The atypical RNA components of cytoplasmic ribosomes from Crithidia fasciculata

RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit of the trypanosomatid flagellate Crithidia fasciculata and analyzed by polyacrylamide gel electrophoresis at 4 °C consisted of one species with a molecular weight of 1.3 × 10⁶ (relative to ribosomal RNA from E. coli MRE 600). When extracted with hot phenol (65 °C), the large ribosomal subunit gave rise to two components with molecular weights of 0.72 and 0.56 × 10⁶. On heating for 60 s, followed by rapid cooling, the single cold-phenol-extracted 1.30 × 10⁶-dalton species completely dissociated into two components of molecular weights 0.72 and 0.56 × 10⁶, present in equimolar amounts. When analyzed by polyacrylamide-agarose gel electrophoresis in the presence of SDS, RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit consisted of three components of molecular weights 1.3, 0.72, and 0.56 × 10⁶, present in apparently equimolar amounts. RNA from the small cytoplasmic ribosomal subunit consisted of one species with a molecular weight of 0.84 × 10⁶, independent of extraction or analytical conditions. It is proposed that under high salt and low temperature conditions, the large ribosomal RNA molecule is held together by its secondary structure, and that denaturing extraction or analytical conditions reveal an otherwise “hidden” lesion present in the molecule in vivo.     

Ribosomal RNA synthesis in mitochondria of Neurospora crassa

Ribosomal RNA synthesis in Neurospora crassa mitochondria has been investigated by continuous labeling with [5-³H]uracil and pulse-chase experiments. A short-lived 32 S mitochondrial RNA was detected, along with two other short-lived components; one slightly larger than large subunit ribosomal RNA, and the other slightly larger than small subunit ribosomal RNA. The experiments give support to the possibility that 32 S RNA is the precursor of large and small subunit ribosomal RNA's. Both mature ribosomal RNA's compete with 32 S RNA in hybridization to mitochondrial DNA. Quantitative results from such hybridization-competition experiments along with measurements of electrophoretic mobility have been used to construct a molecular size model for synthesis of mitochondrial ribosomal RNA's. The large molecular weight precursor (32 S) of both ribosomal RNA's appears to be 2.4 × 10⁶ daltons in size. Maturation to large subunit RNA (1.28 × 10⁶ daltons) is assumed to involve an intermediate ~1.6 × 10⁶ daltons in size, while cleavage to form small subunit RNA (0.72 × 10⁶ daltons) presumably involves a 0.9 × 10⁶ dalton intermediate. In the maturation process ~22% of the precursor molecule is lost. As is the case for ribosomal RNA's, the mitochondrial precursor RNA has a strikingly low G + C content.     

Characterization of the ribosomal RNA precursor in Ilyanassa

Ilyanassa embryos synthesized a high molecular weight, rapidly-labeled RNA species that, as time progressed, diminished in proportion to increasing amounts of nascent ribosomal RNA. We tentatively identify this rapidly-labeled RNA species as the ribosomal RNA precursor. Its molecular weight, determined by acrylamide gel electrophoresis, was 2.4–2.5 × 10⁶; a molecule of intermediate size (1.8 × 10⁶ Daltons) was also detected.     

Minor Components of Rat Liver Ribosomal RNA as Possible Intermediates in the Degradation of 28S RNA in Vivo

The minor RNA components in large ribosomal subunits of rat liver were analyzed by gel electrophoresis. Quantitative analysis showed that these minor components, whose apparent molecular weights ranged from 8.48 × 10⁵ to 11.4 × 10⁵, represented 10 to 13% of the total high-molecular-weight ribosomal RNA.

It was elucidated that they were not artifacts arose during the cell homogenization, sub-cellular fractionation, RNA preparation and gel electrophoresis.

Labeling experiment in vivo showed that they were preferentially present in “old” ribosomes. It was predicted that these minor components were the intermediates in the degradation of 28S ribosomal RNA in vivo.

In the regenerating liver, where the degradation of proteins was reported to be blocked, the relative amount of the minor RNA components was decreased to about a half of that of normal liver. There was, however, no increase in their relative amount in the long-term starved rat liver, where RNA degradation should be going very rapidly.     

