The effect of formycin B on mRNA translation and uptake of purine precursors in Leishmania mexicana

Formycin B is a structural analog of inosine that is a potent inhibitor of Leishmania multiplication. Formycin B is reportedly converted to formycin A nucleotides and incorporated into RNA by the organisms, and it is unclear whether the active form of the drug is the nucleoside itself or its several metabolites. We confirmed that formycin A nucleotides are formed by formycin B-exposed L. mexicana promastigotes, and determined that the intraparasite concentration of Formycin B and its metabolites was 6 times the extracellular formycin B concentration. Formycin B did not significantly inhibit purine nucleoside transport by intact promastigotes or purine base phosphoribosylation by parasite lysates. Thus, the nucleoside does not appear to inhibit these initial steps of purine nucleoside metabolism. Since RNA and protein synthesis in formycin B-treated intact promastigotes was found to be inhibited within 30 minutes, the effect of formycin A metabolites on leishmanial protein synthesis was investigated in in vitro protein synthesis experiments. Messenger RNA from formycin B-treated promastigotes was translated only 40% as efficiently as control promastigote mRNA by rabbit reticulocyte lysates. In addition, when formycin A-5'-triphosphate was preincubated with the rabbit reticulocyte lysates, translation of control mRNA was 86% inhibited. Formycin B toxicity to Leishmania promastigotes appears to be at least partially due to inhibition of protein synthesis by formycin A nucleotides and formycin A containing mRNA.     

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.     

15-deoxyspergualin primarily targets the trafficking of apicoplast proteins in Plasmodium falciparum

15-Deoxyspergualin, an immunosuppressant with tumoricidal and antimalarial properties, has been implicated in the inhibition of a diverse array of cellular processes including polyamine synthesis and protein synthesis. Endeavoring to identify the mechanism of antimalarial action of this molecule, we examined its effect on Plasmodium falciparum protein synthesis, polyamine biosynthesis, and transport. 15-Deoxyspergualin stalled protein synthesis in P. falciparum through Hsp70 sequestration and subsequent phosphorylation of the eukaryotic initiation factor eIF2alpha. However, protein synthesis inhibition as well as polyamine depletion were invoked only by high micromolar concentrations of 15-deoxyspergualin, in contrast to the submicromolar concentrations sufficient to inhibit parasite growth. Further investigations demonstrated that 15-deoxyspergualin in the malaria parasite primarily targets the hitherto underexplored process of trafficking of nucleus-encoded proteins to the apicoplast. Our finding that 15-deoxyspergualin kills the malaria parasite by interfering with targeting of nucleus-encoded proteins to the apicoplast not only exposes a chink in the armor of the malaria parasite, but also reveals new realms in our endeavors to study this intriguing biological process.     

Initiation of endogenous messenger RNA translation on hen oviduct polysomes.

The eIF-2A fraction of reticulocyte ribosomal salt wash is capable of maximally stimulating the translation of endogenous messenger RNA by hen oviduct polysomes. The factor increases the initiation of protein synthesis 2--3-fold when measured by the factor-dependent synthesis of NH2-terminal peptides. The addition to these polysomes of elongation factor, EF-1, also increases protein synthesis but at a distinctly different rate and Mg2+ concentration optimum than the eIF-2A fraction. Moreover, there is no stimulation of NH2-terminal peptide synthesis with EF-1 alone. In contrast, all the known initiation factors are required for the translation of exogenous globulin mRNA on oviduct polysomes. Reticulocyte polysomes isolated by an identical procedure to that used for oviduct polysomes or by standard methods also require all the initiation factors for the translation of either endogenous mRNA or exogenous ovalbumin mRNA. Addition of 7-methylguanosine 5'-monophosphate does not inhibit the factor-dependent stimulation of oviduct polysomes except at high concentrations (1.0 mM) indicating that the sites with which 7-methylguanosine 5'-monophosphate normally competes are already occupied. These findings suggest that the messenger RNA remains bound to the oviduct polysomes or initiation factors. Hence the addition of exogenous factors which are involved with mRNA recognition and binding to the ribosome are not required. It has been previously shown that eIF-2A is capable of binding in vitro the initiatior tRNA to an existing Ado-Urd-Gua-40 S complex and initiating protein synthesis when such a complex is present. These present studies indicate that such an initiation complex may exist within the oviduct cell on membrane-associated polysomes. Under these circumstances eIF-2A mediates binding of the initiator tRNA and initiates protein synthesis.     

