrease amino acid incorporation into endogenous proteins competitively at all concentrations tested. No increase in overall amino acid incorporation is detected when more mRNA is supplied. Similar results are obtained after labeling injected oocytes with leucine, methionine, proline or valine individually. An explanation is presented for the conflicting results obtained when histidine is used as a label.When reticulocyte polysomes are injected, rather than purified globin mRNA, incorporation of amino acids into endogenous proteins remains roughly constant and overall incorporation increases. Similarly, when encephalomyocarditis viral RNA is injected together with either globin mRNA or reticulocyte polysomes, the globin mRNA causes decreased amino acid incorporation into encephalomyocarditis proteins, but the polysomes do not do so. The results demonstrate that different types of mRNA compete for a strictly limited translational capacity which is saturated in the normal oocyte. The limiting component is present in polysomes and is not message-specific. The constraint on protein synthesis in the amphibian oocyte cannot be fully explained by masked mRNA.     

Characteristics of rabbit globin mRNA purification by oligo(dT) cellulose chromatography

We have purified rabbit globin mRNA using oligo(dT)-cellulose and sucrose gradient centrifugation. Both α- and β-globulin mRNA molecules behave heterogeneously with respect to their elution properties during chromatography on oligo(dT)-cellulose. Those fractions eluted at the lowest ionic strength are most active in directing cell-free globin biosynthesis. By making use of hybridization with synthetic [³H]DNA complementary to globin mRNA, we have shown that this technique can be used to quantitate the extent of mRNA purification. Thus, globin mRNA is approximately 90-fold purified from reticulocyte polysomal RNA and originally constituted slightly more than 1% of the polysomal RNA. Since more than 98% of the globin mRNA sequences are bound to oligo(dT)-cellulose, we suggest that most polysomal globin mRNAs contain a poly (A)-rich region and that this region is not of uniform length nor preponderately associated with either the α- or β-globin mRNAs. In addition, we observe that the 9S globin mRNA most resistant to dissociation from oligo (dT)-cellulose is most active in directing globin biosynthesis.     

Inhibition of peptide chain initiation by a nonhemin-regulated translational repressor from Friend leukemia cells.

A nonhemin-regulated translational repressor protein has been purified partially from the postribosomal supernatant fraction of Friend leukemia cells grown in the absence of dimethylsulfoxide. This repressor inhibits protein synthesis in lysates from rabbit reticulocytes or Friend leukemia cells and in a fractionated system using Artemia salina ribosomes, reticulocyte mRNA, and soluble components from reticulocytes. In contrast, the hemin-controlled repressor from reticulocytes does not inhibit protein synthesis in lysates from Friend leukemia cells. The repressor from Friend leukemia cells has no effect on poly(U)-directed synthesis of polyphenylalanine using reticulocyte ribosomes nor on the extension and release of nascent globin chains that were initiated in intact reticulocytes. It does not block completion of peptides on ribosomes isolated from reticulocytes incubated with NaF nor does it inhibit initiation factor-dependent formation of methionylpuromycin, but it inhibits globin mRNA-dependent methionylvaline synthesis. The Friend leukemia cell repressor promotes peptide synthesis-dependent breakdown of polysomes in reticulocyte lysates that appears to involve inhibition of ribosome reattachment to mRNA during peptide chain initiation. It is concluded that the Friend leukemia cell repressor blocks peptide initiation at a point between the addition of methionyl-tRNA^fMet to the ribosomal initiation complex and the NaF-sensitive reaction.     

Competition between α and β globin messenger RNA

Addition to an unfractionated reticulocyte lysate of either α or β globin mRNA or reticulocyte initiation factors does not alter the overall rate of globin synthesis. Addition of β mRNA results in enhanced synthesis of β product and decreased production of α; conversely, addition of α mRNA results in enhanced synthesis of α globin and decreased production of β. We conclude that the amount of any putative α mRNA or β mRNA-specific factor does not normally limit the rate of synthesis of α or β chains; rather, the two mRNAs compete for some non-specific rate-limiting component of chain initiation.     

Effects of Cap Analogue or Cap Removal on the Translation of Rat Brain mRNA In Vitro

Abstract: The role of cap structures in the translation of brain mRNA was examined by measuring protein biosynthesis in vitro in wheat germ and reticulocyte systems programmed by mRNA that was either untreated or oxidized by periodate or from which 5'-terminal 7-methylguanosine (m⁷G) was removed by oxidation and β -elimination. In another series of reactions, amino acid incorporation into polypeptides was measured in the absence and in the presence of varying concentrations of the cap analogue 7-methylguanosine 5'-triphosphate (pppm⁷G). The results indicated that any of the above treatments interfered with brain mRNA translation, the degree of inhibition depending on the translation system used, the concentration of mRNA, and the source of initiation factors. Homologous brain initiation factors were superior to reticulocyte factors in providing a partial relief from inhibition of translation caused by these treatments. It was also found that synthesis of the brain-specific protein S-100 was inhibited by β -elimination of mRNA, by pppm⁷G, or by the presence of capped globin mRNA, indicating that the mRNA for this protein was probably capped.     