Maize mitochondria: purification and characterization of ribosomes and ribosomal ribonucleic Acid

Mitochondria were prepared from etiolated maize shoots (Zea mays L. var. McNair 508) by homogenization followed by differential centrifugation and equilibrium banding in discontinuous sucrose or Renografin-sucrose gradients. Mitochondria prepared by sucrose banding showed better physiological integrity than those prepared by renografin-sucrose banding, although both procedures yielded mitochondria that showed respiratory control and coupling of oxidation to phosphorylation of ADP. Mitochondria prepared by Renografin-sucrose banding were free of dectectable cytoplasmic ribosomal RNA, while sucrose banding resulted in a low level of contamination. Ribosomes isolated from mitochondria sedimented at about 78S, with subunits sedimenting at 60 and 44S. Using Escherichia coli ribosomal RNA as internal standards, the molecular weights of mitochondrial ribosomal RNAs were found to be 0.74 to 0.75 and 1.26 × 10⁶ daltons by polyacrylamide gel electrophoresis, before or after denaturation in formaldehyde. Cytoplasmic ribosomal RNA molecular weights were 0.70 and 1.26 × 16⁶ before denaturation, and 0.68 and 1.5 × 10⁶ after denaturation, suggesting an unusual reaction of the heavy ribosomal RNA to formaldehyde.     

Incorporation of tritiated adenosine into mouse ovum RNA

The total RNA of ovulated mouse ova has been examined by polyacrylamide gel electrophoresis. The amount of RNA present in the two main peaks observed, 28 S and 18 S ribosomal RNA, has been estimated as 0.20 ng.The RNA of ovulated mouse ova was labeled by exposure of growing mouse oocytes to adenosine-8-³H in vivo. For this purpose a small volume of a concentrated solution of the precursor was injected into the ovarian bursa, and ova were collected by superovulation at various subsequent times. The major growth phase of the oocyte is known to lie between 20 and 6 days before ovulation. Significant incorporation into egg RNA was observed when bursal injection was performed between 19 and 7 days, but not between 5 days and 1 day before ovulation.The types of labeled RNA in ova ovulated at five intervals between 19 and 7 days after bursal injection of adenosine-8-³H or uridine-5,6-³H were analyzed by polyacrylamide gel electrophoresis. The distribution of label on the gels demonstrated that the bulk of the label appeared in ribosomal RNA and transfer RNA. In addition labeled heterogeneous RNA was estimated to represent 10–15% of the total incorporation.     

Methylated nucleotide content of mitochondrial ribosomal RNA from hamster cells

We have examined the state of methylation of mitochondrial ribosomal RNA from cultured hamster (BHK-21) cells. Ethidium-sensitive, and hence mitochondrion-specific, methylation levels were determined using multiple isotope techniques and improved purification procedures. The larger mitochondrial rRNA species, 17 S RNA, was found to contain 0.13 methyl group per 100 nucleotides and the smaller, 13 S RNA, 0.37. Methylated nucleotide and base analysis indicated that 17 S RNA contained one ribose-methylated residue (UmUp) per molecule and one unidentified residue; and 13 S RNA contained one methylated cytosine residue, one N⁶-dimethyladenine residue and one thymine residue per molecule. Possible evolutionary implications of these findings have been discussed.     

An unmethylated “3 SE” RNA in hamster mitochondria: A 5 S RNA-equivalent?

A novel low molecular weight (“3 S_E”) RNA associated with hamster cell mitochondria has been partially characterized. It was present at approx. 1:1 molar ratio with structural mitochondrial ribosomal RNA; it was unmethylated; and it resembled other mitochondrial RNA fractions in having a low content of G + C. These findings support the idea that 3 S_E RNA is a mitochondrial equivalent of 5 S ribosomal RNA.     

Identification and partial characterization of multiple discrete polyadenylic acid containing RNA components coded for by HeLa cell mitochondrial DNA

Poly(A)-containing RNA isolated from the components of a HeLa cell mitochondrial lysate which sediment in the polysome region of a sucrose gradient have been analyzed for the presence of discrete species. Eight distinct components have been identified by polyacrylamide gel electrophoresis after formaldehyde treatment. These components, which are highly reproducible in their occurrence and relative amounts under widely varying conditions of isolation, have been characterized as to their sedimentation behavior under denaturing conditions, poly(A) content and homology to separated strands of mitochondrial DNA.One of the discrete components was previously shown to have a sedimentation coefficient of about 7 S in the native state and a molecular weight of about 9.0 × 10⁴, as estimated from its sedimentation rate in formaldehyde. The molecular weights of the other seven components, as derived from sedimentation data, range between 2.6 and 5.3 × 10⁵.The 7 S RNA is complementary to the light mitochondrial DNA strand, while the other seven components are complementary to the heavy strand. Together with the two mitochondrial rRNA species and with mitochondrial 4 S RNA, the eight poly(A)-containing RNA components, if distinct in sequence, would account for about 70% of the single-strand informational content of HeLa mitochondrial DNA.     