Inhibition of protein synthesis in reticulocyte lysates by a double-stranded RNA component in HeLa mRNA

Double-stranded RNA (dsRNA) inhibits protein synthesis initiation in rabbit reticulocyte lysates by the activation of a latent dsRNA-dependent cAMP-independent protein kinase which phosphorylates the α-subunit of the eukaryotic initiation factor eIF-2. In this study, we describe a dsRNA-like component which is present in preparations of HeLa mRNA (poly A⁺) isolated from total cytoplasmic RNA. The inhibitory species in the HeLa cytoplasmic mRNA was detected by (a) its ability to inhibit protein synthesis with biphasic kinetics in reticulocyte lysates translating endogenous globin mRNA, and (b) by the inefficient translation of HeLa cytoplasmic mRNA in a nuclease-treated mRNA-dependent reticulocyte lysate. The inhibitory component was characterized as dsRNA by several criteria including (i) the ability to activate the lysate dsRNA-dependent eIF-2α kinase (dsI); (ii) the prevention of both dsI activation and inhibition of protein synthesis by high levels of dsRNA or cAMP; (iii) the reversal of inhibition by eIF-2; and (iv) the inability to inhibit protein synthesis in wheat germ extracts which lack latent dsI. By the same criteria, the putative dsRNA component(s) appears to be absent from preparations of HeLa mRNA isolated exclusively from polyribosomes.     

The poly(A)-binding protein facilitates in vitro translation of poly(A)-rich mRNA

To investigate the role of the 73-kDa poly(A)-binding protein in protein synthesis, the effect of the addition of homo-polyribonucleotides on the translation of polyadenylated and non-adenylated mRNA was studied in the rabbit reticulocyte lysate. Poly(A) was found to be the most effective polynucleotide in inhibiting duck-globin mRNA translation, whereas it had no effect on the translation of polyribosomal duck-globin mRNP, or on the endogenous synthesis of the rabbit reticulocyte lysate. The translation of poly(A)-free mRNA was not affected by the addition of poly(A). Furthermore, we found that the inhibiting effect of poly(A) can be reversed by addition of purified poly(A)-binding protein. It is thus likely that the 73-kDa poly(A)-binding protein is an essential factor necessary for poly(A)-rich mRNA translation.     

Studies on the possible role of vitreous humor on protein synthesis and morphology of organ cultured adult rabbit lens. II. Epithelial cells.

The protein synthetic activities of epithelial cells of lenses organ-cultured without adhered vitreous humor manifest significant increase compared to the epithelial cells of lenses incubated with attached vitreous humor. This effect is not due to trauma of vitreous removal, as the addition of freeze-dried vitreous humor to the culture medium of lenses without attached vitreous humor could inhibit protein synthesis. However, the protein synthesis inhibitor in the vitreous humor has no visible effect on the lens morphology. It was also found that the factor from vitreous humor has no effect on mRNA production or cell-free protein synthesis. Thus, it seems that the effect on protein synthesis must be mediated via some other pathway.     

Effect of Infection with the Meningopneumonitis Agent on Deoxyribonucleic Acid and Protein Synthesis by Its L-Cell Host

Cycloheximide, which had already been shown to inhibit protein synthesis in Earle's L cells (mouse fibroblasts) without having any effect on the multiplication or protein synthesis in Chlamydia psittaci (strain meningopneumonitis) infecting these host cells, also caused greater than 90% inhibition of deoxyribonucleic acid (DNA) synthesis in L cells after a 3-hr exposure to the drug. L cells infected with the meningopneumonitis agent and treated with cycloheximide were used to follow meningopneumonitis-specific DNA synthesis during intracellular growth of the parasite. The rate at which labeled precursors were incorporated into parasite DNA doubled every 2 hr. The effect of meningopneumonitis infection on L-cell DNA and protein synthesis was investigated in logarithmically growing and in stationary-phase (nondividing) populations of L cells. Host-specific DNA and protein synthesis appeared to be inhibited in infected L cells when compared with logarithmically growing control cells, whereas no inhibition was apparent when the comparison was made with stationary-phase control cells. The maximal amount of protein and DNA synthesis that occurred in meningopneumonitis-infected L cells was equal to the amount of DNA and protein synthesized in logarithmically growing, uninfected L cells. A possible explanation of these results is given.     

