Mutant tRNA Research Articles

Simulation Modeling of A3243g Mutations on tRNALeu (UUR) against Type 2 Diabetes Mellitus using In Silico Method

Mitochondrial disease is a disease that affects the genetic condition of mitochondrial DNA. One of the causes of this disease is the mutation of nucleotide bases A to G at the 3243 nucleotide position of the mtDNA tRNALeu (UUR) gene. The characteristic of the mutation A3243G mtDNA is a heteroplasmic mutation with a relatively low amount of mutant DNA. The mutants that occur can undergo a dimerization process which can reduce aminoacylation. The purpose of this study was to prove in silico the effect of mitochondrial DNA mutation A3243G tRNA leucine on type 2 diabetes mellitus. The stages of the research carried out included structural modeling of tRNALeu and Protein tRNA Synthetase using the homology method, then a native and mutant leucine tRNA model was made. The results of the modeling were assessed and validated. The next step is the dimerization process between mutant tRNAs to see the binding of structures to the tRNA. The next process is docking between the tRNA and protein models. The final step is to simulate molecular dynamics in the native tRNA model, mutants and dimer structures to see the differences in aminoacylation reactions in the three models in mitochondria. This study is expected to provide information about the effect of the A3243G mutation on the causes of type 2 diabetes in mitochondria.

Genetic Interaction of tRNA-Dependent Mistranslation with Fused in Sarcoma Protein Aggregates

High-fidelity protein synthesis requires properly aminoacylated transfer RNAs (tRNAs), yet diverse cell types, from bacteria to humans, show a surprising ability to tolerate errors in translation resulting from mutations in tRNAs, aminoacyl-tRNA synthetases, and other components of protein synthesis. Recently, we characterized a tRNASerAGA G35A mutant (tRNASerAAA) that occurs in 2% of the human population. The mutant tRNA decodes phenylalanine codons with serine, inhibits protein synthesis, and is defective in protein and aggregate degradation. Here, we used cell culture models to test our hypothesis that tRNA-dependent mistranslation will exacerbate toxicity caused by amyotrophic lateral sclerosis (ALS)-associated protein aggregation. Relative to wild-type tRNA, we found cells expressing tRNASerAAA showed slower but effective aggregation of the fused in sarcoma (FUS) protein. Despite reduced levels in mistranslating cells, wild-type FUS aggregates showed similar toxicity in mistranslating cells and normal cells. The aggregation kinetics of the ALS-causative FUS R521C variant were distinct and more toxic in mistranslating cells, where rapid FUS aggregation caused cells to rupture. We observed synthetic toxicity in neuroblastoma cells co-expressing the mistranslating tRNA mutant and the ALS-causative FUS R521C variant. Our data demonstrate that a naturally occurring human tRNA variant enhances cellular toxicity associated with a known causative allele for neurodegenerative disease.

Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells.

Site-specific incorporation of unnatural amino acids (Uaas) in living cells relies on engineered aminoacyl-transfer RNA synthetase-tRNA pairs borrowed from a distant domain of life. Such heterologous suppressor tRNAs often have poor intrinsic activity, presumably due to suboptimal interaction with a non-native translation system. This limitation can be addressed in Escherichia coli using directed evolution. However, no suitable selection system is currently available to do the same in mammalian cells. Here we report virus-assisted directed evolution of tRNAs (VADER) in mammalian cells, which uses a double-sieve selection scheme to facilitate single-step enrichment of active yet orthogonal tRNA mutants from naive libraries. Using VADER we developed improved mutants of Methanosarcina mazei pyrrolysyl-tRNA, as well as a bacterial tyrosyl-tRNA. We also show that the higher activity of the most efficient mutant pyrrolysyl-tRNA is specific for mammalian cells, alluding to an improved interaction with the unique mammalian translation apparatus.

Mitochondrial Cardiomyopathy: The Roles of mt-tRNA Mutations.

