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.

Mechanistic insights into mitochondrial tRNAAla 3'-end metabolism deficiency.

Mitochondrial tRNA 3'-end metabolism is critical for the formation of functional tRNAs. Deficient mitochondrial tRNA 3'-end metabolism is linked to an array of human diseases, including optic neuropathy, but their pathophysiology remains poorly understood. In this report, we investigated the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-associated tRNAAla 5587A>G mutation, which changes a highly conserved adenosine at position 73 (A73) to guanine (G73) on the 3'-end of the tRNA acceptor stem. The m.5587A>G mutation was identified in three Han Chinese families with suggested maternal inheritance of LHON. We hypothesized that the m.5587A>G mutation altered tRNAAla 3'-end metabolism and mitochondrial function. In vitro processing experiments showed that the m.5587A>G mutation impaired the 3'-end processing of tRNAAla precursors by RNase Z and inhibited the addition of CCA by tRNA nucleotidyltransferase (TRNT1). Northern blot analysis revealed that the m.5587A>G mutation perturbed tRNAAla aminoacylation, as evidenced by decreased efficiency of aminoacylation and faster electrophoretic mobility of mutated tRNAAla in these cells. The impact of m.5587A>G mutation on tRNAAla function was further supported by increased melting temperature, conformational changes, and reduced levels of this tRNA. Failures in tRNAAla metabolism impaired mitochondrial translation, perturbed assembly and activity of oxidative phosphorylation complexes, diminished ATP production and membrane potential, and increased production of reactive oxygen species. These pleiotropic defects elevated apoptotic cell death and promoted mitophagy in cells carrying the m.5587A>G mutation, thereby contributing to visual impairment. Our findings may provide new insights into the pathophysiology of LHON arising from mitochondrial tRNA 3'-end metabolism deficiency.

An animal model for mitochondrial tyrosyl-tRNA synthetase deficiency reveals links between oxidative phosphorylation and retinal function.

Mitochondria maintain a distinct pool of ribosomal machinery, including tRNAs and tRNAs activating enzymes, such as mitochondrial tyrosyl-tRNA synthetase (YARS2). Mutations in YARS2, which typically lead to the impairment of mitochondrial protein synthesis, have been linked to an array of human diseases including optic neuropathy. However, the lack of YARS2 mutation animal model makes us difficult to elucidate the pathophysiology underlying YARS2 deficiency. To explore this system, we generated YARS2 knockout (KO) HeLa cells and zebrafish using CRISPR/Cas9 technology. We observed the aberrant tRNATyr aminoacylation overall and reductions in the levels in mitochondrion- and nucleus-encoding subunits of oxidative phosphorylation system (OXPHOS), which were especially pronounced effects in the subunits of complex I and complex IV. These deficiencies manifested the decreased levels of intact supercomplexes overall. Immunoprecipitation assays showed that YARS2 bound to specific subunits of complex I and complex IV, suggesting the posttranslational stabilization of OXPHOS. Furthermore, YARS2 ablation caused defects in the stability and activities of OXPHOS complexes. These biochemical defects could be rescued by the overexpression of YARS2 cDNA in the YARS2KO cells. In zebrafish, the yars2KO larva conferred deficient COX activities in the retina, abnormal mitochondrial morphology, and numbers in the photoreceptor and retinal ganglion cells. The zebrafish further exhibited the retinal defects affecting both rods and cones. Vision defects in yars2KO zebrafish recapitulated the clinical phenotypes in the optic neuropathy patients carrying the YARS2 mutations. Our findings highlighted the critical role of YARS2 in the stability and activity of OXPHOS and its pathological consequence in vision impairments.

