Self-splicing of yeast mitochondrial ribosomal and messenger RNA precursors

We have previously shown linear and circular splicing intermediates resembling intermediates that result from self-splicing of ribosomal precursor RNA of Tetrahymena to be present in mitochondrial RNA. Here we show that splicing of yeast mitochondrial precursor RNA also occurs in vitro in the absence of mitochondrial proteins. The large ribosomal RNA gene, consisting of the intron and part of the flanking exon regions, was inserted behind the SP6 promoter in a recombinant plasmid and was transcribed in vitro. The resulting RNA shows self-catalyzed splicing via incorporation of GTP at the 5'-end of the excised intron, 5'- to 3'-exon ligation, and intron circularization. When purified mitochondrial RNA is incubated under similar conditions with alpha-32P-GTP, the excised ribosomal intron RNA is also labeled, as well as several other RNA species. Some of these RNAs are derived from excised introns from the multiply split gene coding for cytochrome oxidase subunit I.     

Synthesis of ribonucleic acid in the venom gland of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom

1. The incorporation of [5-(3)H]uridine into RNA of the venom gland of Crotalus durissus terrificus was studied after manual extraction (;milking') of the venom. The labelled precursor was injected immediately after milking. 2. The RNA was extracted 1, 2, 4, 6 and 8h after injection of the label and analysed by sucrose-density-gradient centrifugation. 3. The sedimentation analysis showed that 18S rRNA synthesis is higher than 28S rRNA at all time-intervals. The specific radioactivities of both ribosomal components did not reach a plateau even at 8h after injection. 4. An RNA fraction was detected sedimenting between 18S rRNA and 4S tRNA and was called the 10-14S fraction. The specific radioactivity was always higher than that of both classes of rRNA and reached the maximum value at 6h of labelling. 5. The incorporation of the precursor was also studied by radioautography, which helped to elucidate the intracellular origin of the RNA analysed by sucrose-density-gradient centrifugation.     

Synthesis of ribonucleic acid in normal bone in vitro

1. The incorporation of [2-(14)C]uridine into nucleic acids of bone cells was studied in rat and pig trabecular-bone fragments surviving in vitro. 2. The rapid uptake of uridine into trichloroacetic acid-soluble material, and its subsequent incorporation into a crude nucleic acid fraction of bone or purified RNA extracted from isolated bone cells, was proportional to uridine concentration in the incubation medium over a range 0.5-20.0mum. 3. During continued exposure to radioactive uridine, bulk RNA became labelled in a curvilinear fashion. Radioactivity rapidly entered nuclear RNA, which approached its maximum specific activity by 2hr. of incubation; cytoplasmic RNA, and particularly microsomal RNA, was more slowly labelled. The kinetics of labelling and rapid decline of the nuclear/microsomal specific activity ratio were consistent with a precursor-product relationship. 4. Bulk RNA preparations were resolved by zonal centrifugation in sucrose density gradients into components with approximate sedimentation coefficients 28s, 18s and 4s. 5. Rapidly labelled RNA, predominantly nuclear in location, demonstrated a polydisperse sedimentation pattern that did not conform to the major types of stable cellular RNA. Material of highest specific activity, sedimenting in the 4-18s region and insoluble in 10% (w/v) sodium chloride, rapidly achieved its maximum activity during continued exposure to radioactive precursor and decayed equally rapidly during ;chase' incubation, exhibiting an average half-life of 4.3hr. 6. Ribosomal 28s and 18s RNA were of lower specific activity, which increased linearly for at least 6hr. in the continued presence of radioactive uridine. There was persistent but variable incorporation into ribosomal RNA during ;chase' incubation despite rapid decline in total radioactivity of the acid-soluble pool containing RNA precursors.     

