Analysis of polyadenylated RNA from brain synaptosomes and mitochondria

We have isolated RNA from sheep brain synaptosomes and mitochondria separated by an aqueous two-phase system composed of dextran and poly(ethylene glycol). RNA was fractionated through oligo(dT)-cellulose columns and analyzed by electrophoresis through agarose slab gels containing methylmercuric hydroxide and stained with ethidium bromide. The electrophoretic patterns of the poly(A)-containing RNA fraction from synaptosomes and mitochondria are very similar although some high molecular weight RNA species, clearly visible in the synaptosomal fraction, are scarcely detected in the mitochondrial preparations. The electrophoretic analysis of a cleaner RNA preparation from digitonin-treated free mitochondria (mitoplasts) showed that all the poly (A)-RNA species of the synaptosomal preparation are also present in mitoplast. These results strongly suggest that all the discrete poly(A)-RNA species identified in brain synaptosomes are of mitochondrial origin.     

The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells.

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.     

Long-lasting effects of electroconvulsive shock on the pattern of poly(A)-RNA synthesis in rabbit cerebral cortex

The pattern of poly(A)-associated [poly(A)⁺] RNA synthesis was studied in rabbit cerebral cortex in the period following a single electroconvulsive shock (ECS). Labeled uridine was injected into the brain 2 and 4 hr after ECS and the animals sacrificed 1 hr later. Total and poly(A)⁺ RNA were then prepared from cortical nuclei and microsomes and analyzed. The amounts of newly synthesized total and poly(A)⁺ RNA in nuclei and microsomes appeared to be close to the control. However, the pattern of newly synthesized poly(A)⁺ nuclear RNA appeared to be still displaced toward the high molecular weights as it was in the early post-ECS period. The result indicates a long-lasting disturbance of brain poly(A)⁺-RNA metabolism by ECS.     

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.     

Changes in hepatic RNA, poly(A)+RNA, and poly(A)-RNA during the acute phase response to inflammation

The steady state changes in total rat hepatic cytoplasmic RNA, poly(A)+ RNA and poly(A)-RNA were assessed in response to turpentine induced inflammation. From 18 to 24 h after injury, cytoplasmic RNA doubled, while poly(A)+ RNA peaked at 24 h, 3.5 times over control animals. Cell-free translation showed significant increases in messenger RNA levels beginning at 18 h. Gel electrophoresis of translation products revealed significant increases in several polypeptides and a decrease in others. Poly(A)-RNA from control and injured rats translated to an insignificant level and the electrophoretic gel patterns of their proteins were similar. Furthermore, no change had occurred in the 3' poly(A)-sequences during the course of inflammation.     

Synthesis of poly(A)-containing RNA by isolated spinach chloroplasts.

Chloroplasts were isolated from spinach leaves and the intact chloroplasts separated by centrifugation on gradients of silica sol. Chloroplasts prepared in this way were almost completely free of cytoplasmic rRNA. The purified chloroplasts were incubated with 32PO4 in the light. The nucleic acids were then extracted and the RNA was fractionated into poly(A)-lacking RNA and poly(A)-containing RNA (poly(A)-RNA) via oligo(dT)-cellulose chromatography. The poly(A)-RNA had a mean size of approximately 18--20 S as determined by polyacrylamide gel electrophoresis. The poly(A)-RNA was digested with RNase A and RNase T1, and the resulting poly(A) segments were subjected to electrophoresis on a 10% w/v polyacrylamide gel 98% v/v formamide). Radioactivity was incorporated into both poly(A)-RNA and poly(A)-lacking RNA and into the poly(A) segments themselves. The poly(A) segments were between 10 and 45 residues long and alkaline hydrolysis of poly(A) segments followed by descending paper chromatography showed that they were composed primarily of adenine residues. There was no 32PO4 incorporation into acid-insoluble material in the dark. We conclude that isolated chloroplasts are capable of synthesizing poly(A)-RNA.     

Messenger RNA during early embryogenesis in Artemia salina: altered translatability and sequence complexity

