Mitochondrial tRNA Genes Research Articles

Aberrant Mitochondrial tRNA Genes Appear Frequently in Animal Evolution.

Mitochondrial tRNAs have acquired a diverse portfolio of aberrant structures throughout metazoan evolution. With the availability of more than 12,500 mitogenome sequences, it is essential to compile a comprehensive overview of the pattern changes with regard to mt-tRNA repertoire and structural variations. This, of course, requires reanalysis of the sequence data of more than 250,000 mt-tRNAs with a uniform workflow. Here, we report our results on the complete reannotation of all mitogenomes available in the RefSeq database by September 2022 using mitos2. Based on the individual cases of mt-tRNA variants reported throughout the literature, our data pinpoint the respective hotspots of change, i.e. Acanthocephala (Lophotrochozoa), Nematoda, Acariformes and Araneae (Arthropoda). Less dramatic deviations of mt-tRNAs from the norm are observed throughout many other clades. Loss of arms in animal mt-tRNA clearly is a phenomenon that occurred independently many times, not limited to a small number of specific clades. The summary data here provide a starting point for systematic investigations into the detailed evolutionary processes of structural reduction and and loss of mt-tRNAs as well as a resource for further improvements of annotation workflows for mt-tRNA annotation.

Quantifying constraint in the human mitochondrial genome.

Mitochondrial DNA (mtDNA) has an important yet often overlooked role in health and disease. Constraint models quantify the removal of deleterious variation from the population by selection and represent powerful tools for identifying genetic variation that underlies human phenotypes1-4. However, nuclear constraint models are not applicable to mtDNA, owing to its distinct features. Here we describe the development of a mitochondrial genome constraint model and its application to the Genome Aggregation Database (gnomAD), a large-scale population dataset that reports mtDNA variation across 56,434 human participants5. Specifically, we analyse constraint by comparing the observed variation in gnomAD to that expected under neutrality, which was calculated using a mtDNA mutational model and observed maximum heteroplasmy-level data. Our results highlight strong depletion of expected variation, which suggests that many deleterious mtDNA variants remain undetected. To aid their discovery, we compute constraint metrics for every mitochondrial protein, tRNA and rRNA gene, which revealed a range of intolerance to variation. We further characterize the most constrained regions within genes through regional constraint and identify the most constrained sites within the entire mitochondrial genome through local constraint, which showed enrichment of pathogenic variation. Constraint also clustered in three-dimensional structures, which provided insight into functionally important domains and their disease relevance. Notably, we identify constraint at often overlooked sites, including in rRNA and noncoding regions. Last, we demonstrate that these metrics can improve the discovery of deleterious variation that underlies rare and common phenotypes.

Mitochondrial tRNAGlu 14693A > G Mutation, an "Enhancer" to the Phenotypic Expression of Leber's Hereditary Optic Neuropathy.

Leber's hereditary optic neuropathy (LHON), a maternally inherited ocular disease, is predominantly caused by mitochondrial DNA (mtDNA) mutations. Mitochondrial tRNA variants are hypothesized to amplify the pathogenic impact of three primary mutations. However, the exact mechanisms remained unclear. In the present study, the synergistic effect of the tRNAGlu 14693A > G and ND6 14484T > C mutations in three Chinese families affected by LHON is investigated. The m.14693A > G mutation nearly abolishes the pseudouridinylation at position 55 of tRNAGlu, leading to structural abnormalities, decreased stability, aberrant mitochondrial protein synthesis, and increased autophagy. In contrast, the ND6 14484T > C mutation predominantly impairs complex I function, resulting in heightened apoptosis and virtually no induction of mitochondrial autophagy compared to control cell lines. The presence of dual mutations in the same cell lines exhibited a coexistence of both upregulated cellular stress responses to mitochondrial damage, indicating a scenario of autophagy and mutation dysregulation within these dual-mutant cell lines. The data proposes a novel hypothesis that mitochondrial tRNA gene mutations generally lead to increased mitochondrial autophagy, while mutations in genes encoding mitochondrial proteins typically induce apoptosis, shedding light on the intricate interplay between different genetic factors in the manifestation of LHON.

