Functional analysis of the novel mitochondrial tRNA^(Trp)and tRNA^(Ser(AGY))variants associated with type 2 diabetes mellitus

BACKGROUND Mutations in mitochondrial tRNA(mt-tRNA)genes that result in mitochondrial dysfunction play important roles in type 2 diabetes mellitus(T2DM).We previously 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.AIM To assess the potential pathogenicity of T2DM-associated m.A5514G and m.C12237T variants at genetic,molecular,and biochemical levels.METHODS 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-deoxyguanine(8-OhdG),malondialdehyde(MDA),and superoxide dismutase(SOD)were examined in cell lines with and without these mt-tRNA variants.RESULTS 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).CONCLUSION 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.

Novel m.4268T>C mutation in the mitochondrial tRNA^(Ile) gene is associated with hearing loss in two Chinese families

BACKGROUND Herein,we report the genetic,clinical,molecular and biochemical features of two Han Chinese pedigrees with suggested maternally transmitted non-syndromic hearing loss.AIM To investigate the pathophysiology of hearing loss associated with mitochondrial tRNA mutations.METHODS Sixteen 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.RESULTS Three 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 tRNA^(Ile) 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 tRNA^(Ile).The elimination of 6U–67A basepairing may change the tRNA^(Ile) metabolism.Functional mutation was supported by an approximately 64.6%reduction in the level of tRNA^(Ile) 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.CONCLUSION These data show that the novel tRNA^(Ile) 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.

Single-molecule mechanical folding and unfolding kinetics of armless mitochondrial tRNA^(Arg) from Romanomermis culicivorax

The mechanical stability of tRNAs contributes to their biological activities.The mitochondrial tRNAArg from Romanomermis culicivorax is the shortest tRNA ever known.This tRNA lacks D-and T-arms,represents a stem-bulge-stem architecture but still folds into a stable tertiary structure.Although its structure had been reported,studies on its mechanical folding and unfolding kinetic characteristics are lacking.Here,we directly measured the single-molecule mechanical folding and unfolding kinetics of the armless mt tRNAArg by using optical tweezers in different solution conditions.We revealed a two-step reversible unfolding pathway:the first and large transition corresponds to the unfolding of acceptor stem and bulge below 11 pN,and the second and small transition corresponds to the unfolding of anticodon arm at 12 pN-14 pN.Moreover,the free energy landscapes of the unfolding pathways were reconstructed.We also demonstrated that amino acid-chelated Mg^(2+)(aaCM),which mimics the intracellular solution condition,stabilizes the bulge of mitochondrial tRNAArg possibly by reducing the topological constraints or stabilizing the possible local non-canonical base pairings within the bulge region.Our study revealed the solution-dependent mechanical stability of an armless mt tRNA,which may shed light on future mt tRNA studies.

MitochondrialtRNA^(leu(UUR)) Gene Mutation and the DecreasedActivity of Cytochrom e c Oxidase in Preeclam psia

To explore the roles of mitochondria tRNA leu(UUR) gene mutation at nucleotide 3243 and the activity of cytochrome c oxidase in pathogenesis of preeclampsia, 57 patients with preeclampsia and 60 normotension pregnancy women were screened for tRNA leu(UUR) nt3243 A→G mutation with the method of polymers chain reaction (PCR) and restriction fragment length polymorphism. Cytochrome c oxidase activity was determined by measuring the rate of cyanide sensitive oxidation of reduced cytochrome c using luminosity photographer. The results showed that cytochrome c oxidase activity was significantly lower in the preeclampsia group (0.30±0.39/min, n = 32) than that in the controls (0.73±0.54/min, n = 26, P <0.01). The mitochondria DNA mutation at position 3243 was not found in our series. The results suggested that the decreased activity of cytochrome c oxidase might impair the energy production, leading to the mitochondria dysfunction and placenta dysfunction in preeclampsia patients. Mitochondria dysfunction may be involved in the pathogenesis of preeclampsia. The mutation of mitochondria DNA may not be the common contributor of preeclampsia in our series.

Unveiling mitochondrial mysteries:Exploring novel tRNA variants in type 2 diabetes mellitus

The recent study of Ding et al provides valuable insights into the functional implications of novel mitochondrial tRNATrp and tRNASer(AGY)variants in type 2 diabetes mellitus(T2DM).This editorial explores their findings,highlighting the role of mitochondrial dysfunction in the pathogenesis of T2DM.By examining the molecular mechanisms through which these tRNA variants contribute to disease progression,the study introduces new targets for therapeutic strategies.We discuss the broader implications of these results,emphasizing the importance of understanding mitochondrial genetics in addressing T2DM.