Mental disorders in diabetic patients with mitochondrial transfer RNA(Leu) (UUR) mutation at position 3243.
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A new mitochondrial point mutation in the transfer RNA(Leu) gene in a patient with a clinical phenotype resembling Kearns-Sayre syndrome.
OBJECTIVE: To report on the molecular identification of a novel heteroplasmic G-to-A transition at mitochondrial DNA position 3249 in transfer RNA(Leu) gene in a patient with a clinical phenotype resembling Kearns-Sayre syndrome. PATIENT AND METHODS: A 34-year-old patient had been suffering for more than 10 years from progressive visual failure, neurosensorial hearing loss, exercise intolerance, muscle weakness, paresthesia in the lower limbs, and difficulties swallowing. Clinical examination revealed generalized muscle wasting, ptosis, external ophthalmoplegia, and ataxia. Ophthalmologic examination showed dystrophic features in the cornea and retina. In skeletal muscle, morphologic and biochemical studies of the respiratory chain complexes were performed. Polymerase chain reaction, single-strand conformation polymorphism, and direct sequencing were used to screen for mutations in the 22 mitochondrial transfer RNA genes. RESULTS: In skeletal muscle, a significantly decreased catalytic activity of complex I was detected by spectrophotometric analysis and numerous cytochrome c oxidase-negative ragged-red fibers were seen on morphologic examination. A G-to-A substitution 3249 (G3249A) mutation was found in the transfer RNA(Leu) gene of the patient and mutant mitochondrial DNA represented 85% of the total in skeletal muscle but only 45% in leukocytes. The mutation was shown to be present in a small fraction in leukocytes from the unaffected mother and to be absent in leukocytes from the healthy sister. CONCLUSIONS: A causal relationship between a heteroplasmic G3249A transfer RNA(Leu) mutation in a patient suffering from progressive external ophthalmoplegia, retinal dystrophy, ataxia, neurosensorial hearing loss, and muscle wasting is postulated. To our knowledge, the G3249A mutation has never previously been described and was not detected in control subjects.
Adult↗
Cochlear histopathology associated with mitochondrial transfer RNA(Leu(UUR)) gene mutation.
Using dot-blot hybridization and Southern blotting, the authors detected a point mutation at nucleotide pair (np) 3243 in mitochondrial DNA from temporal bone sections of a woman with diabetes and deafness. The mutation could not be detected with agarose gel electrophoresis, suggesting that the degree of heteroplasmy is low. Histologically, there was marked degeneration of the stria vascularis and outer hair cells throughout the cochlea, as well as a reduction of spiral ganglion cells in the base. These findings suggest that the mutation affects these inner ear structures preferentially and that deafness can occur even when the proportion of np 3243 mutation is low.
Cochlea↗
Mitochondrial encephalomyopathy with A to G transition of mitochondrial transfer RNA(Leu(UUR)) 3,243 presenting hypertrophic cardiomyopathy.
In a 24-year-old woman with mitochondrial encephalomyopathy presenting hypertrophic cardiomyopathy, microscopical examination of myocardial biopsy specimen disclosed severe vacuolar degeneration of myocardium and aggregates of enlarged mitochondria with proliferated cristae. Limb muscle biopsy specimen showed "ragged-red fibers" light microscopically and enlarged abnormal mitochondria with markedly increased cristae ultrastructurally. Mitochondrial DNA analysis by polymerase chain reaction (PCR) revealed an A-to-G transition in the mitochondrial transfer RNA(Leu)(UUR) gene at nucleotide position 3,243 which is reported to be associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). However, the clinical features of this case, presenting mainly cardiac abnormalities, were not consistent with the typical MELAS.
Adult↗
Auditory findings in patients with maternally inherited diabetes and deafness harboring a point mutation in the mitochondrial transfer RNA(Leu) (UUR) gene.
Five patients with sensorineural hearing loss, who harbored a point mutation in the mitochondrial transfer RNA (tRNA) gene tRNA(Leu) (UUR), from five unrelated family pedigrees were examined. In these families diabetes and deafness were maternally inherited. Bilateral hearing was more severely impaired at higher frequencies. Audiometric test results revealed that hearing loss involved the cochlea. Hearing gradually deteriorated; the progression rate ranged from 1.5 to 7.9 dB per year. Proportion of mutant mitochondrial DNAs (mtDNAs) in the leukocytes was not related to the rate or degree of hearing loss, although hearing loss appeared at a younger age in patients with higher heteroplasmy. We speculate that after the proportion of damaged mtDNAs, mostly as a result of mutation, exceeds the expression threshold for deficiencies in mitochondrial protein synthesis and oxygen consumption, a drop in adenosine triphosphate level could lead to an imbalance of ion concentration, resulting in cell death in the cochlea.
