Translation Of Endogenous mRNA Research Articles

Microinjection of mRNAs and Oligonucleotides.

Microinjecting lineage tracers into a single blastomere in the normal, intact embryo identifies the repertoire of cell types derived from it. In order to reveal the full developmental potential of that blastomere or identify the mechanisms by which its fate is determined, one needs to modify its gene expression under controlled experimental conditions. One method by which this is easily accomplished in Xenopus is by microinjecting synthetic mRNAs or antisense oligonucleotides into an identified blastomere to target altered gene expression specifically to its lineage. Xenopus blastomeres are robust and tolerate pressure-driven microinjection up to a few hundred cells, and they efficiently translate exogenously supplied mRNAs. Targeted microinjections, described here, significantly reduce off-target effects of the mRNAs or oligonucleotides. Many types of constructs can be synthesized to provide specific information about gene function. For example, microinjecting mRNA encoding the wild-type gene in its normal expression domain or in an ectopic site tests whether it promotes or represses target genes or alters the formation of tissues of interest. Mutant forms of a gene transcript can illuminate the function of different domains of the encoded protein or show the developmental consequences of a mutation found in a human disease. mRNAs encoding dominant-negative forms of a protein can elicit a functional knockdown and thereby establish the necessity for that gene in a developmental process. Microinjecting antisense morpholino oligonucleotides (MOs) that are designed to block either endogenous mRNA translation or splicing is an effective method to reduce the levels of endogenous protein.

The effects of cadmium chloride and sodium selenite on protein synthesis in mouse liver

The study aimed at evaluating the effects of cadmium and selenite ions on protein synthesis and metallothioneins content in mice liver after 2h, 8h, 24h and 14 days of exposure. Our studies revealed that cadmium suppressed protein synthesis after 2h and 24h, but activated after 8h and 14 days. Also, the endogenous mRNA translation were reduced under any exposure to cadmium, meanwhile, metallothioneins content was decreased after 2h, but then was progressively increasing up to 492% after 14 days. Meantime, selenite did not influence metallothioneins content, caused mild activation of protein synthesis, and slightly suppressed the endogenous mRNA translation. The combined treatments with cadmium and selenite favored toward resisting of protein synthesis to cadmium after 2h and 24h of intoxication. Besides, selenite also protected translation against cadmium in cell-free systems, but did not attenuate effects of cadmium on metallothioneins content.

An in vitro system from Plasmodium falciparum active in endogenous mRNA translation.

An in vitro translation system has been prepared from Plasmodium falciparum by saponin lysis of infected-erythrocytes to free parasites which were homogeneized with glass beads, centrifuged to obtain a S-30 fraction followed by Sephadex G-25 gel filtration. This treatment produced a system with very low contamination of host proteins (<1%). The system, optimized for Mg2+ and K+, translates endogenous mRNA and is active for 80 min which suggests that their protein factors and mRNA are quite stable.

Involvement of the 24-kDa cap-binding protein in regulation of protein synthesis in mitosis.

The rate of protein synthesis in metaphase-arrested cells is reduced as compared to interphase cells. The reduction occurs at the translation initiation step. Here, we show that, whereas poliovirus RNA translation is not affected by the mitotic translational block, the translation of vesicular stomatitis virus mRNAs is. In an attempt to elucidate the mechanism by which initiation of protein synthesis is reduced in mitotic cells, we found that the interaction of the mRNA 24-kDa cap-binding protein (CBP) with the mRNA 5' cap structure is reduced in mitotic cell extracts, consistent with their lower translational efficiency. Addition of cap-binding protein complex stimulated the translation of endogenous mRNA in extracts from mitotic but not interphase cells. In addition, we found that the 24-kDa CBP from mitotic cells was metabolically labeled with 32P to a lesser extent than the protein purified from interphase cells. These results are consistent with a hypothesis that the 24-kDa CBP is implicated in the inhibition of protein synthesis in metaphase-arrested cells. Possible mechanisms for this inhibition are offered.

Protein-synthesizing function of the liver in rabbits with experimental myocardial infarction

The content of serum albumin in rabbit blood was found to be lowered within the first day after reproduction of experimental myocardial infarction. The rate and the level of translation of endogenous mRNA were studied in cell-free systems from normal rabbit liver and 6-12-24 h after experimental myocardial infarction. The decrease of the total protein synthesis in the crude cell-free system from the liver of experimental animals was shown to depend on the lack of energy supply rather than on the reduced activity of the protein-synthesizing apparatus. The relative drop of protein synthesis in the cell-free system with saturating concentration of ATP, GTP and creatine phosphate is likely to be connected with a decrease in the proportion of membrane-bound polysomes.

