Characterization of polyadenylated RNA in a protein-producing bacterium, Bacillus brevis 47.

Up to about 50% of the total radioactivity in pulse-labeled RNA in Bacillus brevis 47-5, a high-protein-producing bacterium, was found in the polyadenylated fraction [termed poly(A)-RNA] isolated by adsorption to oligodeoxythymidylic acid-cellulose. Labeled RNA was bound to the cellulose regardless of whether the radioactive precursor was [3H]adenosine or [3H]uridine, showing that the adsorbed material was poly(A)-RNA rather than free poly(A). Poly(A) tracts, isolated after digestion of pulse-labeled RNA with pancreatic and T1 RNases, were homogeneous, with a length of about 95 nucleotides. Susceptibility of the isolated poly(A) tracts to degradation by snake venom phosphodiesterase and polynucleotide phosphorylase indicated that the poly(A) sequences were located directly at the 3'-terminal of the RNA molecules. Comparison of the poly(A)-RNA content in high-protein-producing and nonprotein-producing cells of B. brevis 47 showed much higher levels in the former. Electrophoretic analysis in both denaturing and denaturing polyacrylamide gels of the poly(A)-RNAs showed a heterogeneous population of molecules ranging in size from 23S to 4S. Comparison of the molecular-weight distribution patterns revealed that a significantly greater amount of high-molecular-weight poly(A)-RNA (comigrating with 23S RNA) was present under conditions in which extracellular protein production was high. The possibility that a substantial fraction of the poly(A)-RNA might be involved in the synthesis of extracellular proteins in B. brevis 47 is discussed.     

Stimulation by thyrotropin of synthesis of poly(A)-RNA and non-poly(A)-RNA in rat thyroid tissue

The $\text{in vivo}$ stimulation by thyrotropin of the synthesis of poly(A)-RNA and non-poly(A)-RNA in thyroid tissue was studied in 18 day old male rats. Each rat was injected with 0.25 ml of saline or of thyrotropin (0.25 unit) 4 hr or 8 hr before killing. Rats were injected with ³H-uridine 2 to 4 hr before sampling of thyroid tissue. Poly(A)-RNA and non-poly(A)-RNA were isolated by oligo (dT)-cellulose chromatography. Poly(A)-RNA accounts for about 3.5% of total cellular RNA; the specific activity of labeled poly(A)-RNA was 4–7 fold greater than that of non-poly(A)-RNA. A stimulation of about 40% and 90% over the control values was observed in the incorporation of ³H-uridine into poly(A)-RNA and non-poly(A)-RNA, respectively, in thyroid 4 to 8 hr after hormonal injection. The RNA contents of thyroid from hormone-treated rats did not change during the same time period. The stimulation of synthesis of poly(A)-RNA and non-poly(A)-RNA in thyroid was tissue specific insofar as these phenomena were not seen in liver or brain tissues.     

Stability of polysome-associated polyadenylated RNA from soybean suspension culture cells.

The half-life of polysome-associated, poly(A)-RNA in exponentially growing soybean (Glycine max) suspension culture cells was determined with pulse-chase experiments. Based on a best fit from a computer analysis of the data, two decay components for poly(A)-RNA were found. One component had a half-life of approximately 0.6 h, while the other had a half-life of about 30 h, similar to the doubling time of the cultures. At the beginning of the chase period, the short-lived component represented approximately 90% of the total poly(A)-RNA in the polysomes. This percentage decreased with time so that, under steady-state conditions, the long-lived component probably represented the majority of poly(A)-RNA.     

Newly synthesized extracellular ribonucleic acids in the amphibian gastrula

The possible synthesis of RNA located in the extracellular compartment of Bufo arenarum gastrula was studied using a biochemical method. [3H]adenosine was microinjected into the blastocoel of late blastulae or early gastrulae, which were then dissociated at advanced gastrula stage. RNA was extracted from both, the cellular supernatant and the disaggregated cells, by the Kirby-phenol procedure. Most of the ethanol-precipitable radioactivity was sensitive to RNase and alkaline treatment. The partial characterization of these molecules indicate that the radioactive pattern of total RNA, found in sucrose gradients, the ratio Poly(A)+RNA/Poly(A)-RNA as well as the radioactive pattern of Poly(A) fraction in acrylamide gels were different in samples from cellular and from extracellular origin. Although not conclusive, these results are proposed as a new argument for the existence of an extracellular RNA in the amphibian embryo.     

