On the association of mRNA with the cytoskeleton in uninfected and adenovirus-infected human KB cells

Triton-insoluble cytoskeletons were prepared from uninfected and adenovirus-infected KB cells. Gradient analysis showed that all cellular polyribosomes were present in the cytoskeletons. After disaggregation of the polyribosomes, in vivo or in vitro, most of the messenger RNA (mRNA) remained associated with the cytoskeletal framework. Translation experiments showed that most mRNA species were present in a bound (cytoskeletal), as well as in an unbound state. However, whereas some mRNA species were predominant as unbound mRNP particles, other mRNA species were almost exclusively found in polyribosomes associated with the cytoskeletal framework. Incubation of cytoskeletons in an mRNA-dependent reticulocyte cell-free system revealed synthesis of the same set of polypeptides as took place when using whole cells. Furthermore, the gradual shift from translation of cellular to translation of viral mRNA species during late phase of productive infection with adenovirus could also be followed when cytoskeletons were translated in the cell-free system. These results support the hypothesis that Triton X-100 extraction does not remove actively translating mRNA from the cells, thus suggesting a functional relationship between mRNA translation and mRNA binding to a cytoskeletal framework.     

An investigation of the stability of messenger RNAs in cell-free,translational systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells

The stabilities and translation of Ehrlich ascites tumor cell poly(A)-containing mRNA and mengovirus RNA in fractionated cell-free protein synthesizing systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells were studied. During incubation of the systems about 20% of the input RNA is reduced in size and associated with ribosomes engaged in polypeptide synthesis; the remainder is rapidly degraded by RNases. At the end of active translation, both mRNA and nascent proteins are bound to polysomes which are of the same size as those formed during active protein synthesis. The kinetics of protein synthesis closely follow those of RNA hydrolysis. The stabilities of mengovirus RNA and poly(A)-containing mRNA from Ehrlich ascites tumor cells are the same in both systems.     

Control of protein synthesis in extracts from poliovirus-infected cells. I. mRNA discrimination by crude initiation factors.

By using cell-free systems prepared from uninfected and poliovirus-infected cells, we have been able to demonstrate that crude preparations of initiation factors from infected cells do not stimulate the initiation of translation by polyribosomes containing endogenous host cell mRNA. When tested with polysomes containing endogenous viral mRNA, however, they were able to stimulate initiation of translation nearly as well as uninfected cell initiation factors. The uninfected cell initiation factor preparations were able to stimulate initiation of translation of both cell and viral mRNA. The results indicate an mRNA-specific activity present in crude initiation factor preparations from infected cells. Furthermore, the ability of eIF2 from infected cells to form a ternary complex with GTP and formyl [35S]methionine-tRNAfmet, an mRNA-independent step in initiation, was found not to be deficient. Implications of these data for proposed mechanisms of poliovirus-induced host cell shutoff are discussed.     

Studies on the intracellular segregation of polyribosome-associated messenger ribonucleic acid species in the lactating guinea-pig mammary gland.

1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87--93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.     

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.     

Isolation of giant silk fibroin polysomes and fibroin mRNP particles using a novel ribonuclease inhibitor, hydroxystilbamidine

Hydroxystilbamidine isethionate, a dye capable of binding to both DNA and RNA, has been found to be a powerful inhibitor of cellular ribonucleases. A procedure has been developed that, with the aid of this compound, permits the preparative isolation of giant silk fibroin polyribosomes from the posterior silk gland of Bombyx mori. The polyribosomes contain approximately 45-112 ribosomal particles, as judged by electron microscopy. Treatment of giant fibroin polyribosomes with EDTA releases a particle that sediments at 125S. This mRNP particle contains biologically active silk fibroin mRNA, as judged by cell-free translation in an mRNA-dependent reticulocyte cell-free system.     

RNA isolation from the ribonucleoproteins fixed with formaldehyde

A method for isolating RNA from the polyribosomes and informosomes fixed with formaldehyde was developed. The ribonucleoproteins were obtained by centrifugation in CsCl density gradient. It has been demonstrated that this method makes it possible to obtain full-sized rRNA and mRNA appropriate for molecular hybridization. We succeeded in amplifying 150-nucleotide sequences of individual mRNA and demonstrating the applicability of these RNA preparations for synthesis of labeled probes for RNA arrays. The method proposed is recommended for the search for untranslated mRNA and the study of the changes in translation of individual proteins during early ontogenesis and various pathologies.     

Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries

The coupled regulation of oskar mRNA localization and translation in time and space is critical for correct anteroposterior patterning of the Drosophila embryo. Localization-dependent translation of oskar mRNA, a mechanism whereby oskar RNA localized at the posterior of the oocyte is selectively translated and the unlocalized RNA remains in a translationally repressed state, ensures that Oskar activity is present exclusively at the posterior pole. Genetic experiments indicate that translational repression involves the binding of Bruno protein to multiple sites, the Bruno Response Elements (BRE), in the 3' untranslated region (UTR) of oskar mRNA. We have established a cell-free translation system derived from Drosophila ovaries, which faithfully reproduces critical features of mRNA translation in vivo, namely cap structure and poly(A) tail dependence. We show that this ovary extract, containing endogenous Bruno, is able to recapitulate oskar mRNA regulation in a BRE-dependent way. Thus, the assembly of a ribonucleoprotein (RNP) complex leading to the translationally repressed state occurs in vitro. Moreover, we show that a Drosophila embryo extract lacking Bruno efficiently translates oskar mRNA. Addition of recombinant Bruno to this extract establishes the repressed state in a BRE-dependent manner, providing a direct biochemical demonstration of the critical role of Bruno in oskar mRNA translation. The approach that we describe opens new avenues to investigate translational regulation in Drosophila oogenesis at a biochemical level.     

Small cytoplasmic poly(A)+RNA, homologous to the B2 repetitive sequence of the mouse genome, is present in ribonucleoproteins

Small cytoplasmic poly(A) + RNA homologous to a highly repeated sequence B2 of the mouse genome (scB2 RNA) was not found as free RNA within a cell. This RNA is bound to small (12-18S) ribonucleoprotein particles as well as to heavy RNP particles, apparently informosomes. After deproteinization of the heavy RNP the major part of scB2 RNA molecules cosedimented with heavy RNAs. It seems that scB2 RNA is associated with mRNA in informosomes via short complementary regions. About half of the scB2 RNA molecules was revealed in the cytoskeleton fraction. The possibility that scB2 RNAs are involved in mRNA transport or in the regulation of mRNA translation is discussed.     

Changes in the sequence content of albumin mRNA and in its translational activity in the rat liver with age

To investigate the regulation of age-related changes in albumin synthesis in the rat liver, total postnuclear RNA and polyribosomes, both membrane-bound and free, were prepared from livers of rats of different ages. By the use of a specific complementary DNA probe, the albumin mRNA sequence content was quantitated in these RNA fractions. These studies showed a specific increase in albumin mRNA sequence content in total postnuclear RNA and membrane-bound polyribosomes at between 12 and 24 months of age. Between 24 and 36 months of age, the increase in the amount of albumin mRNA in these two fractions was due only to an increase in liver weight. The increase in albumin mRNA sequence content was not found in the poly(A)⁺ fraction but in the RNA extracted from the void of oligo(dT)-cellulose column chromatography. The isolated polyribosomes were translated in a cell-free system to assess age-related changes in total protein and albumin synthesis due to translational control. No changes with age were found in the translational capacity of membrane-bound and free polyribosomes per RNA unit. Immunoprecipitation of the synthesized albumin in the translation products revealed that albumin synthesis in the cell-free system is not increased proportionally with the elevated albumin mRNA level between 12 and 24 months of age. This indicates that albumin mRNAs present in the livers of old rats are biologically less active than those found in younger animals.     

Characterization and translation of methylated and unmethylated vesicular stomatitis virus mRNA synthesized in vitro by ribonucleoprotein particles from vesicular stomatitis virus-infected L cells.

Ribonucleoprotein particles isolated from extracts of vesicular stomatitis virus (VSV) -infected L cells synthesized in vitro four classes of polyadenylated RNA sedimenting at 29S, 19S, 17S, and 13S. When synthesized in vitro in the presence of the methyl donor S-adenosyl methionine, these RNA species contained the following 5'-terminal structures: (i) m7G5ppp5'AmpAp(70%) ; (ii) m7G5'ppp5'AmpAmpNp (20%) and (iii) pppAp (10%). In the presence of the methylation inhibitor S-adenosylhomocysteine, however, the mRNA contained the 5'-terminal structures G5'ppp5'Ap (80%) and pppAp (20%). The mRNA's synthesized in vitro were translated in the homologous ascites and the heterologous wheat embryo cell-free systems. In both, the products were shown by sodium dodecyl sulfate gel electrophoresis and by immunoprecipitation to contain all five viral proteins, L, G, N, NS, and M. The presumed precursor to the G protein (G*) was also identified by fingerprint analysis. Methylated VSV mRNA was more active in protein synthesis than unmethylated mRNA in both the ascites system and the wheat embryo systems. Addition of S-adenosylmethionine stimulated translation of unmethylated mRNA in the wheat embryo but not in the ascites extract. S-adenosylhomocysteine, however, by preventing mRNA methylation inhibited the translation of unmethylated VSV mRNA in both systems. The mRNA methylating activity present in wheat embryo S-30 extracts was recovered in the ribosome-free supernatant fraction (S-150) and was insensitive to the protein synthesis inhibitor pactamycin.     

Cell-free translation of mammalian myosin heavy-chain messenger ribonucleic acid from growing and fused-L6E9 myoblasts.

