Incorporation of methionine from met-tRNA-Met-F into internal positions of polypeptides by mouse liver polysomes

Two methionine accepting tRNA species corresponding to tRNA_F^Met and tRNA_M^Met from mouse ascites tumor cells were tested for their ability to donate methionine into internal positions of growing polypeptide chains on mouse liver polysomes. Both tRNA species can function in the elongation of polypeptide chains as judged by their ability to incorporate methionine into protein in the absence of chain initiation. The insertion of methionine into internal positions of polypeptide chains from Met-tRNA_F^Met was confirmed by Edman degradation and CNBr cleavage. When both tRNA^Met species were present in saturating concentrations in the cell-free system a strong preference for the incorporation of methionine from Met-tRNA_M^Met became apparent.     

In vitro association of selective +RNA species with 28S RNA of mouse cells

28S RNA prepared either from the poly(A) RNA-depleted fraction of mouse embryo culture cells or from 60S ribosome subunits of adult mouse liver is able to bind selective species of tRNAs in an $\text{in vitro}$ hybridization reaction. The bound tRNA consists predominantly of proline tRNA and, in minor amounts, glycine, alanine, and aspartic acid tRNAs. Quantitative analysis revealed that the hybridization of tRNA may involve a 28S RNA subpopulation, which is present in higher quantity in embryo cells than in adult liver of the mouse.     

Striking differences in mitochondrial tRNA import between different plant species

A systematic comparison of the tRNAs imported into the mitochondria of larch, maize and potato reveals considerable differences among the three species. Larch mitochondria import at least eleven different tRNAs (more than half of those tested) corresponding to ten different amino acids. For five of these tRNAs [tRNA^Phe(GAA), tRNA^Lys(CUU), tRNA^Pro(UGG), tRNA^Ser(GCU) and tRNA^Ser(UGA)] this is the first report of import into mitochondria in any plant species. There are also differences in import between relatively closely related plants; wheat mitochondria, unlike maize mitochondria import tRNA^His, and sunflower mitochondria, unlike mitochondria from other angiosperms tested, import tRNA^Ser(GCU) and tRNA^Ser(UGA). These results suggest that the ability to import each tRNA has been acquired independently at different times during the evolution of higher plants, and that there are few apparent restrictions on which tRNAs can or cannot be imported. The implications for the mechanisms of mitochondrial tRNA Import in plants are discussed.     

Amino acid specificity of the transfer RNA species coded for by HeLa cell mitochondrial DNA

The amino acid specificity of the tRNA species coded for by HeLa cell mitochondrial DNA has been investigated by carrying out hybridizations between amino acid-tRNA complexes labeled in the amino acid and separated mitochondrial DNA strands.The results indicate that there are in HeLa cell mitochondria at least 17 distinct tRNA species hybridizable with mitochondrial DNA, which are specific for 16 amino acids. For 14 of the 16 amino acids, amino-acyl-tRNA synthetase activities distinct from the cytoplasmic ones have been detected in mitochondria. The remaining four amino acids (asparagine, glutamine, histidine and proline) have consistently failed to charge to any detectable extent mitochondrial tRNA species hybridizable with mitochondrial DNA.No obvious relationship appears to exist between the amino acids incorporated into tRNAs hybridizable to mitochondrial DNA and the previously observed pattern of chloramphenicol-sensitive amino acid incorporation by HeLa cell mitochondria.     

Mitochondrial tRNA cleavage by tRNA-targeting ribonuclease causes mitochondrial dysfunction observed in mitochondrial disease

Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrial dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ⁰ cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed.     

Differential effects of 2,4-dinitrophenol and valinomycin (+K+) on uncoupler-stimulated ATPase of human tumor mitochondria

The uncoupler-stimulated mitochondrial ATPase of four human tumors, mouse kidney, brain and fetal liver exhibited a characteristic behavior when preincubated with the H⁺-conducting uncouplers, dinitrophenol, CCCP, S-13 and gramicidin. The ATPase activity was considerably lower with preincubation than without. Preincubation with valinomycin (+K⁺), on the other hand, did not result in a significant decrease of the ATPase activity. These results may be contrasted with those obtained with liver or heart mitochondria, the ATPase activity of which did not suffer any loss when preincubated with dinitrophenol. The effect of preincubation with dinitrophenol on the tumor mitochondria could not be accounted for by dinitrophenol-induced Mg²⁺ efflux, since the differential effects of dinitrophenol and valinomycin (+K⁺) remained even when ATPase activity was determined in presence of Mg²⁺. Small amounts of ATP and ADP in the preincubation mixture containing dinitrophenol protected against the decay of the ATPase activity, implicating the exchangeable adenine nucleotides in the tumor mitochondria. In a model system where liver mitochondria were depleted of their adenine nucleotides, a lower ATPase activity was indeed obtained. However, direct determination of the concentations of adenine nucleotides in dinitrophenol- and valinomycin-treated tumor mitochondria revealed only slight differences.     

