Isolation and characterization of the RNA of membrane-bound ribosomes in Dictyostelium discoideum

The RNA of membrane-bound ribosomes, isolated from Dictyostelium discoideum, represented 13 to 16% of the total ribosomal RNA (rRNA) present throughout growth and development. Membrane-bound ribosomes were released by treatment with sodium deoxycholate and Brij 58. There were no obvious differences in size and base composition between RNAs derived from membrane-bound or free ribosomes. The 17S membrane-bound rRNA and free rRNAs appeared to have similar methyl contents. However, the 25S membrane-bound rRNA contained about 16 to 20% fewer methyl groups than the 17S membrane-bound rRNA and free rRNAs. Free rRNAs turned over rapidly during early development but not during the disaggregation and reaggregation processes. Membrane-bound rRNAs showed very little turnover during the early stages of morphogenesis, but showed rapid turnover during the late stages of development; this class of rRNAs did not turn over during early stages of reaggregation but turned over rapidly during later stages of reaggregation.     

Structure and sequence of the mitochondrial 20S rRNA and tRNA tyr gene of Paramecium primaurelia

The sequence and structure of the large (20s) mitochondrial (mt) rRNA gene and flanking regions from Paramecium primaurelia have been determined. The gene contains two regions of strong homology with other large mt rRNAs: one 44-base region near the 5' end and a 321-base region near the 3' end. Another region of strong homology to both ends of E. coli 23s RNA exists at loci consistent with these regions. The Paramecium gene appears to be 2204 bases in length and contains slightly more homology to E. coli rRNA than its mammalian or fungal counterparts. The gene, located about 1200 bp from the replicative terminal end of the linear mt DNA, is transcribed in the same polarity as replication. Previous R-looping studies detected no large introns within the gene. Here we describe sequences resembling degenerate rRNAs, one of which could represent a small intron. A tRNA tyr gene was found on the same DNA strand, 127 bp downstream from the large rRNA presumptive 3' end. The tRNA is flanked on both sides by short DNA regions of approximately 90% A + T content.     

Probing fungal mitochondrial evolution with tRNA

Sequence data are now available for almost the entire complement of mitochondrial rRNAs from five fungi: Schizosaccharomyces pombe, Saccharomyces cerevisiae, Toropulis glabrata, Aspergillus nidulans and Neurospora crassa. Analysis of these data show that the five mitochondria can be related to a common ancestor. The unusually high similarity between some S. pombe mt tRNAs may be due to a process similar to gene conversion. Using the number of differences between tRNA pairs as a measure of the evolutionary rate the yeast-S. pombe branch has paradoxically a high nuclear rate and a low mt rate of evolution as compared with other branches in the phylogenetic tree. Finally the position of mt tRNA genes in S. pombe is abnormally distinct from gene orders in other mitochondria. All of the above factors must be taken into account when describing the relationship between these mitochondria.     

Mitochondrial proliferation during myogenesis

The mitochondrial (mt) DNA content of muscle cells increases about 4-fold during myogenesis. This is apparently the result of continued mtDNA replication after the myoblasts become post-mitotic and nuclear DNA synthesis ceases. The rate of mtDNA synthesis in prefusion cells exceeds that necessary for growth, indicating mt turnover. Eventually, the mtDNA synthesis drops to about one third the prefusion rate, leading to a stable mtDNA content in muscle tissue.     

Insertional editing in mitochondria of Physarum

RNA produced from a number of genes on the mitochondrial (mt) DNA of Physarum polycephalum have nucleotides inserted at specific sites in their sequence. These insertions are spaced at approximately 25 nucleotide intervals and create open reading frames in mRNA and functional structure in tRNAs and rRNAs. Although most of the insertions at a site are single cytidines; single uridines and certain dinucleotides containing adenosine and guanosine as well as cytidine and uridine are also occasionally inserted at certain sites. This mixed nucleotide insertional RNA editing is unique among currently characterized editing systems.     

Laboratory of Physiological Chemistry, State University Groningen, Netherlands.

To obtain more information about the arrangement of Hind III restriction fragments in the tRNA-rRNA region of the Neurospora crassa mitochondrial (mt) DNA we have cleaved the mtDNA with Hpa I and Hind II. We could construct additional cleavage maps for these enzymes. Hybridization of rRNAs to Hind II fragments confirmed the existence of an intervening region of about 2,300 basepairs in the 24S rRNA (Hahn et al., Cell, in press). About seven tRNA genes, among which the genes for tRNA1Ser and tRNAMetM, are located in a segment of about 5,000 bp separating the 24S and 17S rRNA genes. Another cluster of 14 tRNA genes is found adjacent to the other end of the 24S gene. The genes for tRNALeu1 and tRNAMetF are located in this cluster.     