THE RIBOSOMAL RNA OF HAMSTER-MOUSE HYBRID CELLS

The ribosomal RNA (rRNA) of a series of hamster-mouse somatic cell hybrids was studied. Mouse 28S rRNA was separated from its hamster counterpart by a two-step procedure involving sucrose gradient centrifugation of ribosomes and polyacrylamide gel electrophoresis of rRNA. Both hamster and mouse types of rRNA were synthesized in the 11 hybrids tested, including hybrids containing only about one-half the haploid number of either mouse or hamster chromosomes. It appears that, for both hamster and mouse rRNA, when the chromosomes of one species constituted the majority of the chromosomes of a hybrid, a disproportionately higher percentage of rRNA of that species was present in the hybrid. Some hybrid clones, having a majority of mouse chromosomes, had a mouse rRNA cell concentration approximately four to five times higher than the concentration expected from linear extrapolation of the value found for the mouse parental cell line.     

Isolation of ribosomal RNA precursors from Physarum polycephalum

Ribosomal RNA synthesis in Physarum polycephalum was studied by labeling intact microplasmodia with [³H]uridine. Labeled, high-molecular-weight RNA species were found in a 30,000 S structure released by phenol extraction at room temperature. RNA was released from the structure by further phenol extraction at 65–70 °C. If the labeling period was 15 min or longer, the labeled RNA was seen by polyacrylamide gel electrophoresis to be of two major types, a heterodisperse collection of 45-35 S molecules and a 26 S species. If the labeling was carried out for 30 min in the presence of cycloheximide, the major labeled species had an electrophoretic mobility corresponding to 40 S. Studies of the labeling kinetics, methylation, and base composition of these RNA molecules indicate that they are precursors to ribosomal RNA. The molecular weights of the homogeneous 40 and 26 S precursors are 3.0 × 10⁶ and 1.45 × 10⁶ daltons, respectively, in comparison with molecular weights of 1.29 × 10⁶ and 0.68 × 10⁶ daltons for the completed ribosomal RNA's.     

The properties and function of rapidly-labelled nuclear RNA

Summary Nuclei were isolated from cultured cells of Acer pseudoplatanus L. previously pulse-labelled with [5-³H]uridine or [³²P]phosphate and the properties of the rapidly-labelled RNA were studied. Polyacrylamide gel electrophoresis showed ribosomal RNA precursors and processing intermediates with molecular weights of 3.4, 2.5, 1.4 and 1×10⁶ daltons, together with polydisperse RNA. The relative proportions of ribosomal RNA precursors and polydisperse RNA varied according to the length of the labelling period, but after 30 min approximately 90% of the radioactive RNA was polydisperse. The relationship between this polydisperse RNA and messenger RNA was investigated. The percentage of total nuclear RNA retained by chromatography on oligodeoxythymidylic acid-cellulose columns varied from 6% to 16% depending on the length of the labelling period. This RNA fraction, which has an adenylic acid content of approximately 45%, is assumed to represent RNA with polyadenylic acid sequences attached. A larger proportion of the nuclear polydisperse RNA lacked polyadenylic acid. Both types of polydisperse RNA were similar in size and during polyacrylamide gel electrophoresis migrated as broad peaks with an average molecular weight of approximately 10⁶ daltons. The polydisperse nuclear RNA that lacks polyadenylic acid was found to be similar in nucleotide composition to ribosomal RNA and is assumed to represent growing chains of ribosomal precursor RNA. After short labelling times the majority of the radioactivity incorporated into nuclear RNA is present in molecules of this type. This suggests that the designation of pulse-labelled polydisperse RNA as messenger RNA or precursor to messenger RNA solely on the basis of rapid labelling and size heterogeneity is unsound. The average molecular weight of the polyadenylic acid-containing messenger RNA from the cytoplasm was less than that of the corresponding nuclear RNA (6 and 9×10⁵ daltons respectively). This suggest either that the majority of the nuclear polyadenylic acid-containing RNA does not enter the cytoplasm, or if it does, that it first undergoes a reduction in size.Abbreviations rRNA ribosomal RNA - mRNA messenger - RNA poly(A), polyadenylic acid, poly(A) and poly(A) - RNA RNA with and without poly(A) sequences attached - poly(U) polyuridylic acid - oligo (dT)-cellulose cellulose with oligo deoxythymidylic acid covalently attached - C cytidylic acid - A adenylic acid - G guanylic acid - U uridylic acid     