Identification and characterization of mouse TRMU gene encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase

The nucleotide modification in tRNA plays a pivotal role in the fidelity of translational process. The mutated mitochondrial tRNA (mt tRNA) associated with human diseases often exhibited a defect in nucleotide modification at wobble position of anticodons. Recently, the product of trmU, 5-methylaminomethyl-2-thiouridylate-methyltransferase, has been shown to be one component of enzyme complex for the biosynthesis of mnm5s2U in the wobble position of the bacterial tRNAs. Here we report the identification and characterization of mouse TRMU homolog. A 1532 bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The mouse TRMU gene containing 11 exons encodes a 417 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs related to tRNA modification. The mouse TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver and brain. Furthermore, immunofluorescence analysis of NIH3T3 cells expressing TRMU-GFP fusion protein demonstrated that the mouse Trmu localizes in mitochondria. These observations suggest that the mouse TRMU is a structural and functional homolog of bacterial TrmU, thereby playing a role in the mt tRNA modification and protein synthesis.     

Fractionation of Rat Liver tRNA by Reversed-Phase High Performance Liquid Chromatography: Isolation of ISO-tRNAsPro

Abstract

This paper illustrates the fractionation of cytoplasmic transfer ribonucleic acid from rat liver by reversed-phase high performance liquid chromatography using a gradient of acetonitrile/ammonium acetate. The procedure is fast, highly reproducible, and gives an excellent resolution of the numerous tRNA population: about 50 peaks with area peak percentages ranging from 0.001 to 5 can be monitored. Uncharged tRNA preparations exhibited a chromatographic profile different from aminoacylated tRNA, thus suggesting a possible strategy to distinguish between aminoacylated and nonacylated tRNA species. Moreover, a first approach to map the HPLC peaks was attempted by chromatographing preparations of tRNA which had been aminoacylated with individual ³H-labeled aminoacids. Here is reported the case of tRNA^Pro, which gave three well separated radioactive peaks, most likely corresponding to tRNA^Pro isoacceptor species.     

Mature methyl-deficient tRNA isolated from a mammary adenocarcinoma

A transplantable rat tumor, mammary adenocarcinoma 13762, accumulates tRNA which can be methylated in vitro by mammalian tRNA (adenine-1) methyltransferase. This unusual ability of the tumor RNA to serve as substrate for a homologous tRNA methylating enzyme is correlated with unusually low levels of the A₅₈-specific adenine-1 methyltransferase. The nature of the methyl-accepting RNA has been examined by separating tumor tRNA on two-dimensional polyacrylamide gels. Comparisons of ethidium bromide-stained gels of tumor vs. liver tRNA show no significant quantitative differences and no accumulation of novel tRNAs or precursor tRNAs in adenocarcinoma RNA. Two-dimensional separations of tumor RNA after in vitro [¹⁴C]methylation using purified adenine-1 methyltransferase indicate that about 25% of the tRNA species are strongly methyl-accepting RNAs. Identification of six of the tRNAs separated on two-dimensional gels has been carried out by hybridization of cloned tRNA genes to Northern blots. Three of these, tRNA^Lys3, tRNA^Gln and tRNA^Met_i, are among the adenocarcinoma methyl-accepting RNAs. The other three RNAs, all of which are leucine-specific tRNAs, show no methyl-accepting properties. Our results suggest that low levels of a tRNA methyltransferase in the adenocarcinoma cause selected species of tRNA to escape the normal A₅₈ methylation, resulting in the appearance of several mature tRNAs which are deficient in 1-methyladenine. The methyl-accepting tRNAs from the tumor appear as ethidium bromide-stained spots of similar intensity to those seen for RNA from rat liver; therefore, methyladenine deficiency does not seem to impair processing of these tRNAs.     

