The influence of poly(A)-binding proteins on translation of poly(A)+ RNA in a cell-free system from embryo axes of dry pea seeds

Ribonucleoproteins of the ribosomal fraction of germinated pea embryo axes, containing translationally active mRNA, differ from analogous ribonucleoproteins of dry pea seeds, which contain stored mRNA, by the presence of a 60 kDa protein fraction showing affinity to poly(A). The above protein fraction largely affects the activity of poly(A)⁺ RNA translation in cell-free system. An activating effect is clearly seen at a weight ratio of poly(A)-binding proteins:poly(A)⁺ RNA of 3:1, whereas with an increase in the concentration of these proteins the translational activity drops. The effect of poly(A)-binding proteins containing the 60 kDa fraction on poly(A)⁺ RNA dependent cell-free translation can be efficiently reduced by simultaneous addition of synthetic poly(adenylic acid). It was also proved that activation of translation does not influence its products. It is concluded that poly(A)-binding proteins from the ribosomal fraction of embryo axes of pea seeds, especially the 60 kDa fraction, are involved in regulation of the translational activity of poly(A)⁺ RNA.     

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.     

Cotyledonary mRNA Species and Germinability of Immature Vigna unguiculata Seeds

We have defined four stages in the development of cowpea seeds:I(9–11 days after flowering), II (13–15), HI (17–19)and IV (22–24). Poly A⁺ RNA fractions were prepared fromcotyledons of developing (stages I–IV) and germinating(0, 12, 24 and 48 h after imbibition) seeds. Poly A⁺ RNAs fromstages I–III exhibited high translation activities witha maximum at stage II, and the activity was markedly reducedat stage IV. In cotyledons of germinating seeds, the translationactivity was low until 12 h after the onset of imbibition butrose thereafter. Analysis of in vitro translation products withSDS-polyacrylamide gel electrophoresis and fluorography showedthat the abundant mRNA population underwent a distinct changebetween stages II and III of seed development. Since the mRNApopulation at stage III was very similar to that of stage IV(mature seeds), it appears that, as far as mRNA species areconcerned, the prerequisites for germination are fully availablein the developing seeds by stage III. This assumption was supportedby the fact that immature seeds at stage III exhibited highgermination rates and normal axial growth and produced -amylaseat levels similar to those produced by mature seeds. Severalpolypeptides which have been regarded as translation productsof stored mRNA (poly A⁺ RNA from dry seeds) were detected atearlier stages of germination. (Received September 29, 1988; Accepted January 25, 1989)     

The Messenger RNA Population in the Embryonic Axes of Phaseolus vulgaris During Development and Following Germination

Misra, S. and Bewley, J. D. 1985. The messenger RNA populationin the embryonic axes of Phaseolus vulgaris during developmentand following germination.—J. exp. Bot. 36: 1644–1652. Messenger RNAs were extracted from young, mid-maturation, late-maturation,mature-dry, and 20-h-germinated embryonic axes of Phaseolusvulgaris cv. Taylor's Horticultural. They were translated invitro in a rabbit reticulocyte lysate protein synthesizing system.Analysis of the translation products using two-dimensional polyacrylamidegel electrophoresis indicated that there were substantial changesin the messenger RNA populations of developing and germinatingaxes. The number of polypeptides synthesized increased sharplyat 20–22 d after pollination and then declined. Therewas a parallel increase and decrease in the Poly(A)⁺ contentof the seed axis. The analysis showed that certain messageswere present throughout development and were stored in maturedry seed. These messages were degraded upon subsequent rehydration.Some messages appeared during mid-maturation but declined duringlater stages of development and were absent from the matureseed. In the germinating seed a set of messages unique to germinationappeared. Key words: —Seed development, germination, mRNA, in vitro translation, Phaseolus vulgaris     

Cell-free translation analysis of messenger RNA in echinoderm and amphibian early development

A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(A)⁺ mRNA, poly(A)^? histone mRNA, and poly(A)^? nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)⁺ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)^? nonhistone mRNAS than do embryos. Some differences in the proteins encoded by poly(A)⁺ and poly(A)^? RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)⁺ and poly(A)^? RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous comigration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)^? mRNA, encoding a 70,000–80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)⁺ nonhistone mRNA, poly(A)^? histone mRNA, and poly(A)⁺ histone mRNA. Amphibian ovary RNA appearently lacks an abundant poly(A)^? nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding histone-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay.     

