Kern-Plasma-Transport neusynthetisierter rRNS in Zellen einer Suspensionskultur von Petroselinum sativum

Summary A rapidly labelled rRNA precursor can be detected in callus cells of Petroselinum sativum grown on a liquid synthetic medium. Its molecular weight has been calculated to be 2.3×10⁶. This value agrees with that of the rRNA precursor from other plant material. In order to follow the synthesis and processing of rRNA in time and to correlate single steps in this process with cell organelles it was necessary to obtain pure fractions of nuclei and ribosomes. The isolation method for nuclei is given in detail. The nucleic acids are separated on polyacrylamide gels of low acrylamide concentration. Pulse-chase experiments show that the rRNA precursor is split into two fragments within the nucleus: an 18S and a 25S component. The 18S RNA leaves the nucleus rapidly. It is already found quantitatively in the ribosomal fraction after 30–60 min chase. At that time the 25S RNA is still within the nucleus; it appears much later in the ribosomes. Since the increase in ribosomal label occurs simultaneously with the decrease in nuclear label, it is concluded that there is no degradation of 18S RNA within the nucleus. Apparently there are two distinct transport mechanisms with different kinetics for the two RNA components.     

In vitro cleavage of 45S pre-ribosomal RNA and of giant heterogeneous RNA extracted from human leukemic cells

45S ribosomal precursor RNA and large heterogeneous RNA molecules (>45S) extracted from human leukemic cells were incubated in vitro with purified RNase III, which specifically attacks double-helical RNA regions.About 50% of the ribosomal precursor was cleaved into two major fragments sedimenting at 28S and 32S respectively. A limited number of cleavages was also introduced in about 40% of heterogeneous RNA molecules sedimenting faster than 45S, causing a partial shift to a polydisperse distribution in the 10S–45S range.     

RNA METABOLISM IN HELA CELLS AT REDUCED TEMPERATURE : I. Modified Processing of 45S RNA

Incubation of HeLa cells at suboptimal temperature has been used to study the synthesis of 45S ribosomal RNA precursor and the individual steps of the subsequent processing to 28S RNA. Below 20°C no detectable 45S RNA is formed. The processing of 45S RNA to 32S RNA ceases around 15°C, and the processing of 32S RNA to 28S RNA is inhibited near 25°C. Prolonged incubation at reduced temperature results in further modification of the processing, resulting in the apparent accumulation of 41S RNA. The products of these reactions at reduced temperature appear normal in that the ribosomal RNA made at 27°C can be isolated from functional polyribosomes in the cytoplasm after a short incubation at 37°C.     

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 EFFECT OF PUROMYCIN ON INTRANUCLEAR STEPS IN RIBOSOME BIOSYNTHESIS

Inhibition of protein synthesis by puromycin (100 γ/ml) is known to inhibit the synthesis of ribosomes. However, ribosomal precursor RNA (45S) continues to be synthesized, methylated, and processed. Cell fractionation studies revealed that, although the initial processing (45S → 32S + 16S) occurs in the presence of puromycin, the 16S moiety is immediately degraded. No species of ribosomal RNA can be found to have emerged from the nucleolus. The RNA formed in the presence of puromycin is normal as judged by its ability to enter new ribosomal particles after puromycin is removed. This sequence of events is not a result of inhibition of protein synthesis, for cycloheximide, another inhibitor of protein synthesis, either alone or in combination with puromycin allows the completion of new ribosomes.     

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.     

ULTRASTRUCTURAL AND BIOCHEMICAL STUDIES ON THE PRECURSOR RIBOSOMAL PARTICLES ISOLATED FROM RAT LIVER NUCLEOLI

Sucrose density gradient analyses of pH 5.5 and pH 7.4 extracts from rat liver nucleoli revealed the presence of two broad peaks of approximately 60S and 80S, and 60S and 80–100S, respectively. Ribonucleoprotein (RNP) particles containing precursor ribosomal RNA in these peaks have been characterized by electron microscopy and RNA analyses. Spherical particles only were found in the 60S peak of the pH 5.5 extract, from which 28S RNA and smaller RNA (23S and 18S RNA) exclusively were extracted. In the broad 80S peak of the pH 5.5 extract, about 60% of the particles were spherical while 30% were rodlike. In the RNA species present there were 28S plus smaller RNA (80%) and 35S RNA (20%). The 60Speak of the pH 7.4 extract contained mainly spherical particles (84%), and the RNA species present was mostly 28S plus smaller RNA (89%). In addition to spherical particles (43%), a number of rodlike (31%) and filamentous molecules (26%) were observed in the heavier side of the 80–100S peak of the pH 7.4 extract, from which 45S (14%), 35S (26%), and 28S and smaller RNA (60%) were extracted. Thus the precursor ribosomal particles containing 45S RNA and 35S RNA appear to be filamentous and rodlike molecules, respectively. Folding of loose ribonucleoprotein filaments into compact, spherical, large subparticles may be part of the maturation process of ribosomal large subparticles, in addition to the so-called sequential cleavage of RNA.     

