Small cytoplasmic RNAs from human placental free mRNPs. Structure and their effect on in vitro protein synthesis

A new family of small cytoplasmic RNA species (scRNAs) was found to be associated with human term-placental free messenger ribonucleoprotein particles (mRNPs). Placental scRNAs strongly inhibit translation of both homologous and heterologous mRNAs in a cell-free rabbit reticulocyte system. scRNAs could be resolved into at least four different RNA species. One of the RNA molecules, scRNA species 1, was the most potent protein synthesis inhibitor found among the placental scRNAs. The nucleotide sequence of the scRNA species 1 was determined. In spite of its short length, scRNA species 1 still exhibited a very strong inhibitory effect on the in vitro protein synthesis. scRNAs were found to be complexed with proteins in the form of scRNPs. Proteins of these complexes enhanced the inhibitory effect of scRNAs on in vitro translation. Experiments provided evidence that inhibition of in vitro protein synthesis by the scRNAs is not dependent upon mRNA concentration. However, inhibition can be overcome by increasing the ratio lysate/scRNAs, thus suggesting that scRNAs act on some essential component of the cell-free system. The degree of inhibition is decreased when scRNAs are added after the start of translation, suggesting that scRNAs (or scRNPs) interfere with the initiation stage of translation, probably acting on an initiation factor(s). Placental scRNAs are unique in their size, being smaller than other known scRNAs. Their association with free cytoplasmic repressed mRNPs in human placenta suggests that scRNAs play a role in the regulation of mRNP metabolism and, consequently, in the control of mRNA translation.     

Heat-shock protein synthesis in Chlamydomonas reinhardi. Translational control at the level of initiation of a poly(A)-rich-RNA coded 22-KDa protein in a cell-free system

The effect of temperature on the in vitro translation of control and heat-shock poly(A)-rich RNA, obtained from Chlamydomonas reinhardi cells, incubated for 2 h at 25 degrees C respectively, was studied using the wheat-germ translation system. Incubation of the cells at 42 degrees C induces the synthesis of RNAs coding for several heat-shock proteins, including a 22-kDa major polypeptide as well as several proteins of 45-94 kDa, as demonstrated by run-off translation of polyribosomes isolated from intact cells. However, the high-molecular-mass heat-shock proteins are poorly translated in the wheat-germ system. The poly(A)-rich RNA coding for the 22-kDa heat-induced polypeptide has an apparent sedimentation coefficient higher than that expected from the molecular mass of its translation product, and was preferentially translated in vitro at temperatures above 31 degrees C as compared with pre-existing RNAs. Raising the temperature of translation, slightly inhibited (10%) the runoff translation of polyribosomes isolated from intact cells. However, when initiation was carried out in vitro for a short time at increasing temperatures and translation continued at 25 degrees C in the presence of aurintricarboxylic acid, the 22-kDa heat-shock polypeptides was preferentially translated. Aurintricarboxylic acid did not significantly inhibit incorporation of [35S]methionine when added to polyribosomes isolated from control or heat-shocked cells. From the above data we conclude that the translation of the 22-kDa heat-shock protein is controlled in vitro at the initiation level.     

Mechanism of inhibition of polypeptide chain initiation in heat-shocked Ehrlich cells involves reduction of eukaryotic initiation factor 4F activity