Effects of pancreozymin, urecholine and actinomycin D on the metabolism of ribonucleic acid by canine pancreas

1. Canine pancreas slices were incubated with [6-(14)C]orotic acid and the rate of its incorporation into RNA was measured. RNA was fractionated by shaking homogenates with phenol at 2 degrees , 50 degrees , 65 degrees and 80 degrees . Cytoplasmic RNA was extracted at the lowest temperature and nuclear RNA at the higher temperatures. The samples were centrifuged through sucrose gradients and the E(260) and (14)C-sedimentation patterns determined. Incorporation of orotic acid was very rapid into cytoplasmic 4s RNA. This probably represents end-group turnover. No incorporation into cytoplasmic ribosomal RNA was observed. 2. The nuclear 50 degrees -RNA exhibited two E(260) peaks, at 18s and 28s. This portion of the sample contained but moderate amounts of [(14)C]RNA. The highly labelled material had sedimentation coefficients in the range 35-50s. The nuclear 65 degrees -RNA showed an E(260) peak at 16s. The [(14)C]RNA peak occurred at 25-35s and this portion demonstrated the highest specific activity of any RNA fraction. 3. The 50 degrees -RNA, 65 degrees -RNA and 80 degrees -RNA were hydrolysed and their base compositions were determined. All three samples possess a ribosomal type of composition (G+C)/(A+U)=(1.4-1.7). For this reason they are considered to contain ribosomal precursor RNA as their major constituent. 4. Actinomycin D (0.5mug./ml.) in the incubation medium inhibited incorporation of orotic acid into both nuclear fractions but not into 4s RNA. 5. The cholinergic drug Urecholine inhibited incorporation into the heavy, high-specific-activity portions of the nuclear fractions but did not inhibit incorporation into the ribosomal precursor type of nuclear RNA. A similar result was also obtained with the hormone pancreozymin. Moderate inhibition of incorporation of orotic acid into 4s RNA likewise resulted from the presence of the drug and the hormone.     

Isolierung und Charakterisierung schnell markierter,hochmolekularer RNS aus frei suspendierten Calluszellen der Petersilie (Petroselinum sativum)

Summary By culturing of callus tissue originating from root explants of Petroselinum sativum in a synthetic liquid medium under aeration, freely suspended single cells and small clusters consisting of mostly five cells were obtained. The rapidly dividing cells did not exhibit any morphogenesis. Their nucleic acid metabolism was investigated by pulse experiments with ³²P-orthophosphate. Rapidly labelled RNA was prominently found associated with high molecular RNA. During the fractionation of the total nucleic acids on MAK columns it was eluted after the ribosomal RNA components. Its base ratio, however, differed from the latter in that the AMP content was higher than the GMP content. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis resulted in the separation of the ribosomal RNA from the rapidly labelled RNA, thus proving the higher molecular weight of the latter. Based upon the migration in the gel a sedimentation coefficient of approximately 32S was calculated. The possible function of the heavy rapidly labelled RNA component as precursor of ribosomal RNA is discussed.     

In vitro studies of cerebral cortical RNA and nucleotide metabolism in hypothyroidism.

—Thyroid hormone deficiency induced during the neonatal period in the rat, resulted in an enhanced incorporation of [2-¹⁴C]uridine and [8-¹⁴C]adenosine in vitro into cerebral cortical RNA at 25 days of age. An examination of the acid-soluble pool constituents separated by polyethyleneiminecellulose TLC, revealed that all phosphorylated derivatives were more highly labelled compared to controls. These differences were not apparent at a lower incubation temperature (4°C). When the average specific activity of precursor pool ATP labelled from adenosine was utilized for the calculation of the rate of RNA synthesis, no change was observed in hypothyroidism. The results are compatible with a maturational-dependent increase in nucleoside transport and rate of phosphorylation in hypothyroidism which is reflected in the stimulated incorporation into cerebral RNA. The apparent normal rate of RNA synthesis coupled with a diminished cellular RNA concentration in thyroid hormone deficiency, suggests an increased RNA turnover. Experiments with actinomycin D revealed no apparent difference in the rate of decay of rapidly-labelled (nuclear) RNA. The possibility is discussed that the processing of nuclear RNA, the formation of stable ribosomal complexes and events at the translational level are subject to modification in developing hypothyroid rats.     