Translation of mitochondrial RNA from yeast cytoplasmic petite mutants in anE. coli cell-free system

Mitochondrial RNA from two cytoplasmic ?^? mutants ofS. cerevisiae, which have kept the mitochondrial DNA segment including the ATPase-oligomycin resistance-conferring gene, stimulates protein synthesis in anE. coli cell-free system. SDS-acrylamide gel electrophoresis of the protein product revealed one major peak and two minor ones with apparent molecular weights of around 11,000, 13,500 and 17,000 respectively. The effect is specific since no stimulation is observed with RNA from a ?^? mutant devoid of detectable mitochondrial DNA. These results are interpreted to mean that the mitochondrial DNA of these mutants codes for anin vitro translatable mRNA.     

Occurrence of heat-dissociable ribosomal RNA in insects: the presence of three polynucleotide chains in 26 S RNA from cultured Aedes aegypti cells

When RNA extracted from a mixture of cultured mosquito (Aedes aegypti) and hamster (BHK) cells is heated at 60 °C for five minutes the 26 S mosquito RNA but not the 28 S BHK RNA is converted to 18 S products. These products are not separable from each other or from pre-existent 18 S RNA on 2.4% acrylamide gels and have molecular weights near 0.7 × 10⁶. The large ribosomal RNA from insects belonging to ten different orders shows a similar conversion, although this property is absent in two species of aphid.A. aegypti 26 S RNA dissociates over a narrow temperature range. The reaction equilibrium favours dissociation and is dependent on ionic strength, showing a 6 deg. C change in T_m′ (the temperature of 50% dissociation) with tenfold change in salt concentration. Although the T_m of 26 S RNA from Drosophila melanogaster and A. aegypti is markedly different, reflecting the difference in base composition, the T_m′ of the two RNA species was virtually the same.High molecular weight ribosomal RNA from Escherichia coli, BHK cells and A. aegypti cells was terminally labelled with [³H]isonicotinic acid hydrazide. The specific activities of the large RNA species show the presence of one, two and three polynucleotide chains in 23 S, 28 S and 26 S RNA, respectively. A. aegypti 26 S RNA contains a small, heat-dissociable “IRNA” similar in relative amount and mobility to that found in BHK cells.     

Small molecular weight RNA components in sea urchin embryos

Polyacrylamide gel electrophoresis of RNA from Paracentrotus lividus embryos has shown this material to contain five RNA components of small molecular weight. The components are synthesized early in sea urchin development, simultaneously with tRNA and heterodisperse RNA. After the blastula stage, when synthesis of ribosomal RNA is activated, the labeling incorporated into small molecular weight RNA components constitutes a relatively decreasing proportion of the total labeling in RNA. When labeling is performed prior to the blastula stage, three of the small molecular weight RNA components are labeled to a similar or greater extent than “5” S RNA and the 26-ass RNA. The gel electrophoretic mobilities of the small molecular weight RNA components have been compared with those in Ehrlich ascites cells and found to be different.     

THE EFFECT OF INTRAOCULAR INJECTION OF CORDYCEPIN ON RETINAL RNA SYNTHESIS AND ON RNA AXONALLY TRANSPORTED DURING REGENERATION OF THE OPTIC NERVES OF GOLDFISH