Effects of retinoic acid on protein synthesis in cultured melanoma cells.

Retinoic acid reduces the growth rate of mouse S91 melanoma cells in culture and increases the proportion of cells in the G1 phase of the cell cycle. Because of the integral role protein synthesis has been shown to play in growth control we studied the effect of retinoic acid on the protein synthesis machinery with a cell-free system developed from the melanoma cells. This system was capable of translating endogenous mRNA, exogenous globin mRNA, and the synthetic template poly(U). Of the above activities of the protein synthesis system only the translation of endogenous mRNA was reduced significantly in the cell-free system prepared from retinoic acid-treated cells. Analyses of the amount and function of RNA revealed that treatment with retinoic acid leads to reductions in total RNA content, in the proportion of ribosomes in polysomes, in the amount of poly(A)RNA, and in the amount of polysome-associated mRNA. All these effects of retinoic acid contribute to the decrease in protein synthesis activity of treated cells. Two-dimensional electrophoresis analysis of L-[35S]methionine-labeled proteins produced by untreated and treated cells revealed only a few quantitative differences. We suggest that retinoic acid-induced suppression of protein synthesis activity may be the cause for growth inhibition.

Properties of cerebral polyribosomes and membranes of the rough endoplasmic reticulum after ethanol dependence in rats.

Effects of physical dependence upon ethanol on the polyribosomal properties and the reconstitution of the rough endoplasmic reticulum (RER) of the brain has been examined. The purified free polyribosomes (polysomes), membrane-bound polysomes, and a fraction of RER membrane that has been stripped of polysomes were isolated from rat brain. RNA yields, amino acid incorporation activities, and electron micrographs established the purity of stripped membranes, but no differences were detected following the ethanol treatment. For the polyribosomal fractions, the stability of the mRNA/ribosomal complex was decreased after ethanol dependence as was the in vitro translation of endogenous mRNA. In the reconstitution reaction, the incubation of membranes from ethanol-treated animals with either source of purified bound polysomes resulted in higher yields of protein than when control membranes were used. The above results suggest that ethanol dependence affects the properties of both the RNA and membrane components of the RER of brain.

Purification and properties of a translation inhibitor from wheat germ

A translation inhibitor from wheat germ has been purified more than 400-fold to apparent homogeneity. The inhibitor is a basic protein with a molecular weight of 30 000. This protein effectively blocks protein synthesis in animal cell-free extracts but does not affect protein synthesis in intact cells. Inhibition occurs at a ribosome to inhibitor molar ratio of 100:1, indicating an enzymic mechanism of action. The wheat germ protein inhibits the translation of endogenous mRNA, exogenous mRNA, and poly(uridylic acid) at a step in polypeptide chain elongation and without breakdown of the polysomes. Neither the aminoacylation reaction nor mRNA degradation is affected by the inhibitor. An interesting feature of the inhibition reaction is that it requires, in addition to the wheat germ inhibitor, both ATP and tRNA. The function of these two compounds in the inhibition is presently unknown since neither the hydrolysis of the beta,gamma-pyrophosphate bond of ATP nor a modification of the tRNA can be demonstrated during the reaction.

In vitro inhibition of protein synthesis by purified cores from vaccinia virus.

The mechanism of the shutoff of cellular protein synthesis in vaccinia virus-infected cells has been investigated by using in vitro systems. Purified vaccinia cores cause inhibition of endogenous mRNA translation in nonpreincubated reticulocyte lysates and Ehrlich ascites tumor cell-free systems. Translation of viral mRNA from turnip yellow mosaic virus is also impaired in wheat germ cell-free extracts. The block induced by vaccinia cores in protein synthesis is not due to a decrease in the availability of mRNA but rather to an alteration of the cellular translational machinery. No nucleolytic activity able of digesting mRNA could be detected in purified vaccinia cores with three sensitive tests. There is a lack of inhibition in the poly(Phe)-poly(U) system, which bypasses the normal initiation process. An almost complete disaggregation of polyribosomes in the reticulocyte lysate appears when vaccinia cores are present. These results indicate that mRNA translation in a cell-free system is affected predominantly at the level of polypeptide chain initiation.