Mitochondria are important organelles whose primary role is generating energy through the oxidative phosphorylation (OXPHOS) system. Cardiomyopathy, a common clinical disorder, is frequently associated with pathogenic mutations in nuclear and mitochondrial genes. To date, a growing number of nuclear gene mutations have been linked with cardiomyopathy; however, knowledge about mitochondrial tRNAs (mt-tRNAs) mutations in this disease remain inadequately understood. In fact, defects in mt-tRNA metabolism caused by pathogenic mutations may influence the functioning of the OXPHOS complexes, thereby impairing mitochondrial translation, which plays a critical role in the predisposition of this disease. In this review, we summarize some basic knowledge about tRNA biology, including its structure and function relations, modification, CCA-addition, and tRNA import into mitochondria. Furthermore, a variety of molecular mechanisms underlying tRNA mutations that cause mitochondrial dysfunctions are also discussed in this article.

Expression of Concern: Mitochondrial tRNA mutation and regulation of the adiponectin pathway in maternally inherited hypertension in Chinese Han.

Following publication, the publisher has uncovered conclusive evidence that false identities were used as peer reviewers for this article. These reviewers were not suggested by the authors. These peer reviewers have now been removed. This article is currently under post publication assessment. This expression of concern has been posted while Frontiers awaits the outcome of this assessment and will then be updated accordingly.

Retraction: Mitochondrial tRNA mutations associated with essential hypertension: From molecular genetics to function.

[This retracts the article DOI: 10.3389/fcell.2020.634137.].

Heteroplasmic and homoplasmic m.616T>C in mitochondria tRNAPhe promote isolated chronic kidney disease and hyperuricemia.

Inherited kidney diseases are the fifth most common cause of end-stage renal disease (ESRD). Mitochondrial dysfunction plays a vital role in the progression of inherited kidney diseases, while mitochondrial-transfer RNA (mt-tRNA) variants and their pathogenic contributions to kidney disease remain largely unclear. In this study, we identified the pathogenic mt-tRNAPhe 616T>C mutation in 3 families and documented that m.616T>C showed a high pathogenic threshold, with both heteroplasmy and homoplasmy leading to isolated chronic kidney disease and hyperuricemia without hematuria, proteinuria, or renal cyst formation. Moreover, 1 proband with homoplamic m.616T>C presented ESRD as a child. No symptoms of nervous system evolvement were observed in these families. Lymphoblast cells bearing m.616T>C exhibited swollen mitochondria, underwent active mitophagy, and showed respiratory deficiency, leading to reduced mitochondrial ATP production, diminished membrane potential, and overproduction of mitochondrial ROS. Pathogenic m.616T>C abolished a highly conserved base pair (A31-U39) in the anticodon stem-loop which altered the structure of mt-tRNAPhe, as confirmed by a decreased melting temperature and slower electrophoretic mobility of the mutant tRNA. Furthermore, the unstable structure of mt-tRNAPhe contributed to a shortage of steady-state mt-tRNAPhe and enhanced aminoacylation efficiency, which resulted in impaired mitochondrial RNA translation and a significant decrease in mtDNA–encoded polypeptides. Collectively, these findings provide potentially new insights into the pathogenesis underlying inherited kidney disease caused by mitochondrial variants.

Novel mitochondrial tRNALeu(UUR) 3261A > g mutation in two pedigrees with essential hypertension.

