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.     

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.     

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.     

Biochemical differentiation of lone star tick,Amblyomma americanum (L), salivary glands: Effects of attachment,feeding and mating

Salivary glands of female Amblyomma americanum (L.) are stimulated to differentiate by attachment to a host, subsequent feeding and mating. Incorporation of [³H]uridine into ribosomal and transfer RNAs as well as the synthesis of poly(A⁺)mRNA and protein parallel the pattern of increasing enzymatic activity and secretory ability of the glands. Unfed ticks contained 3.5 ± 0.47 ng poly(A⁺)mRNA/gland pr. By the second day of feeding this had increased more than 5-fold. The greatest amount of poly(A⁺)mRNA found in rapid-feeding phase females (body wt > 100 mg) was 370 ± 80 ng/gland pr. Poly(A⁺)mRNA mass doubles on the final day of feeding, just as the ticks exceeded 100 mg in wt. Ticks attached 1 to 10 days had increasingly greater amounts of salivary monosomes, 60 and 40S ribosomal subunits, and polysomes. Polysomal mass/gland pr also attained its maximum above 100 mg tick wt at the slow/rapid-feeding phase boundary; exceeding by 20 times that of unfed ticks. Degenerating glands from replete ticks continued to synthesize protein. In vitro incorporation of [³H]leucine was greatest within 24 h of attachment. Fluorographs of [³H]leucine labeled protein showed that mating caused a drop in incorporation after the 4th day of feeding. Glands from unmated females attached the same number of days continued to incorporate [³H]leucine at higher levels than those from mated females.     

Hormonal control of tobacco protoplast nucleic acid metabolism during in vitro culture

Tobacco mesophyll protoplasts cultivated in vitro do not synthesize a measurable quantity of chloroplastic ribosomal RNA, but actively synthesize cytoplasmic ribosomal RNA, polyadenylated RNA, and proteins. These syntheses are essentially independent of the presence of hormones in the culture medium and are thus related to the ageing phenomenon induced by isolation from the plant and in-vitro culture. At all stages of culture and in all culture media, protoplasts incorporate low levels of thymidine into their DNA. However, the incorporation of considerable quantities of thymidine, indicative of the S phase, only takes place after 25–30 h and requires the presence of auxin and cytokinin.Abbreviations 6-BA 6-benzyladenine - 2,4-D 2,4 dichlorophenoxyacetic acid - DPC diethylpyrocarbonate - OD optical density; oligo-dT cellulose-oligothymidylic acid-cellulose - poly A⁺ RNA polyadenylated RNA - poly A^- RNA non-polyadenylated RNA - tRNA ribosomal RNA - SDS sodium dodecyl sulphate - TCA trichloroacetic acid - Tris buffer Tris (hydroxymethyl)aminomethane - tRNA transfer RNA     

Interaction between polyamines and nucleic acids or phospholipids

The binding of polyamines to DNA, RNA, and phospholipids has been studied by gel filtration and sucrose density gradient centrifugation. Spermine was found to bind more to a GC-rich DNA. Among RNAs containing double-stranded region [poly(AU), poly(GC), and ribosomal RNA], the binding of spermine was nearly equal. Among the single-stranded RNAs, the binding of spermine was in the order poly(U) > poly(C) > poly(A). An increase in K⁺ or Mg²⁺ concentration resulted in a great decrease in spermine binding to DNA and in a slight decrease in spermine binding to RNA. Therefore, in the presence of more than 2 mm Mg²⁺ and 100 mm K⁺, the binding of spermine to RNA was greater than that to DNA. No significant difference in spermine binding was observed between 16 S ribosomal RNA and 30 S ribosomal subunits, suggesting that ribosomal proteins did not affect significantly the binding of spermine to ribosomal RNA. The binding of spermine to microsomes was dependent on phospholipids. The binding strength was in the order phosphatidylinositol > phosphatidylethanolamine > phosphatidylcholine.     