Functional analysis of the novel mitochondrial tRNATrp and tRNASer(AGY) variants associated with type 2 diabetes mellitus.

Mutations in mitochondrial tRNA (mt-tRNA) genes that result in mitochondrial dysfunction play important roles in type 2 diabetes mellitus (T2DM). We pre-viously reported a large Chinese pedigree with maternally inherited T2DM that harbors novel mt-tRNA Trp A5514G and tRNA Ser(AGY) C12237T variants, however, the effects of these mt-tRNA variants on T2DM progression are largely unknown. To assess the potential pathogenicity of T2DM-associated m.A5514G and m.C12237T variants at genetic, molecular, and biochemical levels. Cytoplasmic hybrid (cybrid) cells carrying both m.A5514G and m.C12237T variants, and healthy control cells without these mitochondrial DNA (mtDNA) variants were generated using trans-mitochondrial technology. Mitochondrial features, including mt-tRNA steady-state level, levels of adenosine triphosphate (ATP), mitochondrial membrane potential (MMP), reactive oxygen species (ROS), mtDNA copy number, nicotinamide adenine dinucleotide (NAD+)/NADH ratio, enzymatic activities of respiratory chain complexes (RCCs), 8-hydroxy-deo-xyguanine (8-OhdG), malondialdehyde (MDA), and superoxide dismutase (SOD) were examined in cell lines with and without these mt-tRNA variants. Compared with control cells, the m.A5514G variant caused an approximately 35% reduction in the steady-state level of mt-tRNA Trp (P < 0.0001); however, the m.C12237T variant did not affect the mt-tRNA Ser(AGY) steady-state level (P = 0.5849). Biochemical analysis revealed that cells with both m.A5514G and m.C12237T variants exhibited more severe mitochondrial dysfunctions and elevated oxidative stress than control cells: ATP, MMP, NAD+/NADH ratio, enzyme activities of RCCs and SOD levels were markedly decreased in mutant cells (P < 0.05 for all measures). By contrast, the levels of ROS, 8-OhdG and MDA were significantly increased (P < 0.05 for all measures), but mtDNA copy number was not affected by m.A5514G and m.C12237T variants (P = 0.5942). The m.A5514G variant impaired mt-tRNA Trp metabolism, which subsequently caused mitochondrial dysfunction. The m.C12237T variant did not alter the steady-state level of mt-tRNA Ser(AGY), indicating that it may be a modifier of the m.A5514G variant. The m.A5514G variant may exacerbate the pathogenesis and progression of T2DM in this Chinese pedigree.

Studies on the Mitochondrial tRNA Gene Variants in Patients with Coronary Artery Disease and Pathogenicity Assessment by in silico Analysis

Studies on the Mitochondrial tRNA Gene Variants in Patients with Coronary Artery Disease and Pathogenicity Assessment by in silico Analysis

Taxonomic review of the Calotesversicolor complex (Agamidae, Sauria, Squamata) in China, with description of a new species and subspecies.

Caloteswangisp. nov., a new species of the agamid genus Calotes Cuvier, 1817, from southern China and northern Vietnam, is described. This species can be distinguished from all known congeners by a combination of morphological characteristics and genetic divergence in the mitochondrial tRNA, ND2, and CO1 genes. Molecular phylogenetic analysis revealed that the new species was formed as a monophyletic group and that considerable genetic divergence existed between its congeners (minimum p-distance, 4.6%). Caloteswangisp. nov. is distinguished by a combination of the following characteristics: average SVL < 90 mm for adult males; 10-14 dorsal eyelid scales; scales on side of neck and adjacent shoulder area pointing obliquely upward; keels on neck scales weakly to strongly developed; fold in front of the shoulder absent; pair of dark triangular patches extending from the front of the shoulder to the jaw angles; and orange coloration of the tongue. Caloteswangisp. nov. is similar to C.irawadi but differs in having scales between the nasal shield and the orbit and a fourth toe with a claw that can reach between the eyes and tympanum (even to the snout when hind the limbs are adpressed forward). Phylogenetic analyses revealed two well-supported subspecies, Lineages A and B in C.wangisp. nov., with mean uncorrected p-distances between them of 2%. We propose that Lineage A, which is mainly from the central and southern Wuzhi Mountains on Hainan Island, is a subspecies, C.w.hainanensisssp. nov. Lineage B mainly comprises individuals from other sites on the island plus the adjacent mainland, and is described as subspecies, C.w.wangissp. nov. A diagnostic key to all Calotes species of China is also provided.

