Human TRUB1 is a highly conserved pseudouridine synthase responsible for the formation of Ψ55 in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro.

Pseudouridine (Ψ) at position 55 in tRNAs plays an important role in their structure and function. This modification is catalyzed by TruB/Pus4/Cbf5 family of pseudouridine synthases in bacteria and yeast. However, the mechanism of TRUB family underlying the formation of Ψ55 in the mammalian tRNAs is largely unknown. In this report, the CMC/reverse transcription assays demonstrated the presence of Ψ55 in the human mitochondrial tRNAAsn, tRNAGln, tRNAGlu, tRNAPro, tRNAMet, tRNALeu(UUR) and tRNASer(UCN). TRUB1 knockout (KO) cell lines generated by CRISPR/Cas9 technology exhibited the loss of Ψ55 modification in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro but did not affect other 18 mitochondrial tRNAs. An in vitro assay revealed that recombinant TRUB1 protein can catalyze the efficient formation of Ψ55 in tRNAAsn and tRNAGln, but not in tRNAMet and tRNAArg. Notably, the overexpression of TRUB1 cDNA reversed the deficient Ψ55 modifications in these tRNAs in TRUB1KO HeLa cells. TRUB1 deficiency affected the base-pairing (18A/G-Ψ55), conformation and stability but not aminoacylation capacity of these tRNAs. Furthermore, TRUB1 deficiency impacted mitochondrial translation and biogenesis of oxidative phosphorylation system. Our findings demonstrated that human TRUB1 is a highly conserved mitochondrial pseudouridine synthase responsible for the Ψ55 modification in the mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro.

A deafness-associated mitochondrial DNA mutation caused pleiotropic effects on DNA replication and tRNA metabolism.

In this report, we investigated the molecular mechanism underlying a deafness-associated m.5783C > T mutation that affects the canonical C50-G63 base-pairing of TΨC stem of tRNACys and immediately adjacent to 5' end of light-strand origin of mitochondrial DNA (mtDNA) replication (OriL). Two dimensional agarose gel electrophoresis revealed marked decreases in the replication intermediates including ascending arm of Y-fork arcs spanning OriL in the mutant cybrids bearing m.5783C > T mutation. mtDNA replication alterations were further evidenced by decreased levels of PolγA, Twinkle and SSBP1, newly synthesized mtDNA and mtDNA contents in the mutant cybrids. The m.5783C > T mutation altered tRNACys structure and function, including decreased melting temperature, conformational changes, instability and deficient aminoacylation of mutated tRNACys. The m.5783C > T mutation impaired the 5' end processing efficiency of tRNACys precursors and reduced the levels of tRNACys and downstream tRNATyr. The aberrant tRNA metabolism impaired mitochondrial translation, which was especially pronounced effects in the polypeptides harboring higher numbers of cysteine and tyrosine codons. These alterations led to deficient oxidative phosphorylation including instability and reduced activities of the respiratory chain enzyme complexes I, III, IV and intact supercomplexes overall. Our findings highlight the impact of mitochondrial dysfunction on deafness arising from defects in mitochondrial DNA replication and tRNA metabolism.

Asymmetrical effects of deafness-associated mitochondrial DNA 7516delA mutation on the processing of RNAs in the H-strand and L-strand polycistronic transcripts.

In this report, we investigated the molecular mechanism underlying a deafness-associated m.7516delA mutation affecting the 5' end processing sites of mitochondrial tRNAAsp and tRNASer(UCN). An in vitro processing experiment demonstrated that m.7516delA mutation caused the aberrant 5' end processing of tRNASer(UCN) and tRNAAsp precursors, catalyzed by RNase P. Using cytoplasmic hybrids (cybrids) derived from one hearing-impaired Chinese family bearing the m.7516delA mutation and control, we demonstrated the asymmetrical effects of m.7516delA mutation on the processing of tRNAs in the heavy (H)-strand and light (L)-strand polycistronic transcripts. Specially, the m.7516delA mutation caused the decreased levels of tRNASer(UCN) and downstream five tRNAs, including tRNATyr from the L-strand transcripts and tRNAAsp from the H-strand transcripts. Strikingly, mutant cybrids exhibited the lower level of COX2 mRNA and accumulation of longer and uncleaved precursors of COX2 from the H-strand transcripts. Aberrant RNA metabolisms yielded variable reductions in the mitochondrial proteins, especially marked reductions in the levels of ND4, ND5, CO1, CO2 and CO3. The impairment of mitochondrial translation caused the proteostasis stress and respiratory deficiency, diminished ATP production and membrane potential, increased production of reactive oxygen species and promoted apoptosis. Our findings provide new insights into the pathophysiology of deafness arising from mitochondrial tRNA processing defects.

Hypertension-associated mitochondrial DNA 4401A>G mutation caused the aberrant processing of tRNAMet, all 8 tRNAs and ND6 mRNA in the light-strand transcript.