Effect of 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole on ribonucleotide metabolism and accumulation of mitochondrial RNA and low-molecular-weight cytoplasmic RNA in HeLa cells

These results provide additional information on the selective inhibition of RNA synthesis by 5,6-dichloro-1-β-d-ribofuranosyl benzimidazole (DRB). DRB only slightly inhibited the poly(A⁺) RNA and ribosomal RNA in the mitochondria (maximal inhibition was ~25%) but severely inhibited the poly(A⁺) RNA in the postmitochondrial supernatant (~95%) and the poly(A⁺) RNA associated with the cytoplasmic membranes (~80%). Separation of the cytoplasmic low-molecular-weight RNAs showed that DRB inhibited the 5.8 S rRNA, a product of RNA polymerase I, by ~95% while there was only a slight inhibition of the 4 S RNAs (~20%) and 5 S RNA (<5%), products of RNA polymerase III. DRB severely inhibited the appearance in the cytoplasm of 28 S rRNA (~95%) and 18 S rRNA (~80%). These results, along with other recent reports (31–34), may suggest that DRB most severely inhibits RNAs that are extensively processed and/or transcribed from genes that contain extensive intervening sequences. These experiments also indicate that the mechanism of DRB inhibition does not involve alterations in ribonucleotide metabolism. DRB did not affect the phosphorylation of any ribonucleotides to triphosphates or the cellular conversion of [³H]uridine to UTP. Also, the size of the UTP and ATP pools in DRB-treated cells was equal to or greater than those in control cells through a period of 240 min. Significant amounts of DRB triphosphate could not be detected in DRB-treated cells suggesting that this may not be the inhibitory form of DRB. Measurements of the specific activity of the UTP pool allowed direct measurements of the accumulation of picomoles of the individual RNAs in the presence of DRB.     

On the Role of Stored mRNA in Protein Synthesis in Embryonic Axes of Germinating Vigna unguiculata Seeds

Polyadenylated (poly A⁺) RNAs were prepared from both dry and incubated embryonic axes of Vigna unguiculata seeds and were translated by a wheat germ translation system. Analysis with gel electrophoresis and fluorography showed that translation products of poly A⁺ RNA from dry embryonic axes were nearly the same as those from 2-hour incubated axes but somewhat different from those of 4- to 24-hour incubated axes, and that translation products remained almost unchanged between the 4- and 24-hour stages of postimbibition. The results indicate the possibility that the stored mRNA (poly A⁺ RNA from dry embryonic axes) directs the protein synthesis required for early stages of germination. This is supported by comparison of the in vitro translation products of poly A⁺ RNAs with those of polysomal RNAs. Experiments with α-amanitin, a specific inhibitor of RNA polymerase II (J. Jendrisak 1980 J Biol Chem 255: 8529-8533), suggested that the synthesis of some of the stored mRNA species is resumed as early as 4 hours after the onset of imbibition.     

Relative amounts of newly synthesized poly(A)+ and poly(A)- messenger RNA during development of Xenopus laevis

The relative amounts of newly synthesized poly(A)⁺ and poly(A)^? mRNA have been determined in developing embryos of the frog Xenopus laevis. Polysomal RNA was isolated and fractionated into poly(A)⁺ and poly(A)^? RNA fractions with oligo(dT)-cellulose. In normal embryos the newly synthesized polysomal poly(A)⁺ RNA has a heterodisperse size distribution as expected of mRNA. The labeled poly(A)^? RNA of polysomes is composed mainly of rRNA and 4S RNA. The amount of poly(A)^? mRNA in this fraction cannot be quantitated because it represents a very small proportion of the labeled poly(A)^? RNA. By using the anucleolate mutants of Xenopus which do not synthesize rRNA, it is possible to estimate the percentage of mRNA which contains poly(A) and lacks poly(A). All labeled polysomal RNA larger than 4S RNA which does not bind to oligo(dT)-cellulose in the anucleolate mutants is considered presumptive poly(A)^? mRNA. The results indicate that about 80% of the mRNA lacks a poly(A) segment long enough to bind to oligo(dT). The poly(A)⁺ and poly(A)^? mRNA populations have a similar size distribution with a modal molecular weight of about 7 × 10⁵. The poly(A) segment of poly(A)⁺ mRNA is about 125 nucleotides long. Analysis of the poly(A)^? mRNA fraction has shown that it lacks poly(A)₁₂₅.     

