Mutation in MTO1 involved in tRNA modification impairs mitochondrial RNA metabolism in the yeast Saccharomyces cerevisiae

The yeast MTO1 gene encodes an evolutionarily conserved protein for the biosynthesis of the 5-carboxymethylaminomethyl group of cmnm⁵s²U in the wobble position of mitochondrial tRNA. However, mto1 null mutant expressed the respiratory deficient phenotype only when coupled with the C1409G mutation of mitochondrial 15S rRNA. To further understand the role of MTO1 in mitochondrial RNA metabolism, the yeast mto1 null mutants carrying either wild-type (P^S) or 15S rRNA C1409G allele (P^R) have been characterized by examining the steady-state levels, aminoacylation capacity of mitochondrial tRNA, mitochondrial gene expression and petite formation. The steady-state levels of tRNA^Lys, tRNA^Glu, tRNA^Gln, tRNA^Leu, tRNA^Gly, tRNA^Arg and tRNA^Phe were decreased significantly while those of tRNA^Met and tRNA^His were not affected in the mto1 strains carrying the P^S allele. Strikingly, the combination of the mto1 and C1409G mutations gave rise to the synthetic phenotype for some of the tRNAs, especially in tRNA^Lys, tRNA^Met and tRNA^Phe. Furthermore, the mto1 strains exhibited a marked reduction in the aminoacylation levels of mitochondrial tRNA^Lys, tRNA^Leu, tRNA^Arg but almost no effect in those of tRNA^His. In addition, the steady-state levels of mitochondrial COX1, COX2, COX3, ATP6 and ATP9 mRNA were markedly decreased in mto1 strains. These data strongly indicate that unmodified tRNA caused by the deletion of MTO1 gene caused the instability of mitochondrial tRNAs and mRNAs and an impairment of aminoacylation of mitochondrial tRNAs. Consequently, the deletion of MTO1 gene acts in synergy with the 15S rRNA C1409G mutation, leading to the loss of COX1 synthesis and subsequent respiratory deficient phenotype.     

Elongator complex influences telomeric gene silencing and DNA damage response by its role in wobble uridine tRNA modification

Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncm⁵U₃₄), 5-methoxycarbonylmethyluridine (mcm⁵U₃₄), and 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U₃₄) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNA^Lys _{s² UUU}, tRNA^Gln _{s² UUG}, and tRNA^Glu _{s² UUC}, which in a wild-type background contain the mcm⁵s²U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U₃₄. Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Δ mutation, which abolish the formation of the 2-thio group of the mcm⁵s²U nucleoside in tRNA^Lys _mcm⁵s²UUU, tRNA^Gln _mcm⁵s²UUG, and tRNA^Glu _mcm⁵s²UUC. These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U₃₄ are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Δ mutants.     

Thiolated tRNAs of Trypanosoma brucei Are Imported into Mitochondria and Dethiolated after Import

All mitochondrial tRNAs in Trypanosoma brucei derive from cytosolic tRNAs that are in part imported into mitochondria. Some trypanosomal tRNAs are thiolated in a compartment-specific manner. We have identified three proteins required for the thio modification of cytosolic tRNA^Gln, tRNA^Glu, and tRNA^Lys. RNA interference-mediated ablation of these proteins results in the cytosolic accumulation non-thio-modified tRNAs but does not increase their import. Moreover, in vitro import experiments showed that both thio-modified and non-thio-modified tRNA^Glu can efficiently be imported into mitochondria. These results indicate that unlike previously suggested the cytosol-specific thio modifications do not function as antideterminants for mitochondrial tRNA import. Consistent with these results we showed by using inducible expression of a tagged tRNA^Glu that it is mainly the thiolated form that is imported in vivo. Unexpectedly, the imported tRNA becomes dethiolated after import, which explains why the non-thiolated form is enriched in mitochondria. Finally, we have identified two genes required for thiolation of imported tRNA^Trp whose wobble nucleotide is subject to mitochondrial C to U editing. Interestingly, down-regulation of thiolation resulted in an increase of edited tRNA^Trp but did not affect growth.     