Ribosomal RNA Turnover in Contact Inhibited Cells

CONTACT inhibition of animal cell growth is accompanied by a decreased rate of incorporation of nucleosides into RNA^1–3. Contact inhibited cells, however, transport exogenously-supplied nucleosides more slowly than do rapidly growing cells^4,5, suggesting that the rate of incorporation of isotopically labelled precursors into total cellular RNA may be a poor measure of the absolute rate of RNA synthesis by these cells. Recently, Emerson⁶ determined the actual rates of synthesis of ribosomal RNA (rRNA) and of the rapidly labelled heterogeneous species (HnRNA) by labelling with ³H-adenosine and measuring both the specific activity of the ATP pool and the rate of incorporation of isotope into the various RNA species. He concluded that contact inhibited cells synthesize ribosomal precursor RNA two to four times more slowly than do rapidly growing cells, but that there is little if any reduction in the instantaneous rate of synthesis of HnRNA by the non-growing cells. We have independently reached the same conclusion from simultaneous measurements on the specific radioactivity of the UTP pool and the rate of ³H-uridine incorporation into RNAs (unpublished work of Edlin and myself). However, although synthesis of the 45S precursor to ribosomal RNA is reduced two to four times in contact inhibited cells, the rate of cell multiplication and the rate of rRNA accumulation are reduced ten times. This suggests either “wastage”⁷ of newly synthesized 45S rRNA precursor, or turnover of ribosomes in contact inhibited cells Two lines of evidence suggest that “wastage” of 45S RNA does not play a significant role in this system. (1) The rate of synthesis of 45S RNA in both growing and contact inhibited cells agrees well with that expected from the observed rates of synthesis of 28S and 18S RNAs (unpublished work of Edlin and myself). Emerson has made similar calculations⁶. (2) 45S RNA labelled with a 20 min pulse of ³H-uridine is converted in the presence of actinomycin D to 28S and 18S RNAs with the same efficiency (approximately 50%) in both growing and contact inhibited cells. These results indicate that, in order to maintain a balanced complement of ribosomal RNAs, contact inhibited cells must turn over their ribosomes. We present evidence here that rRNA is stable in rapidly growing chick cells, but begins to turn over with a half-life of approximately 35–45 h as cells approach confluence and become contact inhibited.     

The pattern of protein and nucleic acid synthesis in germinating spores of Phycomyces blakesleeanus

Summary Synthesis of proteins, RNA and DNA is measured by incorporation of labelled precursors at different times during germination of Phycomyces spores.RNA and protein synthesis increases immediately after activation. DNa synthesis begins at a later stage (± 8 h) of germination when germ tubes are already present. Nuclear division occurs earlier in germination (±4–5 h) and is accompanied by a decrease in RNA synthesis. It can be concluded that at least most of the dormant spores are in the G2 phase of the cell cycle.Analysis of ribosomal RNA after pulse-chase labelling shows only three labelled compounds: a precursor molecule (2.25×10⁶ daltons) and the two mature ribosomal RNA compounds (1.4×10⁶ and 0.7×10⁶ daltons). This suggests that the two rRNAs are formed directly from the precursor molecule. Cycloheximide totally blocks the transformation of the ribosomal precursor molecule into mature rRNA.     

The messenger ribonucleic acid content of Bacillus subtilis 168

Bacillus subtilis 168 messenger RNA was determined by DNA-RNA hybridization techniques, with denatured DNA immobilized upon cellulose nitrate membrane filters. The following results were obtained. (1) Cultures of B. subtilis, growing exponentially in enriched glucose-salts medium at 37 degrees , incorporated [5-(3)H]uracil into both ribosomal and messenger RNA fractions without the kinetic delay expected from the presence of the intracellular nucleotide pools. (2) However short the time of labelling with exogenous labelled uracil (down to 7sec.), 32-36% of the rapidly labelled RNA was messenger RNA and 68-64% was an RNA with the hybridization characteristics of ribosomal RNA. Analysis of the apparent nucleotide base composition of total (32)P-labelled rapidly labelled RNA and the two RNA fractions separated by hybridization at a DNA/RNA ratio 5:1 confirmed this finding. Of the rapidly labelled RNA, 31% readily hybridized with DNA at low DNA/RNA ratios and had an apparent base composition like that of the DNA, whereas 69% was hybridized only at low efficiency at low DNA/RNA ratios and had a composition identical with that of ribosomal RNA. (3) In cultures dividing every 48min. at 37 degrees , kinetic analysis of RNA labelled over a 20min. period showed that the average life-time of messenger RNA was 2.7-3.0min. and that its amount was 3.0% of the total RNA. (4) The hybridization of (3)H-labelled randomly labelled RNA with DNA at a DNA/RNA ratio 5:1 showed that 2.9% of the randomly labelled RNA had the characteristics of messenger RNA. (5) Experiments carried out as described by Pigott & Midgley (1968) indicated that hybridization at low DNA/RNA ratios (5:1) effectively accounted for all the messenger RNA in a given specimen. The efficiency coefficient of RNA hybridization lay within the range of 90-95% input, if an excess of DNA sites was offered for RNA binding. (6) These measurements are compared with other results obtained by different methods, and reasons for any major disagreement are suggested.     