Determination of N-(9-( -D-ribofuranosyl)purin-6-ylcarbamoly)threonine at the picomole level in transfer-RNA

An assay has been developed for quantitation of the modified nucleoside, t⁶A, in tRNA at the pmole level. For tRNA from a variety of species, the content of t⁶A was found to be 0.18–0.25 mole %. These values lend support to the suggestion that t⁶A is located at the 3′-end of the anticodon in tRNA's whose codons begin with adenosine. Essentially no t⁶A was found in Mycoplasma sp. (Kid) tRNA which is deficient in many modified nucleosides. In the rat, no organ specific differences were found. The amount of t⁶A in Novikoff hepatoma tRNA was essentially the same as in tRNA from normal rat liver.     

Conservatism of sites of tRNA loci among the linkage groups of severalDrosophila species

Summary The sites of seven tRNA genes (Arg-2, Lys-2, Ser-2b, Ser-7, Thr-3, Thr-4, Val-3b) were studied by in situ hybridization.¹²⁵I-labeled tRNA probes fromDrosophila melanogaster were hybridized to spreads of polytene chromosomes prepared from fourDrosophila species representing different evolutionary lineages (D. melanogaster, Drosophila hydei, Drosophila pseudoobscura, andDrosophila virilis). Most tRNA loci occurred on homologous chromosomal elements of all four species. In some cases the number of hybridization sites within an element varied and sites on nonhomologous elements were found. It was observed that both tRNA ₂ ^Arg and tRNA ₂ ^Lys hybridized to the same site on homologous elements in several species. These data suggest a limited amount of exchange among different linkage groups during the evolution ofDrosophila species.     

Functional changes in rat liver tRNA following aflatoxin B1 administration

Administration of aflatoxin B₁ (3 mg/kg body wt) to rats leads to strong inhibition of the acceptor activity of liver tRNA as measured by charging with [¹⁴C]-chorella protein hydrolysate. The maximum inhibition occurs 2 h after treatment. At increasing intervals after treatment, the inhibition appears to be gradually relieved, till control values are restored by 72 h. The charging experiment using several [¹⁴C]-amino acids separately shows pronounced inhibition of acceptor activity of all tRNA species, although the degree of inhibition varies with individual species. Preliminary results seem to rule out the possibility of hypermethylation of tRNA or damage to the CCA terminus as probable causes. The resultant functional changes may be attributed to a covalent interaction of aflatoxin B₁-metabolite with tRNA.     

Methyl-deficient mammalian transfer RNA: II. Homologous methylation in vitro of liver tRNA from normal and ethionine-fed rats: ethionine effect on 5-methyl-cytidine synthesis in vivo

Following hydroxyapatite chromatography, rat liver tRNA methylase activity was assayed with liver tRNA from normal rats and with methyl-deficient liver tRNA from ethionine-fed rats. The difference in homologous methylation between normal and methyl-deficient tRNA was maximal in certain fractions in presence of cadaverine, and much less in presence of Mg⁺⁺ or Mg⁺⁺ plus cadaverine. These methylase fractions, which contained endogenous tRNA, were used for preparative homologous methylation of added normal and methyl-deficient tRNA in presence of 30 mM cadaverine. The ¹⁴C-methylated tRNA was digested with RNase T₂ and the resulting methylated mononucleotides were characterized and quantitated after twodimensional thinlayer chromatography and autoradiography. The major products of homologous tRNA methylation were m⁵C and m¹A. However, the methylase fraction used here did not catalyze the formation of m⁶₂A with m⁶₂A-deficient tRNA as substrate.- In addition to the previously described, analytically detectable m⁶₂A-deficiency, a partial m⁵C-deficiency was demonstrated in liver tRNA from ethionine-fed rats by measuring the methylacceptance in vitro. In presence of cadaverine, with the methylase fraction used here, methyl-deficient tRNA from ethionine-fed rats was a twofold more efficient methyl-acceptor in vitro than normal liver tRNA, while endogenous tRNA isolated from the methylase fraction was a threefold more efficient methyl-acceptor than normal liver tRNA. Homologous methylation of normal tRNA, as observed here, has not been described before.     