Detection of mtDNA Mutations Using the Amplification Refractory Mutation System-Polymerase Chain Reaction Method in Type II Diabetes Mellitus Patients in Publich Health Center Poasia

Background: Diabetes Mellitus (DM) is a heterogeneous disease caused by hereditary and ecological factors. One form of diabetes mellitus that is related to genetic factors is type 2 DM. type 2 diabetec melitus caused by dysfunction of insulin secretion, due to the presence of inhibition in the production of Adenosine Triphosphate (ATP) necessary in the process of its secretion by cells β glands of the pancreas. The dysfunction is related to the mutation of A to G at the 3243rd nucleotide position of the mitochondrial DNA tRNA gene. The purpose of the study was to find out whether there was a heteroplasmic mtDNA mutation in the genes of respondents to type 2 diabetes mellitus using the ARMS-PCR method. Methods: The type of research used is descriptive research, the sample used in this study is a blood sample in respondents of type 2 diabetes mellitus. The method chosen in this study is the Amplification Refractory Mutation System (ARMS)-PCR Method which is a PCR application that uses a specific primer. Results: According to this study, 20 of the 20 samples evaluated using the ARMS-PCR method were positive for the heteroplasmic A3243G mutation, which is defined by the presence of DNA bands measuring 200 bp on both tubes. These pathogenic mutations are inherited maternally and can cause a variety of disorders. Conclusion: After analyzing all samples, this study has concluded that they all contain hetero plasma mutations. The suggestions provided in this study are expected to be useful for future researchers who employ samples from other public health centers and hospitals.

Conserved spatiotemporal expression landscape of dominant tRNA genes in human and mouse

Transfer RNAs are integral for protein synthesis and the interpretation of the information contained in DNA. To date, a few methods, including custom microarrays and custom targeted sequencing, have been used to quantify tRNA. However, methods using available RNA-sequencing data have not yet been reported. We created a bioinformatics pipeline to quantify the highly expressed tRNAs in RNA-Seq effectively, demonstrated by the preserved ratio of the expression levels of two massively duplicated tRNAAla genes in mouse. Using this quantification, we examined the tRNA expression with relation to tissue type and developmental stage in both human and mouse. Heart exhibited the highest overall tRNA expression for both human and mouse. Furthermore, tRNA expression grew to a peak before decreasing steadily with developmental stage, a trend that was conserved in both human and mouse. The two mitochondrial tRNA genes, tRNASer(TCA)(m) and tRNALeu(TTA)(m), which partly contribute to these trends, have been attributed to various human diseases. The tissue-specific high expression of tRNAGln(CAG) and tRNAGln(CAA) in human brains, especially in hindbrain and cerebellum, suggests their important roles in neurological disorders. In summary, our approach revealed conserved spatiotemporal expression of highly expressed tRNAs in both human and mouse. Our method can be applied to other RNA-Seq data to examine the roles of these tRNAs in different human diseases or scientific studies.

