Intranuclear distribution of rat liver ribosomal RNA]

A method is described for the isolation of pure liver nuclei with minimal cytoplasmic contaminants, loss of nuclear RNA and degradation of nuclear RNA. The RNA components are extracted in three distinct fractions by subsequent treatment with phenol at 4 degrees, 50 degrees and 85 degrees C. The total and 14C-orotate labelled RNA components in the three nuclear RNA fractions are characterized by nucleotide composition, poly(A)-RNA content and agar-gel electrophoresis. The results show that the RNA in three fractions correspond to the nucleosol, nucleolus and chromatin compartments of the nucleus. The nuclear HnRNA components are exclusively in the 85 degrees C RNA. Nuclear ribosomal RNA is extracted in the 4 degrees C and 50 degrees C RNA fractions. These two nuclear RNA fractions are distinct in constituent pre-rRNA species and the rate of labelling of their rRNA components. The amount of the pre-rRNA and rRNA species is determined. The results show that the nucleolus-nucleosol and nucleosol-cytoplasm transitions of ribosomal subparticles are markedly slower processes than the preceeding steps of ribosome biogenesis.     

Cellular DNA replication is independent of the synthesis or accumulation of ribosomal RNA

We have used an antibody against RNA polymerase I to investigate the role of rRNA synthesis and/or accumulation in the control of cell proliferation. The antibody was microinjected directly into the nuclei of quiescent Swiss 3T3 cells that were subsequently stimulated with serum. Under the experimental conditions used, the microinjection of the antibody against RNA polymerase I (RNA pol I) caused a 50–70% decrease in nucleolar RNA synthesis that lasted for at least 17 h, a >90% inhibition in the accumulation of nucleolar RNA, and a 70% inhibition in the accumulation of total cellular RNA. A control IgG, similarly microinjected into Swiss 3T3 cells had no inhibitory effect on either the synthesis or accumulation of nucleolar and cellular RNA. Despite the dramatic effect on the synthesis and accumulation of ribosomal RNA (rRNA) the antibody against RNA pol I was totally ineffective in inhibiting the entry into S phase of serum-stimulated Swiss 3T3 cells. Cells depleted of cellular RNA by metaphase arrest also entered S phase with subnormal amounts of cellular RNA. The results of these experiments clearly indicate that a normal rate of nucleolar RNA synthesis, and a normal rate of accumulation of total cellular RNA are not a prerequisite for the entry of cells into S phase.     

Functional defects in transfer RNAs lead to the accumulation of ribosomal RNA precursors

Normal expression and function of transfer RNA (tRNA) are of paramount importance for translation. In this study, we show that tRNA defects are also associated with increased levels of immature ribosomal RNA (rRNA). This association was first shown in detail for a mutant strain that underproduces tRNA(Arg2) in which unprocessed 16S and 23S rRNA levels were increased several-fold. Ribosome profiles indicated that unprocessed 23S rRNA in the mutant strain accumulates in ribosomal fractions that sediment with altered mobility. Underproduction of tRNA(Arg2) also resulted in growth defects under standard laboratory growth conditions. Interestingly, the growth and rRNA processing defects were attenuated when cells were grown in minimal medium or at low temperatures, indicating that the requirement for tRNA(Arg2) may be reduced under conditions of slower growth. Other tRNA defects were also studied, including a defect in RNase P, an enzyme involved in tRNA processing; a mutation in tRNA(Trp) that results in its degradation at elevated temperatures; and the titration of the tRNA that recognizes rare AGA codons. In all cases, the levels of unprocessed 16S and 23S rRNA were enhanced. Thus, a range of tRNA defects can indirectly influence translation via effects on the biogenesis of the translation apparatus.     

Intranuclear maturation pathways of rat liver ribosomal ribonucleic acids.