Selective inhibition of viral protein accumulation in interferon-treated cells; nondiscriminate inhibition of the translation of added viral and cellular messenger RNAs in their extracts

Treatment of monolayers of mouse L cells with 7.5 vesicular stomatitis virus plaque reduction units/ml of mouse interferon (specific activity 2 × 10 7 NIH mouse reference standard units/mg protein) results in a 95% decrease in the reovirus yield and, as shown earlier, an about 80% decrease in double-stranded and an about 60% decrease in single-stranded reo RNA accumulation in cells infected with 10 plaque-forming units of virus/cell. Interferon at this concentration does not affect the rate of accumulation of host proteins in uninfected or reovirus-infected cells. It inhibits reo virion protein accumulation in infected cells. In control cells the rate of reo virion protein accumulation increases about 2-fold between 10 and 14 hr after infection. In cells treated with interferon it remains at the same low inhibited level. Reo virion proteins were assayed in cell extracts by immunoprecipitation with antisera specific for various size classes or by gel electrophoresis. These results reveal the selective nature of the inhibition of protein accumulation in interferon-treated, virus-infected cells. The effect of treating L cells with interferon was also tested on the capacity of their extracts to translate various mRNAs. The translation of endogenous mRNA, of mRNA in L cell polysomes (added as such) and of polyuridylic acid is at most only slightly inhibited whereas that of added natural mRNAs (including reovirus and encephalomyocarditis virus mRNA, rabbit globin mRNA, and a mixture of L cell mRNAs) is strongly inhibited. The dominant nature of the inhibitor(s) is indicated by the fact that translation of added mRNA is impaired in a mixture of extracts from interferon-treated and from control cells. Thus an effect of interferon treatment of cells is manifested in subcellular systems. However, the selectivity of the effect in vivo, is not well reflected in vitro.

Properties of the ribosomal salt wash from unfertilized and fertilized sea urchin eggs and its effect on natural mRNA translation

The acrylamide gel pattern of a ribosomal salt-wash fraction (Fraction RSW) prepared from unfertilized sea urchin eggs was compared with the pattern of a similar preparation from fertilized eggs. Significant differences were found indicating that as early as 2 min after fertilization a marked change occurs at the ribosomal level. Fraction RSW from unfertilized eggs is highly inhibitory on the translation of endogenous mRNA in cell-free systems prepared either from sea urchin or from rabbit reticulocytes. By contrast, Fraction RSW from fertilized eggs, at least at the concentration used in these experiments, does not significantly affect the endogenous protein synthesis. Furthermore, it was shown that Fraction RSW prepared from unfertilized eggs inhibits protein synthesis by interfering with the elongation process. The possibility of a simultaneous inhibitory effect at the initiation step cannot be ruled out.

Availability of mRNA for translation during normal and transcription-blocked development

Cell-free incorporation of amino acids into proteins has been studied in systems prepared from developing sea urchins. The incorporation given by preparations from normal embryos has been compared at several stages of development, pre- and post-gastrulation. These experiments were run in parallel with studies on systems from embryos whose RNA synthesis has been blocked, ab initio, with dactinomycin. 1. 1. Incorporation was followed with l-[ 14C]leucine when translation of endogenous mRNA was to be examined, and with l-[ 14C]phenylalanine when poly U was employed as artificial messenger. In the latter cases, incorporation was a measure of peptide-bond synthesis potential, while the incorporation of leucine in optimized systems lacking poly U provided a measure of the endogenous messengers available for translation at each stage. 2. 2. In systems prepared from embryos exposed continuously to dactinomycin, the endogenous messenger RNA was all of “maternal” origin, i.e., synthesized during oogenesis and surviving into the period of embryogenesis. 3. 3. By the test employed, mRNA availability increases very rapidly during the first cleavage cycle of normal embryos. From the very low values characteristic of unfertilized eggs, the incorporation rates rise until at telophase they are close to those given by blastula preparations. The remainder of the increase has appeared by the 32-cell stage. From blastula to gastrula, some further increases occur. 4. 4. Preparations from embryos with transcription blocked give an essentially identical type of changing incorporation up to the blastula stage. Thereafter, failure of morphogenesis is accompanied by a steady decline in available endogenous mRNA, with values approaching zero at the time that normal embryos would be gastrulating. 5. 5. This decline in messenger availability cannot be ascribed to nonspecific injury by the drug to the cells nor to their potential for peptide-bond synthesis, since that potential, as measured by poly U translation, is unaffected.