The protein of M(r) 21,000 constituting the prosome-like particle of duck erythroblasts is homologous to apoferritin.

In duck erythroblasts, two major populations of untranslated messenger (m) RNP can be separated by sucrose gradient centrifugation in low ionic strength. One of these contains globin mRNA associated to protein factors, among them the prosomes. The other, sedimenting in the 35S zone, contains non-globin mRNA. From this '35S' mRNP, a new RNP particle called the prosome-like particle was isolated and characterized [Akhayat, O., Infante, A. A., Infante, D., Martins de Sa, C., Grossi de Sa, M.-F. & Scherrer, K. (1987) Eur. J. Biochem. 170, 23-33]. The PLP is a multimer of a protein of M(r) 21,000, and contains small RNA species. The particle is tightly associated with repressed mRNA and inhibits in vitro protein synthesis. We show here that the protein of M(r) 21,000, constituting the prosome-like particle, is apoferritin. Different approaches confirm the RNP character of this particle and provide evidence that some of its RNA species are tRNA. The hypothesis is discussed as to whether (apo-)ferritin might serve other functions in addition to iron storage.     

Analysis of the control of citrulline synthesis in isolated rat-liver mitochondria

Irradiation of chicken muscle cells with ultraviolet light (254 nm) to cross-link RNA and protein moieties was used to examine the polypeptide complements of cytoplasmic mRNA-protein complexes (mRNP). The polypeptides of translationally active mRNP complexes released from polysomes were compared to the repressed nonpolysomal cytoplasmic (free) mRNP complexes. In general, all of the polypeptides present in free mRNPs were also found in the polysomal mRNPs. In contrast to polysomal mRNPS, polypeptides of Mr 28 000, 32 000, 46 000, 65 000 and 150 000 were either absent or present in relatively smaller quantities in free mRNP complexes. On the other hand, the relative proportion of polypeptides of Mr 130 000 and 43 000 was higher in free mRNPs than in polysomal mRNP complexes. To examine the role of cytoplasmic mRNP complexes in protein synthesis or mRNA metabolism, the changes in these complexes were studied following (a) inhibition of mRNA synthesis and (b) heat-shock treatment to alter the pattern of protein synthesis. Actinomycin D was used to inhibit mRNA synthesis in chick myotubes. The possibility of newly synthesized polypeptides of cytoplasmic mRNP complexes being assembled into these complexes in the absence of mRNA synthesis was examined. These studies showed that the polypeptides of both free and polysomal mRNP complexes can bind to pre-existing mRNAs, therefore suggesting that polypeptides of mRNP complexes can be exchanged with a pool of RNA-binding proteins. In free mRNP complexes, this exchange of polypeptides is significantly slower than in the polysomal mRNP complexes. Heat-shock treatment of chicken myotubes induces the synthesis of three polypeptides of Mr = 81 000, 65 000 and 25 000 (heat-shock polypeptides). Whether this altered pattern of protein synthesis following heat-shock treatment could affect the polypeptide composition of translationally active polysomal mRNPs was examined. The results of these studies show that, compared to normal cells, more newly synthesized polypeptides were assembled into polysomal mRNPs following heat-shock treatment. A [35S]methionine-labeled polypeptide of Mr = 80 000 was detected in mRNPs of heat-shocked cells, but not of normal cells. This polypeptide was, however, detected by AgNO3 staining of the unlabeled polypeptide of mRNP complexes of normal cells. These results, therefore, suggest that the assembly of newly synthesized 80 000-Mr polypeptide to polysomal mRNPs was enhanced following induction of new heat-shock mRNAs. The results of these studies reported here have been discussed in relation to the concept that free mRNP complexes are inefficiently translated in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.     

Cell-free translation in lysates from Spodoptera frugiperda (Lepidoptera: Noctuidae) cells

1. Conditions for in vitro translation of mRNA in cell-free extracts from cultured Spodoptera frugiperda cells were defined. 2. Incorporation of [35S]methionine into acid-precipitable material increased for approximately 1 hr, and was sensitive to the protein synthesis inhibitors pactamycin and cycloheximide. 3. Micrococcal nuclease-treated lysate, primed with purified rabbit globin mRNA, synthesized a major protein with the size of full length globin, indicating that the lysate supported correct initiation and elongation of polypeptides.     