Biosynthesis and amino terminal acetylation of cat hemoglobin B

Acetylation of the amino-terminal serine of the β chains of cat hemoglobin B (HbB) occurs during synthesis of hemoglobin in a mRNA-dependent protein synthesizing system from rabbit reticulocyte lysate in the presence of acetyl-CoA and cat reticulocyte mRNA. The process occurs after peptide chain growth of about 30 amino acid residues. When endogenous acetyl-CoA was removed from the rabbit reticulocyte lysate by pretreatment with oxalacetate and citrate synthase, nonacetylated HbB (HbB′) was synthesized. Thus, β^B globin chain synthesis goes to completion in the absence of acetylation even though the latter normally occurs during nascent chain growth. When HbB′ was incubated with acetyl-CoA in a rabbit reticulocyte lysate, hemoglobin with properties identical to those of HbB was produced. Thus, the selective amino terminal acetylation of β^B globin also occurs in the completed hemoglobin.     

Defective initiation on natural messenger RNA by cell free systems from Krebs ascites cells incubated at elevated temperatures.

1. Cell-free systems prepared from Krebs II ascites cells incubated at 45 degrees C have a much lower endogenous activity than those from cells incubated at 37 degrees C. The endogenous activity is mainly due to completion of polypeptide chains initiated in the intact cell. The low activity of the 45 degrees C system is due to a lesion in initiation in cells incubated at 45 degrees C. 2. Cell-free systems from cells incubated at 45 degrees C can translate efficiently poly (U) at 8 mM Mg2+. However, they initiate poorly on globin mRNA, indicating that these systems reflect the situation in the intact cell. 3. The lesion in globin mRNA translation in 45 degrees C systems can be overcome by addition of reticulocyte initiation factors. At saturation concentrations of factors, the response of a 45 degrees C system is restored to almost normal. 4. 45 degrees C systems from 40-S initiation complexes with methionyl tRNAfmet almost as efficiently as normal, but their ability for form 80-S complexes with globin mRNA is impaired, unless they are supplied with exogenous initiation factors.     

Message stability in injected frog oocytes: long life of mammalian alpha and beta globin messages

The stability of messenger RNA for rabbit and mouse α and β globin has been tested by injection into living frog oocytes, which were subsequently cultured for up to two weeks. [³H]histidine was added to the culture medium at various times and incorporated into haemoglobin whose synthesis was measured by Sephadex and carboxymethylcellulose chromatography. α Globin mRNA is translated only 20% as efficiently as β globin mRNA after injection into oocytes; the same messages are translated with almost equal efficiency if tested in a reticulocyte cell-free system, or if injected into oocytes as unpurified reticulocyte polysomes. For up to two weeks, injected haemoglobin mRNA was about as stable as the mRNAs of the host oocyte. The injected mRNAs for α and β mouse globins also had a similar stability. A fall in the absolute rate of amino acid incorporation into haemoglobin was observed in oocytes that were labelled for several days, but this could be wholly accounted for by a decrease in the efficiency of the oocytes' translational system. The synthesis of β globin from each molecule of injected β mRNA takes place over a longer period of time, when the mRNA is injected into oocytes, than would have been the case if it had remained in the reticulocytes from which it was prepared. We conclude that α and β globin mRNA molecules are very stable in oocytes, and we suggest that the translational life of a message may be determined in part by the kind of cell in which it operates.     

A precursor of globin messenger RNA

The size of pulse-labeled globin messenger RNA nucleotide sequences was investigated, to determine whether newly transcribed globin mRNA molecules are larger than steady-state globin mRNA. Molecular hybridization techniques were used to compare directly the sedimentation of steady-state (unlabeled) and pulse-labeled (radioactive) globin mRNA sequences in the same analytical sucrose gradient. In gradients containing 98% formamide, radioactive globin mRNA sequences from mouse fetal liver cells labeled for 15 to 20 minutes with [³H]uridine sediment in a broad band with a peak at approximately 14 S, while steady-state globin mRNA sediments at 10 S. The large radioactive RNA can be recovered from one gradient and recentrifuged in a second gradient, in which it again sediments in a broad band with a peak at 14 S. The large radioactive RNA is cleaved to 10 S during a 75-minute “chase” with either actinomycin D or unlabeled uridine plus cytidine. The estimated half-life of the precursor is 45 minutes or less under these conditions. A covalent RNA precursor larger than 18 S with a similar turnover rate is not observed.     