Changes in Gene Expression during Tomato Fruit Ripening

Total proteins from pericarp tissue of different chronological ages from normally ripening tomato (Lycopersicon esculentum Mill. cv Rutgers) fruits and from fruits of the isogenic ripening-impaired mutants rin, nor, and Nr were extracted and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Analysis of the stained bands revealed increases in 5 polypeptides (94, 44, 34, 20, and 12 kilodaltons), decreases in 12 polypeptides (106, 98, 88, 76, 64, 52, 48, 45, 36, 28, 25, and 15 kilodaltons), and fluctuations in 5 polypeptides (85, 60, 26, 21, and 16 kilodaltons) as normal ripening proceeded. Several polypeptides present in ripening normal pericarp exhibited very low or undetectable levels in developing mutant pericarp. Total RNAs extracted from various stages of Rutgers pericarp and from 60 to 65 days old rin, nor, and Nr pericarp were fractionated into poly(A)⁺ and poly(A)⁻ RNAs. Peak levels of total RNA, poly(A)⁺ RNA, and poly(A)⁺ RNA as percent of total RNA occurred between the mature green to breaker stages of normal pericarp. In vitro translation of poly(A)⁺ RNAs from normal pericarp in rabbit reticulocyte lysates revealed increases in mRNAs for 9 polypeptides (116, 89, 70, 42, 38, 33, 31, 29, and 26 kilodaltons), decreases in mRNAs for 2 polypeptides (41 and 35 kilodaltons), and fluctuations in mRNAs for 5 polypeptides (156, 53, 39, 30, and 14 kilodaltons) during normal ripening. Analysis of two-dimensional separation of in vitro translated polypeptides from poly(A)⁺ RNAs isolated from different developmental stages revealed even more extensive changes in mRNA populations during ripening. In addition, a polygalacturonase precursor (54 kilodaltons) was immunoprecipitated from breaker, turning, red ripe, and 65 days old Nr in vitro translation products.     

Reduction of turgor induces rapid changes in leaf translatable RNA

The turgor of pea (Pisum sativum) leaves was reduced by exposing excised pea shoots to a stream of 23°C air for 20 min. Poly(A)⁺ RNA was isolated from control and wilted shoots, translated in vitro and radiolabeled translation products separated by electrophoresis on two-dimensional (isoelectric focusing-sodium dodecyl sulfate) polyacrylamide gels. This analysis showed that the levels of several poly(A)⁺ RNAs increased in wilted plants. Most of the poly(A)⁺ RNAs induced in wilted plants did not accumulate in response to heat shock or exogenously applied ABA even though endogenous ABA levels were found to increase in shoots 30 min after wilting and by 4 h had increased 50-fold (1 versus 0.02 microgram per gram fresh weight). A λgt10 cDNA library was constructed using poly(A)⁺ RNA from wilted shoots which had been incubated for 4 hours. Differential screening of the library identified four clones corresponding to poly(A)⁺ RNAs which are induced in wilted shoots.     

Stored polyadenylated RNA and loss of vigour in germinating wheat embryos

Loss of vigour in wheat seed is associated with lesions affecting the rate of disappearance of stored poly A⁺ RNA (presumptive mRNA) in the germinating embryo when germination takes place at a sub-optimal temperature. During germination in the presence of α-amanitin and consequent of de novo polyA⁺ RNA biosynthesis, the wheat embryo can degrade up to 70% of the stored poly A⁺ RNA of the quiescent embryo before any significant reduction in the rate of protein biosynthesis in the embryo becomes apparent. It is possible that two subpopulations of poly A⁺ RNA species exist in wheat embryos during early germination, one population being degraded rapidly upon rehydration of the embryo whilst the other population supports protein biosynthesis in the initial germination stages prior to degradation.     