A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine

In many prokaryotes and in organelles asparagine and glutamine are formed by a tRNA-dependent amidotransferase (AdT) that catalyzes amidation of aspartate and glutamate, respectively, mischarged on tRNA^Asn and tRNA^Gln. These pathways supply the deficiency of the organism in asparaginyl- and glutaminyl-tRNA synthtetases and provide the translational machinery with Asn-tRNA^Asn and Gln-tRNA^Gln. So far, nothing is known about the structural elements that confer to tRNA the role of a specific cofactor in the formation of the cognate amino acid. We show herein, using aspartylated tRNA^Asn and tRNA^Asp variants, that amidation of Asp acylating tRNA^Asn is promoted by the base pair U₁–A₇₂ whereas the G₁–C₇₂ pair and presence of the supernumerary nucleotide U_20A in the D-loop of tRNA^Asp prevent amidation. We predict, based on comparison of tRNA^Gln and tRNA^Glu sequence alignments from bacteria using the AdT-dependent pathway to form Gln-tRNA^Gln, that the same combination of nucleotides also rules specific tRNA-dependent formation of Gln. In contrast, we show that the tRNA-dependent conversion of Asp into Asn by archaeal AdT is mainly mediated by nucleotides G₄₆ and U₄₇ of the variable region. In the light of these results we propose that bacterial and archaeal AdTs use kingdom-specific signals to catalyze the tRNA-dependent formations of Asn and Gln.     

Mtu1 defects are correlated with reduced osteogenic differentiation

Accumulating evidence has revealed that mitochondria dynamics and function regulation is essential for the successful mesenchymal stem cell (MSC) differentiation. In the present study, the researchers reported for the first time that Mtu1 defects are correlated with reduced osteogenic differentiation. Using in vitro cultured bone marrow MSCs and stromal cell line MS5, we demonstrated that depressed Mtu1 expression was associated with reduced 2-thiouridine modification of the U34 of mitochondrial tRNA^Gln, tRNA^Glu, and tRNA^Lys, which led to respiratory deficiencies and reduced mitochondrial ATP production, and finally suppressed osteogenic differentiation. As expected, these Mtu1-deficient mice exhibited obvious osteopenia. Therefore, our findings in this study provide new insights into the pathophysiology of osteopenia.Subject terms: Cell biology, Post-translational modifications, Mesenchymal stem cells     

Structural relationship between tRNA2 Lys and tRNA4 Lys from mouse lymphoma cells

Summary Mouse lymphoma cells have three major isoaccepting lysine tRNAs. Two of these isoacceptors, tRNA₂ ^Lys and tRNA₄ ^Lys, were sequenced by rapid gel or chromatogram readout methods. They have the same primary sequence but differ in two modified nucleotides. tRNA₄ ^Lys has an unmodified uridine replacing one dihydrouridine and an unidentified nucleotide, t⁶A^*, replacing t⁶A. This unidentified nucleotide is not a hypomodified form of t⁶A. Thus, tRNA4ys is not a simple precursor of tRNA₂ ^Lys. Both tRNAs have an unidentified nucleotide, U^**, in the third position of the anticodon. Also, partial sequences of minor homologs of tRNA₂ ^Lys and tRNA₄ ^Lys were obtained. The distinctions between tRNA₂ ^Lys and tRNA₄ ^Lys may be part of significant cellular roles as illustrated by the differential effects of these isoacceptors on the synthesis by lysyl-tRNA synthetase of diadenosine-5,5-P₁,P₄-tetraphosphate, a putative signal in DNA replication.     

Chromosomal assignment of a large tRNA gene cluster (tRNALeu,tRNAGln, tRNALys,tRNAArg, tRNAGly) to 17p13.1

Summary A cluster of tRNA genes (tRNA _UAG ^Leu , tRNA _CUG ^Gln , tRNA _UUU ^Lys , tRNA _UCU ^Arg ) and an adjacent tRNA _GCC ^Gly have been assigned to human chromosome 17p12–p13.1 by in situ hybridization using a 4.2 kb human DNA fragment for tRNA^Leu, tRNA^Gln, tRNA^Lys, tRNA^Arg, and, for tRNA^Gly, 1.3 kb and 0.58 kb human DNA fragments containing these genes as probes. This localization was confirmed and refined to 17p13.100–p13.105 using a somatic cell hybrid mapping panel. Preliminary experiments with the biotiny lated tRNA Leu, Gln, Lys, Arg probe and metaphase spreads from other great apes suggest the presence of a hybridization site on the long arm of gorilla (Gorilla gorilla) chromosome 19 and the short arm of orangutan (Pongo pygmaeus) chromosome 19 providing further support for homology between HSA17, GGO19 and PPY19.     

