Intermolecular hybridization of 5S rRNA with 18S rRNA: identification of a 5'-terminally-located nucleotide sequence in mouse 5S rRNA which base-pairs with two specific complementary sequences in 18S rRNA.

Eukaryotic 5S rRNA hybridizes specifically with 18S rRNA in vitro to form a stable intermolecular RNA:RNA hybrid. We have used 5S rRNA/18S rRNA fragment hybridization studies coupled with ribonuclease digestion and primer extension/chain termination analysis of 5S rRNA:18S rRNA hybrids to more completely map those mouse 5S rRNA and 18S rRNA sequences responsible for duplex formation. Fragment hybridization analysis has defined a 5'-terminal region of 5S rRNA (nucleotides 6-27) which base-pairs with two independent sequences in 18S rRNA designated Regions 1 (nucleotides 1157-1180) and 2 (nucleotides 1324-1339). Ribonuclease digestion of isolated 5S rRNA:18S rRNA hybrids with both single-strand- and double-strand-specific nucleases supports the involvement of this 5'-terminal 5S rRNA sequence in 18S rRNA hybridization. Primer extension/chain termination analysis of isolated 5S rRNA:18S rRNA hybrids confirms the base-pairing of 5S rRNA to the designated Regions 1 and 2 of 18S rRNA. Using these results, 5S rRNA:18S rRNA intermolecular hybrid structures are proposed. Comparative sequence analysis revealed the conservation of these hybrid structures in higher eukaryotes and the same but smaller core hybrid structures in lower eukaryotes and prokaryotes. This suggests that the 5S rRNA:16S/18S rRNA hybrids have been conserved in evolution for ribosome function.     

RNase L-independent specific 28S rRNA cleavage in murine coronavirus-infected cells

We characterized a novel 28S rRNA cleavage in cells infected with the murine coronavirus mouse hepatitis virus (MHV). The 28S rRNA cleavage occurred as early as 4 h postinfection (p.i.) in MHV-infected DBT cells, with the appearance of subsequent cleavage products and a decrease in the amount of intact 28S rRNA with increasing times of infection; almost all of the intact 28S rRNA disappeared by 24 h p.i. In contrast, no specific 18S rRNA cleavage was detected in infected cells. MHV-induced 28S rRNA cleavage was detected in all MHV-susceptible cell lines and all MHV strains tested. MHV replication was required for the 28S rRNA cleavage, and mature cytoplasmic 28S rRNA underwent cleavage. In certain combination of cells and viruses, pretreatment of virus-infected cells with interferon activates a cellular endoribonuclease, RNase L, that causes rRNA degradation. No interferon was detected in the inoculum used for MHV infection. Addition of anti-interferon antibody to MHV-infected cells did not inhibit 28S rRNA cleavage. Furthermore, 28S rRNA cleavage occurred in an MHV-infected mouse embryonic fibroblast cell line derived from RNase L knockout mice. Thus, MHV-induced 28S rRNA cleavage was independent of the activation of RNase L. MHV-induced 28S rRNA cleavage was also different from apoptosis-related rRNA degradation, which usually occurs concomitantly with DNA fragmentation. In MHV-infected 17Cl-1 cells, 28S rRNA cleavage preceded DNA fragmentation by at least 18 h. Blockage of apoptosis in MHV-infected 17Cl-1 cells by treatment with a caspase inhibitor did not block 28S rRNA cleavage. Furthermore, MHV-induced 28S rRNA cleavage occurred in MHV-infected DBT cells that do not show apoptotic signs, including activation of caspase-3 and DNA fragmentation. Thus, MHV-induced 28S rRNA cleavage appeared to differ from any rRNA degradation mechanism described previously.     

Accelerated rates of ribosomal RNA synthesis during growth of contracting heart cells in culture