Essential hypertension (EH) was associated with mitochondrial tRNA mutations. This study was designed to assess the association between EH and mitochondrial dysfunction. A total of 30 individuals from two different Chinese families exhibit maternally inherited EH were assessed for genetic, clinical, and biochemical phenotypes pertaining to EH and mitochondrial functionality. These analyses included assessments of tRNALeu(UUR) 3261A > G mutation status, mitochondrial membrane permeability, mitochondria-associated ATP and reactive oxygen species (ROS) generation, and electron transport chain functionality. EH was detected in 6 total analyzed members of the two families assessed in the present study, with its initial age of onset and presentation varying among patients. These patients with EH exhibited the tRNALeu(UUR) 3261A > G mutation and were of the B5 and D4 Eastern Asian mitochondrial haplogroups. This 3261A > G mutation was predicted to result in disruption of normal tRNALeu(UUR) activity owing to the destabilization of conserved base pairing (30A-40U). Consistent with this prediction, we found that cybrid cell lines exhibiting this 3261A > G mutation exhibited a ~49.05% decrease in baseline tRNALeu(UUR) levels. These cells additionally exhibited ~44.81% reductions in rates of mitochondrial translation. To facilitate future molecular diagnosis, the 3261A > G mutation should be included in the list of hereditary risk factors. Our findings will aid in the counseling of EH families.

The Mitochondrial tRNAPhe 625G>A Mutation in Three Han Chinese Families With Cholecystolithiasis

In this study, we assessed three Chinese families with inherited cholecystolithiasis and conducted the clinical, genetic, and molecular characterization of these subjects. Eight of eighteen matrilineal relatives had a clinical phenotype in these three families. Sequence analysis of complete mitochondrial genomes in these probands identified the homoplasmic tRNAPhe 625 G > A mutation and distinct sets of mtDNA polymorphisms belonging to haplogroups H2, F4b, and M10a. The 625G > A mutation disturbed the classic G-C base-pairings at a highly conserved position 49 in the T-stem of mitochondrial tRNAs. Molecular dynamics simulation showed that the structure of tRNAphe with 625 G > A mutation was noticeably remodeled while compared with the isoform of the wild type. The occurrence of tRNAPhe 625 G > A mutation in these various genetically unrelated subjects strongly indicates that this mutation is involved in the pathogenesis of cholecystolithiasis. This is the first evidence that tRNA mutations are associated with cholecystolithiasis, and it provided more insights into the genetic mechanism of cholecystolithiasis.

Measuring the tolerance of the genetic code to altered codon size.

Translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet codon, rather than quadruplet codon, genetic code? Here, we investigate the tolerance of the Escherichia coli genetic code to tRNA mutations that increase codon size. We found that tRNAs from all 20 canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs). Many of these selectively incorporate a single amino acid in response to a specified four-base codon, as confirmed with mass spectrometry. However, efficient quadruplet codon translation often requires multiple tRNA mutations. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These may constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results suggest that the triplet codon code was selected because it is simpler and sufficient, not because a quadruplet codon code is unachievable. These data provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.

Structural basis for impaired 5′ processing of a mutant tRNA associated with defects in neuronal homeostasis

SignificanceUnderstanding and treating neurological disorders are global priorities. Some of these diseases are engendered by mutations that cause defects in the cellular synthesis of transfer RNAs (tRNAs), which function as adapter molecules that translate messenger RNAs into proteins. During tRNA biogenesis, ribonuclease P catalyzes removal of the transcribed sequence upstream of the mature tRNA. Here, we focus on a cytoplasmic tRNAArgUCU that is expressed specifically in neurons and, when harboring a particular point mutation, contributes to neurodegeneration in mice. Our results suggest that this mutation favors stable alternative structures that are not cleaved by mouse ribonuclease P and motivate a paradigm that may help to understand the molecular basis for disease-associated mutations in other tRNAs.