Mitochondrial impairment and synaptic dysfunction are associated with neurological defects in iPSCs-derived cortical neurons of MERRF patients

BackgroundMyoclonic epilepsy with ragged-red fibers (MERRF) syndrome is a rare inherited mitochondrial disease mainly caused by the m.8344A > G mutation in mitochondrial tRNALys gene, and usually manifested as complex neurological disorders and muscle weakness. Currently, the pathogenic mechanism of this disease has not yet been resolved, and there is no effective therapy for MERRF syndrome. In this study, MERRF patients-derived iPSCs were used to model patient-specific neurons for investigation of the pathogenic mechanism of neurological disorders in mitochondrial disease.MethodsMERRF patient-derived iPSCs were differentiated into excitatory glutamatergic neurons to unravel the effects of the m.8344A > G mutation on mitochondrial bioenergetic function, neural-lineage differentiation and neuronal function. By the well-established differentiation protocol and electrophysiological activity assay platform, we examined the pathophysiological behaviors in cortical neurons of MERRF patients.ResultsWe have successfully established the iPSCs-derived neural progenitor cells and cortical-like neurons of patients with MERRF syndrome that retained the heteroplasmy of the m.8344A > G mutation from the patients’ skin fibroblasts and exhibited the phenotype of the mitochondrial disease. MERRF neural cells harboring the m.8344A > G mutation exhibited impaired mitochondrial bioenergetic function, elevated ROS levels and imbalanced expression of antioxidant enzymes. Our findings indicate that neural immaturity and synaptic protein loss led to the impairment of neuronal activity and plasticity in MERRF neurons harboring the m.8344A > G mutation. By electrophysiological recordings, we monitored the in vivo neuronal behaviors of MERRF neurons and found that neurons harboring a high level of the m.8344A > G mutation exhibited impairment of the spontaneous and evoked potential-stimulated neuronal activities.ConclusionsWe demonstrated for the first time the link of mitochondrial impairment and synaptic dysfunction to neurological defects through impeding synaptic plasticity in excitatory neurons derived from iPSCs of MERRF patients harboring the m.8344A > G mutation. This study has provided new insight into the pathogenic mechanism of the tRNALys gene mutation of mtDNA, which is useful for the development of a patient-specific iPSCs platform for disease modeling and screening of new drugs to treat patients with MERRF syndrome.

Rewiring of Aminoacyl-tRNA Synthetase Localization and Interactions in Plants With Extensive Mitochondrial tRNA Gene Loss.

The number of tRNAs encoded in plant mitochondrial genomes varies considerably. Ongoing loss of bacterial-like mitochondrial tRNA genes in many lineages necessitates the import of nuclear-encoded counterparts that share little sequence similarity. Because tRNAs are involved in highly specific molecular interactions, this replacement process raises questions about the identity and trafficking of enzymes necessary for the maturation and function of newly imported tRNAs. In particular, the aminoacyl-tRNA synthetases (aaRSs) that charge tRNAs are usually divided into distinct classes that specialize on either organellar (mitochondrial and plastid) or nuclear-encoded (cytosolic) tRNAs. Here, we investigate the evolution of aaRS subcellular localization in a plant lineage (Sileneae) that has experienced extensive and rapid mitochondrial tRNA loss. By analyzing full-length mRNA transcripts (PacBio Iso-Seq), we found predicted retargeting of many ancestrally cytosolic aaRSs to the mitochondrion and confirmed these results with colocalization microscopy assays. However, we also found cases where aaRS localization does not appear to change despite functional tRNA replacement, suggesting evolution of novel interactions and charging relationships. Therefore, the history of repeated tRNA replacement in Sileneae mitochondria reveals that differing constraints on tRNA/aaRS interactions may determine which of these alternative coevolutionary paths is used to maintain organellar translation in plant cells.

Deep exome sequencing identifies enrichment of deleterious mosaic variants in neurodevelopmental disorder genes and mitochondrial tRNA regions in bipolar disorder.