Comparative patterns of synthesis of polyadenylated and nonpolyadenylated RNA in various brain regions following intraventricular administration of 3H-uridine into adult rats

Measurements of populations of unlabeled RNA indicate that the absolute concentrations and relative proportions of poly(A)-RNA and of nonpoly(A)-RNA, relative to total cellular RNA are similar in three brain regions. The incorporation of ³H-uridine into poly(A)-RNA and nonpoly(A)-RNA was measured in cerebrum, diencephalon, and midbrain-hindbrain from 15 min through 8.0 hr after intraventricular injection of the precursor into adult rat brains. Incorporation of ³H-uridine into poly(A)-RNA was very rapid and reached maximum levels of specific activity within 30 to 120 minutes, depending upon locus, after injection of the precursor. The specific activity of nonpoly(A)-RNA increased with time, but remained lower than that of poly(A)-RNA throughout the 8.0 hr period. Regionally differential synthesis occurred both in poly(A)-RNA and nonpoly(A)-RNA in the several brain regions. Establishment of the time kinetics of brain RNA synthesis should provide useful basis for selection of the conditions for labeling pulses for further studies of $\text{in vivo}$ RNA metabolism.     

The metabolic behaviour of nuclear and cytoplasmic non-polyadenylated RNAs with an affinity for poly(adenylic acid) from Friend murine leukaemia cells

Using poly(A)-Sepharose and poly(U)-Sepharose affinity chromatography, various classes of nuclear RNA can be distinguished in Friend leukaemia cells. One of these contains a poly(A) tract (poly(A)+-RNA) and another lacks a poly(A) tract but has an affinity for poly(A)-Sepharose (poly(A)-u+-RNA). The stability of these two particular nuclear RNA classes was examined by using a 'pulse-chase' technique involving D-glucosamine treatment. Nuclear poly(A)-u+-RNA was found to decay as a single component with a half-life of about 12 min. In contrast, nuclear poly(A)+-RNA appears to consist of at least two distinct metabolic components with half-lives of about 22 min and 120 min. Furthermore, poly(A)-u+-RNA is transported from the nuclei much more rapidly than the poly(A)+-RNA. The 'pulse-chase' approach also allowed a quantitative estimate to be made of the conversion of nuclear poly(A)+-RNA and poly(A)-u+-RNA to cytoplasmic poly(A)-RNA and poly(A)-u+-RNA.     

Size and turnover of polyadenylic acid-containing ribonucleic acids in a fragile mutant of Saccharomyces cerevisiae.

Ribonucleic acid-containing polyadenylic acid [poly(A)+-RNA] was studied in lysates from an osmotic-sensitive mutant of Saccharomyces cerevisiae characterized by low nuclease activity. The poly(A)+-RNA fraction, analyzed by electrophoresis in polyacrylamide-formamide gels, constitutes a heterogeneous population of molecules, with molecular weights ranging from 0.2 X 10(6) to 3 X 10(6) and having an average of 1.2 X 10(6). The turnover rate of poly(A)+-RNA was determined by the decay of radioactivity after a cold uracil chase, and the observed half-life of 21 min corresponds to about 10% of the cell doubling time. Poly(A)+-RNA was analyzed by gel electrophoresis under denaturing and non-denaturing conditions. A correlation was established between the apparent secondary structure and the turnover rate of poly(A)+-RNA species.     