Contractile activity of neonatal cardiac myocytes stimulated hypertrophic growth as compared with nonbeating cells that were depolarized with 50 mM KCl. Growth of contracting myocytes was associated with an increased rRNA content as measured by the total RNA/DNA ratio. The fractional rates of rRNA synthesis (K8) and rRNA degradation were determined in contracting and nonbeating myocytes to assess their relative contributions in increasing rRNA content during growth. The values for K8 were calculated from the specific radioactivity of 3'-[3H]UMP in 18 and 28 S rRNA after purification by hybridization to cloned rDNA. The cellular [3H]UTP pool served as the precursor for rRNA synthesis in myocytes that were labeled with 50 microM [3H]uridine. K8 values for 18 and 28 S rRNA in contracting myocytes were accelerated by 59 and 53%, respectively, after 3 days as compared with nonbeating myocytes. Calculations of the rate of cellular rRNA synthesis, which took into account the increased content of myocyte rRNA, revealed that synthesis of both 18 and 28 S rRNA was accelerated 2-fold after 2 days of contraction. The derived values for degradation of 18 and 28 S rRNA were increased marginally in contracting myocytes, but cellular rRNA degradation rates averaged 57% higher. The difference between cellular rates of rRNA synthesis and degradation in contracting myocytes accounted for the 30% increase in rRNA content. These data demonstrated that increased rRNA content in contracting myocytes resulted from acceleration of the fractional rate of rRNA synthesis.     

Bacterial ribosomal RNA in pieces

The exact knowledge on the ribosomal RNA (rRNA) structure is an important prerequisite for work with rRNA sequences in bioinformatic analyses and in experimental research. Most available rRNA sequences of bacteria are based on gene sequences and on similarity analyses using Escherichia coli rRNA as a standard. Therefore, it is often overlooked that many bacteria harbour mature rRNA 'in pieces'. In some cases, the processing steps during the fragmentation lead to the removal of rRNA segments that are usually found in the ribosome. In this review, the current knowledge on the mechanisms of rRNA fragmentation and on the occurrence of fragmented rRNA in bacteria is summarized, and the physiological implications of this phenomenon are discussed.     

Starved Tetrahymena Synthesize and Maintain an Excess of 5s rRNA in Their Cytoplasm1

We have measured the rate of accumulation of newly synthesized 5s ribosomal RNA (5s rRNA) in Tetrahymena thermophila cells in early log phase growth and in cells that had been starved in a dilute salt solution. From these measurements we have determined the rates of synthesis and levels of accumulation of 5s rRNA relative to 5.8s rRNA in these two different cell populations. In growing cells 5s rRNA is transcribed and accumulated in a 1:1 molar ratio when compared with 5.8s rRNA. In contrast, in starved cells, 5s rRNA is produced at a rate which is about 15% higher than that seen for 5.8s rRNA. This excess 5s rRNA accumulates in the cytoplasm in a non-ribosomal form and is maintained in the cell as long as the cell remains in a starved condition. The role this excess 5s rRNA may play in the control of 5s rRNA gene expression is discussed.     

Cloning of a gene involved in rRNA precursor processing and 23S rRNA cleavage in Rhodobacter capsulatus.

In Rhodobacter capsulatus wild-type strains, the 23S rRNA is cleaved into [16S] and [14S] rRNA molecules. Our data show that a region predicted to form a hairpin-loop structure is removed from the 23S rRNA during this processing step. We have analyzed the processing of rRNA in the wild type and in the mutant strain Fm65, which does not cleave the 23S rRNA. In addition to the lack of 23S rRNA processing, strain Fm65 shows impeded processing of a larger 5.6-kb rRNA precursor and slow maturation of 23S and 16S rRNAs from pre-23S and pre-16S rRNA species. Similar effects have also been described previously for Escherichia coli RNase III mutants. Processing of the 5.6-kb precursor was independent of protein synthesis, while the cleavage of 23S rRNA to generate 16S and 14S rRNA required protein synthesis. We identified a DNA fragment of the wild-type R. capsulatus chromosome that conferred normal processing of 5.6-kb rRNA and 23S rRNA when it was expressed in strain Fm65.     

Post-transcriptional regulation of ribosome accumulation during myoblast differentiation

The synthesis, accumulation and stability of rRNA were examined in embryonic quail myoblasts differentiating in cell culture. Quail myoblasts initially divide rapidly in culture, and accumulate 28S and 18S rRNA and ribosomes at a rate which maintains a constant ribosome content during cell division. After these myoblasts fuse, cell division ceases and ribosomes accumulate in fibers, but at a reduced rate which is only one fourth that in dividing myoblasts. Measurements of rRNA stability by 3H-methyl-methionine pulse-chase analysis show that 28S and 18S rRNA formed by fibers turn over with half-lives of 45 hr, and rRNA formed by myoblasts remains stable until fusion and then also turns over in fibers. Turnover of rRNA in fibers accounts for only half the reduction in ribosome accumulation following myoblast fusion. Measurements of the incorporation of 3H-adenosine into rRNA and ATP pools show that the rates of synthesis of rRNA precursor do not decrease after myoblast fuse, but half the rRNA molecules synthesized by fibers are degraded during processing. Degradation of rRNA during processing reduces the rate of formation of 28S and 18S rRNA, and together with rRNA turnover quantitatively accounts for the reduced rate of ribosome accumulation in fibers.     