An mRNA-dependent reticulocyte cell-free protein synthesizing system very efficient in the translation of myosin heavy-chain mRNA from a rat myogenic cell line is described. This system exhibits a high degree of fidelity with regard to the spectrum and relative proportion of the different proteins synthesized from a sample of cytoplasmic RNA as compared to the proteins synthesized in vivo by the cells from which the RNA is prepared. The main feature of this system is the use of a K+ and Cl- concentration similar to those of the reticulocyte cytoplasm. Using this system, myosin heavy chain, identified by low-salt precipitation, electrophoretic mobility, and partial peptide analysis, represents 17% of the total protein synthesis when cytoplasmic RNA from well-fused L6E9 cells is used. Furthermore, when RNA preparations from growing myoblasts, that when analyzed in other cell-free translational systems seem not to contain any myosin heavy-chain mRNA, are tested in the system reported here, they are proven to contain high amounts of translatable myosin heavy-chain mRNA.     

Regulation of mRNA utilization in mouse erythroleukemia cells induced to differentiate by exposure to dimethyl sulfoxide.

Mouse erythroleukemia cells contain several abundant mRNA species that occur to a considerable extent as untranslated molecules. For two of these species, which code for polypeptides P40 and P21, the proportion of molecules engaged in translation decreases rapidly after exposure of the cells to dimethyl sulfoxide. The extent of utilization of a third species, the P36 mRNA, is not altered. The rate of production of the P40 mRNA does not appear to be affected in the dimethyl sulfoxide-treated cells. The P21 mRNA appears to be produced in increasing amounts, leading to a large accumulation of untranslated molecules in the cytoplasm. The mRNA for actin remains nearly fully utilized during this process, but its intracellular concentration decreases, thus resulting in a reduction in the amounts present in polysomes. The results indicate that some mRNA species in mouse tumor cells are subject to a translational repression process that can serve to regulate selectively the extent of expression of the corresponding genes.     

Translational activity of messenger ribonucleic acid isolated from unstimulated and phytohaemagglutinin-activated lymphocytes.

Purified cytoplasmic poly(A)+ RNA isolated from unstimulated pig lymphocytes has the same ability to direct translation in a range of cell-free systems as the corresponding mRNA from 20h phytohaemagglutinin-activated lymphocytes. Additional methylation of the mRNA is not required for maximum protein synthesis in the wheat-germ cell-free system. Misleading results are obtained if the mRNA preparations used are not adequately purified, and a method suitable for routine assessment of the degree of purification achieved is described. Cell-free protein-synthesizing systems from unstimulated lymphocytes translate added lymphocyte mRNA with lower efficiency than do comparable systems from phytohaemagglutinin-activated lymphocytes, whatever the source of the mRNA used.     

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.     

Synthesis of large subunit of ribulosebisphosphate carboxylase by thylakoid-bound polyribosomes from spinach chloroplasts

Intact chloroplasts were isolated from developing first leaves of spinach. The chloroplasts were broken and separated into an extensively washed membrane (thylakoid) fraction and a soluble (stroma) fraction. The membrane fraction contained polyribosomes with properties similar to those of thylakoid-bound polyribosomes of other organisms. The distribution of mRNA for large-subunit ribulosebisphosphate carboxylase (LS) was determined by translating RNA from chloroplasts, thylakoids, and stroma in a wheat germ cell-free translation system. LS translation product was identified by immunoprecipitation with antibody to LS from spinach, electrophoresis of the immunoprecipitated product, and fluorography. At least 44% of translatable chloroplast LS-mRNA was in the washed thylakoid fraction. Thylakoid-bound LS-mRNA was in polyribosomes since LS was produced by thylakoids in an Escherichia coli cell-free translation system under conditions where initiation did not take place. Our results demonstrate that membrane-bound polyribosomes can synthesize the stroma-localized polypeptide LS, and suggest that the thylakoids may be an important site of its synthesis.     

Studies on the form and synthesis of messenger ribonucleic acid in the rat ventral prostate gland, including its tissue-specific stimulation by androgens

1. When prostate polyribosomes are labelled with radioactive precursors in vivo and subsequently dissociated with sodium dodecyl sulphate, a heterogeneous 6-15S RNA species may be identified that possesses all of the distinctive properties of mRNA. 2. Apart from the selective incorporation of 5'-fluoro-orotic acid into this 6-15S RNA component, it is bound by nitrocellulose filters under experimental conditions where only poly(A)-rich species of RNA are specifically retained. Most importantly, however, only the 6-15S RNA fraction is capable of promoting the incorporation of amino acids into peptide linkage in an mRNA-depleted cell-free system derived from ascites-tumour cells. 3. With the development of a simpler method for labelling the total RNA fraction of the prostate gland in vitro, the poly(A)-enriched RNA fraction may be readily isolated by adsorption and elution from oligo(dT)-cellulose. The synthesis of the poly(A)-enriched 6-15S RNA fraction is stringently controlled by androgens in a highly tissue- and steroid-specific manner. 4. From an analysis of the proteins synthesized in the ascites cell-free system in the presence of the poly(A)-rich RNA fraction, it appears that protein synthesis in the prostate gland is stimulated in a rather general way, even during the earliest phases of the androgenic response. This conclusion may require modification when more specific means of analysis are available than those used in the present investigation. 5. The implications of these findings to the mechanism of action of androgens are discussed.