Modification of mitochondrial ribosomal RNA from hamster cells: the presence of GmG and late-methylated UmGmU in the large subunit (17S) RNA

The methylated residues of the large subunit RNA (17 S) of hamster cell mitochondrial ribosomes have been characterized and quantitated. Digestion of 17 S RNA with alkali or ribonuclease T₂ yielded approximately one equivalent of GmpGp, a fractional equivalent of GmpUp and slightly less than an equivalent of UmpGmpUp. Pulse-labeling experiments indicated that the Um residue of UmpGmpUp was methylated relatively late, and that the GmpUp was derived from a partially methylated precursor to UmpGmpUp. No ψp was detected in 17 S RNA or in the small subunit (13 S) ribosomal RNA. We propose that the UmpGmpUp of 17 S RNA is homologous to a “universal” UmpGmp ψp sequence found in eukaryotic 28 S rRNA and possibly to similar, but incompletely methylated, sequences in fungal mitochondrial ribosomal RNA and in bacterial ribosomal RNA.     

Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells. In vivo and in vitro methylation

Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-³H]methionine; (2) in vivo incorporation of [³²P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [³H]deoxycytidine; (4) in vitro methylation of DNAs with ³H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, $\text{BHK21}\text{C13}$, $\text{C13}\text{B4}$ (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers.Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 10⁶) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells.Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from ³H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.     

Methylation status of 13S ribosomal RNA from hamster mitochondria: the presence of a novel riboside, N4-methylcytidine.

The ribosomal RNA ("13S" RNA) of the small ribosomal subunit of hamster cell mitochondria has been found to have a distinctive pattern of methylated residues. Each molecule contained, on the average, approximately one residue of m4Cp, m5Cp and m5Up, and two residues of m62Ap. The natural occurrence of m4Cp has not previously been reported; we propose that this nucleotide is homologous to its ribose-methylated congener, m4Cmp, which is characteristic of bacterial 16S ribosomal RNA. We detected neither m4Cp nor m4Cmp in the hamster cell cytoplasmic ribosomal RNA. This is the first documentation of a modified residue present in mitochondrial RNA but absent from the cytoplasmic RNA of the same cells.     

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.     

Genome-Wide Methylation Patterns in Salmonella enterica Subsp. enterica Serovars

The methylation of DNA bases plays an important role in numerous biological processes including development, gene expression, and DNA replication. Salmonella is an important foodborne pathogen, and methylation in Salmonella is implicated in virulence. Using single molecule real-time (SMRT) DNA-sequencing, we sequenced and assembled the complete genomes of eleven Salmonella enterica isolates from nine different serovars, and analysed the whole-genome methylation patterns of each genome. We describe 16 distinct N⁶-methyladenine (m6A) methylated motifs, one N⁴-methylcytosine (m4C) motif, and one combined m6A-m4C motif. Eight of these motifs are novel, i.e., they have not been previously described. We also identified the methyltransferases (MTases) associated with 13 of the motifs. Some motifs are conserved across all Salmonella serovars tested, while others were found only in a subset of serovars. Eight of the nine serovars contained a unique methylated motif that was not found in any other serovar (most of these motifs were part of Type I restriction modification systems), indicating the high diversity of methylation patterns present in Salmonella.     

Comparative analysis of the complete mitochondrial genomes of three rockfishes (Scorpaeniformes,Sebastiscus) and insights into the phylogenetic relationships of Sebastidae

Mitochondrial genome is a powerful molecule marker to provide information for phylogenetic relationships and revealing molecular evolution in ichthyological studies. Sebastiscus species, a marine rockfish, are of essential economic value. However, the taxonomic status and phylogenetic relationships of Sebastidae have been controversial so far. Here, the mitochondrial genomes (mitogenomes) of three species, S. tertius, S. albofasciatus, and S. marmoratus, were systemically investigated. The lengths of the mitogenomes’ sequences of S. tertius, S. albofasciatus, and S. marmoratus were 16910, 17056, and 17580 bp, respectively. It contained 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNA (tRNA) genes, and one identical control region (D-loop) among the three species. The genetic distance and K_a/K_s ratio analyses indicated 13 PCGs were suffering purifying selection and the selection pressures were different from certain deep-sea fishes, which were most likely due to the difference in their living environment. The phylogenetic tree was constructed by Bayesian Inference (BI) and Maximum Likelihood (ML). Most interestingly, the results indicated that Sebastidae and Scorpaenidae were grouped into a separate branch, so the taxonomic status of Sebastidae should be classified into subfamily Sebastinae. Our results may lead to a taxonomic revision of Scorpaenoidei.     