Almost all living organisms studied respond to elevated temperature with a marked inhibition of overall protein synthesis but increased synthesis of a specific set of proteins, the so-called heat-shock proteins. We have prepared a cell-free protein synthesizing system (lysate) from heat-shocked Ehrlich ascites tumor cells that reflects the inhibition of protein synthesis in intact cells at elevated temperatures. We have isolated and partially purified a stimulator of the heat-shocked cell lysate from Ehrlich cells. Through four purification steps, the stimulator is chromatographically identical to eukaryotic initiation factor 4F (eIF-4F), an initiation factor which specifically binds mRNA cap structure. Therefore, we have tested the effects of highly purified reticulocyte eIF-4F on the heat-shocked cell lysate. Protein synthesis is strongly stimulated by addition of highly purified eIF-4F. Synthesis in the heat-shocked lysate is more inhibited at high (70 mM) KCl concentrations, than at lower concentrations, and stimulation by eIF-4F is correspondingly greater at higher KCl concentrations, so that the rate of protein synthesis is returned to control (non-heat-shocked lysate) levels at all KCl concentrations. Furthermore, at 70 mM KCl, in heat-shocked lysates, synthesis of the 68-kDa heat-shock protein is much less inhibited than synthesis of the bulk of non-heat-shock proteins, and eIF-4F stimulates synthesis of 68-kDa protein to a much lesser extent than non-heat-shock proteins. Thus, addition of purified eIF-4F reverses the effects of elevated temperatures on Ehrlich cells that are reflected in lysates. Therefore, we propose that the inhibition of translation in heat-shocked Ehrlich cells is the result of inactivation of eIF-4F function.     

Studies on rat liver catalase. X. Effect of hemin and an inhibitor on the translation of catalase messenger RNA1.

Rat liver catalase mRNA was translated in a rabbit reticulocyte lysates and wheat germ cell-free system in the presence or absence of hemin and/or a translational inhibitor prepared from reticulocytes, liver cells, and wheat germs. Failure to add hemin to the lysates, or the addition of a hemin-regulated translational inhibitor (HRI) to the hemin-supplemented lysates caused a repressed translation. A preparation of inhibitor from rat liver showed activity similar to that of HRI for this translating system. The translation repression by rat liver inhibitor was reversed by eIF-2 (initiation factor) or GTP, but ATP enhanced the repression. The translation of catalase mRNA in the wheat germ system was not affected by the addition of hemin. An inhibitor prepared from wheat germ extracts, as well as the rat liver inhibitor, markedly decreased the rate of translation. eIF-2, GTP, and ATP behaved in the manner described above. Catalase synthesis in a cell-free system derived from rat liver (using endogenous mRNA) was not influenced by either hemin or the inhibitor. The possibilities are discussed that the synthesis of catalase in liver cells is controlled by a translational inhibitor at the level of chain initiation, and that the formation of the inhibitor from its inactive proinhibitor is regulated by the amount of heme.     

Evidence that the 59-kDa protein synthesis initiation factor from wheat germ is functionally similar to the 80-kDa initiation factor 4B from mammalian cells

The results of this investigation show that the 59-kDa protein synthesis initiation factor from wheat germ, designated eukaryotic initiation factor (eIF)-4G by Browning et al. (Browning, K.S., Maia, D.M., Lax, S.R., and Ravel, J.M. (1987) J. Biol. Chem. 262, 539-541), cross-links to the 5'-terminal cap of oxidized mRNA in the presence of eIF-4A, eIF-4F, and ATP, stimulates the RNA-dependent ATPase activities of eIF-4A and a mixture of eIF-4A and eIF-4F, and stimulates the unwinding activities of eIF-4A, eIF-4F, and a mixture of eIF-4A and eIF-4F. These findings strongly suggest that the 59-kDa factor from wheat germ is the functional equivalent of the 80-kDa protein synthesis initiation factor, eIF-4B, from mammalian cells. Recent reports indicate that the wheat germ initiation factor which contains two subunits of 80 and 28 kDa and which was given the designation "eIF-4B" by Lax et al. (Lax, S.R., Lauer, S.J., Browning, K. S., and Ravel, J.M. (1986) Methods Enzymol. 118, 109-128) is an isozyme form of eIF-4F and not the functional equivalent of mammalian eIF-4B. On the basis of functional characteristics we propose that the designation for the wheat germ factor containing the 80- and 28-kDa polypeptides be changed from eIF-4B to eIF-(iso)4F and the designation for the 59-kDa factor be changed from eIF-4G to eIF-4B.     