Affinity labelling of rat liver ribosomal protein S26 by heptauridylate containing a 5′-terminal alkylating group

Heptauridylate bearing a radioactive alkylating [¹⁴C]-4-(N-2-chloroethyl-N-methylamino)benzylamine attached to the 5-phosphate via amide bond, was bound to ribosomes and small ribosomal subunits from rat liver which thereby were coded to bind N-acylated Phe tRNA. After completion of the alkylating reaction and subsequent hydrolysis of the phosphamide bond ribosomal proteins were isolated. Radioactivity was found covalently associated preferentially with protein S26 and, to a very small extent, with proteins S3 and S3a. The affinity labelling reaction could be abolished by (pU)₁₄ and poly(U). From the results it is concluded that ribosomal protein S26 is located at the mRNA binding site of rat liver ribosomes.     

Structural and functional aspects of oligo(uridylic acid)-containing and poly(adenylic acid)-containing ribonucleic acids from rat liver

Uridylate tracts were released from rat liver mRNA by nuclease digestion and terminally-labeled invitro with ³²P using polynucleotide kinase. The pattern of fragments released from A⁺ and U⁺ mRNAs were the same as judged by electrophoresis on urea-polyacrylamide gels. The bulk of the fragments were in the size range 20–40 residues, but larger components (up to 70 residues in length) could also be seen. The ability of U⁺ and A⁺ mRNAs to direct the synthesis of proteins in a rabbit reticulocyte cell-free system was evaluated. The translation products were resolved by two-dimensional polyacrylamide gel electrophoresis. A comparison of the proteins made by the two classes of RNA showed that the U⁺ mRNA fraction represents a subset of A⁺ mRNA species, although the proportions of the protein products were quite different and several proteins were found to be unique to the U⁺ mRNA class.     

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.     

Mobilization of newly synthesized RNAs into polysomes inXenopus laevis embryos

Summary The mobilization of newly synthesized 18S and 28S rRNAs, 4S RNA and poly(A)⁺ RNA into polysomes was studied in isolated cells ofXenopus laevis embryos between cleavage and neurula stages. Throughout these stages, 4S RNA and poly(A)⁺ RNA were mobilized immediately following their appearance in the cytoplasm. 18S rRNA however, stayed in the ribosomal subunit fraction for about 30 min until the 28S rRNA appeared, when the two rRNAs were mobilized together at an equimolar ratio. This mobilization, at a 1:1 molar ratio, appeared to be realized at initiation monome formation. Thus, the efficiency of the mobilization of two newly synthesized rRNAs, shortly after their arrival at the cytoplasm, differed considerably but difference disappeared once steady state was reached.The contribution of newly synthesized 18S and 28S rRNAs to polysomes remains small throughout early development. around 3% of newly synthesized 4S RNA is polysomal which is the same distribution observed for unlabeled 4S RNA. Less than 10% of the newly synthesized cytoplasmic poly(A)⁺ RNA was mobilized into polysomes during cleavage, but in later stages the proportion increased to around 20%–25%. These results show that newly synthesized RNAs are utilized for protein synthesis at characteristic rates soon after they are synthesized during early embryonic development. On the basis of the data presented here and elsewhere we discuss quantitative aspects of the utilization of newly synthesized and maternal RNAs during early embryogenesis.     