Isolation of messenger ribonucleoproteins from mammalian cells

EDTA-dissociated polysomes from normally growing and adenovirus-infected KB cells were fractionated by affinity chromatography on oligo(dT)-cellulose into an unadsorbed fraction containing the ribosomal subunits and a second fraction which bound to the adsorbent. The latter fraction, recovered from the oligo(dT)-cellulose by elution with formamide in a buffer containing 0.2 m-NaCl, was shown to contain messenger RNA together with protein. Evidence was obtained that a substantial part (50%) of ³⁵S-labelled protein was associated with the mRNA when eluted from the oligo(dT)-cellulose, and that up to 75% of the labelled protein was mRNA-associated after dilution of the eluted material in buffer not containing formamide. As judged from the characteristic gel electrophoresis pattern of adenovirus-specific mRNA derived from such messenger ribonucleoprotein complexes, the mRNA remained intact during the isolation procedure. Analysis of the fraction containing the majority of the messenger ribonucleoprotein complexes on sodium dodecyl sulfate-polyacrylamide gels showed a specific pattern of labelled polypeptides, which in the case of material from uninfected cells consisted of four major labelled polypeptides; approximate molecular weights 56,000, 68,000, 78,000 and 130,000. When the material from the adenovirus infected cells was analysed a set of four polypeptides, which migrated identically to those of the uninfected cells, was found. However, in addition to this apparently common set of polypeptides, the material from the infected cells, harvested late in the infectious cycle, contained one extra polypeptide with an approximate molecular weight of 110,000.     

Nascent ribosomal and messenger RNA in DNA-membrane-complexes of Escherichia coli

Summary From Escherichia coli, DNA-membrane-complexes have been isolated which contain about 40% of the ribosomes, about 95% of the DNA and nearly all of the nascent RNA. The kinetic data on pulse labeled RNA show an average time of turnover of about 60 sec both for nascent messenger- and nascent ribosomal RNA. A proportion of the polysomes with nascent messenger RNA as well as most of the nascent ribosomal RNA is found in association with membranes, as has been shown by subfractionations of the DNA-membrane-complex involving treatment with DNase and desoxycholate. In this early transient stage, ribosomal precursor RNA already acquires some ribosomal proteins, as has been shown by arginine pulse label. Data on partial release of DNA from the DNA-membrane-complex by treatment with extremely low doses of DNase indicate that messenger RNA synthesis occurs in clusters on the DNA.The results support models in which, at any given time, RNA synthesis proceeds mainly in sections of the DNA close to the membrane. Thereby the DNA is linked to the membrane via nascent RNA contained in ribosomal precursors as well as via nascent messenger RNA on membrane-bound polysomes.     

RNA as an enzyme

The catalytic activity of ribonucleic acid is reviewed, with the intervening sequence (IVS) of the ribosomal RNA precursor of Tetrahymena serving as a major example. The IVS catalyzes its own excision from the precursor RNA and at the same time ligation of the flanking sequences, a reaction termed self-splicing. The excised IVS RNA can act as an enzyme to catalyze sequence-specific cleavage and ligation reactions on substrate RNA molecules. The RNA polymerization activity of the IVS supports the possibility that RNA catalysis could have been important in establishing a prebiotic self-replicating system. Other systems in which RNA catalysis has been found include related group I IVSs, group II IVSs, ribonuclease P, and certain plant infectious RNAs.     