The complete mitochondrial genome of Arctic Calanus hyperboreus (Copepoda,Calanoida) reveals characteristic patterns in calanoid mitochondrial genome

Copepoda is the most diverse and abundant group of crustaceans, but its phylogenetic relationships are ambiguous. Mitochondrial (mt) genomes are useful for studying evolutionary history, but only six complete Copepoda mt genomes have been made available and these have extremely rearranged genome structures. This study determined the mt genome of Calanus hyperboreus, making it the first reported Arctic copepod mt genome and the first complete mt genome of a calanoid copepod. The mt genome of C. hyperboreus is 17,910 bp in length and it contains the entire set of 37 mt genes, including 13 protein-coding genes, 2 rRNAs, and 22 tRNAs. It has a very unusual gene structure, including the longest control region reported for a crustacean, a large tRNA gene cluster, and reversed GC skews in 11 out of 13 protein-coding genes (84.6%). Despite the unusual features, comparing this genome to published copepod genomes revealed retained pan-crustacean features, as well as a conserved calanoid-specific pattern. Our data provide a foundation for exploring the calanoid pattern and the mechanisms of mt gene rearrangement in the evolutionary history of the copepod mt genome.     

ERB1, the yeast homolog of mammalian Bop1, is an essential gene required for maturation of the 25S and 5.8S ribosomal RNAs

We have recently shown that the mammalian nucleolar protein Bop1 is involved in synthesis of the 28S and 5.8S ribosomal RNAs (rRNAs) and large ribosome subunits in mouse cells. Here we have investigated the functions of the Saccharomyces cerevisiae homolog of Bop1, Erb1p, encoded by the previously uncharacterized open reading frame YMR049C. Gene disruption showed that ERB1 is essential for viability. Depletion of Erb1p resulted in a loss of 25S and 5.8S rRNAs synthesis, while causing only a moderate reduction and not a complete block in 18S rRNA formation. Processing analysis showed that Erb1p is required for synthesis of 7S pre-rRNA and mature 25S rRNA from 27SB pre-rRNA. In Erb1p-depleted cells these products of 27SB processing are largely absent and 27SB pre-rRNA is under-accumulated, apparently due to degradation. In addition, depletion of Erb1p caused delayed processing of the 35S pre-rRNA. These findings demonstrate that Erb1p, like its mammalian counterpart Bop1, is required for formation of rRNA components of the large ribosome particles. The similarities in processing defects caused by functional disruption of Erb1p and Bop1 suggest that late steps in maturation of the large ribosome subunit rRNAs employ mechanisms that are evolutionarily conserved throughout eukaryotes.     

DIFFERENT TURNOVER RATES OF BRAIN RIBOSOMAL RIBONUCLEIC ACIDS IN MALE AND FEMALE RATS

Abstract— Following a single intracranial injection of [5-³H]orotic acid, the decay dynamics were determined for rRNAs of whole brain in male and female Wistar-inbred albino rats aged 2.5-3.5 months. The turnover rate for male brain rRNAs was significantly lower than in females (mean half-lives being, respectively, 12.2 ± 2.2 (S.D.M.) days, and 7.4 ± 1.3 days in four regression measurements). This difference was apparently not related to the turnover rate of acid-soluble brain nucleotides, which turned over much faster and at a similar rate in rats of both sexes; it also could not be connected with brain levels of rRNAs or DNAs, which were quite similar For males and females. The results are discussed in terms of possible sex hormone determination of brain RNA metabolic patterns especially in males.     

Extrachromosomal DNA in the Apicomplexa.

Malaria and related apicomplexan parasites have two highly conserved organellar genomes: one is of plastid (pl) origin, and the other is mitochondrial (mt). The organization of both organellar DNA molecules from the human malaria parasite Plasmodium falciparum has been determined, and they have been shown to be tightly packed with genes. The 35-kb circular DNA is the smallest known vestigial plastid genome and is presumed to be functional. All but two of its recognized genes are involved with genetic expression: one of the two encodes a member of the clp family of molecular chaperones, and the other encodes a conserved protein of unknown function found both in algal plastids and in eubacterial genomes. The possible evolutionary source and intracellular location of the plDNA are discussed. The 6-kb tandemly repeated mt genome is the smallest known and codes for only three proteins (cytochrome b and two subunits of cytochrome oxidase) as well as two bizarrely fragmented rRNAs. The organization of the mt genome differs somewhat among genera. The mtDNA sequence provides information not otherwise available about the structure of apicomplexan cytochrome b as well as the unusually fragmented rRNAs. The malarial mtDNA has a phage-like replication mechanism and undergoes extensive recombination like the mtDNA of some other lower eukaryotes.     