The maturation of pre-rRNA (precursor to rRNA)in liver nuclei is studied by agar/ureagel electrophoresis, kinetics of labelling in vivo with [14C] orotate and electron-microscopic observation of secondary structure of RNA molecules. (1) Processing starts from primary pre-rRNA molecules with average mol. wt. 4.6X10(6)(45S) containing the segments of both 28S and 18S rRNA. These molecules form a heterogeneous peak on electrophoresis. The 28S rRNA segment is homogeneous in its secondary structure. However, the large transcribed spacer segment (presumably at the 5'-end) is heterogeneous in size and secondary structure. A minor early labelled RNA component with mol.wt. about 5.8X10(6) is reproducibly found, but its role as a pre-rRNA species remains to be determined. (2) The following intermediate pre-rRNA species are identified: 3.25X10(6) mol.wt.(41S), a precursor common to both mature rRNA species ; 2.60X10(6)(36S) and 2.15X10(6)(32S) precursors to 28S rRNA; 1.05X10(6) (21S) precursor to 18S rRNA. The pre-rRNA molecules in rat liver are identical in size and secondary structure with those observed in other mammalian cells. These results suggest that the endonuclease-cleavage sites along the pre-rRNA chain are identical in all mammalian cells. (3) Labelling kinetics and the simultaneous existence of both 36S and 21S pre-rRNA reveal that processing of primary pre-rRNA in adult rat liver occurs simultaneously by at least two major pathways: (i) 45S leads to 41S leads to 32S+21S leads to 28S+18S rRNA and (ii) 45S leads to 41S leads to 36S+18S leads to 32S leads to 28S rRNA. The two pathways differ by the temporal sequence of endonuclease attack along the 41 S pre-rRNA chain. A minor fraction (mol.wt.2.9X10(6), 39S) is identified as most likely originating by a direct split of 28S rRNA from 45S pre-rRNA. These results show that in liver considerable flexibility exists in the order of cleavage of pre-rRNA molecules during processing.     

Mitochondrial small ribosomal RNA is a component of germinal granules in Xenopus embryos

Mitochondrial large rRNA (mtlrRNA) and small rRNA (mtsrRNA) have been identified as components of germinal granules in Drosophila. We have previously reported that mtlrRNA is present on the germinal granules in Xenopus embryos. Here we report that mtsrRNA is also a common component of the germinal granules. Extra-mitochondrial mtsrRNA is localized on the surface of germinal granules in germ plasm from four-cell to blastula stage, then disappears until the completion of gastrulation. This temporal and spatial distribution pattern is identical to that of mtlrRNA. During the stages when both mitochondrial rRNAs are present around the germinal granules, mitochondrial-type ribosomes, typified by their smaller size, were also present on the surface of the germinal granules.     

Intranuclear accumulation of plant tubulin in response to low temperature

Summary. Concurrently with cold-induced disintegration of microtubular structures in the cytoplasm, gradual tubulin accumulation was observed in a progressively growing proportion of interphase nuclei in tobacco BY-2 cells. This intranuclear tubulin disappeared upon rewarming. Simultaneously, new microtubules rapidly emerged from the nuclear periphery and reconstituted new cortical arrays, as was shown by immunofluorescence. A rapid exclusion of tubulin from the nucleus during rewarming was also observed in vivo in cells expressing GFP-tubulin. Nuclei were purified from cells that expressed GFP fused to an endoplasmic-reticulum retention signal (BY-2-mGFP5-ER), and green-fluorescent protein was used as a diagnostic marker to confirm that the nuclear fraction was not contaminated by nuclear-envelope proteins. These purified, GFP-free nuclei contained tubulin when isolated from cold-treated cells, whereas control nuclei were void of tubulin. Furthermore, highly conserved putative nuclear-export sequences were identified in tubulin sequences. These results led us to interpret the accumulation of tubulin in interphasic nuclei, as well as its rapid nuclear export, in the context of ancient intranuclear tubulin function during the cell cycle progression. Correspondence and reprints: Department of Plant Physiology, Faculty of Science, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic.     

On the evolution of ribosomal RNA

Despite the availability of a rapidly growing ribosomal RNA database that now includes organisms in all three primary lines of descent (eubacteria, archaebacteria, and eukaryotes), theoretical treatment of the evolution of the ribosomal RNAs has lagged behind that of the protein genes. In this paper a theory is developed that applies current views of protein gene evolution to the ribosomal RNAs. The major topics addressed are the variability in size, gene arrangement, and processing of the rRNAs among the three primary lines of descent. Among the conclusions are that the rRNAs of eukaryotes retain some primitive features that were probably present in the rRNAs of the earliest cell (the progenote) and that the genes coding for the three major rRNA species were probably originally unlinked.     

Ribosome-dependent conversion of polyA-containing heterogenous nuclear RNA into smaller RNA molecules.

The polyA-containing heterogenous nuclear RNA fraction separated from total rat liver nRNA by gel filtration on Sepharose 4B followed by affinity chromatography on polyU-Sepharose and containing predominantly the 45S components becomes enzymatically bound to homologous 80S ribosomes and polyribosomes at 0 degree C. If 80S ribosomes or polyribosomes with bound poly-a-containing HnRNA are subjected to a further incubation at 37 degree C, the original 45S RNA is gradually converted into smaller RNA species of 10- 35S which remain bound to the particle. This ribosome-dependent cleavage of larger HnRNA species into smaller RNA molecules may represent the ultimate step of mRNA maturation.     