The absence of ribothymidine in specific eukaryotic transfer RNAs. I. Glycine and threonine tRNAs of wheat embryo

Ribothymidine, generally considered a universal nucleotide in tRNA, is completely absent in five specific wheat embryo tRNAs. These consist of two species of glycine tRNA and three species of threonine tRNA. These tRNAs, all extensively purified, are acceptable substrates for $\text{E. coli}$ - ribothymidine forming-uracil methylase, which produces one mole of ribothymidine per mole of tRNA. These five tRNAs account for about 90% of the wheat embryo tRNAs which are substrates for this methylase. Nucleotide sequence analysis of one of these tRNAs, tRNA^Gly_I, confirmed both the complete absence of ribothymidine at position 23 from the 3′end, and the presence of uridine at that site instead. In addition, it is shown that methylation with $\text{E. coli}$ uracil methylase quantitatively converts uridine at position 23 to ribothymidine, while no other uridine in the molecule is affected.Using $\text{E. coli}$ uracil methylase as an assay we have detected this class of ribothymidine lacking tRNA, in each case consisting of a few specific species, in other higher organisms, such as wheat seedling, fetal calf liver and beef liver, in addition to wheat embryo. We could not detect this class of tRNA in $\text{E. coli}$ or yeast tRNA.     

Sequences of three transfer RNAs from mosquito mitochondria

The sequences of three transfer RNAs from mosquito cell mitochondria, tRNA_UCG^Arg, tRNA_GUC^Asp, and tRNA_GAU^Ile, determined using a combination of rapid ladder and fingerprinting procedures are reported. These were compared with hamster mitochondrial tRNA_UCG^Arg and tRNA_GUC^Asp determined similarly, and a bovine mitochondrial tRNA_GAU^Ile determined using a somewhat different approach. The primary sequences of the mosquito tRNAs were 35 to 65% homologous to the corresponding mammalian mitochondrial species, and bore little homology to “conventional” (bacterial or eucaryotic cytoplasmic) tRNA. The modification status of the mosquito mitochondrial tRNAs resembled that of mammalian mitochondrial tRNA. The results contribute to the generalization that metazoan mitochondrial tRNA constitutes a distinctive, albeit loosely structured, phylogenetic group.     

Tissue-specific expression atlas of murine mitochondrial tRNAs

Mammalian mitochondrial tRNA (mt-tRNA) plays a central role in the synthesis of the 13 subunits of the oxidative phosphorylation complex system (OXPHOS). However, many aspects of the context-dependent expression of mt-tRNAs in mammals remain unknown. To investigate the tissue-specific effects of mt-tRNAs, we performed a comprehensive analysis of mitochondrial tRNA expression across five mice tissues (brain, heart, liver, skeletal muscle, and kidney) using Northern blot analysis. Striking differences in the tissue-specific expression of 22 mt-tRNAs were observed, in some cases differing by as much as tenfold from lowest to highest expression levels among these five tissues. Overall, the heart exhibited the highest levels of mt-tRNAs, while the liver displayed markedly lower levels. Variations in the levels of mt-tRNAs showed significant correlations with total mitochondrial DNA (mtDNA) contents in these tissues. However, there were no significant differences observed in the 2-thiouridylation levels of tRNA^Lys, tRNA^Glu, and tRNA^Gln among these tissues. A wide range of aminoacylation levels for 15 mt-tRNAs occurred among these five tissues, with skeletal muscle and kidneys most notably displaying the highest and lowest tRNA aminoacylation levels, respectively. Among these tissues, there was a negative correlation between variations in mt-tRNA aminoacylation levels and corresponding variations in mitochondrial tRNA synthetases (mt-aaRS) expression levels. Furthermore, the variable levels of OXPHOS subunits, as encoded by mtDNA or nuclear genes, may reflect differences in relative functional emphasis for mitochondria in each tissue. Our findings provide new insight into the mechanism of mt-tRNA tissue-specific effects on oxidative phosphorylation.     

Methionine on the rise: how mitochondria changed their codon usage

The tRNA specific for methionine (tRNA^Met) of human mitochondria contains a formyl‐cytosine at the wobble position of the anticodon to facilitate its binding to AUG, AUA and (in one instance) to AUU. In this issue of The EMBO Journal, Haag et al identify a two‐step enzyme pathway facilitating the modification of the tRNA. Sequential reactions of the methyltransferase NSUN3 and the dioxygenase ALKBH1/ABH1 are important to render the tRNA as able to recognize the non‐canonical methionine codons AUA and AUUs, a property critical for efficient protein synthesis in human mitochondria.