Cytoplasmic methylation of mature 5.8 S ribosomal RNA

Levels of 2-O-methylation were determined in ribosomal 5·8 S RNAs from whole cells and both the nuclear and cytoplasmic fractions of rat liver, rat kidney cells in culture (NRK) and HeLa cells. All 5·8 S RNA molecules contained the alkali stable Gm-Cp dinucleotide at position 77 but only whole cell rat liver RNA contained large amounts (0·7 mol) of Um at position 14. All nuclear 5·8 S RNA fractions were largely undermethylated at this site. In contrast, cytoplasmic 5.8 S RNA from rat liver and, to a lesser degree, NRK cells contained significantly more Um; up to 80% of the molecules from rat liver contained the methylated residue. These results indicate that mature 5·8 S RNA can be methylated in the cytoplasm. When labeling kinetics were examined in NRK cells, the methylation at residue 14 was found to increase as a function of the time spent in the cytoplasm, confirming that this modification is, unlike other ribosomal RNA methylations, in part or largely cytoplasmic.     

Mapping of mitochondrial 4 S RNA genes in Xenopus laevis by electron microscopy

The distribution of sites hybridizing with mitochondrial 4 S RNA molecules on mitochondrial DNA of Xenopus laevis has been mapped in relation to the ribosomal RNA genes and EcoRI restriction endonuclease sites. RNA molecules linked to ferritin were employed for this purpose. We have obtained evidence for 15 4 S RNA sites on the H-strand and six sites on the L-strand of X. laevis mtDNA^∥. An indication of the possible existence of one additional site on the H-strand and four additional sites on the L-strand has been obtained. One 4 S RNA site is located in the gap between the two rRNA genes, and one site flanks each outside end of the rRNA genes. The other 4 S RNA sites are distributed almost evenly throughout both strands of the mtDNA. A comparison with the map of 4 S RNA sites on the mtDNA of HeLa cells (Angerer et al., 1976) suggests considerable evolutionary conservation of site organization.     

Ribosomal RNA sequence conservation and gene number in the larval brine shrimp

The haploid genome size of Artemia is determined to be about 0.9·10¹², as evidenced both by Feulgen microspectrophotometry of individual diploid class nuclei, which are but one of five polyploid classes present within the larvae, and by analysis of the reassociation kinetics of the isolated single copy DNA component. Polysomes isolated from 24-h incubation stage larvae contain an average of 10 ribosomes per messenger RNA molecule. Their rRNAs are found to have sedimentation coefficients of 18 S and 26 S, corresponding to molecular weights of 0.70·10⁶ and 1.40·10⁶, respectively, as determined by polyacrylamide electrophoresis and also by sucrose density centrifugation. Denaturation in glyoxal followed by agarose gel electrophoresis shows that unlike deuterostome rRNAs, Artemia 26 S rRNA contains a cryptic nick about midway in the molecule, which is not found in the 18 S molecule. Isolated rRNAs were labelled in vitro with ¹²⁵I and hybridized with filter-immobilized DNA to saturation, which occurred at 0.051% for Xenopus, and at 0.074% for Artemia. From these results, it is calculated that in the haploid Artemia genome there are about 320 copies of the (18 S + 26 S) ribosomal RNA genes. Reciprocal heterologous hybridizations between these two species show that they share about 30% homology between their rDNA coding sequences.     

A Flexible peptide tether controls accessibility of a unique C-terminal RNA-binding domain in leucyl-tRNA synthetases

A unique C-terminal domain extension is required by most leucyl-tRNA synthetases (LeuRS) for aminoacylation. In one exception, the enzymatic activity of yeast mitochondrial LeuRS is actually impeded by its own C-terminal domain. It was proposed that the yeast mitochondrial LeuRS has compromised its aminoacylation activity to some extent and adapted its C terminus for a second role in RNA splicing, which is also essential. X-ray crystal structures of the LeuRS-tRNA complex show that the 60 residue C-terminal domain is tethered to the main body of the enzyme via a flexible peptide linker and allows interactions with the tRNA^Leu elbow. We hypothesized that this short peptide linker would facilitate rigid body movement of the C-terminal domain as LeuRS transitions between an aminoacylation and editing complex or, in the case of yeast mitochondrial LeuRS, an RNA splicing complex. The roles of the C-terminal linker peptide for Escherichia coli and yeast mitochondrial LeuRS were investigated via deletion mutagenesis as well as by introducing chimeric swaps. Deletions within the C-terminal linker of E. coli LeuRS determined that its length, rather than its sequence, was critical to aminoacylation and editing activities. Although deletions in the yeast mitochondrial LeuRS peptide linker destabilized the protein in general, more stable chimeric enzymes that contained an E. coli LeuRS C-terminal domain showed that shortening its tether stimulated aminoacylation activity. This suggested that limiting C-terminal domain accessibility to tRNA^Leu facilitates its role in protein synthesis and may be a unique adaptation of yeast mitochondrial LeuRS that accommodates its second function in RNA splicing.     