Secondary structure maps of ribosomal RNA. II. Processing of mouse L-cell ribosomal RNA and variations in the processing pathway

Secondary structure mapping in the electron microscope was applied to ribosomal RNA and precusor ribosomal RNA molecules isolated from nucleoli and the cytoplasm of mouse L-cells. Highly reproducible loop patterns were observed in these molecules. The polarity of L-cell rRNA was determined by partial digestion with 3′-exonuclease. The 28 S region is located at the 5′-end of the 45 S rRNA precursor. Together with earlier experiments on labeling kinetics, these observations established a processing pathway for L-cell rRNA. The 45 S rRNA precursor is cleaved at the 3′-end of the 18 S RNA sequence to produce a 41 S molecule and a spacer-containing fragment (24 S RNA). The 41 S rRNA is cleaved forming mature 18 S rRNA and a 36 S molecule. The 36 S molecule is processed through a 32 S intermediate to the mature 28 S rRNA. This pathway is similar to that found in HeLa cells, except that in L-cells a 36 S molecule occurs in the major pathway and no 20 S precusor to 18 S RNA is found. The processing pathway and its intermediates in L-cells are analogous to those in Xenopus laevis, except for a considerable size difference in all rRNAs except 18 S rRNA.The arrangement of gene and transcribed spacer regions and of secondary structure loops, as well as the shape of the major loops were compared in L-cells, HeLa cell and Xenopus rRNA. The over-all arrangement of regions and loop patterns is very similar in the RNA from these three organisms. The shapes of loops in mature 28 S RNA are also highly conserved in evolution, but the shapes of loops in the transcribed spacer regions vary greatly. These observations suggest that the sequence complementarity that gives rise to this highly conserved secondary structure pattern may have some functional importance.     

On the interpretation of small-angle x-ray solution scattering from spherical viruses.

The intermediates in the ribosome assembly in exponentially growing Escherichia coli have been identified by centrifuging a crude lysate, pulse-labeled with a radioactive RNA base, through a sucrose gradient and analyzing for precursor rRNA in the gradient fractions by gel electrophoresis. The major intermediate in the assembly of the 50 S subunit cosediments with the mature subunit, whereas two minor precursor species sediment between the 30 S and 50 S peaks. The assembly of the 30 S subunit proceeds via a minor intermediate sedimenting slightly behind the mature subunit and a major precursor particle that cosediments with the mature 30 S subunit.The fraction of the rRNA contained in these precursor particles was determined by direct determination of the amount of rRNA in the precursor particles, and from the labeling kinetics of their rRNA. The direct estimation indicated that about 2% of the total 23 S type RNA, and 3 to 5% of the total 16 S type RNA is harboured in precursor particles. In the kinetic experiments the specific activity of the nucleoside triphosphates and of the different ribosomal particles was followed after addition of a radioactive RNA precursor to the growth medium. The results were compared with a digital simulation of the flow of isotopes through the assembly pathways. This method indicated that approximately 2% of the total 23 S type RNA, as well as 2% of the total 16 S type RNA, is contained in the precursor particles.     

Synthesis and maturation of ribosomal ribonucleic acids in isolated HeLa cell nuclei. A tracer study on the topology of the 45S precursor of ribosomal ribonucleic acids

The synthesis and processing of RNA by isolated HeLa cell nuclei was studied at low ionic strength in the presence of alpha-amanitin. The RNA polymerase reaction, with endogenous template and enzyme, rapidly reaches a plateau dependent on the amount of nuclei. Evidence is presented that incorporation of [(3)H]UMP proceeds only in growing RNA chains, whereas initiation of new RNA chains is arrested. The product formed contains all the main components of the 45S pre-rRNA (precursor of rRNA) maturation pathway (45S, 32S and 20S pre-rRNA; 28S and 18S rRNA). Most of the labelled material is in the mature rRNA components and their immediate precursors, even at very short times of incubation (2min). Small, but definite, 5S and 4S RNA peaks are also observed. At shorter incubation times a substantial amount of [(3)H]UMP is incorporated into RNA molecules in the 24S and 10-16S zones. This RNA material is considered to represent the non-conserved segments of 45S pre-rRNA in the process of nucleolytic degradation. A model for the tracer study of the topology of 45S pre-rRNA, on arrest of rRNA initiation, is discussed. The experimental evidence obtained supports the following structure of 45S pre-rRNA: 5'-end-28S rRNA unit-18S rRNA unit-nonconserved segment-3'-end.     