A novel fluorescent reporter sensitive to serine mis-incorporation

ABSTRACT High-fidelity translation was considered a requirement for living cells. The frozen accident theory suggested that any deviation from the standard genetic code should result in the production of so much mis-made and non-functional proteins that cells cannot remain viable. Studies in bacterial, yeast, and mammalian cells show that significant levels of mistranslation (1–10% per codon) can be tolerated or even beneficial under conditions of oxidative stress. Single tRNA mutants, which occur naturally in the human population, can lead to amino acid mis-incorporation at a codon or set of codons. The rate or level of mistranslation can be difficult or impossible to measure in live cells. We developed a novel red fluorescent protein reporter that is sensitive to serine (Ser) mis-incorporation at proline (Pro) codons. The mCherry Ser151Pro mutant is efficiently produced in Escherichia coli but non-fluorescent. We demonstrated in cells and with purified mCherry protein that the fluorescence of mCherry Ser151Pro is rescued by two different tRNASer gene variants that were mutated to contain the Pro (UGG) anticodon. Ser mis-incorporation was confirmed by mass spectrometry. Remarkably, E. coli tolerated mistranslation rates of ~10% per codon with negligible reduction in growth rate. Conformational sampling simulations revealed that the Ser151Pro mutant leads to significant changes in the conformational freedom of the chromophore precursor, which is indicative of a defect in chromophore maturation. Together our data suggest that the mCherry Ser151 mutants may be used to report Ser mis-incorporation at multiple other codons, further expanding the ability to measure mistranslation in living cells.

The C nucleotide at the mature 5' end of the Escherichia coli proline tRNAs is required for the RNase E cleavage specificity at the 3' terminus as well as functionality.

Proline tRNA 3′-maturation in Escherichia coli occurs through a one-step RNase E endonucleolytic cleavage immediately after the CCA determinant. This processing pathway is distinct from the 3′-end maturation of the other tRNAs by avoiding the widespread use of 3′ → 5′ exonucleolytic processing, 3′-polyadenylation and subsequent degradation. Here, we show that the cytosine (C) at the mature 5′-terminus of the proK and proL tRNAs is required for both the RNase E cleavage immediately after the CCA determinant and their functionality. Thus, changing the C nucleotide at the mature 5′-terminus of the proL and proK tRNAs to the more common G nucleotide led to RNase E cleavages 1–4 nucleotides downstream of the CCA determinant. Furthermore, the 5′-modified mutant tRNAs required RNase T and RNase PH for their 3′-maturation and became substrates for polyadenylation and degradation. Strikingly, the aminoacylation of the 5′-modified proline tRNAs was blocked due to the change in the recognition element for prolyl-tRNA-synthetase. An analogous modification of the pheV 5′-mature terminus from G to C nucleotide did not support cell viability. This result provides additional support for the importance of first nucleotide of the mature tRNAs in their processing and functionality.

Novel m.4268T>C mutation in the mitochondrial tRNAIle gene is associated with hearing loss in two Chinese families.

BACKGROUNDHerein, we report the genetic, clinical, molecular and biochemical features of two Han Chinese pedigrees with suggested maternally transmitted non-syndromic hearing loss. AIMTo investigate the pathophysiology of hearing loss associated with mitochondrial tRNA mutations.METHODSSixteen subjects from two Chinese families with hearing loss underwent clinical, genetic, molecular, and biochemical evaluations. Biochemical characterizations included the measurements of tRNA levels using lymphoblastoid cell lines derived from five affected matrilineal relatives of these families and three control subjects.RESULTSThree of the 16 matrilineal relatives in these families exhibited a variable seriousness and age-at-onset (8 years) of deafness. Analysis of mtDNA mutation identified the novel homoplasmic tRNAIle 4268T>C mutation in two families both belonging to haplogroup D4j. The 4268T>C mutation is located in a highly conserved base pairing (6U–67A) of tRNAIle. The elimination of 6U–67A base-pairing may change the tRNAIle metabolism. Functional mutation was supported by an approximately 64.6% reduction in the level of tRNAIle observed in the lymphoblastoid cell lines with the 4268T>C mutation, in contrast to the wild-type cell lines. The reduced level of tRNA was below the proposed threshold for normal respiration in lymphoblastoid cells. However, genotyping analysis did not detect any mutations in the prominent deafness-causing gene GJB2 in any members of the family. CONCLUSIONThese data show that the novel tRNAIle 4268T>C mutation was involved in maternally transmitted deafness. However, epigenetic, other genetic, or environmental factors may be attributed to the phenotypic variability. These findings will be useful for understanding families with maternally inherited deafness.