Maternal mRNAs are regulated by diverse P body-related mRNP granules during early Caenorhabditis elegans development

Processing bodies (P bodies) are conserved mRNA-protein (mRNP) granules that are thought to be cytoplasmic centers for mRNA repression and degradation. However, their specific functions in vivo remain poorly understood. We find that repressed maternal mRNAs and their regulators localize to P body-like mRNP granules in the Caenorhabditis elegans germ line. Surprisingly, several distinct types of regulated granules form during oocyte and embryo development. 3' untranslated region elements direct mRNA targeting to one of these granule classes. The P body factor CAR-1/Rap55 promotes association of repressed mRNA with granules and contributes to repression of Notch/glp-1 mRNA. However, CAR-1 controls Notch/glp-1 only during late oogenesis, where it functions with the RNA-binding regulators PUF-5, PUF-6, and PUF-7. The P body protein CGH-1/Rck/Dhh1 differs from CAR-1 in control of granule morphology and promotes mRNP stability in arrested oocytes. Therefore, a system of diverse and regulated RNP granules elicits stage-specific functions that ensure proper mRNA control during early development.     

Purification of histone messenger ribonucleoprotein particles from HeLa cell S-phase polysomes. Characterization of associated proteins

We have purified HeLa histone mRNA from polysomes of S-phase cells which had been synchronized by hydroxyurea treatment. This mRNA was shown to direct the in vitro synthesis of all five histones which amount to at least 90-95% of its total translational activity. Polysomal histone mRNP was also purified and identified by cell-free translation and hybridization to a clone of histone DNA from E. esculentus. The protein moiety of this mRNP contained three prominent species of molecular weight 86,000, 73,000 and 53,000 daltons. The presence of the 73,000 species previously assessed to be bound to poly(A) is discussed in view of the fact that histone mRNA does not contain a pail. As globin mRNA, histone mRNA as well as histone mRNP were translated with equal efficiency in cell-free extracts from either S-phase or hydroxyurea blocked HeLa cells.     

The presence of protein kinase activity and acceptors of phosphate groups in nonpolysomal cytoplasmic messenger ribonucleoprotein complexes of embryonic chicken muscle

Nonpolysomal cytoplasmic (free) mRNA.protein (mRNP) complexes of embryonic chicken muscle were purified by a combination of oligo(dT)-cellulose chromatography and sucrose density gradient centrifugation. The protein moieties of the purified mRNP complex were analyzed by two-dimensional gel electrophoresis using separation according to charge in the first dimension and molecular weight in the second. Sixteen polypeptides of Mr = 27,000 to 75,000 were present in the mRNP complex. These mRNP polypeptides displayed different electrophoretic migration properties than those of ribosomal proteins. A protein kinase activity was found associated with the mRNP. This enenzyme was able to transfer phosphate group(s) from ATP to at least three acidic mRNP polypeptides of Mr = 27,000, 38,000, and 73,000 and one basic polypeptide of Mr = 75,000. Among these, the Mr = 38,000 acidic polypeptide was the best acceptor of phosphate groups.     

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.     

Poly(A+)mRNA sequences in free mRNP and polysomes of mouse ascites cells

The poly(A⁺)RNA of the free mRNP of mouse Taper ascites cell contains a very reduced number of different mRNA sequences compared to the polysome poly(A⁺)RNA. By the technique of mRNA:cDNA hybridization we have determined that the free mRNP contains approximately 400 different mRNA sequences while the polysomes contain about 9000 different mRNAs. The free mRNP poly(A⁺)RNA sequences are present in two abundance classes, the abundant free mRNP class containing 15 different mRNA sequences and the less abundant free mRNP class containing 400 different mRNAs. The polysome poly(A⁺)RNA consists of three abundance classes of 25, 500 and 8500 different mRNA sequences.Despite its intracellular location in RNP structures not directly involved in protein synthesis the poly(A⁺)RNA purified from the free RNP of these cells was a very effective template for protein synthesis in cell-free systems. Cell-free translation products of free mRNP and polysome poly(A⁺)RNAs were analyzed by two-dimensional gel electrophoresis. This analysis confirmed the hybridization result that the free mRNP poly(A⁺)RNA contained fewer sequences than polysomal poly(A⁺)RNA. The abundant free RNP-mRNA directed protein products were a subset of the polysome mRNA-directed protein products. The numbers of more abundant products of cell-free protein synthesis directed by the free RNP-mRNA and polysomal mRNA were in general agreement with the hybridization estimates of the number of sequences in the abundant classes of these two mRNA populations.     