Adult↗
High resolution nuclear magnetic resonance study of base pairing in the native and denaturated conformers of transfer RNA Leu 3 .
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Adenine↗
Investigation on the mitochondrial transfer RNA(Leu)(UUR) in blood cells from patients with cluster headache.
Various mutations in the mitochondrial tRNA(Leu)(UUR) gene give rise to a variety of neurological disorders. Among these, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS syndrome) are frequently associated with a tRNA(Leu)(UUR) mutation at nucleotide position 3243 of the mitochondrial DNA. A supplementary clinical feature seen in these patients is headache in early life. Recently, a tRNA(Leu)(UUR) mutation at nucleotide position 3243 has been found in a patient presenting with cluster headache. This led us to examine the mitochondrial genomes of 22 patients presenting with cluster headache. None of the patients harboured the reported tRNA(Leu)(UUR) mutation or any other length variations of the mtDNA. Cluster headache is most likely not causally associated with the A3243G mutation of the mitochondrial DNA.
Cluster Headache↗
Mitochondrial DNA transfer RNA mutation Leu(UUR)A-->G 3260: a second family with myopathy and cardiomyopathy.
A family with maternally inherited myopathy and cardiomyopathy is described. Mitochondrial DNA analysis showed a heteroplasmic point mutation at position 3260 in the leucine transfer RNA gene, previously reported in a large Italian family with a similar phenotype. This observation confirms pathogenicity of this mutation and suggests phenotypic specificity.
Adult↗
Two transfer RNA sequences abut the large ribosomal RNA gene in Tetrahymena mitochondrial DNA: tRNA(leu) (anticodon UAA) and tRNA(met) (anticodon CAU).
The sequence of a 1,427 base pair restriction fragment, HaeIII fragment 6, of the ciliate protozoan Tetrahymena mitochondrial DNA, is presented. The first 780 nucleotide sequence aligns well with the terminal segment of the large rDNA sequence of Paramecium mitochondria. Immediately abutting this rDNA termination sequence, a tRNA sequence was found with anticodon UAA for leucine. The derived tRNA sequence is 81 bases long without the 3' CCA end, has a high G + C content of 48.1%, and can be folded into a normal cloverleaf structure with mostly conserved bases and normal stems and loops. The tRNA sequence found at an analogous position of the Paramecium mitochondrial DNA is tRNA(tyr). Following a highly A + T rich sequence of 300 base pairs, another tRNA-like sequence is present; this putative tRNA has only 67 bases with anticodon CAT (Met) and forms standard aminoacyl, anticodon and T psi C stems with a conventional T psi C loop. However, the DHU loop and stem are unusually short and irregular; the base at position 8 is G instead of T; and the base following the anticodon, which is normally a purine, is T. The significance of these tRNA structures is discussed.
Animals↗
The exchange of the discriminator base A73 for G is alone sufficient to convert human tRNA(Leu) into a serine-acceptor in vitro.
Transfer RNA (tRNA) identify is maintained by the highly specific interaction of a few defined nucleotides or groups of nucleotides, called identity elements, with the cognate aminoacyl-tRNA synthetase, and by nonproductive interactions with the other 19 aminoacyl-tRNA synthetases. Most tRNAs have a set of identity elements in at least two locations, commonly in the anticodon loop or in the acceptor stem, and at the discriminator base position 73. We have used T7 RNA polymerase transcribed tRNAs to demonstrate that the sole replacement of the discriminator base A73 of human tRNA(Leu) with the tRNA(Ser)-specific G generates a complete identity switch to serine acceptance. The reverse experiment, the exchange of G73 in human tRNA(Ser) for the tRNA(Leu-specific A, causes a total loss of serine specificity without creating any leucine acceptance. These results suggest that the discriminator base A73 of human tRNA(Leu) alone protects this tRNA against serylation by seryl-tRNA synthetase. This is the first report of a complete identity switch caused by an exchange of the discriminator base alone.
Acylation↗
Gene for cytochrome c oxidase subunit II in the mitochondrial DNA of Culex quinquefasciatus and Aedes aegypti (Diptera: Culicidae).