Kinetic study of protein unfolding and refolding using urea gradient electrophoresis

When total cytoplasmic RNA from mouse Friend cells is fractionated using oligo(dT)-cellulose or poly(U)-Sepharose chromatography, approximately 20% of the messenger RNA activity (as measured in the reticulocyte lysate cell-free system) remains in the unbound fraction, even though this contains < 0.5% of the poly(A) (as measured by titration with poly(U)). This RNA, operationally defined as poly(A)^?, is found almost entirely in polysome structures in vivo. Its major translation products, as shown by one-dimensional sodium dodecyl sulphate-containing gels, are the histones and actin. Two-dimensional gels (isoelectric focusing: sodium dodecyl sulphate/gel electrophoresis) show that, with the exception of the mRNAs coding for histones, poly(A)^? mRNA encodes similar proteins to poly(A)⁺ mRNA, though in very different abundances. This is directly confirmed by the arrest of the translation of the abundant poly(A)^? mRNAs after hybridization with a complementary DNA transcribed from poly(A)⁺ RNA.RNA sequences which are rare in the poly(A)⁺ RNA are also found in poly(A)^? RNA, as shown by hybridizing a cDNA transcribed from poly(A)⁺ RNA to total and poly(A)^? polysomal RNA. That this does not simply represent a flow-through of poly(A)⁺ RNA is indicated by (i) the lack of poly(A) by hybridizing to poly(U) in this fraction, (ii) the fact that further passage through poly(U)-Sepharose does not remove the hybridizing sequences, (iii) the very different quantitative distribution of proteins encoded by poly(A)⁺ and poly(A)^? RNAs. We also think that it does not result from removal of poly(A) from polyadenylated RNAs during extraction because RNAs prepared using the minimum of manipulations give similar results. The distribution of both total mRNA and α and β globin mRNAs between poly(A)⁺ and poly(A)^? RNA does not change significantly during the dimethyl sulphoxide-induced differentiation of Friend cells.     

Characterization of poly(A+)RNA in free messenger ribonucleoprotein and polysomes of mouse Taper ascites cells

Cytoplasmic extracts of mouse Taper ascites cells were centrifuged on sucrose gradients to give 0–80 S, monosome, and polysome fractions. CsCl equilibrium density centrifugation of formaldehyde-fixed material from the 0–80 S fraction demonstrated that the messenger RNA in the 0–80 S fraction was in the form of free ribonucleoprotein. The size of the poly(A⁺)RNA and the size of the poly(A) segments of these molecules were shown to be very similar in both the free mRNP² and polysome fractions. The labeling kinetics of the free mRNP poly(A⁺)RNA was similar to that of the polysomal poly(A⁺)RNA.The free mRNP poly(A⁺)RNA efficiently stimulated protein synthesis in the wheat germ cell-free system, supporting the view that it was mRNA. Two-dimensional gel electrophoresis was used to analyze the proteins whose synthesis was directed by free mRNP and polysomal poly(A⁺)RNA. The free mRNP poly(A⁺)RNA directed the synthesis of a simpler set of abundant protein products than did the polysomal poly(A⁺)RNA. Most of the free mRNP abundant protein products were also present in the polysomal products, though obvious quantitative differences were evident, indicating that each individual mRNA had its own characteristic distribution between polysomes and the translationally inactive RNP form.     

The isolation of plasmids containing dna complementary to messenger rna for variant surface glycoproteins of Trypanosoma brucei

We have isolated poly(A)⁺ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)⁺ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs.The total translation products from the four poly(A)⁺ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins.We have made duplex DNA copies of these poly(A)⁺ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)⁺ RNA showed that 7–10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)⁺ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation.Poly(A)⁺ RNA from each of the variants only hybridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-terminal half of the VSGs.     

Isolation and cell-free translation of chick lens crystallin mRNA during normal development and transdifferentiation of neural retina

Messenger RNA has been isolated from day-old chick lens. Size characterization and heterologous cell-free translation demonstrate that the predominant species of mRNA present code for α-, β- and δ-crystallins. Total polysomal RNA and polysomal RNA which did not bind to oligo (dT)-cellulose translate in the cell-free system to give a crystallin profile qualitatively similar to that of poly(A)⁺ mRNA. RNA from postribosomal supernatant which binds to oligo(dT)-cellulose also translates to give crystallins, but the products are enriched for β-crystallins. Messenger RNAs isolated from 15-day embryo lens fiber and lens epithelium cells give products on translation which reflect the different protein compositions of these two cell types, as do mRNAs isolated from chick lenses at various developmental stages. Messenger RNAs were isolated from freshly excised 8-day embryo neural retina and from this tissue undergoing transdifferentiation into lens cells in cell culture. Cell-free translation demonstrates no detectable crystallin mRNAs in the freshly excised material, but by 42 days in cell culture, crystallin mRNAs are the most prominent species.     