Bipolar disorder (BD) is a global medical issue, afflicting around 1% of the population with manic and depressive episodes. Despite various genetic studies, the genetic architecture and pathogenesis of BD have not been fully resolved. Besides germline variants, postzygotic mosaic variants are proposed as new candidate mechanisms contributing to BD. Here, we performed extensive deep exome sequencing (DES, ~300×) and validation experiments to investigate the roles of mosaic variants in BD with 235 BD cases (194 probands of trios and 41 single cases) and 39 controls. We found an enrichment of developmental disorder (DD) genes in the genes hit by deleterious mosaic variants in BD (P = 0.000552), including a ClinVar-registered pathogenic variant in ARID2. An enrichment of deleterious mosaic variants was also observed for autism spectrum disorder (ASD) genes (P = 0.000428). The proteins coded by the DD/ASD genes with non-synonymous mosaic variants in BD form more protein-protein interaction than expected, suggesting molecular mechanisms shared with DD/ASD but restricted to a subset of cells in BD. We also found significant enrichment of mitochondrial heteroplasmic variants, another class of mosaic variants, in mitochondrial tRNA genes in BD (P = 0.0102). Among them, recurrent m.3243 A > G variants known as causal for mitochondrial diseases were found in two unrelated BD probands with allele fractions of 5-12%, lower than in mitochondrial diseases. Despite the limitation of using peripheral tissues, our DES investigation supports the possible contribution of deleterious mosaic variants in the nuclear genome responsible for severer phenotypes, such as DD/ASD, to the risk of BD and further demonstrates that the same paradigm can be applied to the mitochondrial genome. These results, as well as the enrichment of heteroplasmic mitochondrial tRNA variants in BD, add a new piece to the understanding of the genetic architecture of BD and provide general insights into the pathological roles of mosaic variants in human diseases.

Abstract 228: Recurrent mitochondrial mutations in thyroid cancer

Abstract Despite the importance of mitochondrial function on the bioenergetics and metabolism of the cell, understanding the functional impact of somatic mitochondrial mutations has been difficult. Additionally, small cohorts, as used in previous studies, lack the power to estimate the prevalence of specific recurrent mutations. We evaluated 177540 advanced cancer patients, across 84 different tumor types, who were sequenced as part of routine clinical care on the FoundationOne®CDx platform. Off-target sequencing reads were mapped to the mitochondrial genome (rCRS) and variant calling was performed. We had a median read depth of 18 and filtering by allele frequency (&amp;lt;= 95% and &amp;gt;= 5%) led to the identification almost 400,000 predicted somatic mitochondrial variants. Over 80% of patients had at least one mitochondrial variant detected, with an average of 2.2 mitochondrial variants detected per patient. We next assessed each recurrent mitochondrial short variant to look for associations with specific cancers. The alteration with the strongest enrichment was G3224A, a mutation in the mitochondrial leucine tRNA gene (MT-TL1), which had a prevalence of 3.6% (51/1426) in thyroid cancer samples, as compared to 0.05% (85/176114) in all other disease types (OR=76.8, P&amp;lt;2.2e-16). The G3244A mutation has been previously identified as occurring in thyroid cancer, specifically Hurthle cell carcinomas, as well as in the mitochondrial disease MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Thyroid cancer samples in our dataset with G3244A were found to be associated with thyroid follicular carcinomas (7/124, 5.6%) and Hurthle cell carcinomas (7/57, 12.2%). Additional recurrent MT-TL1 mutations were identified in the thyroid cancer cohort at positions 3242 (9/1426) and 3248 (5/1426). We also identified a mutation in the NADH dehydrogenase subunit MT-ND1. G3380A has been previously shown to occur in thyroid and colorectal cancer and results in a R25Q mutation that is thought to affect ND1 function. G3380A was identified in 26/1426 (1.8%) of thyroid cancer patients. The majority of G3380A variants occurred in thyroid anaplastic carcinomas 23/26 (88.5%). Our dataset has allowed us to identify and determine the prevalence of rare mitochondrial short variant alterations in cancer. Further research is needed to investigate associations of these alterations with other clinical features. Citation Format: Julia F. Hopkins, Zoe Fleischmann, Dexter X. Jin, Jonathan Keith Killian, Meagan Montesion. Recurrent mitochondrial mutations in thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 228.

A Multisystem Mitochondrial Disease Caused by a Novel MT-TL1 mtDNA Variant: A Case Report.