Screening for Mitochondrial tRNA Mutations in 318 Patients with Dilated Cardiomyopathy

Objectives: Dilated cardiomyopathy (DCM) is a complex cardiovascular disease with unknown etiology. Although nuclear genes play active roles in DCM, mitochondrial dysfunction was believed to be involved in the pathogenesis of DCM. The objective of this study was to analyze the association between mitochondrial tRNA (mt-tRNA) mutations and DCM. Materials and Methods: We performed a mutational analysis of mt-tRNA genes in a cohort of 318 patients with DCM and 200 age- and gender-matched control subjects. To further assess their pathogenicity, phylogenetic analysis and mitochondrial functions including mtDNA copy number, ATP, and ROS were analyzed. Results: Seven possible pathogenic mutations, i.e., MT-TL1 3302A>G, MT-TI 4295A>G, MT-TM 4435A>G, MT-TA 5655T>C, MT-TH 12201T>C, MT-TE 14692A>G, and MT-TT 15927G>A, were identified in the DCM group but were absent in controls. These mutations occurred at extremely conserved nucleotides of corresponding tRNAs and led to the failure in tRNAs metabolism. Moreover, a significant reduction in ATP and mtDNA copy number and a marked increase in ROS level were observed in polymononuclear leukocytes (PMNs) derived from the DCM patients carrying these mt-tRNA mutations, suggesting that these mutations may cause mitochondrial dysfunction that was responsible for DCM. Conclusions: Our data indicated that mt-tRNA mutations may be the molecular basis for DCM, which provides novel insights into the pathophysiology of DCM that manifested as mitochondrial dysfunction.

The Mitochondrial tRNAAsp T7561C, tRNAHis C12153T, and A12172G Mutations May Be Associated with Essential Hypertension in a Han Chinese Pedigree

Objectives: Mutations in mitochondrial tRNA (mt-tRNA) are the important causes for maternally inherited hypertension; however, the pathophysiology of mt-tRNA mutations in clinical expression of hypertension remains poorly understood. Material and Methods: In this study, we report the molecular features of a Han Chinese pedigree with maternally transmitted essential hypertension. The entire mitochondrial genomes are PCR amplified and sequenced. Moreover, phylogenetic analysis, haplogroup analysis, as well as pathogenicity scoring system are used to assess the potential roles for mtDNA mutations. Results: Strikingly, among 10 matrilineal relatives, 3 suffer from variable degree of hypertension at different age at onset. Sequence analysis of the complete mitochondrial genomes suggests the presence of three possible pathogenic mtDNA mutations: tRNA^Asp T7561C, tRNA^His C12153T, and A12172G, together with a set of variants belonging to East Asian mitochondrial haplogroup M7a. Interestingly, the T7561C mutation occurs at position 44 in the variable region of tRNA^Asp, while the C12153T and A12172G mutations are localized at extremely conserved nucleotides in the D-arm and anticodon stem of tRNA^His gene, respectively, which are critical for tRNA steady-state level and function. Conclusions: Mitochondrial T7561C, C12153T, and A12172G mutations may lead to the failure in tRNAs metabolism and cause mitochondrial dysfunction that is responsible for hypertension. However, the homoplasmy form of mt-tRNA mutations, incomplete penetrance of hypertension suggests that T7561C, C12153T, and A12172G mutations are insufficient to produce the clinical phenotype; hence, other risk factors such as environmental factors, nuclear genes, and epigenetic modifications may contribute to the phenotypic manifestation of maternally inherited hypertension in this Chinese pedigree.

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.

The Effect of tRNA[Ser]Sec Isopentenylation on Selenoprotein Expression.