Extreme ends of the genome are conserved and rearranged in the defective interfering RNAs of semliki forest virus

We have analyzed Semliki Forest virus defective interfering RNA molecules, generated by serial undiluted passaging of the virus in baby hamster kidney cells. The 42 S RNA genome (about 13 kb ²) has been greatly deleted to generate the DI RNAs, which are heterogeneous both in size (about 2 kb) and sequence content. The DI RNAs offer a system for exploring binding sites for RNA polymerase and encapsidation signals, which must have been conserved in them since they are replicated and packaged. In order to study the structural organization of DI RNAs, and to analyze which regions from the genome have been conserved, we have determined the nucleotide sequences of (1) a 2.3 kb long DI RNA molecule, DI309, (2) 3′-terminal sequences (each about 0.3 kb) of two other DI RNAs, and (3) the nucleotide sequence of 0.4 kb at the extreme 5′ end of the 42 S RNA genome.The DI309 molecule consists of a duplicated region with flanking unique terminal sequences. A 273-nucleotide sequence is present in four copies per molecule. The extreme 5′-terminal nucleotide sequence of the 42 S RNA genome is shown to contain domains that are conserved in the two DI RNAs of known structure: DI309, and the previously sequenced DI301 (Lehtovaara et al., 1981). Here we report which terminal genome sequences are conserved in the DI RNAs, and how they have been modified, rearranged or amplified.     

Insights into the Catalytic Mechanism of 16S rRNA Methyltransferase RsmE (mU1498) from Crystal and Solution Structures

RsmE is the founding member of a new RNA methyltransferase (MTase) family responsible for methylation of U1498 in 16S ribosomal RNA in Escherichia coli. It is well conserved across bacteria and plants and may play an important role in ribosomal intersubunit communication. The crystal structure in monomer showed that it consists of two distinct but structurally related domains: the PUA (pseudouridine synthases and archaeosine‐specific transglycosylases)-like RNA recognition and binding domain and the conserved MTase domain with a deep trefoil knot. Analysis of small-angle X-ray scattering data revealed that RsmE forms a flexible dimeric conformation that may be essential for substrate binding. The S‐adenosyl‐l‐methionine (AdoMet)-binding characteristic determined by isothermal titration calorimetry suggested that there is only one AdoMet molecule bound in the subunit of the homodimer. In vitro methylation assay of the mutants based on the RsmE-AdoMet-uridylic acid complex model showed key residues involved in substrate binding and catalysis. Comprehensive comparisons of RsmE with closely related MTases, combined with the biochemical experiments, indicated that the MTase domain of one subunit in dimeric RsmE is responsible for binding of one AdoMet molecule and catalytic process while the PUA-like domain in the other subunit is mainly responsible for recognition of one substrate molecule (the ribosomal RNA fragment and ribosomal protein complex). The methylation process is required by collaboration of both subunits, and dimerization is functionally critical for catalysis. In general, our study provides new information on the structure-function relationship of RsmE and thereby suggests a novel catalytic mechanism.     

Complete sequence of bovine mitochondrial DNA conserved features of the mammalian mitochondrial genome

We present here the complete 16,338 nucleotide DNA sequence of the bovine mitochondrial genome. This sequence is homologous to that of the human mitochondrial genome (Anderson et al., 1981) and the genes are organized in virtually identical fashion. The bovine mitochondrial protein genes are 63 to 79% homologous to their human counterparts, and most of the nucleotide differences occur in the third positions of codons. The minimum rate of base substitution that accounts for the nucleotide differences in the codon third positions is very high: at least 6 × 10^?9 changes per position per year. The bovine and human mitochondrial transfer RNA genes exhibit more interspecies variation than do their cytoplasmic counterparts, with the “TΨC” loop being the most variable part of the molecule. The bovine 12 S and 16 S ribosomal RNA genes, when compared with those from human mitochondrial DNA, show conserved features that are consistent with proposed secondary structure models for the ribosomal RNAs. Unlike the pattern of moderate-to-high homology between the bovine and human mitochondrial DNAs found over most of the genome, the DNA sequence in the bovine D-loop region is only slightly homologous to the corresponding region in the human mitochondrial genome. This region is also quite variable in length, and accounts for the bulk of the size difference between the human and bovine mitochondrial DNAs.     

Topography of 16 S RNA in 30 S subunits and 70 S ribosomes accessibility to cobra venom ribonuclease

A ribonuclease extracted from the venom of the cobra Naja oxiana, which shows an unusual specificity for double-stranded RNA regions, was used to obtain new insight on the topography of Escherichia coli ribosomal 16 S RNA in the 30 S subunit and in the 70 S couple. ³²P-labeled 30 S subunits or reconstituted 70 S tight couples containing ³²P-labeled 16 S RNA have been digested under progressively stronger conditions. The cleavage sites have been precisely localized and the chronology of the hydrolysis process studied.The enzyme cleaves the 16 S RNA within 30 S subunits at 21 different sites, which are not uniformly distributed along the molecule. These results provide valuable information on the 16 S RNA topography and evidence for secondary structure features.The binding of the 50 S subunit markedly reduces the rate of the 16 S RNA hydrolysis and provides protection for several cleavage sites. Four of them are clustered in the 3′-terminal 200 nucleotides of the molecule, one in the middle (at position 772) and one in the 5′ domain (at position 336). Our results provide further evidence that the 3′-terminal and central regions of the RNA chain are close to each other in the ribosome structure and lie at the interface of the two subunits. They also suggest that the 5′ domain is probably not involved exclusively in structure and assembly.