Sequence complexity of messenger RNA in cotyledons of developing pea (Pisum sativum) seeds

The hybridization kinetics of poly(A)⁺-RNA preparations from the cotyledons of developing pea (Pisum sativum seeds to complementary DNAs have shown that the number of distinct sequences in poly(A)⁺ -RNA decreases from ca 20 000 at the early stage of cotyledon development to ca 200 at a late stage of cotyledon development. The decrease in sequences is accounted for entirely by the disappearance of ‘rare’ poly(A)⁺ -RNAs (< 10³ copies/cell) as seed development proceeds. There is an increase (1–6) in very abundant poly(A)⁺-RNA sequences (? 5 × 10⁵ copies/cell) from early- to mid-developmental stages, concomitantly with the increase in the synthesis of seed-specific storage protein polypeptides. In agreement with the continuing synthesis of most of these polypeptides to the end of seed development, the number of very abundant poly(A)⁺-RNAs is maintained to the late cotyledon development stage. Abundant poly(A)⁺-RNA sequences (ca 10⁴ sequences/cell) increase from 80 to 180 during development, possibly corresponding to the polypeptides which are not storage proteins but are known to be accumulated in pea seeds. Hybridization of single-copy pea genomic DNA sequences to poly(A)⁺-RNA from developing seeds showed that ca 5 % of the single-copy sequences were present in mRNA from mid-development cotyledons. In addition, hybridization of cDNA prepared against poly(A)⁺-RNA from nuclei of early development cotyledons to the corresponding cytoplasmic polysomal poly(A)⁺-RNA showed that the cytoplasmic poly(A)⁺-RNA contained ca 50 % of the sequences present in the nuclei. These results are discussed and interpreted in the light of existing results from similar systems.     

Complexity and characterization of polyadenylated RNA in the mouse brain

The complexity of nuclear RNA, poly(A)hnRNA, poly(A)mRNA, and total poly(A)RNA from mouse brain has been measured by saturation hybridization with nonrepeated DNA. These DNA populations were complementary, respectively, to 21, 13.5, 3.8, and 13.3% of the DNA. From the RNA Cot required to achieve half-sturation, it was estimated that about 2.5–3% of the mass of total nuclear RNA constituted most of the complexity. Similarly, complexity driver molecules constituted 6–7% of the mass of the poly(A)hnRNA. 75–80% of the poly(A)mRNA diversity is contained in an estimated 4–5% of the mass of this mRNA. Poly(A)hnRNA constituted about 20% of the mass of nuclear RNA and was comprised of molecules which sedimented in DMSO-sucrose gradients largely between 16S and 60S. The number average size of poly(A)hnRNA determined by sedimentation, electron microscopy, or poly(A) content was 4200–4800 nucleotides. Poly(A)mRNA constituted about 2% of the total polysomal RNA, and the number average size was 1100–1400 nucleotides. The complexity of whole cell poly(A)RNA, which contains both poly(A)hnRNA and poly(A)mRNA populations, was the same as poly(A)hnRNA. This implies that cytoplasmic polyadenylation does not occur to any apparent qualitative extent and that poly(A)mRNA is a subset of the poly(A)hnRNA population. The complexity of poly(A)hnRNA and poly(A)mRNA in kilobases was 5 × 10⁵ and 1.4 × 10⁵, respectively. DNA which hybridized with poly(A)mRNA renatures in the presence of excess total DNA at the same rate as nonrepetitive tracer DNA. Hence saturation values are due to hybridization with nonrepeated DNA and are therefore a direct measure of the sequence complexity of poly(A)mRNA. These results indicate that the nonrepeated sequence complexity of the poly(A)mRNA population is equal to about one fourth that observed for poly(A)hnRNA.     

RNA from callus cultures and leaves of Nicotiana tabacum

MAK column chromatography has been used to analyse RNA from normal and crown gall callus cultures and leaves of Nicotiana tabacum. To determine the elution behaviour of well-defined DNA-like RNAs with different GC content, complementary RNAs (c-RNA) synthesized on Agrobacterium tumefaciens DNA and crown gall DNA were used. The elution profile of the RNA from all three tissues followed a similar pattern. By salt gradient elution the RNA in the tRNA region showed a remarkably high CMP content which was significantly higher for the normal tissues than for crown gall tissue. RNA from the callus cultures contained more DNA-like RNA (D-RNA) with a higher turnover rate than RNA from leaves. Because of its relatively low poly A content, measured as RNase A + T₁ resistance, as well as its high turnover rate, the salt-eluted D-RNA is thought to be heterogeneous nuclear RNA (Hn-RNA) and not mRNA. RNA molecules that might represent the mRNA population, having intramolecular poly A tracts, were subsequently eluted by a salt gradient, a low salt buffer and with the chaotropic agent guanidine thiocyanate, which removed tenaciously bound (TB-RNA) in two fractions, α and β. Crown gall RNA showed both a different labelling behaviour and a higher poly A content in the α and β fractions compared to the normal tissues. c-RNAs may be eluted at different salt concentrations because of their different GC content. They give rise to a considerable fraction of TB-RNA which in the presence of tobacco leaf RNA was split into fractions similar to α and β. No fraction was found amongst these RNAs which did have intramolecular poly A tracts.     