Translation of maternal messenger ribonucleoprotein particles from sea urchin in a cell-free system from unfertilized eggs and product analysis

Maternal mRNP particles were isolated from the postribosomal supernatant fluid of unfertilized sea urchin eggs. They were translated in a cell-free system derived from unfertilized eggs. The translation of these particles required the presence of 12 mM MgCl₂, which is considered very high. The same high Mg²⁺ requirement was observed when mRNP particles were translated in a cell-free system from morula embryos. In contrast, mRNA extracted from mRNP particles is translated at 3 mM MgCl₂. This concentration of Mg²⁺ is known to be optimal for initiation of mRNA translation. Likewise, a rabbit globin mRNA is faithfully translated into α and β globin chains in a cell-free system from eggs at 3, but not at 12, mM MgCl₂. The translational products directed by mRNP or by mRNA derived from mRNP were examined in two gel systems and were found to be very similar. In both cases, histones were identified as part of the translational product. This indicated that the translation of mRNP in high Mg²⁺ is not due to nonspecific binding of these particles to ribosomes. The rates of globin synthesis in a cell-free system derived from eggs is comparable to that of morula ribosomes and to that reported for translation of globin with mouse liver and reticulocyte ribosomes, indicating that unfertilized sea urchin egg ribosomes do not possess a translational inhibitor and that no deficiency in initiation factors for mRNA translation could explain the low rate of protein synthesis in unfertilized sea urchin eggs.     

Globin mRNA translation on Artemia salina ribosomes with components from Friend leukemia cells.

Globin mRNA can be translated with relatively high efficiency in a fractionated cell-free system containing ribosomes prepared from cytst of Artemia salina. These ribosomes have unusually low endogenous activity for peptide synthesis in the absence of added mRNA. The system requires components from the postribosomal supernatant and from the 0.5 M KCl ribosomal wash fraction. Both these fractions were derived from either rabbit reticulocytes or unstimulated Friend leukemia cells that produce little or no hemoglobin. The activity of mRNA and enzyme fractions from rabbit reticulocytes and Friend leukemia cells were tested in this system in vitro for their ability to direct the synthesis of the alpha and beta chains of globin. The alpha:beta chain ratio synthesized from mRNA in the rabbit reticulocyte salt wash fraction was 4:1. The corresponding value for the 9-S mRNA fraction from the salt-washed reticulocyte ribosomes was 1:4, thus these two fractions appear to provide sources enriched in either alpha or beta globin mRNA. Under all conditions tested, the ratio and amounts of peptides formed in vitro appear to reflect mRNA composition. Globin mRNA from dimethysulfoxide-stimulated Friend leukemia cells when translated in vitro produced alpha and beta chains in a ratio of 1:1. These peptides are formed in the same ratio in the intact cells.     

Inhibition of protein synthesis by double-stranded RNA in reticulocyte lysates: evidence for activation of an endoribonuclease.

Double-stranded RNA is a potent inhibitor of protein synthesis in rabbit reticulocyte lysates. Three lines of evidence suggest that at least part of this inhibitory activity is due to activation of a nuclease which degrades mRNA: (1) In the presence of emetine reticulocyte polysomes are partially degraded to structures containing 1–3 ribosomes; (2) 34S Mengo-virus RNA is degraded to fragments sedimenting at less than 18S; (3) The template activity of globin mRNA extracted from the lysates is reduced by 90% when compared to appropriate controls. The ability of double-stranded RNA to activate a nuclease in the reticulocyte system is very similar to that observed in extracts from interferon treated cells and probably involves formation of the unusual oligonucleotide pppA^2′ p^5′ A^2′ p^5′ A.     

Nonuniform size distribution of nascent peptides: The role of messenger RNA

The origin of the nonuniform size distribution of nascent rabbit globin peptides has been investigated in the reticulocyte lysate and wheat germ cell-free protein synthesis systems. Increasing the concentrations of the cellular components involved in protein synthesis failed to alter the elution pattern observed upon chromatographic analysis of reticulocyte lysate nascent chains. Nascent chains isolated from a globin messenger RNA-directed wheat germ cell-free system showed a nonuniform size distribution of nascent peptides similar to that of the rabbit reticulocyte nascent chains. These observations indicate that the nonuniformity of the globin nascent chains arises from a unique property of the messenger RNA being translated and not from limiting concentrations of a component or components of the reticulocyte protein synthesis system.