Mitochondrial tRNA (MTT) genes are hotspot for mitochondrial DNA mutation and are responsible of half mitochondrial disease. MTT mutations are associated with a broad spectrum of phenotype often with complex multisystem involvement and complex genotype-phenotype correlations. MT-TL1 mutations, among which the m.3243A>G mutation is the most frequent, are associated with myopathy, maternal inherited diabetes and deafness, MELAS, cardiomyopathy, and focal segmental glomerulosclerosis. Here we report the case of an Italian 49-years old female presenting with encephalomyopathy, chronic proteinuric kidney disease and a new heteroplasmic m.3274_3275delAC MT-TL1 gene mutation. Our case demonstrates a systemic mitochondrial disease caused by the heteroplasmic m.3274_3275delAC MT-TL1 gene mutation, not yet described in the literature. A mitochondrial disease should be suspected in case of complex multisystem phenotypes, including steroid-resistant nephrotic syndrome with multisystemic involvement.

Comparative analysis of tRNA genes in the mitochondrial genome of Parasitidae (Parasitiformes: Mesostigmata)

ABSTRACT The mitochondrial genome of Parasitus fimetorum was sequenced to study the Parasitidae tRNA gene secondary structure in Parasitiformes. tRNAscan-SE and ARWEN identified and inferred the secondary structures of 22 tRNA genes of Parasitus fimetorum, which were compared to 22 tRNA genes in sequenced Parasitidae mitochondrial genomes. The results showed that most tRNA genes of Parasitidae had a typical cloverleaf secondary structure; the base substitutions or mismatches occurred during the folding process, and the stem region showed less base variation than the loop region, showing high conservation. The base composition showed a clear preference for A + T. Relative synonymous codon usage (RSCU) analysis showed that the third codon of tRNA genes in Parasitidae species preferred to use codons that ended with A or U. ENC-plot analysis showed that most genes were distributed under the standard curve, indicating that codon usage in Parasitidae species was more influenced by natural selection pressure. Phylogenetic analysis showed that Parasitidae is a monophyletic group. This study complements the research on mitochondrial tRNA genes in Parasitidae, which has far-reaching implications for understanding the importance of tRNA gene sequences and secondary structures in comparative genomics and facilitates further studies on the evolution of species in the Parasitidae.

The Evolutionary Dynamics of the Mitochondrial tRNA in the Cichlid Fish Family.

Simple SummaryCichlids are a unique example of fish diversity and species richness which have been explained by sympatric speciation at different freshwater sources in Africa. The mitochondria contribute to cell vitality by providing energy. It contains a circular genome with an established translation system that is spatially independent of the cytosolic counterpart. The current study aimed to investigate the evolutionary dynamics of the mitochondrial tRNA and its role in cichlids’ diversity. The available cichlid mitogenomes in the public database were filtered, in addition to newly sequenced accessions from a specific cichlid group known as the haplotilapiine lineage that is widely distributed in the Egyptian sector of the Nile River. Based on the comparative analysis of mitogenomic data, we identified 22 tRNA genes, in which a single gene was D-armless, while the cloverleaf secondary structure subdivided into stem-loop formations was predicted and used to define the levels of genetic divergence for the remained tRNAs. Peculiarly, in cichlids, the formation known as “T-arm” showed the lowest polymorphism levels among other structures in contrast to other organisms (e.g., scorpions). Comparing the whole family to the specific haplotilapiine lineage showed that the tryptophan tRNA was the most conserved tRNA, with signatures of possible purifying selection.The mitochondrial transfer RNA genes (tRNAs) attract more attention due to their highly dynamic and rapidly evolving nature. The current study aimed to detect and evaluate the dynamics, characteristic patterns, and variations of mitochondrial tRNAs. The study was conducted in two main parts: first, the published mitogenomic sequences of cichlids mt tRNAs have been filtered. Second, the filtered mitochondrial tRNA and additional new mitogenomes representing the most prevalent Egyptian tilapiine were compared and analyzed. Our results revealed that all 22 tRNAs of cichlids folded into a classical cloverleaf secondary structure with four domains, except for trnSGCU, missing the D domain in all cichlids. When consensus tRNAs were compared, most of the mutations were observed in the trnP at nucleotide levels (substitutions and indels), in contrast to trnLUAA. From a structural perspective, the anticodon loop and T-loop formations were the most conserved structures among all parts of the tRNA in contrast to the A-stem and D-loop formations. The trnW was the lowest polymorphic unneutral tRNA among all cichlids (both the family and the haplotilapiine lineage), in contrast with the neutral trnD that was extremely polymorphic among and within the haplotilapiine lineage species compared to other cichlids species. From a phylogenetic perspective, the trnC was extremely hypervariable and neutral tRNA in both haplotilapiine lineage and cichlids but was unable to report correct phylogenetic signal for the cichlids. In contrast to trnI and trnY, less variable neutral tRNAs that were able to cluster the haplotilapiine lineage and cichlids species as previously reported. By observing the DNA polymorphism in the coding DNA sequences (CDS), the highest affected amino acid by non-synonymous mutations was isoleucine and was equally mutated to valine and vice versa; no correlation between mutations in CDS and tRNAs was statistically found. The current study provides an insight into the mitochondrial tRNA evolution and its effect on the cichlid diversity and speciation model at the maternal level.