The cytochrome c oxidase subunit II (COII) gene between transfer RNA for Leu and Lys in the mitochondrial DNA of Culex quinquefasciatus Say and Aedes aegypti (L.) was amplified by the polymerase chain reaction (PCR) technique. Both the gene order and direction of transcription were identical to Anopheles and Drosophila. Nucleotide sequences of the PCR-amplified COII genes in these two mosquitoes exhibited 88% homology, and the frequency of transition was very close to that of transversion. The homology of deduced amino acid sequences of COII between these two mosquitoes was 95%. Two highly conserved segments of COII proteins were found in mosquitoes, fruit flies, locust, and honeybee. These segments contain the major amino acid residues of cytochrome c oxidase involved in electron transport and ligand binding. The amino acid residues are located at the positions similar to those of the mammalian enzymes. Two sets of the phylogenetic trees of a similar pattern were generated by comparing the divergences of nucleotide and amino acid sequences of COII. The branch lengths of the trees estimated by amino acid and nucleotide sequences showed different evolution rates of Aedes and Culex from their common ancestor.
Aedes↗
The pathogenic U3271C human mitochondrial tRNA(Leu(UUR)) mutation disrupts a fragile anticodon stem.
The U3271C mutation affecting the human mitochondrial transfer RNA(Leu(UUR)) (hs mt tRNA) is correlated with diabetes and mitochondrial encephalopathies. We have explored the relationship between the structural effects of this mutation and its impact on function using chemical probing experiments and in vitro aminoacylation assays to investigate a series of tRNA constructs. Chemical probing experiments indicate that the U3271C substitution, which replaces an AU pair with a CA mispair, significantly destabilizes the anticodon stem. The introduction of a compensatory A3261G mutation reintroduces base pairing at this site and restores the structure of this domain. In fact, the anticodon stem of the A3261G/U3271C mutant appears more structured than wild-type (WT) hs mt tRNA(Leu(UUR)), indicating that the entirely AU stem of the native tRNA is intrinsically weak. The results of the chemical probing experiments are mirrored in the aminoacylation activities of the mutants. The U3271C substitution decreases aminoacylation reactivity relative to the WT tRNA due to an increase in K(m) for the pathogenic mutant. The binding defect is a direct result of the structural disruption caused by the pathogenic mutation, as the introduction of the stabilizing compensatory mutation restores aminoacylation activity. Other examples of functional defects associated with the disruption of weak domains in hs mt tRNAs have been reported, indicating that the effects of pathogenic mutations may be amplified by the fragile structures that are characteristic of this class of tRNAs.
Anticodon↗
Participation of the intron in the reaction catalyzed by the Xenopus tRNA splicing endonuclease.
Introns have generally been assumed to be passive in the transfer RNA splicing reaction. Experiments have now been done showing that the endonuclease is able to cut a precursor provided that a base in the single-stranded loop of the intron can pair with the base of the 5' exon situated at the position that immediately follows the anticodon stem (position 33 in the yeast tRNA isoacceptor pre-tRNA(Leu)3, position 32 in yeast pre-tRNA(Phe)). The elucidation of the role of the intron reveals that in addition to the conserved bases, there are positions in the mature domain which, although not necessarily occupied by the same base in all pre-tRNA's, nevertheless have a fundamental role in the splicing reaction. These positions are termed cardinal positions.
Animals↗
New genes for tRNA(Phe), tRNA(Leu), and tRNA(Thr) from Saccharomyces cerevisiae.
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Base Sequence↗
Only one nucleotide insertion to the long variable arm confers an efficient serine acceptor activity upon Saccharomyces cerevisiae tRNA(Leu) in vitro.
Several tRNA species have a long variable arm composed of over ten nucleotides, which are relevant to those specific to serine, leucine and tyrosine in prokaryotes, while there are only serine and leucine-specific tRNAs in eukaryotes. To clarify the evolutionary aspects of the identity determination mechanism of these tRNAs, the tRNA(Ser) recognition in Saccharomyces cerevisiae was studied. Unmodified tRNA(Leu) transcript had serylation ability of low efficiency, but native tRNA(Leu) did not, indicating that some modification of tRNA(Leu) serves as a negative identity determinant for seryl-tRNA synthetase. Changing the discriminator base did not seriously affect the serine accepting efficiency. The tRNA(Leu) transcript possessing the variable arm of tRNA(Ser) was efficiently aminoacylated with serine. Eventually, it was found that only one nucleotide insertion to the variable arm of tRNA(Leu) was sufficient to confer an efficient serine accepting activity. The mode of serine tRNA recognition is similar to that in Escherichia coli in that the end of the long variable arm, but not the anticodon or discriminator base, is important. However, S. cerevisiae seryl-tRNA synthetase adopts a substantially different mechanism for rejection of tRNA(Leu) from that of its E. coli counterpart.
Base Sequence↗
The discriminator bases G73 in human tRNA(Ser) and A73 in tRNA(Leu) have significantly different roles in the recognition of aminoacyl-tRNA synthetases.