Limitation of reticulocyte transfer RNA in the translation of heterologous messenger RNAs.

The effect of various tRNAs on protein synthesis was investigated using a tRNA-dependent cell-free system from Ehrlich ascites cells. Ascites cell tRNA and rabbit liver tRNA were found to promote efficient translation of globin mRNA, oviduct mRNA, and encephalomycarditis (EMC) viral RNA. In contrast, reticulocyte tRNA participated efficiently only in the translation of globin mRNA; the translation of oviduct mRNA AND EMC viral RNA in the presence of reticulocyte tRNA resulted in the synthesis of relatively few large mature proteins and the accumulation of discrete, smaller polypeptides. These results suggest that isoaccepting tRNA species required for the synthesis of ovalbumin and EMC viral protein (but not hemoglobin) are probably functionally absent in reticulocyte tRNA, causing a premature, nonrandom termination of synthesis of these proteins. This provides preliminary evidence that variations in tRNA populations, frequently observed between different cell types, are large enough to define and perhaps regulate the proteins that the cell is capable of synthesizing.     

Molecular modifications associated with Aging of globin messenger RNA in vitro.

Using polyacrylamide gel elution-electrophoresis in aqueous medium, highly purified rabbit globin mRNA can be fractionated into several populations of molecules differing by their mean poly(A) content. Both alpha and beta globin mRNA are heterogenous with respect to their electrophoretic mobilities. With the conditions used no separation of alpha and beta globin mRNA occurs during electrophoresis. From the specific radioactivity distribution in the different mRNA fractions one can conclude that the polyadenylate sequence at the 3' end of globin mRNA molecules becomes shorter with aging. This shortening occurs on alpha as well as beta, globin mRNAs and the extent of heterogeneity in poly(A) content is similar for both globin mRNAs. Furthermore, using two different methods of mRNA fractionation (polyacrylamide gel elution-electrophoresis and elution of poly (U)-Sepharose-bound mRNA at increasing temperatures) it is shown that old mRNA molecules differ from relatively young messages in their ability to direct cell-free globin synthesis. Modifications reducing template activity in vitro thus seem to take place during globin mRNA aging.     

Possible involvement of messenger RNA-associated proteins in protein synthesis

Two distinct forms of globin messenger RNA were isolated from mouse spleen cells infected with Friend erythroleukemia virus: polyribosomal messenger ribonucleoprotein particles (15S mRNP), and their corresponding protein-free mRNAs obtained by chemical deproteinization. The translation efficiencies of both messenger forms were assayed in a Krebs II ascites cell-free system. Selective removal of RNA-binding proteins from the ascites cell lysate did not affect globin synthesis when the mRNA was supplied as 15S mRNP; deproteinized mRNA however was not translated. Only in the presence of two fractions of RNA-binding proteins was the protein-free mRNA translated. Some of the RNA-binding proteins have the same molecular weights and isoelectric points as the principal proteins of 15S mRNP.     

Ribonucleoprotein organization of eukaryotic RNA: XV. Different nucleoprotein structures of globin messenger RNA sequences in nuclear and polyribosomal ribonucleoprotein particles

Heterogeneous nuclear RNA and polyribosomal messenger RNA are both complexed with specific sets of proteins in the cell, forming ribonucleoprotein complexes known as hnRNP and mRNP, respectively. In the present investigation, the nucleoprotein structures of globin mRNA sequences in hnRNP and mRNP were probed by digestion with nuclease, under conditions in which RNA-protein rearrangements were shown not to occur. Mild digestion with pancreatic RNAase of a Friend erythroleukemia cell RNP fraction containing both hnRNP and mRNP resulted in a preferential depletion of globin mRNA-homologous sequences, as measured by hybridization of the surviving RNA with globin complementary DNA. Hypersensitivity to nuclease typifies 65% of the globin mRNA-homologous sequences, with the other 35% remaining relatively nuclease-resistant. Removal of polyribosomal mRNP by release with EDTA, followed by re-isolation of hnRNP on a sucrose gradient eliminated the nuclease-hypersensitive class of globin mRNA sequences in favor of the relatively nuclease-resistant class. These results suggest that mRNA sequences are more nuclease-sensitive in polyribosomal mRNP than they are in nuclear hnRNP particles. The implication is that mRNA sequences undergo a significant change in RNP structure at some point during their movement from nucleus to cytoplasm.     