Mitochondrial tRNA processing defects were associated with human diseases but their pathophysiology remains elusively. The hypertension-associated m.4401A>G mutation resided at a spacer between mitochondrial tRNAMet and tRNAGln genes. An in vitro processing experiment revealed that the m.4401A>G mutation caused 59% and 69% decreases in the 5' end processing efficiency of tRNAGln and tRNAMet precursors, catalyzed by RNase P, respectively. Using human umbilical vein endothelial cells-derived cybrids, we demonstrated that the m.4401A>G mutation caused the decreases of all 8 tRNAs and ND6 and increases of longer and uncleaved precursors from the Light-strand transcript. Conversely, the m.4401A>G mutation yielded the reduced levels of tRNAMet level but did not change the levels of other 13 tRNAs, 12 mRNAs including ND1, 12S rRNA and 16S rRNA from the Heavy-strand transcript. These implicated the asymmetrical processing mechanisms of H-strand and L-strand polycistronic transcripts. The tRNA processing defects play the determined roles in the impairing mitochondrial translation, respiratory deficiency, diminishing membrane potential, increasing production of reactive oxygen species and altering autophagy. Furthermore, the m.4401A>G mutation altered the angiogenesis, evidenced by aberrant wound regeneration and weaken tube formation in mutant cybrids. Our findings provide new insights into the pathophysiology of hypertension arising from mitochondrial tRNA processing defects.

A coronary artery disease-associated tRNAThr mutation altered mitochondrial function, apoptosis and angiogenesis.

The tissue specificity of mitochondrial tRNA mutations remains largely elusive. In this study, we demonstrated the deleterious effects of tRNAThr 15927G>A mutation that contributed to pathogenesis of coronary artery disease. The m.15927G>A mutation abolished the highly conserved base-pairing (28C-42G) of anticodon stem of tRNAThr. Using molecular dynamics simulations, we showed that the m.15927G>A mutation caused unstable tRNAThr structure, supported by decreased melting temperature and slower electrophoretic mobility of mutated tRNA. Using cybrids constructed by transferring mitochondria from a Chinese family carrying the m.15927G>A mutation and a control into mitochondrial DNA (mtDNA)-less human umbilical vein endothelial cells, we demonstrated that the m.15927G>A mutation caused significantly decreased efficiency in aminoacylation and steady-state levels of tRNAThr. The aberrant tRNAThr metabolism yielded variable decreases in mtDNA-encoded polypeptides, respiratory deficiency, diminished membrane potential and increased the production of reactive oxygen species. The m.15927G>A mutation promoted the apoptosis, evidenced by elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 3, 7, 9 and PARP. Moreover, the lower wound healing cells and perturbed tube formation were observed in mutant cybrids, indicating altered angiogenesis. Our findings provide new insights into the pathophysiology of coronary artery disease, which is manifested by tRNAThr mutation-induced alterations.

Leber's hereditary optic neuropathy is potentially associated with a novel m.5587T>C mutation in two pedigrees.

Mitochondrial (mt)DNA mutations have been revealed to be associated with Leber's hereditary optic neuropathy (LHON). The present study conducted clinical, genetic and molecular evaluations of two Han Chinese families. A total of 4 (3 men and 1 female) out of 14 matrilineal relatives in the families exhibited visual impairment with variable severity and age of onset. The average age of onset of visual loss was 20.5 years old. Molecular analysis of the complete mitochondrial genome in these pedigrees demonstrated that the three primary mutations associated with LHON were not detected; however, the homoplasmic m.5587T>C mutation was identified, which was localized at the end of the mitochondrially encoded transfer (t)RNA alanine gene and may alter the tertiary structure of this tRNA. Subsequently, this structural alteration may result in tRNA metabolism failure. In addition, distinct sets of mtDNA polymorphisms belonging to haplogroup F1 were detected in both families tested. The findings of the present study suggested that the m.5587T>C mutation may be involved in the pathogenesis of visual impairment. In addition, the mtDNA variant m.15024G>A(p.C93H) in the mitochondrially encoded cytochrome B gene was detected in both families, which exhibited evolutionary conservation, indicating it may serve a potential modifying role in the development of visual impairment associated with m.5587T>C mutation in these families. Furthermore, other modifying factors, including nuclear modifier genes, and environmental and personal factors may also contribute to the development of LHON in subjects carrying this mutation.

Coronary heart disease is associated with a mutation in mitochondrial tRNA.

Coronary heart disease (CHD) is the leading cause of death worldwide. Mitochondrial genetic determinant for the development of CHD remains poorly explored. We report there the clinical, genetic, molecular and biochemical characterization of a four-generation Chinese family with maternally inherited CHD. Thirteen of 32 adult members in this family exhibited variable severity and age-at-onset of CHD. Mutational analysis of their mitochondrial genomes identified the tRNA(Thr) 15927G>A mutation belonging to the Eastern Asian haplogroup B5. The anticipated destabilization of a highly conserved base-pairing (28C-42G) by the 15927G>A mutation affects structure and function of tRNA(Thr). Northern analysis revealed ≈80% decrease in the steady-state level of tRNA(Thr) in the mutant cell lines carrying the 15927G>A mutation. The 15927G>A mutation changed the conformation of tRNA(Thr), as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. In addition, ∼39% reduction in aminoacylated efficiency of tRNA(Thr) was observed in mutant cells derived from this Chinese family. An in vivo mitochondrial protein labeling analysis showed ∼53% reduction in the rate of mitochondrial translation in mutant cells. The impaired mitochondrial protein synthesis leads to defects in overall respiratory capacity or malate/glutamate-promoted respiration or succinate/glycerol-3-phosphate-promoted respiration, or N,N,N',N'-tetramethyl-pphenylenediamine/ascorbate-promoted respiration in mutant cells. An increasing production of reactive oxygen species was observed in the mutant cells carrying the 15927G>A mutation. These results provide the direct evidence that the tRNA(Thr) 15927G>A mutation is associated with CHD. Our findings may provide new insights into pathophysiology and intervention targets of this disorder.