Construction and Initial Characterization of Escherichia coli Strains with Few or No Intact Chromosomal rRNA Operons

The Escherichia coli genome carries seven rRNA (rrn) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these rrn deletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Delta7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the rrn deletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.     

Characterization of Vibrio fischeri rRNA operons and subcloning of a ribosomal DNA promoter.

Analysis of rRNA genes in Vibrio fischeri indicates the presence of eight rRNA gene sets in this organism. It was found that the genes for 5S rRNA, 16S rRNA, and 23S rRNA are organized in operons in the following order: 5' end 16S rRNA 23S RNA 5S rRNA 3' end. Although the operons are homologous, they are not identical with regard to cleavage sites for various restriction endonucleases. A DNA library was constructed, and three ribosomal DNA clones were obtained. One of these clones contained an entire rRNA operon and was used as a source for subcloning. The promoter region which leads to plasmid instability was successfully subcloned into pHG165. The terminator region was subcloned into pBR322.     

Localization and structure of endonuclease cleavage sites involved in the processing of the rat 32S precursor to ribosomal RNA.

The initial endonuclease cleavage site in 32 S pre-rRNA (precursor to rRNA) is located within the rate rDNA sequence by S1-nuclease protection mapping of purified nucleolar 28 S rRNA and 12 S pre-rRNA. The heterogeneous 5'- and 3'-termini of these rRNA abut and map within two CTC motifs in tSi2 (internal transcribed spacer 2) located at 50-65 and 4-20 base-pairs upstream from the homogeneous 5'-end of the 28 S rRNA gene. These results show that multiple endonuclease cleavages occur at CUC sites in tSi2 to generate 28 S rRNA and 12 S pre-rRNA with heterogeneous 5'- and 3'-termini, respectively. These molecules have to be processed further to yield mature 28 S and 5.8 S rRNA. Thermal-denaturation studies revealed that the base-pairing association in the 12 S pre-rRNA:28 S rRNA complex is markedly stronger than that in the 5.8 S:28 S rRNA complex. The sequence of about one-quarter (1322 base-pairs) of the 5'-part of the rat 28 S rDNA was determined. A computer search reveals the possibility that the cleavage sites in the CUC motifs are single-stranded, flanked by strongly base-paired GC tracts, involving tSi2 and 28 S rRNA sequences. The subsequent nuclease cleavages, generating the termini of mature rRNA, seem to be directed by secondary-structure interactions between 5.8 S and 28 S rRNA segments in pre-rRNA. An analysis for base-pairing among evolutionarily conserved sequences in 32 S pre-rRNA suggests that the cleavages yielding mature 5.8 S and 28 S rRNA are directed by base-pairing between (i) the 3'-terminus of 5.8 S rRNA and the 5'-terminus of 28 S rRNA and (ii) the 5'-terminus of 5.8 S rRNA and internal sequences in domain I of 28 S rRNA. A general model for primary- and secondary-structure interactions in pre-rRNA processing is proposed, and its implications for ribosome biogenesis in eukaryotes are briefly discussed.     

Analysis of 23S rRNA genes in metagenomes - a case study from the Global Ocean Sampling Expedition

As an evolutionary marker, 23S ribosomal RNA (rRNA) offers more diagnostic sequence stretches and greater sequence variation than 16S rRNA. However, 23S rRNA is still not as widely used. Based on 80 metagenome samples from the Global Ocean Sampling (GOS) Expedition, the usefulness and taxonomic resolution of 23S rRNA were compared to those of 16S rRNA. Since 23S rRNA is approximately twice as large as 16S rRNA, twice as many 23S rRNA gene fragments were retrieved from the GOS reads than 16S rRNA gene fragments, with 23S rRNA gene fragments being generally about 100 bp longer. Datasets for 16S and 23S rRNA sequences revealed similar relative abundances for major marine bacterial and archaeal taxa. However, 16S rRNA sequences had a better taxonomic resolution due to their significantly larger reference database.Reevaluation of the specificity of previously published PCR amplification primers and group specific fluorescence in situ hybridization probes on this metagenomic set of non-amplified 23S rRNA sequences revealed that out of 16 primers investigated, only two had more than 90% target group coverage. Evaluations of two probes, BET42a and GAM42a, were in accordance with previous evaluations, with a discrepancy in the target group coverage of the GAM42a probe when evaluated against the GOS metagenomic dataset.     