Mutations in the Escherichia coli 23S rRNA increase the rate of peptidyl-tRNA dissociation from the ribosome

We have studied in vivo the phenotypes of 23S rRNA mutations G2582A, G2582U, G2583C, and U2584C, which are located at the A site of Escherichia coli 50S ribosomal subunit. All mutant rRNAs incorporated into 50S ribosomal subunits. Upon sucrose gradient fraction of cell lysates, 23S rRNAs mutated at G2582 to A and G2583 to C accumulated in the 50S and 70S fractions and were under-represented in the polysome fraction. Induction of 23S rRNAs mutated at G2582 and G2583 lead to a drastic reduction in cell growth. In addition, mutations G2582A and G2583C reduced to one-third the total protein synthesis but not the RNA synthesis. Finally, we show that 23S rRNA mutations G2582A, G2582U, and G2583C cause a significant increase in peptidyl-tRNA drop-off from ribosomes, thereby reducing translational processivity. The results clearly show that tRNA-23S rRNA interaction has an essential role in maintaining the processivity of translation.     

Comparison of the nucleotide sequence of soybean 18S rRNA with the sequences of other small-subunit rRNAs

We present the sequence of the nuclear-encoded ribosomal small-subunit RNA from soybean. The soybean 18S rRNA sequence of 1807 nucleotides (nt) is contained in a gene family of approximately 800 closely related members per haploid genome. This sequence is compared with the ribosomal small-subunit RNAs of maize (1805 nt), yeast (1789 nt), Xenopus (1825 nt), rat (1869 nt), and Escherichia coli (1541 nt). Significant sequence homology is observed among the eukaryotic small-subunit rRNAs examined, and some sequence homology is observed between eukaryotic and prokaryotic small-subunit rRNAs. Conserved regions are found to be interspersed among highly diverged sequences. The significance of these comparisons is evaluated using computer simulation of a random sequence model. A tentative model of the secondary structure of soybean 18S rRNA is presented and discussed in the context of the functions of the various conserved regions within the sequence. On the basis of this model, the short base-paired sequences defining the four structural and functional domains of all 18S rRNAs are seen to be well conserved. The potential roles of other conserved soybean 18S rRNA sequences in protein synthesis are discussed.     

3'-Terminal sequence of wheat mitochondrial 18S ribosomal RNA: further evidence of a eubacterial evolutionary origin.

We have determined the sequences of the 3'-terminal approximately 100 nucleotides of [5' -32P]pCp-labeled wheat mitochondrial, wheat cytosol, and E. coli small sub-unit rRNAs. Sequence comparison demonstrates that within this region, there is a substantially greater degree of homology between wheat mitochondrial 18S and E. coli 16S rRNAs than between either of these and wheat cytosol 18S rRNA. Moreover, at a position occupied by 3-methyluridine in E. coli 16S rRNA, the same (or a very similar) modified nucleoside is present in wheat mitochondrial 18S rRNA but not in wheat cytosol 18S rRNA. Further, E. coli 16S and 23S rRNAs hybridize extensively to wheat mitochondrial 18S and 26S rRNA genes, respectively, but wheat cytosol 18S and 26S rRNAs do not. No other mitochondrial system studies to date has provided comparable evidence that a mitochondrial rRNA is more closely related to its eubacterial homolog than is its counterpart in the cytoplasmic compartment of the same cell. The results reported here provide additional support for the view that plant mitochondria are of endosymbiotic, specifically eubacterial, origin.     

Mutations in the Escherichia coli23S rRNA Increase the Rate of Peptidyl-tRNA Dissociation from the Ribosome

We have studied in vivothe phenotypes of 23S rRNA mutations G2582A, G2582U, G2583C, and U2584C, which are located at the A site of Escherichia coli50S ribosomal subunit. All mutant rRNAs incorporated into 50S ribosomal subunits. Upon sucrose gradient fractionation of cell lysates, 23S rRNAs mutated at G2582 to A and G2583 to C accumulated in the 50S and 70S fractions and were underrepresented in the polysome fraction. Induction of 23S rRNAs mutated at G2582 and G2583 lead to a drastic reduction in cell growth. In addition, mutations G2582A and G2583C reduced to one-third the total protein synthesis but not the RNA synthesis. Finally, we show that 23S rRNA mutations G2582A, G2582U, and G2583C cause a significant increase in peptidyl-tRNA drop-off from ribosomes, thereby reducing translational processivity. The results clearly show that tRNA–23S rRNA interaction has an essential role in maintaining the processivity of translation.