Effects on protein synthesis of injecting synthetic polyribonucleotides into living cells

Micro-injection into the oocytes and eggs of Xenopus laevis was used to ascertain the effects of synthetic polyribonucleotides on protein synthesis in living cells. Poly(U) and poly(A) were not translated detectably, nor did they change the rate of endogenous protein synthesis. The same was true of poly(G,U), poly(A,G,U), poly(A,C,G,U), G-U-G-(U)(n), A-(U)(n) and AUG. In contrast, A-U-G-(U)(n) was a potent inhibitor of protein synthesis in the cell. This might be because it is initiated normally but lacks a termination codon, or because it inhibits the translation of other molecules in some way not dependent on its normal initiation. Poly(G,U), poly(A,G,U) and poly(A,C,G,U) inhibited haemoglobin synthesis when they were injected into the oocyte with haemoglobin mRNA. The synthetic polyribonucleotides did not inhibit the translation of the natural mRNA when the two sorts of molecules were injected at different times. It is suggested that the synthetic RNA molecules compete with the natural mRNA for a pre-initiation factor in limited supply.     

Control of ribonucleic acid function by oligonucleoside methylphosphonates

Oligodeoxyribonucleoside methylphosphonates contain nonionic 3'-5' linked methylphosphonate internucleotide bonds in place of the normal charged phosphodiester linkage of natural nucleic acids. These oligomers are resistant to nuclease hydrolysis, can pass through the membranes of mammalian cells in culture and can form stable hydrogen-bonded complexes with complementary nucleotide sequences of cellular RNAs such as mRNA. The oligomers are readily synthesized on insoluble polymer supports. Their chainlength and nucleotide sequence can be determined by chemical sequencing procedures. Oligonucleoside methylphosphonates which are complementary to the 5'-end, initiation codon region, or coding region of rabbit globin mRNA inhibit translation of the mRNA in rabbit reticulocyte lysates and globin synthesis in rabbit reticulocytes. This inhibition is due to the interaction of the oligomers with mRNA and the extent of inhibition is influenced by the secondary structure of the mRNA and the location of oligomer binding site on the mRNA. Oligomers complementary to the initiation codon regions of N, NS and G protein mRNAs of Vesicular stomatitis virus (VSV) inhibit virus protein synthesis in VSV-infected Mouse L-cells. These oligomers do not affect L-cell protein synthesis or growth. Virus protein synthesis and growth can also be selectively inhibited by oligonucleoside methylphosphonates which are complementary to the donor or acceptor splice junctions of virus pre mRNA. An oligomer complementary to the donor splice junction of SV40 large T antigen mRNA inhibits T-antigen synthesis in SV40-infected African green monkey kidney cells but does not inhibit overall cellular protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a protein synthesis system from rat liver. Translation of endogenous and exogenous messenger RNA

An in vitro system prepared from rat liver postmitochondrial supernatant exhibits a high rate of protein synthesis for an extended period of time. This system initiates translation of either endogenous or exogenous mRNA, incorporates Met at a rate of 13 pmol/mg of postmitochondrial supernatant protein/min, maintains this rate for at least 90 min, and performs several rounds of translation/mRNA molecule. Up to 50% of the activity is due to reinitiation of protein synthesis using endogenous mRNA. In addition, 60-70% of the protein synthesized was released from ribosomes into the medium. Addition of globin mRNA stimulates protein synthesis and results in the synthesis of a protein that comigrates with authentic rabbit globin. Black beetle virus mRNA 2 also stimulates protein synthesis and results in synthesis of a protein with molecular weight corresponding to that of the mature viral protein. With endogenous rat liver mRNA this system synthesizes a large number of proteins.     

Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum.

Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific mRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis inhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of inhibitors, albeit at a diminished rate relative to that of the uninhibited control.     

Evidence for simultaneous derepression of messenger RNA and the guanine nucleotide exchange factor in fertilized sea urchin eggs

Translational control was studied in extracts of Lytechinus pictus eggs and zygotes. We showed that neither mRNA nor initiation factors alone limit translation in these lysates; rather they are together rate limiting. Added globin mRNA was translated in egg and zygote lysates but overall protein synthesis did not increase significantly as the added RNA competed with the endogenous message. The lysates mimicked the in vivo response, since microinjection of globin mRNA into L. pictus eggs similarly competed with endogenous mRNAs. A number of translational components were used to determine if they would stimulate protein synthesis in these lysates. The addition of globin polyribosomes increased the level of protein synthesis. The majority of this increase was due to reinitiation of the globin mRNA, and under these conditions the level of endogenous protein synthesis in both egg and zygote extracts did not change. The addition of crude initiation factors alone did not appreciably alter the rate of protein synthesis in the egg lysates. However, in the presence of added mRNA, these initiation factors stimulated translation two- to fourfold. Of all the initiation factors tested, only the guanine nucleotide exchange factor (GEF, eIF-2B, RF) significantly increased protein synthesis when globin mRNA was present. The addition of an unfractionated initiation factor preparation further stimulated protein synthesis in the presence of added GEF and mRNA, suggesting that a component other than mRNA and GEF was also limiting in these egg lysates. Other initiation factors, including eIF-2, eIF-4A, eIF-4B, and eIF-4F, did not substitute for the component in the unfractionated initiation factor preparation. We propose that alkalinization of the cytoplasm and the subsequent activation of initiation factors and mRNAs contribute to the large stimulation of protein synthesis in echinoid eggs after fertilization. Furthermore, we discuss the possibility that the increase in NADPH at the expense of NAD+, which occurs within 3 min after fertilization, may lead to the activation of GEF.     

Injected mRNA does not increase protein synthesis in unfertilized, fertilized, or ammonia-activated sea urchin eggs

We have investigated whether the rate of protein synthesis in unfertilized and fertilization-activated sea urchin eggs is limited by the availability of mRNA by injecting eggs, zygotes, and ammonia-activated eggs with globin mRNA. Message-injected and buffer-injected cells were labeled with radioactive amino acids and the proteins separated on a polyacrylamide gel. The relative amounts of newly synthesized globin and endogenous proteins were obtained by scanning the gel fluorograph. Globin mRNA is translated poorly in Strongylocentrotus droebachiensis eggs and does not significantly increase or decrease endogenous protein synthesis. In zygotes and ammonia-activated eggs, however, globin mRNA is translated well and appears to compete with endogenous mRNAs for the limiting component of the translational machinery as it is released. Our results are consistent with the hypothesis that either ribosomes or recruitment factors are gradually activated after fertilization or ammonia treatment, that such components are the rate-limiting factor, and that they impart the typical sigmoidal increase in protein synthesis rate observed in fertilized eggs before the first cleavage.     

Eukaryotic initiation factor 4A stimulates translation in microinjected Xenopus oocytes

The injection of heterologous mRNA into fully grown Xenopus oocytes results not only in the synthesis of the heterologous protein but also in a reciprocal decrease in the synthesis of endogenous proteins. This indicates that injected and endogenous mRNAs compete for some component which is rate-limiting for translation in oocytes. We have attempted to identify this rate-limiting translational component. We find that heterologous and homologous polysomes compete with endogenous mRNAs as effectively as naked mRNA, indicating that polysomes do not contain detectable levels of the rate-limiting factor. In addition, we have used micrococcal nuclease digestion and a mRNA-specific oligonucleotide to destroy the mRNA component of polysomes. The remaining polysome factors, when injected into oocytes, failed to stimulate translation. When several eukaryotic translation initiation factors were injected into oocytes, initiation factor 4A consistently increased general oocyte protein synthesis by about twofold. It is possible that the availability of eIF-4A in oocytes is a key factor in limiting the overall rate of protein synthesis.