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos. V. Inability of inhibitor to repress 5S RNA synthesis.

A specific inhibitor of ribosomal RNA (rRNA) synthesis was partially purified from an acid-soluble fraction of Xenopus laevis blastulae. Effects of this inhibitor on 5S rRNA synthesis of isolated neurula cells of the same species were investigated. The results show that the synthesis of both 5S rRNA and 4S RNA proceeds normally when both 18 and 28S rRNA are almost completely inhibited. Failure of the inhibitor to suppress 5S rRNA synthesis suggests that it plays an important role in the regulation of 18 and 28S rRNA synthesis during development and that the synthesis of 5S rRNA is not coordinated to that of 18 and 28S rRNA.     

Cloning and restriction analysis of ribosomal RNA genes from Mycobacterium smegmatis

A genomic library of Mycobacterium smegmatis DNA was constructed in phage EMBL3. A clone (gamma HB85) containing rRNA genes was isolated using as probes, fragments of E. coli rRNA cistron B. This cloned DNA fragment was mapped by restriction analysis and was shown to contain one complete set of rRNA genes (rRNA A). The physical mapping of the second set of rRNA genes of M. smegmatis (rRNA B) was done by restriction analysis of total chromosomal DNA. The two sets of rRNA genes showed highly conserved restriction sites within the respective sets but not in the flanking regions. The two rRNA sets of genes are organised like in the other eubacteria in the order 16S-23S-5S.     

Heterogeneous rRNA molecules encoded by Streptomyces coelicolor M145 genome are all expressed and assembled into ribosomes

The Streptomyces coelicolor M145 genome harbors six copies of divergent rRNA operons that differ at ~0.2% and ~0.6% of the nucleotide positions in small subunit (SSU) and large subunit (LSU) rRNA genes, respectively. When these rRNA genes are expressed, a single cell may harbor three different kinds of SSU rRNA and five kinds of LSU rRNA. Primer extension analyses revealed that all of the heterogeneous rRNA molecules are expressed and assembled into ribosomes. This finding and the maintenance of the intragenomic variability of rRNA operons imply the existence of functional divergence of rRNA species in this developmentally complex microorganism.     

向日葵rRNA基因克隆及其染色体定位研究

该研究以内葵杂3号三交种为材料,采用同源序列法克隆了5SrRNA和18SrRNA基因并进行了序列测定,测得片段长度分别为515bp和1808bp。以5SrRNA、18SrRNA和45SrRNA基因为探针,分别与内葵杂3号三交种染色体进行荧光原位杂交（FISH）分析。结果表明：45SrRNA和18SrRNA基因均得到3对杂交信号且位点分布相同,分别位于第3对和第10对染色体及第2对随体染色体的短臂末端;5SrRNA基因的信号位点共有2对,分布在第7对和第10对染色体短臂端部。     

5S rRNA sequences of myxobacteria and radioresistant bacteria and implications for eubacterial evolution

5S rRNA sequences were determined for the myxobacteria Cystobacter fuscus, Myxococcus coralloides, Sorangium cellulosum, and Nannocystis exedens and for the radioresistant bacteria Deinococcus radiodurans and Deinococcus radiophilus. A dendrogram was constructed by using weighted pairwise grouping based on these and all other previously known eubacterial 5S rRNA sequences, and this dendrogram showed differences as well as similarities compared with results derived from 16S rRNA analyses. In the dendrogram, Deinococcus 5S rRNA sequences clustered with 5S rRNA sequences of the genus Thermus, as suggested by the results of 16S rRNA analyses. However, in contrast to the 16S rRNA results, the Deinococcus-Thermus cluster divided the 5S rRNA sequences of the alpha subdivision of the class Proteobacteria from the 5S rRNA sequences of the beta and gamma subgroups of the Proteobacteria. The myxobacterial 5S rRNA sequence data failed to confirm the existence of a delta subgroup of the class Proteobacteria, which was suggested by the results of 16S rRNA analyses.