Preformed and newly synthesized messenger RNA in germinating wheat embryos

In 6 h germinated wheat (Triticum aestivum L. cv. Cama) embryos, more than half of the messenger RNAs are actively involved in translation. Neither preformed nor newly synthesized poly A⁺-RNA is translated preferentially. Germination in the presence of cordycepin showed that the half-life of the templates is about 2 h and that the newly synthesized messengers are essential to support protein synthesis in the embryo from the first hours of germination. Most of the messenger RNAs in 6 h germinated embryos are newly synthesized. The polypeptides coded for by either the endogenous messenger ribonucleoproteins or purified poly A⁺-RNA from both dry and germinated embryos are qualitatively identical; minor quantitative differences can however be observed.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - mRNP messenger ribonucleoprotein - poly A⁺-RNA polyadenylic acid containing RNA - PB polysome buffer - GM germination medium     

Regulation of Gene Expression in Developing Zea mays Embryos: Protein Synthesis during Embryogenesis and Early Germination of Maize

Polypeptides synthesized in dissected embryos of Zea mays at different stages of embryogenesis and early germination have been characterized by their migration in two-dimensional gel electrophoresis. This analysis has been carried out with in vivo labeled polypeptides from excised embryos and with proteins synthesized in vitro in the rabbit reticulocyte system directed by poly(A⁺) RNA isolated from the different developmental stages. We have identified three main sets of expressed polypeptides: (a) embryonic set: this group of polypeptides is synthesized in young and mature embryos but not in early germination; (b) maturation set: this group of polypeptides is not present in young embryos and appears during the maturation period. Some of these polypeptides are still present in early germination while others disappear from stored mRNAs in dry embryos. One particular group from this set can be induced prematurely in young embryos by incubation with abscisic acid; and (c) germination set: this group of polypeptides is not expressed in the maturation period and appears after brief imbibition of the dry embryos.     

Preformed mRNA in Cotyledons of Ungerminated Seeds of Cicer arietinum L

Polyadenylated RNA was isolated from total RNA extracted from cotyledons of ungerminated or 18-hour-germinated chick-pea seeds by affinity chromatography on oligo(dT)-cellulose. Both poly(A)-containing RNA fractions exhibited a template activity when assayed in two cell-free translation systems, wheat germ extracts, and nuclease-treated reticulocyte lysates. Translation of preformed mRNA from cotyledons of dry seeds was completely abolished in the presence of several inhibitors of polypeptide chain initiation and also in the presence of the two “cap” analogues m⁷ GTP and m⁷ GMP. The patterns of polypeptides synthesized by translation of poly(A)-containing RNAs from cotyledons of ungerminated or 18-hour-germinated seeds, in the wheat germ system, analyzed by electrophoresis and autoradiography, were similar but not identical. It is concluded that cotyledons of dry Cicer arietinum L. seeds contain preformed mRNA.     

Use of a cloned library for the study of abundant poly(A)+RNA during Xenopus laevis development

Over 200 cloned sequences from recombinant DNA libraries prepared from Xenopus laevis embryonic poly(A)⁺RNA have been analyzed by colony hybridization with [³²P]cDNA prepared from poly(A)⁺RNA from several stages of development. The period of early embryogenesis extending through the beginning of gastrulation (stage 10) is marked by the relative constancy of the abundant poly(A)⁺RNA population. Between the gastrula and tailbud stages (stage 24) there is a dramatic change in the pattern of abundant poly(A)⁺RNA species; the new pattern remains fairly constant for at least 2 days of development to the late prefeeding tadpole stages (stage 41). We have also compared nonpolysomal and polysomal poly(A)⁺RNA populations at two different stages. In stage 10 (early gastrula) postribosomal (free ribonucleoprotein) and polysomal poly(A)⁺RNA populations partly overlap; however, many cloned sequences occur in quite different concentrations in one fraction or the other. Among the sequences that are predominantly nonpolysomal at gastrula few become predominantly polysomal at tailbud stages. Thus, we have no evidence for a major recruitment of abundant nonpolysomal RNAs into polysomes with progressing development. We rather observe a general pattern in which a cloned sequence that is nonpolysomal in one stage of development tends to be nonpolysomal (if detectable at all) in other stages as well.     