RNA from developing embryos of Artemia salina (5, 10, and 20 h after re-initiation of development) was translated 3-10 times more efficiently in a rabbit reticulocyte lysate cell-free protein synthesizing system than RNA from dormant gastrulae. The latter did not appear to contain any significant amount of translation inhibitor activity. Ninety percent of the translatable activity in dormant gastrulae was recovered as poly(A)--RNA, whereas 80% of that in post-gastrular developing embryos was present as poly(A)+-RNA. The size of most polypeptides coded for by dormant gastrular RNA was less than 130,000 daltons whereas the size of those coded for by developing embryonic RNA was up to 200,000 daltons, which correlated with a corresponding shift to poly A-containing RNA of higher molecular weight. Two major polypeptides of about 37,000 daltons coded for by dormant gastrular RNA disappeared at 20 h after resumption of development. Hybridization of complementary DNA (cDNA) to a 1000-fold excess of the homologous poly(A)+-RNA revealed the presence of three complexity classes of mRNA. Forty-five percent, 30%, and 25% of RNA in dormant gastrulae were present as high, middle, and low abundance classes comprising about 10, 80, and 9700 species, respectively whereas in the nauplii there were 10, 150, and 7900 species of high, middle, and low abundancy sequences, respectively. Heterologous hybridizations using cDNA complementary to highly abundant messenger population of nauplii (isolated by chromatography on hydroxyapatite) to poly(A)+-RNA from dormant cysts showed considerably divergence in this class of messengers from the two developmental stages. Re-initiation of development of dormant Artemia gastrulae is thus characterized by a "re-programming" seen as a simultaneous and rapid increase in the polyadenylation and translatability of poly(A)+-RNA accompanied by a qualitative change in its sequence complexity.     

Synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis

The synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis was studied. A significant amount of RNA puls-labelled with 3H-uridine is polyadenylated. With the beginning of RNA synthesis in outgrowing spores labelled poly(A)-containing RNA was detected. The amount of poly(A)-RNA during the outgrowth and first cell division remains constant. Besides poly(A)-RNA the synthesis of tRNA and rRNA occurs. These results indicate a simultaneous activation of synthesis of tRNA, rRNA as well as of poly(A)-containing RNA during outgrowth of B. subtilis spores.     

The identification of globin messenger ribonucleic acid in newt erythropoietic cells.

Polyadenylated [poly(A)+]-RNA isolated from newt (Triturus cristatus) erythropoietic cells contained two main species sedimenting at 9S and 25S, and minor amounts of a 15-20S component. The 9S poly(A)+-RNA fraction induced synthesis of newt haemoglobin and globins in frog oocytes and in an mRNA-dependent rabbit reticulocyte lysate, confirming its identity as newt globin mRNA. Translation of 9S globin mRNA in reticulocyte lysate was concentration-dependent, the patterns of globin synthesis suggesting both preferential utilization and unequal amounts of the different globin mRNA subspecies. Globin mRNA activity was also evident in the 25S poly(A)+-RNA fraction whose localization in polyribosomes excluded its function as a nuclear globin mRNA precursor. Denaturation in formamide and estimation of its relative methyl content indicated that the 25S poly(A)+-RNA fraction contained equimolar amounts of 9S globin mRNA and 26S rRNA. Translation of the 25S fraction in reticulocyte lysate was less efficient than that of comparable amounts of 9S globin mRNA and induced a pattern of globin synthesis similar to that obtained with subsaturating amounts of 9S mRNA. The 25S mRNA-rRNA complex was considered to be a non-physiological aggregate generated by extraction of RNA in the presence of buffers of moderate to high ionic strength.     

Isolation and translation in vitro of poly(A)+RNA from the free-living nematode Panagrellus silusiae.

Polysomal RNA was isolated from the free-living nematode Panagrellus silusiae. Passage of this RNA through a cellulose column resulted in the fractionation of the input RNA into poly(A)-RNA (ca. 97.5% of the total) and poly(A)+ RNA (ca. 2.5% of the total). RNase digestion, followed by polyacrylamide gel electrophoresis, revealed that the poly(A)+ RNA contained poly(A) tracts that ranged from 75 to 104 nucleotides in length with a mean value of about 90 residues. There was no evidence of poly(A) sequences in the poly(A)- RNA fraction. Poly(A)+ RNA gave a 25- to 50-fold stimulation (over background) of amino acid incorporation in the wheat germ cell-free protein-synthesizing system. At least 26 proteins were evident after electrophoresis in cylindrical sodium dodecyl sulfate-polyacrylamide gels. Poly(A)-RNA was capable of stimulating protein synthesis in vitro with about five discrete proteins being produced. In summary, the properties of mRNA from a simple organism such as P. silusiae are very similar to those of more complex eukaryotes.     

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.     