Evidence for the regulation of protein synthesis by a wheat germ phosphoprotein factor

A 48-kilodalton phosphoprotein, termed T-protein or pT, isolated from wheat germ and purified to homogeneity is found to inhibit the translation of tobacco mosaic virus (TMV) RNA in both wheat germ and reticulocyte lysates. The translation of TMV RNA in both systems was inhibited over 80% by 8 microM pT. There was no evidence to indicate that the reticulocyte lysate also contained a pT-like protein. pT was rapidly phosphorylated in the wheat germ and reticulocyte lysates. Although the relationship between pT phosphorylation and inhibition of protein synthesis is not known, there is evidence to indicate that complete phosphorylation of pT is not required for inhibition. Furthermore, no significant differences in the kinetics of inhibition of protein synthesis between prephosphorylated and unmodified pT were observed. Investigation of the mechanism of inhibition indicated that neither the aminoacylation of tRNA nor the elongation of nascent polypeptide chains was affected by pT. On the other hand, pT was found to prevent the formation of the 80S initiation complex. This action of pT was not due to the binding of pT to the ribosomes. However, the effect of pT was found to vary with the concentrations and types of mRNA used in the translational system. These results suggest that pT may interact with specific region(s) of the mRNA and prevent its translation. Alternatively, pT could block the translation of mRNA by binding to one or more of the initiation factors that interact with mRNA to facilitate mRNA binding to the 43S preinitiation complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of mRNA cap-binding protein from Drosophila melanogaster embryos.

A protein with specific affinity for the mRNA cap structure was purified both from the postribosomal supernatant and from the ribosomal high-salt wash of Drosophila melanogaster embryos by m7GTP-Sepharose chromatography. This protein had an apparent molecular mass of 35 kilodaltons (kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size very different from those of the cap-binding proteins that have been characterized thus far. Drosophila 35-kDa cap-binding protein (CBP) could also be isolated from the ribosomal high-salt wash as part of a salt-stable protein complex consisting of polypeptides of 35, 72, and 140 to 180 kDa. Polyclonal antibodies against Drosophila 35-kDa CBP neither reacted with eucaryotic initiation factor 4E from rabbit reticulocytes nor affected mRNA translation in a rabbit reticulocyte cell-free system. However, in a cell-free system from Drosophila embryos, mRNA translation was specifically inhibited by these antibodies. The requirement of 35-kDa CBP for mRNA translation in Drosophila was diminished under ionic conditions in which the importance of mRNA cap structure recognition was reduced. Despite the structural differences between Drosophila 35-kDa CBP and mammalian initiation factor 4E, both proteins were functionally interchangeable in the in vitro translation system from Drosophila embryos.     

Evidence that the 5'-untranslated leader of mRNA affects the requirement for wheat germ initiation factors 4A, 4F, and 4G

The efficiency of translation of alfalfa mosaic virus (AMV) RNA 4, barley alpha-amylase (B alpha A) mRNA, and two chimeric mRNAs, AMV 4-B alpha A and B alpha A-AMV 4 (in which the 5' leader sequences of the two mRNAs were interchanged), was measured in an S30 extract from wheat germ and a fractionated system from wheat germ in which translation could be made dependent upon initiation factor (eIF) 3, 4A, 4F, or 4G. In the S30 system, AMV RNA 4 and the chimeric mRNA AMV 4-B alpha A are translated much more efficiently than B alpha A mRNA and the chimeric mRNA B alpha A-AMV 4. When the S30 system was supplemented with high amounts of purified eIF-3, eIF-4A, eIF-4F, and eIF-4G, B alpha A and B alpha A-AMV 4 mRNAs were translated as efficiently as AMV RNA 4 and AMV 4-B alpha A mRNA. These findings indicated that the mRNAs containing the B alpha A leader sequence required higher amounts of one or more of the initiation factors (eIF-3, eIF-4A, eIF-4F, and eIF-4G) for efficient translation. Determination of the amounts of the initiation factors required for translation in the fractionated system showed that AMV RNA 4 required 2-4-fold lower amounts of eIF-3, eIF-4A, eIF-4F, and eIF-4G than did B alpha A mRNA. Replacement of the B alpha A leader sequence with that of AMV RNA 4 decreased the amounts of eIF-4A, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4F required. Replacement of the AMV RNA 4 leader sequence with that of B alpha A mRNA increased the amounts of eIF-4F, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4A required. These data strongly suggest that the amounts of the factors required are affected not only by the 5' leader itself but also by interactions between the 5' leader and a region(s) of the mRNA 3' to the initiation codon.     