The effect of chromosomal polytenization on the rates of RNA synthesis and decay in the salivary glands of the blowfly, Calliphora erythrocephala

The rates of total RNA synthesis and accumulation have been measured in the polytenic salivary gland cells of the blowfly, Calliphora erythrocephala, by three methods: (1) injecting larvae with [2-³H]adenosine and determining its flow into the cellular ATP pool and RNA, (2) measuring the increase in glandular RNA optically, and (3) measuring the rate of flow of ATP out of the cellular pool. The size of the steady-state pool of rapidly turning over RNA and its half-life, were calculated from these kinetic data and, also, by an independent measurement of the steady-state content of nuclear RNA. These parameters were compared at a number of developmental stages which differed in degree of chromosomal polytenization. The results indicate that these polytenic cells synthesize RNA at a rate approximately 10³ times those of other diploid eukaryotic cells. This rate is independent of the increase in chromosomal polyteny that accompanies larval development. Approximately 67% of the newly synthesized salivary gland RNA is an unstable component with an average first-order half-life of 20–25 min. The remainder is a long-lived species with an estimated average first-order half-life of about 30 hr.     

Poly(A) length,cytoplasmic adenylation and synthesis of poly(A)+ RNA in early mouse embryos

The poly(A) content of early mouse embryos fluctuates widely: after a transient increase in the one-cell embryo, there is a 70% drop in the two-cell and an approximately fivefold increase between the two-cell and early blastocyst stages (L. Pikó and K. B. Clegg, 1982, Dev. Biol.89, 362–378). To shed light on the significance of these changes, we analyzed the size distribution of total poly(A) from embryos at different stages of development by gel electrophoresis and hybridization with [³H]poly(U). The number-average size of poly(A) tracts varies only slightly, from 61 to 77 nucleotides, indicating that the changes in poly(A) content are due primarily to changes in the number of poly(A) sequences, i.e., the number of poly(A)+ mRNA. From these data, the number of poly(A)+ mRNA can be estimated as follows: ovulated egg, 1.7 × 10⁷; one-cell embryo, 2.4 × 10⁷; late two-cell, 0.7 × 10⁷; late eight-cell, 1.3 × 10⁷; and early blastocyst, 3.4 × 10⁷. These results suggest the elimination of the bulk of maternal poly(A)+ mRNA at the two-cell stage, to be replaced by newly synthesized mRNA derived from the embryonic genome. To study the synthesis of poly(A)+ mRNA, we cultured mouse embryos in vitro with [³H]adenosine and analyzed the labeled poly(A)+ RNA as to molecular size, length of the poly(A) tail, and relative distribution of label in poly(A) vs internal locations. We observed an active incorporation of label into large-molecular-weight (average size about 2 kb) poly(A)+ RNA at all stages from the one-cell to the blastocyst. However, in the one-cell embryo, about 70% of the label was localized in the poly(A) tail, suggesting cytoplasmic polyadenylation, and only about 30% was localized in the remainder of the molecule, suggesting the complete new synthesis of a small amount of poly(A)+ RNA. Differences in the size distribution of the labeled poly(A) as compared with the total poly(A) in the one-cell embryo indicate that the labeling is not due to a general turnover of poly(A) tails, but rather to the polyadenylation of previously nonpolyadenylated, stored RNA. Significant new synthesis of poly(A)+ RNA is evident from the two-cell stage onward and most likely accounts for the sharp rise in the number of poly(A)+ RNA molecules by the early blastocyst stage.     