The metabolism of a poly(A) minus mRNA fraction in HeLa cells

About 30% of HeLa cell mRNA lacks poly(A) when labeled in the presence of different rRNA inhibitors. Our method of RNA fractionation precludes contamination of the poly(A)^? mRNA with large amounts of poly(A)⁺ sequences. The poly(A)^? species is associated with polyribosomes, has an average sedimentation value equal to or greater than poly(A)⁺ mRNA, and behaves like the poly(A)⁺ mRNA in its sensitivity to EDTA and puromycin release from polyribosomes. There is very little, if any, hybridization at Rot values characteristic of abundant RNA sequences between the poly(A)^? RNA fractions from total cytoplasm or from polyribosomes and ³H-cDNA made to poly(A)⁺ RNA. This indicates that poly(A)^? mRNA does not arise from poly(A)⁺ mRNA by nonadenylation, deadenylation, or degradation of random abundant mRNA sequences. The rate of accumulation of poly(A)^? mRNA larger than 9S in the cytoplasm parallels the accumulation of poly(A)^? mRNA. The poly(A)^? mRNA is maintained as approximately 30% of the total labeled mRNA in a short (90 min) and in a long (20 hr) time period. These data indicate that poly(A)^? mRNA is not short-lived nuclear or cytoplasmic heterogeneous RNA contamination, and that the half-life of the poly(A)^? mRNA may parallel that of the poly(A)⁺ mRNA. Cordycepin appears to almost completely (95%) inhibit poly(A)⁺ mRNA while only partially (60%) inhibiting the poly(A)^? mRNA. The origin of the cordycepin-insensitive mRNA has not been ascertained.     

Identification of the protein cross-linked to 3′-terminus of 5 S RNA in rat liver ribosomal 60 S subunits

To cross-link the 3′-terminus of 5 S RNA to its neighbouring proteins, ribosomal 60 S subunits of rat liver were oxidized with sodium periodate and reduced with sodium borohydride. 5 S RNP was then isolated by EDTA treatment followed by sucrose density-gradient centrifugation and subjected to SDS-polyacrylamide gel electrophoresis. The protein with a slower mobility than the L5 protein, which was thought to be cross-linked 5 S RNP, was labeled with ¹²⁵I, treated with RNAase, and analyzed by two-dimensional polyacrylamide gel electrophoresis, followed by radioautography. A radioactive spot located anodically from L5 protein was observed, suggesting that it is the L5 protein-oligonucleotide complex. When analyzed by SDS slab polyacrylamide gel electrophoresis followed by radioautography, the peptide pattern of the α-chymotrypsin digest of this ¹²⁵I-labeled protein-oligonucleotide complex was similar to that of the digest of ¹²⁵I-labeled L5 protein. The results indicate that L5 protein binds to the 3′-terminal region of 5 S RNA in rat liver 60 S subunits.     

Poly(A)+ RNA synthesis in germinating pollen ofNicotiana tabacum L

Poly(A)⁺RNA is synthesized during the first hours of pollen germination and is rapidly incorporated into polysomal structures. After a 2-h pulse with uracil-¹⁴C, 42% of the transcribed fraction of polysomal RNA is polyadenylated. Following 4 h of germination the amount of the newly-made poly(A)⁺RNA decreases steadily at the rate of about 14% per h, whereas that of rapidly-labelled poly(A)⁻RNA continues to grow. Beginning 1 h of cultivation the ratio of poly(A)⁻/poly(A)⁺RNA increases exponentially. Similarly as in non-polyadenylated mRNA the main portion of the synthesized polysomal poly(A)⁺RNA sediments at a rate of 4 to 14 S and its mean size decreases slightly with the time of labelling. RNA isolated from nuclei and cell wall containing pollen tube fraction differed from the polysomal one in higher apeoific radioactivity and the polyadenylated RNA exhibited higher size distribution. The comparison of the results with earlier observations suggests the involvement of poly(A)in mRNA translation in pollen tubes.     

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.