Initiation factors that bind mRNA. A comparison of mammalian factors with wheat germ factors

Three mammalian eukaryotic initiation factors (eIF) are required for the ATP-dependent binding of mRNA to the 40 S ribosomal subunit. These three factors, eIF-4A, eIF-4B, and eIF-4F, have also been isolated from wheat germ. Three assays were used to measure the ability of the wheat germ factors to interact with and/or substitute for the mammalian factors. Two assay systems were used to measure partial reactions involving the interaction of the three factors, ATP, and mRNA: 1) RNA-dependent ATP hydrolysis and 2) cross-linking of the factors to the 5' cap of oxidized mRNA. A third assay system was used to measure the ability of the factors to support initiation of protein synthesis. The results of the ATP hydrolysis and cross-linking experiments indicate that the wheat germ factors can interact with or substitute for the mammalian factors. Wheat germ eIF-4A appears to be functionally equivalent to mammalian eIF-4A. Wheat germ eIF-4B and eIF-4F appear to be isozymes possessing functions similar to mammalian eIF-4F. Wheat germ eIF-4B does not appear to be a functional equivalent to the mammalian eIF-4B. In a complete translation system from wheat germ, mammalian factors partially substitute for wheat germ factors, whereas the wheat germ factors are ineffective in the mammalian system.     

Selection of AUG initiation codons differs in plants and animals.

The influence of the nucleotide at position -3 relative to the AUG initiation codon on the initiation of protein synthesis was studied in two different in vitro translation systems using synthetic mRNAs. The four mRNAs, transcribed from cDNAs directed by an SP6 promoter, were identical except for mutations at nucleotide -3. In each case, translation of mRNAs produced a single protein of Mr = 12,600. Relative translational efficiencies showed a hierarchy in the reticulocyte lysate system (100, 85, 61 and 38% for A, G, U and C in position -3, respectively) but no differences in the wheat germ system. Differential mRNA degradation or polypeptide chain elongation were excluded as causes of the differences observed in translation in the reticulocyte lysate. mRNA competition increased the differences observed in translational efficiencies in reticulocyte lysate but showed no effect in wheat germ. Analysis of 61 plant and 209 animal mRNA sequences revealed qualitative and quantitative differences between the consensus sequences surrounding AUG initiation codons. Whereas the consensus sequence for animals was CACCAUG that for plants was AACAAUGGC. Both the structural and functional findings suggest that the factors which select AUG initiation codons in plants and animals differ significantly.     

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.     

Effect of capping upon the mRNA properties of satellite tobacco necrosis virus ribonucleic acid.

The mRNA guanyltransferase-mRNA methyltransferases of vaccinia virions can be used to introduce a 5'-terminal m7g(5')pp(5')Apm... capping group onto the RNA of satellite tobacco necrosis virus (STNV RNA) to yield intact capped STNV RNA. Studies with an in vitro system from wheat germ and limiting quantities of capped and uncapped STNV RNA show that the rates and extents of formation of initiation complexes of protein synthesis by intact capped and uncapped STNV RNA are identical, suggesting that 5'-terminal cap groups cannot function in the translation of STNV RNA. Also, the cap analogue pm7G equally inhibits the initiation and the translation of limiting quantities of both capped and uncapped STNV RNA. These contrasting observations suggest that the wheat germ system contains a pm7G sensitive protein and that STNV RNA has a tertiary structure that restricts the function of an added 5'-terminal capping group. This theory is supported by observations that fragmented capped STNV RNA is better at forming initiation complexes than is equally fragmented uncapped STNV RNA.     