Transfer RNA[Ser]Sec carries multiple post-transcriptional modifications. The A37G mutation in tRNA[Ser]Sec abrogates isopentenylation of base 37 and has a profound effect on selenoprotein expression in mice. Patients with a homozygous pathogenic p.R323Q variant in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder, and hence we wondered whether selenoprotein expression was impaired. Patient fibroblasts with the homozygous p.R323Q variant did not show a general decrease in selenoprotein expression. However, recombinant human TRIT1R323Q had significantly diminished activities towards several tRNA substrates in vitro. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Mass-spectrometry revealed that hypermodification of U34 to mcm5Um occurs independently of isopentenylation of A37 in tRNA[Ser]Sec. Western blotting and 75Se metabolic labeling showed only moderate effects on selenoprotein levels and 75Se incorporation. A detailed analysis of Trit1-deficient liver using ribosomal profiling demonstrated that UGA/Sec re-coding was moderately affected in Selenop, Txnrd1, and Sephs2, but not in Gpx1. 2′O-methylation of U34 in tRNA[Ser]Sec depends on FTSJ1, but does not affect UGA/Sec re-coding in selenoprotein translation. Taken together, our results show that a lack of isopentenylation of tRNA[Ser]Sec affects UGA/Sec read-through but differs from a A37G mutation.

Mutational Screening for Mitochondrial tRNA Mutations in 150 Children with High Myopia

Background: Myopia is a very common eye disease with an unknown etiology. Increasing evidence shows that mitochondrial dysfunction plays an active role in the pathogenesis and progression of this disease. Objectives: The purpose of this study was to analyze the relationship between mitochondrial tRNA (mt-tRNA) variants and high myopia (HM). Methods: The entire mt-tRNA genes of 150 children with HM, as well as 100 healthy subjects, were PCR-amplified and sequenced. To assess the pathogenicity, we used the phylogenetic conservation analysis and pathogenicity scoring system. Results: We identified six candidate pathogenic variants: tRNALeu (UUR) T3290C, tRNAIle A4317G, tRNAAla G5591A, tRNASer (UCN) T7501C, tRNAHis T12201C, and tRNAThr G15915A. However, these variants were not identified in controls. Further phylogenetic analysis revealed that these variants occurred at the positions, which were very evolutionarily conserved and may have structural-functional impacts on the tRNAs. Subsequently, these variants may lead to the impairment of mitochondrial translation and aggravated mitochondrial dysfunction, which play an active role in the phenotypic expression of HM. Conclusions: Our results suggested that variants in mt-tRNA genes were the risk factors for HM, which provided valuable information for the early detection and prevention of HM.

Genomic Changes and Genetic Divergence of Vibrio alginolyticus Under Phage Infection Stress Revealed by Whole-Genome Sequencing and Resequencing.

Bacteriophages (phages) and their bacterial hosts were the most abundant and genetically highly diverse organisms on the earth. In this study, a series of phage-resistant mutant (PRM) strains derived from Vibrio alginolyticus were isolated and Infrequent-restriction-site PCR (IRS-PCR) was used to investigate the genetic diversity of the PRM strains. Phenotypic variations of eight PRM strains were analyzed using profiles of utilizing carbon sources and chemical sensitivity. Genetic variations of eight PRM strains and coevolved V. alginolyticus populations with phages were analyzed by whole-genome sequencing and resequencing, respectively. The results indicated that eight genetically discrepant PRM stains exhibited abundant and abundant phenotypic variations. Eight PRM strains and coevolved V. alginolyticus populations (VE1, VE2, and VE3) contained numerous single nucleotide variations (SNVs) and insertions/indels (InDels) and exhibited obvious genetic divergence. Most of the SNVs and InDels in coding genes were related to the synthesis of flagellar, extracellular polysaccharide (EPS), which often served as the receptors of phage invasion. The PRM strains and the coevolved cell populations also contained frequent mutations in tRNA and rRNA genes. Two out of three coevolved populations (VE1 and VE2) contained a large mutation segment severely deconstructing gene nrdA, which was predictably responsible for the booming of mutation rate in the genome. In summary, numerous mutations and genetic divergence were detected in the genomes of V. alginolyticus PRM strains and in coevolved cell populations of V. alginolyticus under phage infection stress. The phage infection stress may provide an important force driving genomic evolution of V. alginolyticus.