Aberrant RNA processing contributes to the pathogenesis of mitochondrial diseases in trans-mitochondrial mouse model carrying mitochondrial tRNALeu(UUR) with a pathogenic A2748G mutation.

Mitochondrial tRNAs are indispensable for the intra-mitochondrial translation of genes related to respiratory subunits, and mutations in mitochondrial tRNA genes have been identified in various disease patients. However, the molecular mechanism underlying pathogenesis remains unclear due to the lack of animal models. Here, we established a mouse model, designated ‘mito-mice tRNALeu(UUR)2748’, that carries a pathogenic A2748G mutation in the tRNALeu(UUR) gene of mitochondrial DNA (mtDNA). The A2748G mutation is orthologous to the human A3302G mutation found in patients with mitochondrial diseases and diabetes. A2748G mtDNA was maternally inherited, equally distributed among tissues in individual mice, and its abundance did not change with age. At the molecular level, A2748G mutation is associated with aberrant processing of precursor mRNA containing tRNALeu(UUR) and mt-ND1, leading to a marked decrease in the steady-levels of ND1 protein and Complex I activity in tissues. Mito-mice tRNALeu(UUR)2748 with ≥50% A2748G mtDNA exhibited age-dependent metabolic defects including hyperglycemia, insulin insensitivity, and hepatic steatosis, resembling symptoms of patients carrying the A3302G mutation. This work demonstrates a valuable mouse model with an inheritable pathological A2748G mutation in mt-tRNALeu(UUR) that shows metabolic syndrome-like phenotypes at high heteroplasmy level. Furthermore, our findings provide molecular basis for understanding A3302G mutation-mediated mitochondrial disorders.

Mitochondrial tRNA variants in 811 Chinese probands with Leber’s hereditary optic neuropathy

Leber’s hereditary optic neuropathy (LHON) is the maternal inheritance of eye disorder. LHON-linked mitochondrial DNA (mtDNA) mutations affect the ND1, ND4 or ND6 genes encoding essential subunits of complex I. However, the role of mitochondrial tRNA defects in the pathogenesis of LHON is poorly understood. In this report, Sanger sequence analysis of 22 mitochondrial tRNA genes identified 139 variants in a cohort of 811 Han Chinese probands and 485 control Chinese subjects. Among these, 32 (4 known and 28 novel/putative) tRNA variants in 71 probands may contribute to pathogenesis of LHON, as these exhibited (1) present in < 1% of controls; (2) evolutionary conservation; (3) potential and significance of structural and functional modifications. Such variants may have potentially compromised structural and functional aspects in the processing of tRNAs, structure stability, tRNA charging, or codon-anticodon interactions during translation. These 32 variants presented either singly or with multiple mutations, with the primary LHON-linked ND1 3640G > A, ND4 11778G > A or ND6 14484 T > C mutations in the probands. The thirty-eight pedigrees carrying only one of tRNA variants exhibited relatively low penetrances of LHON, ranging from 5.7% to 42.9%, with an average of 19%. Strikingly, the average penetrances of optic neuropathy among 33 Chinese families carrying both a known/putative tRNA variant and a primary LHON-associated mtDNA mutation were 40.1%. These findings suggested that mitochondrial tRNA variants represent a significant causative factor for LHON, accounting for 8.75% cases in this cohort. These new insights may lead to beneficial applications in the pathophysiology, disease management, and genetic counseling of LHON.