On the biosynthesis of bacterial ribosomal RNA

By comparison of the fingerprints of 5S and 23S ribosomal RNAs from Bacillus licheniformis with that of the precursor of 23S ribosomal RNA, it can be shown that 5S RNA is not a part of the precursor of 23S ribosomal RNA.     

Dynamics of ribosomal RNA structure

The structural dynamics of ribosomal 5S RNAs have been investigated by probing single strandedness through enzymatic cleavage and chemical modification. This comparative study includes 5S rRNAs from E. coli, B. stearothermophilus, T. thermophilus, H. cutirubrum, spinach chloroplast, spinach cytomplasm, and Artemia salina. The structural studies support a unique tertiary interaction in eubacterial 5S rRNAs, involving nucleotides around positions 43 and 75. In addition long range structural effects are demonstrated in E. coli 5S rRNA due to the conversion of C to U at position 92.     

Metastable secondary structures in ribosomal RNA molecular hysteresis in the acid-base titration of Escherichia coli ribosomal RNA

The metastable conformational states which underlie the hysteresis displayed by Escherichia coli ribosomal RNA in its pH titration in the acid range have been analyzed in terms of acid-stable RNA secondary structures. Sedimentation measurements show that the phenomenon is intramolecular, so that analysis of the hysteresis loops can, in principle, reveal details of molecular architecture. Hysteresis cycles obtained spectrophotometrically and potentiometrically were compared for RNA in solutions of different ionic strengths and ionic compositions. The effect is much smaller at lower ionic strength and disappears in the absence of magnesium ions. The curve followed upon addition of acid appears to reflect the equilibrium state of the system at each pH value. On the “base branch” of the loop, a slow absorbance change (complete in hours) was observed after the pH was raised by addition of a portion of base. This slow process is attributed to the annealing of “mismatched” multihelical regions of the ribosomal RNA. Certain regions, however, remain in metastable configurations for days and it is these long-lived non-equilibrium structures that underlie the hysteresis. Titration at 35 °C gave hysteresis loops of the same size and shape as at 20 °C; indeed, we found that the metastabilities are not removed even at 80 °C. Ultraviolet light absorbance difference spectra at 80 °C between solutions at the same pH, but on different branches of the cycle, give insight into the nature of the metastable conformation(s).Our experimental observations lead us to propose that the hysteresis is due to the formation at acidic pH of double-helical structures involving protonated guanine and adenine base pairs. The G.G pairs seem especially important to account for the very high thermal stability, as well as for the fact that the structures formed at a given pH value as acid is added dissociate only at higher pH values when the solution is titrated with base. Titrations of transfer RNA, along with literature data on 16 S rRNA primary structure, imply that the metastable regions in rRNA may consist of perhaps 10 to 15 base pairs.     

RNA:RNA interactions in the large subunit ribosomal RNA of Euglena gracilis

In Euglena gracilis, the cytoplasmic large subunit (LSU) rRNA is composed of 14 discrete small RNA species that must somehow interact in the functional ribosome. We have isolated native complexes of Euglena rRNA and show here that the largest of these complexes contains eight of the 14 LSU rRNA species. Several of these small rRNA species are able to associate in vitro to reform an isolated domain of LSU rRNA structure.     

A naked plant-specific RNA ten-fold smaller than the smallest known viral RNA: the viroid

Viroids are subviral plant pathogens at the frontier of life. They are solely composed by a single-stranded circular RNA of 246-401 nt with a compact secondary structure. Viroids replicate autonomously when inoculated into their host plants and incite, in most of them, economically important diseases. In contrast to viruses, viroids do not code for any protein and depend on host enzymes for their replication, which in some viroids occurs in the nucleus and in others in the chloroplast, through a rolling-circle mechanism with three catalytic steps. Quite remarkably, however, one of the steps, cleavage of the oligomeric head-to-tail replicative intermediates to unit-length strands, is mediated in certain viroids by hammerhead ribozymes that can be formed by their strands of both polarities. Viroids induce disease by direct interaction with host factors, the nature of which is presently unknown. Some properties of viroids, particularly the presence of ribozymes, suggest that they might have appeared very early in evolution and could represent 'living fossils' of the precellular RNA world that presumably preceded our current world based on DNA and proteins.