Abstract— The presence of relatively large amounts of RNA has been demonstrated in regenerating axons of the goldfish optic nerve. Previous experiments have suggested that this R NA may be composed of only small molecular weight 4S RNA. The present experiments were performed in order to see if inhibiting RNA transport by intraocular injections of cordycepin causes a selective depletion of 4S RNA arriving in the contralateral optic tectum, and thus add further evidence that 4S RNA is axonally transported. Optic nerves were crushed in a group of goldfish and 18 days later 10.0 /tg of cordycepin was injected into the right eye followed 3 h later by injections of [³H]uridine into the same eye. Six days later the amount of axonally transported [³H]RNA was decreased by 89% compared with non-cordycepin treated controls. The effect of cordycepin on retinal RNA synthesis was shown by autoradiography to be primarily on retinal ganglion cell RNA synthesis with lesser effects on other cellular elements of the retina. SDS polyacrylamide gel electrophoresis at both 1 and 6 days after intraocular injections of cordycepin and [³H]uridine, showed that cordycepin blocks the retinal synthesis of ribosomal RNAs but appeared to have little effect on the synthesis of 4S RNA. When transported RNA in the tectum was fractionated by gel electrophoresis 6 days after injection, it was found that the amount of ribosomal RNA was decreased by approx 70% as a result of cordycepin pretreatment. This correlated well with the effect of cordycepin on the transport of available RNA precursors (also decreased by approx 70%) and is consistent with the contention that in these experiments ribosomal RNA is synthesized in the tectum itself and is not axonal. The amount of [³H] 4S RNA arriving in the tectum, however, was decreased by greater than 90% suggesting that its presence in the tectum was not entirely dependent on the availability of ³H precursors for local synthesis in the tectum. These results are consistent with data suggesting that 4S RNA is the predominant, if not the only, RNA species axonally transported during regeneration of goldfish optic nerves.     

Characteristics and intracellular distribution of messengerlike RNA in encysted embryos of Artemia salina

Homogenates of dormant cysts of Artemia salina were fractionated by differential centrifugation. RNA was prepared from the various fractions and tested for stimulatory activity in a [¹⁴C]leucine incorporating Escherichia coli system. The highest specific activity was found in the RNA extracted from a cytoplasmic fraction sedimenting at 15,000 g. Some activity was associated with the soluble and crude ribosomal fractions, while the RNA extracted from the crude nuclear fraction was less active.The 15,000 g sediment was purified by centrifugation in a sucrose density gradient. The active material formed a characteristic, colored band at a buoyant density of about 1.17 g/ml. The banding fraction was mainly composed of endoplasmic vesicles and mitochondria. The specific activity of the extracted RNA was further increased when the 15,000 g sediment was treated with buffered 20–100 mM EDTA (with or without 0.1% Triton X-100) before banding.Sedimentation analysis of the active RNA from the purified 15,000 g fractions revealed three distinct absorption peaks at 28 S, 18 S, and 16 S, apparently representing cytoplasmic and mitochondrial rRNA. The 28 S and 18 S peaks were reduced by EDTA treatment, but only to a certain limit. By gel electrophoresis a number of additional components were resolved, including 4 S and 5 S RNA. The template activity showed a heterodisperse distribution with a maximum at 17–20 S, not correlated with the 16 S peak. Isolated 18 S and 28 S rRNA had very low activity.The experiments suggest that in Artemia cysts an appreciable amount of messengerlike RNA is associated with mitochondria and/or endoplasmic vesicles carrying ribosomal monomers.     

Ribonucleoprotein organization of polyadenylate sequences in HeLa cell heterogeneous nuclear RNA.

Ribonucleoprotein particles containing heterogeneous nuclear RNA (Pederson, 1974) were isolated from HeLa cells and digested with ribonucleases A and T₁ at high ionic strength. The nuclease-resistant material, comprising 9.4% of the initial acid-insoluble [³H]adenosine radioactivity, was further fractionated by poly(U)-Sepharose chromatography. The bound fraction eluted from the column with 50% formamide and banded in cesium sulfate gradients (without aldehyde fixation) at a buoyant density characteristic of ribonucleoprotein (1.45 g/cm³). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this material revealed two Coomassie blue-stained bands. The major polypeptide had a molecular weight of 74,000 a less prominent band had a molecular weight of 86,000. The RNA components contained 74.4 mol % AMP and 17.7 mol % UMP. Polyacrylamide gel electrophoresis of the RNA, labeled with [³H]adenosine, demonstrated the presence of molecules 150 to 200 nucleotides in length (poly(A)), as well as molecules 20 to 30 nucleotides long (oligo(A)). Both poly(A) and oligo(A) sequences have previously been identified in HeLa heterogeneous nuclear RNA. These data demonstrate that both the poly (A) and oligo(A) sequences in HeLa heterogeneous nuclear RNA exist in vivo tightly complexed with specific proteins.