The majority of calf muscle cell messenger RNAs contain poly(A)

Previous studies from our laboratory have investigated messenger RNA metabolism in calf muscle cells in tissue culture. The analysis of mRNA was based on its poly(A) content. We have now examined directly the proportion of mRNA which contains poly(A) in these cells. After separation of poly(A)+ -and poly(A) - -RNA on oligo(dT) -cellulos, the two fractions were translated in a reconstituted, heterologous cell-free protein-synthesizing system and the products were compared with those from the translation of total RNA. The great majority of mRNA form either prefusion or postfusion cultures was poly(A)- containing; quantitative determinations show that about 70-90% of the actin mRNA is poly(A)-containing. In order to determine if a large fraction of the calf muscle mRNA can be translated by a heterologous cell-free system, [3H]-POLY(A)+ -RNA was added to reticulocyte lysates and the formation of initiation complexes was followed. These experiments suggest that the bulk of calf muscle cell mRNA would be utilized in such a system and justify the use of cell-free systems to examine the poly(A) content of total mRNA. Thus, differential polyadenylation does not seem to be an important aspect of mRNA metabolism in cultured muscle cells. The previous study of mRNA in these cells, based on poly(A) content, is apparently a valid measure of overall mRNA metabolism.     

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.     

Isolation and partial purification of the major abundant class rat seminal vesicle poly(A+)-messenger RNA

Total poly(A(+))-RNA (poly(A(+))-RNA(tot)) was isolated from rat seminal vesicle and its size distribution determined by 70% formamide 5-25% sucrose density analysis. One major peak was resolved in the 10-13 S region and accounted for approximately 35% of the total poly(A(+))-RNA applied. Preparative 1% SDS, 5-20% linear sucrose density gradients also resolved a single major peak in the 11S region (poly(A(+))(11S). Analysis of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) under denaturing conditions on 2% agarose gel electrophoresis demonstrated two major components in both poly(A(+))-RNA populations. Size estimations for these components are 620 and 540 NT respectively. (3)H-cDNA was made to both poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S). Back-hybridization of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) to their respective (3)H-cDNA revealed a highly abundant class representing 41% and 85% of the sequences in their respective (3)H-cDNA's. The highly abundant class corresponded to 3-5 sequences present in 30,000-50,000 copies/cell. Invitro translation of poly(A(+))-RNA(11S) resulted in two major polypeptides coded for by the 620 NT long and 540 NT long poly(A(+))-RNA respectively.Images     

Spironolactone antagonism of aldosterone action on Na+ transport and RNA metabolism in toad bladder epithelium.

In earlier studies, aldosterone increased the incorporation of precursors into a class of cytoplasmic RNA with the characteristics of messenger RNA (mRNA), in toad bladder epithelium. In the present studies, this effect was analyzed further with a competitive antagonist, spironolactone (SC-9420). Paired hemibladders were labeled with 3H-uridine (30 min pulse - 140 min chase), with or without aldosterone (3.5 x 10(-8) M, 7 X 10(-8) M) in the presence or absence of SC-9420 (7 X 10(-6) M, 2.5 X 10(-5) M) at molar ratios of 200:1 to 280:1. Cytoplasmic RNA, either the total phenol-SDS extract or polyadenylated-RNA (poly(A)(+)-RNA) obtained by oligo-deoxythymidylate-cellulose (oligo(dT)-cellulose) chromatography was analyzed in linear 5 -- 20% sucrose gradients. Eight sets of experiments were completed in which the short-circuit current (scc) was monitored for 180 min and the incorporation of 3H-uridine (30 min pulse -- 150 min chase) was simultaneously determined on pools of epithelia from 5 to 10 hemibladders. The fractional change in scc correlated linearly with the fractional change in 3H-uridine of 12S cytoplasmic RNA (r=0.95, p less than 0.001). The poly(A)(+)-RNA fraction had no detectable rRNA or tRNA and gave a heterogeneous pattern, typical of mRNA, in the sucrose gradients. In the presence of exogenous aldosterone, SC-9420 inhibited the incorporation of 3H-uridine into poly(A)(+)-RNA (particularly 12S). These results support the inference that induction of mRNA mediates the action of aldosterone on Na+ transport.     

Isolation and characteristics of poly(A+) RNA in the lactating bovine udder

Total cellular poly(A+)-RNA was isolated from a lactating cow mammary gland. The poly(A+)-RNA molecules exhibit a heterogeneous distribution from 500 to 5000 nucleotides (average size--1600 nucleotides) and are made up of three main fractions (1550, 950 and 600 nucleotides) possessing a high template activity during translation in vitro. Optimal conditions for poly(A+)-RNA translation in a cell-free protein-synthesizing system from wheat embryos were elaborated. Immunochemical analysis of translation products revealed that 30% of the synthesized polypeptides are precipitated by immunoglobulins against cow milk proteins. Using hybridization with homologous cDNA, the kinetic complexity and heterogeneity of total cellular poly(A+)-RNA were investigated. This population was shown to consist of four classes differing in the diversity of their nucleotide sequences and the number of copies per cell. The total amount of the poly(A+)-RNA species in the cells of a lactating cow mammary gland is 9200, i.e., 0.46% of the genome complexity.