The recognition of human tRNA(Leu) or tRNA(Ser) by cognate aminoacyl- tRNA synthetases has distinct requirements. Only one base change (A73-->G) in tRNA(Leu) is required to generate an efficient serine acceptor in vitro, whereas several changes in three structural domains (the acceptor stem, DHU loop and long extra arm) of tRNA(Ser) are necessary in order to produce a leucine acceptor. Hence, the molecular basis for the discrimination between human tRNA(Ser) and tRNA(Leu) by the seryl-tRNA synthetase depends almost exclusively on a highly specific recognition of the discriminator base G73. In order to elucidate the specific role of the functional groups of this base in discrimination, tRNA(Ser) constructs were made which contain the artificial base analogues 2-aminopurine riboside or inosine at the discriminator position 73. Aminoacylation of these constructs by a HeLa S100 extract showed that molecules with 2-aminopurine riboside, but not with inosine, in position 73 could be serylated at low efficiency. However, the 2-aminopurine riboside and the inosine derivatives of tRNA(Ser) were equally efficient competitive inhibitors of serylation, whereas tRNAs(Ser) with any other natural base at position 73 did not competitively inhibit serylation of tRNA(Ser). This was in contrast to leucylation of tRNA(Leu), where tRNA(Leu) transcripts with any other nucleotide in the discriminator position acted as strong competitive inhibitors. These results suggest that the discriminator bases in human tRNA(Ser) and tRNA(Leu) play completely different roles in recognition of the tRNAs by their cognate aminoacyl-tRNA synthetases.
Amino Acyl-tRNA Synthetases↗
Deficiency of 1-methylguanosine in tRNA from Salmonella typhimurium induces frameshifting by quadruplet translocation.
The trmD gene encodes the tRNA(m1G37)methyltransferase, which methylates guanosine (G) to 1-methylguanosine (m1G) at position 37 of tRNAs that read CUN (leucine), CCN (proline), and CGG (arginine) codons. A mutant, trmD3, has previously been isolated, which at high temperature lacks m1G in tRNA, and this deficiency was correlated with a +1 frameshifting activity. In this study, the mechanism of this trmD3-induced frameshift involving mutant tRNA(Pro) and tRNA(Leu) species has been investigated. Potential frameshifting sites for proline tRNAs, CCC-N, were efficiently suppressed in the mutant strain. Hybrid beta-galactosidases encoded by plasmid constructs containing the sites CCC-U and CCC-A were subjected to amino-terminal sequencing. The protein sequences demonstrated that a quadruplet translocation had occurred and that a proline was inserted at these sites, suggesting that a tRNA(Pro) deficient in m1G is the frameshifting agent. Therefore, a mechanism involving a quadruplet codon-anticodon interaction is favoured for trmD3-dependent +1 frameshifting. Of the four potential sites for tRNA(Leu) (CCU-N), two, CCU-U and CCU-C, were significantly suppressed in the trmD3 mutant. Thus, species of tRNA(Leu) may also act as +1 frameshift suppressors. No -1 frameshifting activity was found with the trmD3 mutant.
Amino Acid Sequence↗
Recognition nucleotides of Escherichia coli tRNA(Leu) and its elements facilitating discrimination from tRNASer and tRNA(Tyr).
In order to study how Escherichia coli leucyl-tRNA synthetase recognizes tRNA(Leu) and discriminates it from the other two class II tRNAs, tRNA(Ser) and tRNA(Tyr), various mutations were introduced into class II tRNA transcripts. The discriminator base A73, but not the anticodon sequence, was found to serve as a critical recognition element of tRNA(Leu). A base substitution at the invariant nucleotide A14, but not at any of the other nucleotides characteristic of the E. coli tRNA(Leu) isoacceptors among the three class II tRNAs, caused significantly damaged aminoacylation with leucine. A two base-pair deletion in the long variable arm also resulted in no significant decrease of activity. Transplanting the three tertiary elements characteristic of E. coli tRNA(Leu) (i.e. the location of the G18G19 sequence in the D-loop, the A15 U48 base-pair and the stem pairing pattern of the long variable arm) besides the discriminator base change introduced the leucine charging activity in terms of Vmax/Km, up to 0.1 of that for the normal sequence of tRNA(Leu) into both tRNA(Ser) and tRNA(Tyr). These results indicate that A73 and A14 (or its vicinity) are involved in recognition by leucyl-tRNA synthetase, and that several tertiary elements play a significant role in the discrimination of tRNA(Leu) from the other two class II tRNAs.
Base Sequence↗