Synthesis and transport of various RNA species in developing embryos of Xenopus laevis.

Embryonic cells of Xenopus laevis were labeled for varying lengths of time, and their nuclear and cytoplasmic RNAs were analyzed, with the following results. (1) The synthesis of small nuclear RNAs (snRNAs) is detected from blastula stage on. (2) The initiation of 4 S and 5 S RNA syntheses occurs at blastula stage. However, while the former is transported into the cytoplasm immediately after its synthesis, the latter remains within the nucleus, until its transport starts later, concomitantly with that of 28 S rRNA. (3) As soon as “blastula” cells start to synthesize 40 S rRNA precursor at 5th hr of cultivation, 18 S rRNA is transported first; the transport of 28 S rRNA begins 2 hr later. (4) On a per-cell basis, poly(A)-RNA is synthesized in blastula stage at a much higher rate than in the later stages. About one-third of the total blastula poly(A)-RNA, and about one-fifth in the case of tailbud cells, is transported quickly into the cytoplasm. Then, it appears that the RNAs which are synthesized at early embryonic stages are transported to the cytoplasm without delays, except for 5 S RNA and snRNAs.     

Differential accumulation of two size classes of poly(A) associated with messenger RNA during oogenesis in Xenopus laevis

The RNA of full-grown oocytes of Xenopus laevis contains two distinct size classes of poly(A), designated poly(A)_S and poly(A)_L, which contain 15–30 (mean = 20) and 40–80 (mean = 61) A residues, respectively. Both poly(A)_L and poly(A)_S are associated with RNA which is heterogeneous in size. The two classes of poly(A)⁺ RNA can be separated by affinity chromatography: Only poly(A)_L⁺ RNA binds to oligo(dT)-cellulose under appropriate conditions, but up to 50% of the poly(A)_S⁺ RNA can be isolated from the void fraction by binding to poly(U)-Sepharose. Both classes of poly(A)⁺ RNA are active as messenger RNA in an in vitro system and yield identical patterns of in vitro protein products. Previtellogenic oocytes contain almost exclusively poly(A)_L, which accumulates up to vitellogenesis but remains almost constant in amount (molecules/oocyte) during vitellogenesis and in the full-grown oocyte. Poly(A)_S accumulates (molecules/oocyte) from early vitellogenesis up to the full-grown oocyte. The total number of poly(A)⁺ RNA molecules per oocyte increases throughout oogenesis from 2 × 10¹⁰/previtellogenic oocyte [80–90% poly(A)_L] to 20 × 10¹⁰/full-grown oocyte (25–40% poly(A)_L). It is argued that poly(A)_S is protected from degradation in the oocyte, thus stabilizing the “maternal” poly(A)⁺ mRNA.     

Characterization of the E.coli poly(A) polymerase: nucleotide specificity, RNA-binding affinities and RNA structure dependence

Polyadenylation of RNA molecules in bacteria and chloroplasts has been implicated as part of the RNA degradation pathway. The polyadenylation reaction is performed in Escherichia coli mainly by the enzyme poly(A) polymerase I (PAP I). In order to understand the molecular mechanism of RNA polyadenylation in bacteria, we characterized the biochemical properties of this reaction in vitro using the purified enzyme. Unlike the PAP from yeast nucleus, which is specific for ATP, E.coli PAP I can use all four nucleotide triphosphates as substrates for addition of long ribohomopolymers to RNA. PAP I displays a high binding activity to poly(U), poly(C) and poly(A) ribohomopolymers, but not to poly(G). The 3′-ends of most of the mRNA molecules in bacteria are characterized by a stem–loop structure. We show here that in vitro PAP I activity is inhibited by a stem–loop structure. A tail of two to six nucleotides located 3′ to the stem–loop structure is sufficient to overcome this inhibition. These results suggest that the stem–loop structure located in most of the mRNA 3′-ends may function as an inhibitor of polyadenylation and degradation of the corresponding RNA molecule. However, RNA 3′-ends produced by endonucleolytic cleavage by RNase E in single-strand regions of mRNA molecules may serve as efficient substrates for polyadenylation that direct these molecules for rapid exonucleolytic degradation.