PltRNAdb: Plant transfer RNA database.

Transfer RNAs (tRNAs) are intermediate-sized non-coding RNAs found in all organisms that help translate messenger RNA into protein. Recently, the number of sequenced plant genomes has increased dramatically. The availability of this extensive data greatly accelerates the study of tRNAs on a large scale. Here, 8,768,261 scaffolds/chromosomes containing 229,093 giga-base pairs representing whole-genome sequences of 256 plant species were analyzed to identify tRNA genes. As a result, 331,242 nuclear, 3,216 chloroplast, and 1,467 mitochondrial tRNA genes were identified. The nuclear tRNA genes include 275,134 tRNAs decoding 20 standard amino acids, 1,325 suppressor tRNAs, 6,273 tRNAs with unknown isotypes, 48,475 predicted pseudogenes, and 37,873 tRNAs with introns. Efforts also extended to the creation of PltRNAdb (https://bioinformatics.um6p.ma/PltRNAdb/index.php), a data source for tRNA genes from 256 plant species. PltRNAdb website allows researchers to search, browse, visualize, BLAST, and download predicted tRNA genes. PltRNAdb will help improve our understanding of plant tRNAs and open the door to discovering the unknown regulatory roles of tRNAs in plant genomes.

Proteomic Analysis of m.8296A>G Variation in the Mitochondrial tRNALys Gene

Variation in the mitochondrial tRNA^Lys gene at position 8296 was previously found to be associated with maternally inherited diabetes mellitus and deafness, hypertrophic cardiomyopathy, myoclonic epilepsy with ragged-red fibers and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. The pathogenicity of the m.8296A>G variation is unclear. In this study, we aimed to analyze the mitochondrial proteome in a patient with m.8296A>G variation to elucidate the effects of this mutation at the protein level. Whole-exome sequencing and mitochondrial genome analysis were performed in a patient with sensorineural hearing impairment, cognitive impairment, leukodystrophy, migraine-like headaches, and gastrointestinal dysmotility. Mitochondrial genome analysis identified a homoplasmic m.8296A>G variation in the mitochondrial tRNA^Lys gene in the proband and unaffected mother. Global mitochondrial proteome analysis was carried out in the muscle mitochondria of the index patient and a control subject. Comparative muscle mitochondrial proteome analysis revealed a total of 13 nuclear-encoded mitochondrial proteins differently expressed with respect to the control. Ten of the 13 proteins were downregulated. Most of the proteins were involved in ATP synthesis and Krebs cycle and have strong interactions with each other. We considered the m.8296A>G variation to be pathogenic with variable penetrance for our patient’s phenotype, and this variation led to different expressions of nuclear-encoded proteins involved in energy metabolism.

In Vivo Analysis of Mitochondrial Protein Synthesis in Saccharomyces cerevisiae Mitochondrial tRNA Mutants.

I describe here a protocol for the analysis of mitochondrial protein synthesis as a useful tool to characterize the mitochondrial defects associated with mutations in mitochondrial tRNA genes. The yeast Saccharomyces cerevisiae mutants, bearing human equivalent pathogenic mutations, were used as a simple model for analysis. The mitochondrial proteins were labeled by L[35S]-methionine incorporation in growing cells, extracted from purified mitochondria, and fractionated by SDS-polyacrylamide gel electrophoresis followed by autoradiography. By this method, it is possible to distinguish different protein synthesis profiles in the analyzed mitochondrial tRNA mutants.