Cloning, Sequencing and Analysis of the 16S-23S rDNA Intergenic Spacers (IGSs) of Two Strains of Vibrio vulnificus

According to the conserved sequences flanking the 3′ end of the 16S and the 5′ end of the 23S rDNAs, PCR primers were designed, and the 16S-23S rDNA intergenic spacers (IGSs) of two strains of Vibrio vulnificus were amplified by PCR and cloned into pGEM-T vector. Different clones were selected to be sequenced and the sequences were analyzed with BLAST and the software DNAstar. Analyses of the IGS sequences suggested that the strain ZSU006 contains five types of polymorphic 16S-23S rDNA intergenic spacers, namely, IGS^GLAV, IGS^GLV, IGS^lA, IGS^G and IGS^A; while the strain CG021 has the same types of IGSs except lacking IGS^A. Among these five IGS types, IGS^GLAV is the biggest type, including the gene cluster of tRNA^Glu - tRNA^Lys - tRNA^Ala - tRNA^Val; IGS^GLV includes that of tRNA^Glu-tRNA^Lys-tRNA^Val; IGS^AG, tRNA^Ala-tRNA^Glu; IGS^IA, tRNA^Ile-tRNA^Ala; IGS^G, tRNA^Glu and IGS^A, tRNA^Ala. Intraspecies multiple alignment of all the IGS sequences of these two strains with those of V. vulnificus ATCC27562 available at GenBank revealed several highly conserved sequence blocks in the non-coding regions flanking the tRNA genes within all of strains, most notably the first 40 and last 200 nucleotides, which can be targeted to design species-specific PCR primers or detection probes. The structural variations of the 16S-23S rDNA intergenic spacers lay a foundation for developing diagnostic methods for V. vulnificus.     

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.     

Coevolution of Specificity Determinants in Eukaryotic Glutamyl- and Glutaminyl-tRNA Synthetases

The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNA^Gln for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNA^Gln and tRNA^Glu with glutamate. This ancient GluRS also separately differentiated to exclude tRNA^Gln as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but they play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNA^Gln and tRNA^Glu recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p:GluRS complex upon the diauxic shift to respiratory conditions.     

Identification of transfer RNA suppressors in Escherichia coli. II. Duplicate genes for tRNA2Gln.

The number of gene copies for tRNA₂^Gln in λpsu⁺2 was determined by genetic and biochemical studies. The transducing phage stimulates the production of the su⁺2 (amber suppressor) and su°2 glutamine tRNAs and methionine tRNA_m. When the su⁺2 amber suppressor was converted to an ochre suppressor by single-base mutation, the phage stimulated ochre-suppressing tRNA₂^Gln, instead of the amber-suppressing tRNA₂^Gln. From the transducing phage carrying the ochre-suppressing allele, strains carrying both ochre and amber suppressors were readily obtainable. These phages stimulated both ochre-suppressing and amber-suppressing tRNA₂^Gln, but not the non-suppressing form. We conclude that the original transducing phage carries two tRNA₂^Gln genes, one su⁺2 and one su°2. The transducing phage carrying two suppressors, ochre and amber, segregates one-gene derivatives that encode only one or the other type of suppressor tRNA. These derivatives apparently arise by unequal recombination involving the two glutamine tRNA genes in the parental phage. This segregation is not accompanied by the loss of the tRNA_m^Met gene. Based on these results, it is suggested that Escherichia coli normally carries in tandem two identical genes specifying tRNA₂^Gln at 15 minutes on the bacterial chromosome. su⁺2 mutants may arise by single-base mutations in the anticodon region of either of these two, leaving the other intact. By double mutations, tRNA₂^Gln genes could also become ochre suppressors. A tRNA_m^Met gene is located near, but not between, these two tRNA₂^Gln genes.     

Identification of transfer RNA suppressors in Escherichia coli. I. Amber suppressor su+2, an anticodon mutant of tRNA2Gln.