Role of cytoplasmic mRNP proteins in translation.

Polyribosomal and free mRNPs from rabbit reticulocytes were isolated and characterized. Translation of mRNPs was studied in the rabbit reticulocyte and wheat germ cell-free systems. Both classes of mRNPs were active in rabbit reticulocyte lysates. However, considerable differences between mRNPs and mRNA have been revealed. High concentrations of mRNA in the form of mRNP did not inhibit protein biosynthesis, whereas the same amounts of deproteinized mRNA caused inhibition of this process. Polyribosomal mRNPs and deproteinized mRNA, but not free mRNPs, are active in the wheat germ cell-free translation system. Translation of free mRNPs in this system can be restored by addition of 0.5 M KCl-wash of rabbit reticulocyte ribosomes. These results suggest the existence of a special repressor/activator regulatory system which controls mRNA distribution between free mRNPs and polyribosomes in rabbit reticulocytes. This regulatory system should include: i) a translation repressor associated with mRNA within free mRNPs, preventing its translation; and ii) a translation activator associated with ribosomes, overcoming the effect of the repressor. Both classes of cytoplasmic mRNPs contain a major 50 kDa protein (p50). The content of this protein per mol of mRNA in free mRNPs is twice as much as in polyribosomal ones. The method of p50 isolation has been developed and some properties of this protein were investigated. It has been shown that small amounts of p50 stimulate, whereas high amounts inhibit mRNA translation. We suggest that p50 has a dual role in protein biosynthesis. In polyribosomal mRNPs (p50:mRNA approximately 2:1, mol/mol), this protein promotes the translation process.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Turnip mosaic virus VPg interacts with Arabidopsis thaliana eIF(iso)4E and inhibits in vitro translation

The interaction between turnip mosaic virus (TuMV) viral protein linked to the genome (VPg) and Arabidopsis thaliana eukaryotic initiation factor (iso)4E (eIF(iso)4E) was investigated to address the influence of potyviral VPg on host cellular translational initiation. Affinity chromatographic analysis showed that the region comprising amino acids 62-70 of VPg is important for the interaction with eIF(iso)4E. In vitro translation analysis showed that the addition of VPg significantly inhibited translation of capped RNA in eIF(iso)4E-reconstituted wheat germ extract. This result indicates that VPg inhibits cap-dependent translational initiation via binding to eIF(iso)4E. The inhibition by VPg of in vitro translation of RNA with wheat germ extract did not depend on RNase activity. Our present results may indicate that excess VPg produced at the encapsidation stage shuts off cap-dependent translational initiation in host cells by inhibiting complex formation between eIF(iso)4E and cellular mRNAs.     

Effects of ribosomal wash factors and spermidine on endogenous and exogenous mRNA stimulated protein synthesis in the wheat germ cell-free system.

Differential effects of Mg2+, spermidine, and reticulocyte ribosomal wash factors on the translation of endogenous, myeloma, and globin mRNA's have been observed in studies with the wheat germ cell-free protein synthesizing system. Spermidine stimulated globin mRNA translation but not the translation of endogenous wheat germ messages, and the polyamine actually inhibited the translation of myeloma mRNA. Ribosomal wash factors, on the other hand, stimulated endogenous and myeloma mRNA dependent protein synthesis in an Mg2+-dependent fashion but inhibited globin mRNA translation. The combination of ribosomal wash factors and spermidine was either stimulatory or inhibitory depending on the Mg2+ concentration and the message. It was further observed that translation of exogenous myeloma mRNA proceeded for only 60 min at 25 degrees C under all conditions tested in this study, while translation of endogenous wheat germ messages continued for longer periods of time. No differential effects of spermidine on the synthesis of high molecular weight myeloma proteins were observed.