Species of RNA synthesized during early germination in the radicle of the lentil embryo

RNA synthesis is activated in cells of the plant embryo very soon after the start of imbibition by the seed. This study was undertaken to determine whether synthesis of one particular RNA or all the major RNA species was initially activated in the radicle of lentil embryos ( Vicia lens ). Two different methods were used after incorporation of radioactive precursor to identify the newly synthesized RNA species. First, RNA was extracted and analyzed using gel electrophoresis, gel filtration, affinity chromatography and base composition analysis. The second method was to localize the labelled RNA molecules within cells using autoradiography of sections of embryonic radicles. The data indicate that newly synthesized heterogeneous nuclear RNA and possibly messenger RNA, transfer RNA, 5S ribosomal RNA and precursor of ribosomal RNA are detectable 3 h after the start of imbibition of the decoated embryo and before completion of initial water uptake. It is concluded that synthesis of all major species of RNA is simultaneously initiated in the radicle of the germinating embryo.     

Developmental changes in ribosomal ribonucleic Acid and fraction I protein in wheat leaves

In light-grown wheat (Triticum aestivum L.) seedlings, the amount of chloroplast and cytoplasmic ribosomal RNA increased to a maximum in the first leaf near the end of its growth and declined by about 60% in the following 3 days. While total ribosomal RNA was declining, labeled uracil was still incorporated into cytoplasmic ribosomal RNA, but the rate of incorporation into chloroplast ribosomal RNA fell by more than 80%, as did the incorporation of labeled leucine into fraction I protein. Either there is greater replacement of cytoplasmic ribosomal RNA than chloroplast ribosomal RNA in mature leaves, or chloroplasts are able to repress the incorporation of exogenous precursor when there is no net synthesis of RNA.     

Ribonucleic acid synthesis in the renal cortex at the initiation of compensatory growth.

The mechanisms responsible for the increase in RNA per cell during the first 48h of renal compensatory growth were studied in the renal cortex. Unilaterally nephrectomized, sham-operated or non-operated rats were used. Incorporation into RNA of labelled precursors was studied in vivo and in vitro. Sham-operation produced significant changes in precursor incorporation, absolute amounts of UTP and RNA, and the rate of RNA synthesis. At 6h after surgery, the amount of RNA decreased in sham-operated controls, whereas that in growing cortex remained unchanged. Incorporation into RNA in vivo was greater in the growing cortex, although the rate of RNA synthesis was not increased. At 24h, precursor incorporation into RNA and UTP and RNA synthesis were all increased in the growing cortex. In contrast with results obtained in vivo, slices of growing cortex incorporated less labelled precursor into RNA than did cortex slices from sham-operated controls, from 3 to 48h. Maximal differences were found from 6 to 24h. An attempt was made to equalize endogenous precursor pool sizes by increasing the concentration of unlabelled uridine in the media; incorporation differences were narrowed significantly. Serum from nephrectomized animals did not increase precursor incorporation into RNA in vitro. An increase in RNA synthesis is an important factor in RNA accretion in the renal cortex beyond 12h of compensatory growth. This is accompanied by increased UTP content and preceded by expansion of other pools. The amount of labelled precursor incorporated into RNA is greatly influenced by its delivery rate to the growing kidney in vivo and by intracellular dilution of expanded precursor pools in vitro.     

RIBOSOME SYNTHESIS IN TETRAHYMENA PYRIFORMIS

The cellular site of synthesis of ribosomal RNA in Tetrahymena pyriformis was studied by analyzing the purified nuclear and cytoplasmic RNA from cells pulse labeled with uridine-³H. The results of studies using zonal centrifugation in sucrose density gradients show that the ribosomal RNA is synthesized in the nucleus as a large precursor molecule sedimenting at 35S. The 35S molecule undergoes rapid transformation through two main nuclear intermediates, sedimenting at about 30S and 26S. The smaller ribosomal RNA (17S) appears first in the cytoplasm and it seems to be absent from the nucleus. The apparent delay in the appearance of the larger ribosomal RNA (26S) in the cytoplasm is due to the presence of a larger pool of its precursors in the nucleus as indicated by pulse-chase experiments. The newly synthesized ribosomal RNA's appear in the cytoplasm as discrete 60S and 45S ribonucleoprotein particles, before their incorporation into the polysomes.     