Inhibition of protein synthesis in vesicular stomatitis virus infected Chinese hamster ovary cells: role of virus mRNA-ribonucleoprotein particle

Although host protein synthesis is preferentially inhibited, there is a steady decline in the ability of Chinese hamster ovary (CHO) cells infected with vesicular stomatitis virus (VSV) to synthesize both host and viral proteins. We previously reported finding an mRNA-ribonucleoprotein particle (mRNP) that contained all five VSV mRNAs and viral N protein exclusively. This particle apparently regulates translation by sequestering a majority of the VSV mRNA made late in infection and thus rendering it unavailable for protein synthesis. In the present investigation the mRNP was also shown to inhibit in vitro protein synthesis in rabbit reticulocyte and wheat germ lysates programmed with mRNA isolated from VSV-infected cells. The synthesis of eIF-2 X GTP X Met-tRNA (ternary) complex, the first step in initiation of protein synthesis, was markedly inhibited by the mRNP. The inhibition was partially reversed by addition of purified eIF-2 to the inhibited lysate or ternary complex formation reaction. These results indicate a dual role of the mRNP in regulating protein synthesis during infection. Nucleocapsid also inhibited in vitro protein synthesis, although this inhibition was not reversed by eIF-2. Nucleocapsid did not inhibit ternary complex formation in vitro. Consequently, nucleocapsid may also regulate in vivo protein synthesis, but by a mechanism different from the mRNP.     

Direct microinjection of rabbit globin mRNA into mouse 3T3 cells. Analysis of the polypeptides synthesized in vivo

3T3 cells grown attached to 9 mm² coverslips have been microinjected in the cytoplasm with total rabbit globin mRNA and the polypeptides synthesized after injection have been labelled with [³⁵S]-methionine under conditions in which the product of as few as 100 cells could be analysed by high resolution two-dimensional gel electrophoresis followed by 10 days' fluorography. Microinjection of rabbit globin mRNA results in the synthesis of a basic polypeptide of mol. wt 15 K that is not present in control cells, and that co-migrates with purified [³H]leucine-labelled globin as determined by high resolution two-dimensional gel electrophoresis (NEPHGE). Visual inspection of the fluorograms revealed that the injection of globin mRNA (up to 14000 molecules/cell) does not alter significantly the relative intensity of the major acidic (IEF) and basic (NEPHGE) polypeptides synthesized by the cells.     

Inhibition of cell-free protein synthesis by low-molecular-weight RNAs from free cytoplasmic ribonucleoprotein particles

Free cytoplasmic messenger ribonucleoprotein (mRNP) particles from rat liver were treated with EDTA and separated into two populations of RNP particles with sedimentation maxima of 20 S and 35 S respectively. The 20-S and 35-S RNP particles, treated with 0.5 M KCl, have protein-to-RNA ratios of 0.31:1 and 5.7:1 respectively. Whereas 20-S and 35-S RNP particles exhibit a similar protein complement of seven major polypeptides, the low-molecular-weight RNA components of the two particle populations are different. A characteristic set of distinct low-molecular-weight RNAs is found for 20-S and 35-S RNP particles. When the individual low-molecular-weight RNAs of 20-S and 35-S RNP particles isolated from preparative polyacrylamide gels were assayed for their capability to inhibit protein synthesis in vitro, several potent translational inhibitory RNAs were detected. In particular, the low-molecular-weight RNAs of 147, 203 and 263 nucleotides in length associated with the 35-S RNP particles turned out to be strong inhibitors of protein synthesis.     

Evidence for the protection of specific RNA sequences in globin messenger ribonucleoprotein particles.

Purified 15 S globin mRNA-protein (mRNP) complexes obtained by EDTA dissociation of duck reticulocytes polyribosomes were digested with the calcium dependant Staphylococcus aureus nuclease (EC 3. 1. 4. 7.). 25% of the globin mRNA sequences were resistant to extensive nuclease digestion as determined by TCA precipitation of the digested 15 S particles labelled in vivo with tritiated uridine. Polyacrylamide gel electrophoresis of the RNA from nuclease digested 15 S particles showed that the protected oligoribonucleotides were distributed into two distinct size classes of 25,000 and 12,000 MW. Comparison between in vitro iodine-labelled 9 S globin mRNA extracted from Staphylococcal nuclease digested 15 S mRNP particles was carried out by fingerprinting. Mapping of T1 ribonuclease digests by high-voltage electrophoresis and homochromatography showed that specific oligoribonucleotides were protected against nuclease attack by proteins of the 15 S mRNP.