In order to isolate the gene for amber suppressor su⁺2 (SupE) in Escherichia coli, a non-defective su⁺2-transducing phage lambda was isolated in three steps: first, deletion derivatives of F′su⁺2 gal (λ) were selected, linking su⁺2 to the right-hand prophage attachment site, att^λPB′; second, these F′-factors were relysogenized by λ and defective transducing phages, λdsu⁺2, were produced by induction; and third, non-defective λpsu⁺2 transducing phages were produced by recombination of λdsu⁺2 isolates with λ. Upon infection by λpsu⁺2, the production of transferRNAs accepting glutamine and methionine was markedly stimulated. Fingerprint analysis of these tRNAs revealed that they consisted of normal tRNA₂^Gln, mutant tRNA₂^Gln and tRNA_m^Met. The mutant tRNA₂^Gln carried a singlebase alteration from G to A at the 3′-end of the anticodon. The production of tRNA₁^Gln was not stimulated by the infection of λpsu⁺2. We conclude that the wild-type allele of su⁺2 (SupE) is the structural gene for tRNA₂^Gln, and the su⁺2 amber suppressor was derived by a single base mutation, changing the anticodon from CUG to CUA, in one of the multi-copy genes for tRNA₂^Gln. The fact that λpsu⁺2 also induces the production of tRNA_m^Met suggests that this tRNA is encoded in the same chromosomal region of E. coli as is tRNA₂^Gln.     

Gene rearrangements in gekkonid mitochondrial genomes with shuffling,loss, and reassignment of tRNA genes

Background

Vertebrate mitochondrial genomes (mitogenomes) are 16–18 kbp double-stranded circular DNAs that encode a set of 37 genes. The arrangement of these genes and the major noncoding region is relatively conserved through evolution although gene rearrangements have been described for diverse lineages. The tandem duplication-random loss model has been invoked to explain the mechanisms of most mitochondrial gene rearrangements. Previously reported mitogenomic sequences for geckos rarely included gene rearrangements, which we explore in the present study.

Results

We determined seven new mitogenomic sequences from Gekkonidae using a high-throughput sequencing method. The Tropiocolotes tripolitanus mitogenome involves a tandem duplication of the gene block: tRNA^Arg, NADH dehydrogenase subunit 4L, and NADH dehydrogenase subunit 4. One of the duplicate copies for each protein-coding gene may be pseudogenized. A duplicate copy of the tRNA^Arg gene appears to have been converted to a tRNA^Gln gene by a C to T base substitution at the second anticodon position, although this gene may not be fully functional in protein synthesis. The Stenodactylus petrii mitogenome includes several tandem duplications of tRNA^Leu genes, as well as a translocation of the tRNA^Ala gene and a putative origin of light-strand replication within a tRNA gene cluster. Finally, the Uroplatus fimbriatus and U. ebenaui mitogenomes feature the apparent loss of the tRNA^Glu gene from its original position. Uroplatus fimbriatus appears to retain a translocated tRNA^Glu gene adjacent to the 5’ end of the major noncoding region.

Conclusions

The present study describes several new mitochondrial gene rearrangements from Gekkonidae. The loss and reassignment of tRNA genes is not very common in vertebrate mitogenomes and our findings raise new questions as to how missing tRNAs are supplied and if the reassigned tRNA gene is fully functional. These new examples of mitochondrial gene rearrangements in geckos should broaden our understanding of the evolution of mitochondrial gene arrangements.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-930) contains supplementary material, which is available to authorized users.     

Loss of Anticodon Wobble Uridine Modifications Affects tRNALys Function and Protein Levels in Saccharomyces cerevisiae

In eukaryotes, wobble uridines in the anticodons of tRNA^Lys _UUU, tRNA^Glu _UUC and tRNA^Gln _UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm⁵s²U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm⁵ side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm⁵s²U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm⁵U and s²U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm⁵s²U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA^Lys _UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm⁵ and s² wobble uridine modification that could account for growth impairment and mainly originates from tRNA^Lys _UUU hypomodification and malfunction.     

The role of the catalytic domain of E. coli GluRS in tRNA discrimination

Discrimination of tRNA^Gln is an integral function of several bacterial glutamyl-tRNA synthetases (GluRS). The origin of the discrimination is thought to arise from unfavorable interactions between tRNA^Gln and the anticodon-binding domain of GluRS. From experiments on an anticodon-binding domain truncated Escherichia coli (E. coli) GluRS (catalytic domain) and a chimeric protein, constructed from the catalytic domain of E. coli GluRS and the anticodon-binding domain of E. coli glutaminyl-tRNA synthetase (GlnRS), we show that both proteins discriminate against E. coli tRNA^Gln. Our results demonstrate that in addition to the anticodon-binding domain, tRNA^Gln discriminatory elements may be present in the catalytic domain in E. coli GluRS as well.