Ribosomal ribonucleic acid and ribosomal precursor ribonucleic acid in Anacystis nidulans

The RNA of the blue-green alga Anacystis nidulans contains three ribosomal RNA species with molecular weights of 0.56x10(6), 0.9x10(6), and 1.1x10(6) if the RNA is extracted in the absence of Mg(2+). The 0.9x10(6)mol.wt. rRNA is extremely slowly labelled in (32)P-incorporation experiments. This rRNA may be a cleavage product of the 1.1x10(6)mol.wt. rRNA from the ribosomes of cells in certain physiological states (e.g. light-deficiency during growth). The cleavage of the 1.1x10(6)mol.wt. rRNA during the extraction procedure can be prevented by the addition of 10mm-MgCl(2). (32)P-pulse-labelling studies demonstrate the rapid synthesis of two ribosomal precursor RNA species. One precursor RNA migrating slightly slower than the 1.1x10(6)mol.wt. rRNA appears much less stable than the other precursor RNA, which shows the electrophoretic behaviour of the 0.7x10(6)mol.wt. rRNA. Our observations support the close relationship between bacteria and blue-green algae also with respect to rRNA maturation. The conversion of the ribosomal precursor RNA species into 0.56x10(6)- and 1.1x10(6)-mol.wt. rRNA species requires Mg(2+) in the incubation medium.     

The nature of the changes in the pattern of RNA synthesis by the juvenile hormone analogue altosid

The effect of various concentrations of Altosid and actinomycin D under defined conditions on housefly metamorphosis was investigated with three strains of houseflies. The morphogenetic response varied with the strains and the length of time which the larvae were exposed to the juvenile hormone analogue. De novo RNA synthesis was studied with (2-¹⁴C)-glycine. Methods were developed for the isolation of nuclear, soluble, and ribosomal RNA. The procedure presented provides a DEAE-cellulose chromatographic method for the removal of high molecular weight RNA from DEAE at a neutral pH. Labelling of the RNAs was increased in the presence of the juvenile hormone analogue indicating an increase in the rate of RNA synthesis. The higher incorporation of the labelled precursor into nuclear RNA demonstrates that cytoplasmic RNA is derived from the nuclei.     

The pattern of methylation of RNA in peripheral nerve of the chick during development

The pattern of the methylation of RNA was investigated in organ cultures of the sciatic nerve of the chicken. Nerve tissue from 14-day embryos, 17-day embryos and 3-day- old chicks was incubated with [methyl-³H]methionine or with [2-¹⁴C]uridine and [methyl-³H]methionine simultaneously for various periods of time. Subsequently, RNA was extracted from the tissues and the purified preparations were fractionated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the rapidly labelled RNA changed during the three developmental stages. The incorporation of both uridine and the methyl groups from methionine was highest in the‘heavy’RNA species of the 14-day embryonic nerve during the 0.5 and 1.0 h incubation periods. In contrast, in the nerves of 3-day-old chicks during a 0.5 h pulse with both precursors, methylation was almost entirely limited to the transfer RNA species. Furthermore, the incorporation of uridine in the nerves from 3-day-old animals revealed the presence of a heterogeneous population of rapidlylabelled, unmethylated species of RNA, most of which migrated between the smaller ribosomal RNA and transfer RNA components of the bulk RNA. The pattern of uridine incorporation and the methylation of the rapidly-labelled RNA of the 17-day embryonic nerve represented a transitional state between that of the 14-day embryos and that of the 3-day-old chicks. The 17-day embryonic stage of development corresponded to the phase of the onset of rapid deposition of myelin lipids in the sciatic nerve. Pulse-chase experiments on the embryonic nerves indicated that a number of methylated precursors of ribosomal RNA and labile, heterogeneous, probably DNA-like RNA were synthesized.