Cell-free translation of messenger RNA from oocytes of Xenopus laevis

The poly(A)⁺ RNA which accumulates during oogenesis in the amphibian Xenopus laevis is shown to be functional mRNA; the RNA was active in the mRNA-dependent “shift assay” for initiation sites in the rabbit reticulocyte lysate, and was an efficient template for protein synthesis in the wheat-germ cell-free system. Analysis of the in vitro protein products showed no differences between the coding properties of poly(A)⁺ RNA extracted from oocytes at all stages of development from previtellogenesis to maturity. In previtellogenic oocytes, the in vitro products of polysomal and of mRNP-associated poly(A)⁺ RNA were also identical. Neither was there any evidence for changes in the coding properties of the poly(A)⁺ mRNA of the oocyte. However, the patterns of oocyte in vivo protein synthesis changed markedly during early vitellogenesis. We conclude that the mRNP-associated poly(A)⁺ RNA present in mature oocytes constitutes the stored maternal mRNA, and that during oogenesis the coding composition of the poly(A)⁺ mRNA synthesised does not change markedly, while some form of translational control operates to direct the changing pattern of protein synthesis.     

Cell-free synthesis of epoxide hydrase by liver poly (A+)-RNA isolated from phenobarbital treated rats

Total liver RNA has been isolated from normal and 8 day phenobarbital treated rats by guanidine thiocyanate β-mercaptoethanol extraction and fractionated by oligo (dT)-cellulose chromatography to yield poly (A⁺)-RNA. Poly (A⁺)-RNA from normal and phenobarbital treated rats have similar translational activity in the rabbit reticulocyte cell-free system. However major alterations occurred in the polypeptide products directed by these two classes of RNA. The translation products directed by 8 day phenobarbital poly(A⁺)-RNA were immunoprecipitated with rabbit IgG prepared against purified rat liver epoxide hydrase. The immunoprecipitate was subjected to SDS-polyacrylamide gel electrophoresis and the radioactive products detected by fluorography. Analysis of the fluorogram revealed that the major immunoprecipitable product co-electrophoresed with purified epoxide hydrase. These data suggest that the primary translation product of epoxide hydrase messenger RNA has the same molecular weight as the mature form of the enzyme.     

Cell-free synthesis of opiate binding sites

A procedure is described for the detection of opiate binding sites synthesized during in vitro translation of various mRNA preparations. RNA were isolated from membrane bound polysomes which were prepared from NG 108-15 hybridoma, C6BU1 glioma cells, as well as from N18TG2, NB2aAg and NB41A3 neuroblastoma cells. Polyadenylated [poly(A)⁺] RNA were purified, translated in vitro in a rabbit reticulocyte lysate and the translation products assayed for their ability to bind [³H] bremazocine. Bound and free ligands were separated by column chromatography. After translation of poly(A)⁺ RNA obtained from NG 108-15 cells we demonstrated a stereospecific, saturable binding of [³H]bremazocine (displaced by levorphanol and not by dextrorphan) with a K_d of 2.4 ± 1.0 nM. The total amount of opiate binding sites synthesized was 6.2 ± 0.5 fmol per μg of poly(A)⁺ RNA. Opiate binding sites were undetectable at zero time and a plateau was reached after translation had proceeded for 20 min. Five time less opiate binding sites were synthesized when the poly(A)⁺ RNA purified from N18TG2 neuroblastoma cells were used under the same experimental conditions. There was no detectable binding of opiate ligands with poly(A)⁺ RNA obtained from C6BU1 glioma cells, NB2aAg or NB41A3 neuroblastoma cells.