Complex I mutations synergize to worsen the phenotypic expression of Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternal inheritance of eye disease because of the mitochondrial DNA (mtDNA) mutations. We previously discovered a 3866T>C mutation within the gene for the ND1 subunit of complex I as possibly amplifying disease progression for patients bearing the disease-causing 11778G>A mutation within the gene for the ND4 subunit of complex I. However, whether and how the ND1 mutation exacerbates the ND4 mutation were unknown. In this report, we showed that four Chinese families bearing both m.3866T>C and m.11778G>A mutations exhibited higher penetrances of LHON than 6 Chinese pedigrees carrying only the m.3866T>C mutation or families harboring only the m.11778G>A mutation. The protein structure analysis revealed that the m.3866T>C (I187T) and m.11778G>A (R340H) mutations destabilized the specific interactions with other residues of ND1 and ND4, thereby altering the structure and function of complex I. Cellular data obtained using cybrids, constructed by transferring mitochondria from the Chinese families into mtDNA-less (ρ°) cells, demonstrated that the mutations perturbed the stability, assembly, and activity of complex I, leading to changes in mitochondrial ATP levels and membrane potential and increasing the production of reactive oxygen species. These mitochondrial dysfunctions promoted the apoptotic sensitivity of cells and decreased mitophagy. Cybrids bearing only the m.3866T>C mutation displayed mild mitochondrial dysfunctions, whereas those harboring both m.3866T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions. These suggested that the m.3866T>C mutation acted in synergy with the m.11778G>A mutation, aggravating mitochondrial dysfunctions and contributing to higher penetrance of LHON in these families carrying both mtDNA mutations.

Characterization of aminoglycoside resistance and virulence genes among Enterococcus spp. isolated from a hospital in China.

This study investigated the aminoglycoside resistance phenotypes and genotypes, as well as the prevalence of virulence genes, in Enterococcus species isolated from clinical patients in China. A total of 160 enterococcal isolates from various clinical samples collected from September 2013 to July 2014 were identified to the species level using the VITEK-2 COMPACT system. The antimicrobial susceptibilities of the identified Enterococcus strains were determined by the Kirby-Bauer (K-B) disc diffusion method. PCR-based assays were used to detect the aminoglycoside resistance and virulence genes in all enterococcal isolates. Of 160 Enterococcus isolates, 105 were identified as E. faecium, 35 as E. faecalis, and 20 isolates were classified as "other" Enterococcus species. High-level aminoglycoside resistance (HLAR) for gentamicin, streptomycin, and both antibiotics was identified in 58.8, 50, and 34.4% of strains, respectively. The most common virulence gene (50.6% of isolates) was efaA, followed by asa1 (28.8%). The most prevalent aminoglycoside resistance genes were aac(6')-Ie-aph(2''), aph(2')-Id, aph(3')-IIIa, and ant(6')-Ia, present in 49.4%, 1.3%, 48.8% and 31.3% of strains, respectively. Overall, E. faecium and E. faecalis were most frequently associated with hospital-acquired enterococcal infections in Zhejiang Province. All aminoglycoside resistance genes, except aph(2'')-Id, were significantly more prevalent in HLAR strains than amongst high level aminoglycoside susceptible (HLAS) strains, while there was no significant difference between HLAR and HLAS strains in regard to the prevalence of virulence genes, apart from esp, therefore, measures should be taken to manage infections caused by multi-drug resistant Enterococcus species.

The clinical diagnostic significance of cerebrospinal fluid D-lactate for bacterial meningitis.

BACKGROUND: To study the clinical and laboratory significance of D­lactate in the diagnosis of bacterial meningitis (BM). METHODS: The levels of D­lactate, L­lactate, IL-6, IL-8, and other biochemical markers were determined in 83 CSF samples from different types of meningitis and the controls. RESULTS: The CSF values of D­lactate, L­lactate, IL-6, IL-8, erythrocytes, leukocytes, and protein were higher in patients with BM than those in the controls and patients with viral meningitis. The levels of D­lactate, L­lactate, IL-6, and erythrocytes in the BM group were higher than those in the tuberculous meningitis group. At the cutoff 12.8 µmol/l, D­lactate showed the diagnostic sensitivity of 94.7%. D­lactate gave the area under the curve (AUC) 0.905, which was higher than those of other markers. Using multiple marker detection, the AUC reached 0.956, which was the highest among all the parameters. Pearson correlation analysis revealed that D­lactate was positively correlated to IL-6 and L­lactate (r=0.727, 0.789 and P=0.000, 0.000, respectively). CONCLUSIONS: THE CSF concentrations of D­lactate are significantly increased in the presence of BM. Measurement of D­lactate provides a rapid diagnosis and differential diagnosis for BM. Combination of D­lactate with other biochemical markers improves the specificity.