The relationship between ribosomal repeat length and genome size in Vicia

The organization of the ribosomal RNA genes was examined in several species of Vicia in an attempt to determine whether a relationship exists between genome size and ribosomal repeat length. Species within this genus exhibit a sevenfold variation in haploid DNA content. Our data suggest that species with an intermediate genome size maintain one predominant Eco RI class of ribosomal repeat of about 9 kilobases (kb). In contrast, the smallest and largest genomes of Vicia possess one major and several minor classes. The possible relationship between repeat classes among species is discussed. We examined the species with the smallest (V. villosa) and largest (V. faba) genomes in closer detail by R-loop analysis of a satellite DNA from Hoechst 33258 dye-CsCl gradients. Heterogeneity was found in the length of the ribosomal repeat for both species, but no appreciable difference was observed in the distribution of these lengths, which averaged 11–12 kb. This heterogeneity is associated with the nontranscribed spacer region. Intervening sequences were not found in either the 25S or 18S coding regions of the ribosomal repeat of either of these two plants. A putative ribosomal RNA precursor of 7 kb was identified for both species.     

Abnormal maturation of precursor 16S RNA in a ribosomal assembly defective mutant of E. coli

The precursor and mature 16S ribosomal RNAs from a novel thermosensitive ribosomal assembly defective mutant of E. coli, in which genetic evidence suggests that the ribosomal protein S4 is altered, have been isolated and characterised by finger-printing methods. The precursor 16S RNA, which is accumulated at 42°, appears to be identical with that present in wild-type strains, and with that previously described by other workers. However, the mature 16S RNA, which is contained in apparently normal functional 30S ribosomal particles synthesised at the growth-permissive temperature of 30°, is incompletely trimmed and has either one or two additional nucleotides at its 5′-terminus. This might be due either to an accumulation of two late intermediates in the maturation process, or to mis-trimming of the RNA. Both possibilities suggest that the change in the protein S4 is not only responsible for the thermosensitive character of ribosomal assembly in this mutant, but also causes an alteration in the trimming site, affecting its recognition by the enzyme involved in the maturation.     

The action of α-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids

α-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of α-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of α-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10–20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by α-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.     

RNA metabolsim during vegetative growth and morphogenesis of the cellular slime mold Dictyostelium discoideum

During vegetative growth of the cellular slime mold Dictyostelium discoideum, RNA is rapidly labeled by radioactive precursor and both the 25 S and the 17 S ribosomal RNA species appear in the cytoplasm 6–7 min after the onset of labeling. Thirty minutes after further incorporation of radioactive RNA precursors has been blocked, less than 10% of the label in RNA is associated with the nuclear fraction. After aggregation of the slime mold amoebae, RNA appears in the cytoplasm at a reduced rate, the small ribosomal subunit appearing in the cytoplasmic fraction more slowly than the larger ribosomal subunit. Some labeled RNA remains in the nuclei of developing cells long after the incorporation of ³H-uridine is blocked.     

THE SELECTIVE INTERRUPTION OF NUCLEOLAR RNA SYNTHESIS IN HELA CELLS BY CORDYCEPIN

Cordycepin is an analogue of adenosine lacking the 3'-OH. When incorporated into a growing RNA molecule, cordycepin prevents further elongation, thus producing a prematurely terminated RNA molecule. When HeLa cells are exposed to low concentrations of cordycepin, DNA and protein synthesis are unaffected during short exposure periods. The synthesis of completed ribosomal and ribosomal-precursor (45S) RNA is significantly depressed. Partially completed 45S ribosomal precursor molecules accumulate in the nucleolus. 18S ribosomal RNA can be cleaved from these incomplete precursors, while 32S ribosomal precursor cannot be produced from partially snythesized 45S molecules. The synthesis of transfer RNA is also reduced in the presence of cordycepin. The synthesis of the nuclear heterogeneous RNA species is unaffected by the drug while the cytoplasmic heterogeneous RNA is slightly reduced.