Contribution of a mitochondrial tyrosyl-tRNA synthetase mutation to the phenotypic expression of the deafness-associated tRNASer(UCN) 7511A>G mutation

Nuclear modifier genes have been proposed to modify the phenotypic expression of mitochondrial DNA mutations. Using a targeted exome-sequencing approach, here we found that the p.191Gly>Val mutation in mitochondrial tyrosyl-tRNA synthetase 2 (YARS2) interacts with the tRNASer(UCN) 7511A>G mutation in causing deafness. Strikingly, members of a Chinese family bearing both the YARS2 p.191Gly>Val and m.7511A>G mutations displayed much higher penetrance of deafness than those pedigrees carrying only the m.7511A>G mutation. The m.7511A>G mutation changed the A4:U69 base-pairing to G4:U69 pairing at the aminoacyl acceptor stem of tRNASer(UCN) and perturbed tRNASer(UCN) structure and function, including an increased melting temperature, altered conformation, instability, and aberrant aminoacylation of mutant tRNA. Using lymphoblastoid cell lines derived from symptomatic and asymptomatic members of these Chinese families and control subjects, we show that cell lines harboring only the m.7511A>G or p.191Gly>Val mutation revealed relatively mild defects in tRNASer(UCN) or tRNATyr metabolism, respectively. However, cell lines harboring both m.7511A>G and p.191Gly>Val mutations displayed more severe defective aminoacylations and lower tRNASer(UCN) and tRNATyr levels, aberrant aminoacylation, and lower levels of other tRNAs, including tRNAThr, tRNALys, tRNALeu(UUR), and tRNASer(AGY), than those in the cell lines carrying only the m.7511A>G or p.191Gly>Val mutation. Furthermore, mutant cell lines harboring both m.7511A>G and p.191Gly>Val mutations exhibited greater decreases in the levels of mitochondrial translation, respiration, and mitochondrial ATP and membrane potentials, along with increased production of reactive oxygen species. Our findings provide molecular-level insights into the pathophysiology of maternally transmitted deafness arising from the synergy between tRNASer(UCN) and mitochondrial YARS mutations.

Nuclear modifier genes have been proposed to modify the phenotypic expression of mitochondrial DNA mutations. Using a targeted exome-sequencing approach, here we found that the p.191Gly>Val mutation in mitochondrial tyrosyl-tRNA synthetase 2 (YARS2) interacts with the tRNA Ser(UCN) 7511A>G mutation in causing deafness. Strikingly, members of a Chinese family bearing both the YARS2 p.191Gly>Val and m.7511A>G mutations displayed much higher penetrance of deafness than those pedigrees carrying only the m.7511A>G mutation. The m.7511A>G mutation changed the A4:U69 base-pairing to G4:U69 pairing at the aminoacyl acceptor stem of tRNA Ser(UCN) and perturbed tRNA Ser(UCN) structure and function, including an increased melting temperature, altered conformation, instability, and aberrant aminoacylation of mutant tRNA. Using lymphoblastoid cell lines derived from symptomatic and asymptomatic members of these Chinese families and control subjects, we show that cell lines harboring only the m.7511A>G or p.191Gly>Val mutation revealed relatively mild defects in tRNA Ser(UCN) or tRNA Tyr metabolism, respectively. However, cell lines harboring both m.7511A>G and p.191Gly>Val mutations displayed more severe defective aminoacylations and lower tRNA Ser(UCN) and tRNA Tyr levels, aberrant aminoacylation, and lower levels of other tRNAs, including tRNA Thr , tRNA Lys , tRNA Leu(UUR) , and tRNA Ser(AGY) , than those in the cell lines carrying only the m.7511A>G or p.191Gly>Val mutation.

Furthermore, mutant cell lines harboring both m.7511A>G and p.191Gly>Val mutations exhibited greater decreases in the levels of mitochondrial translation, respiration, and mitochondrial ATP and membrane potentials, along with increased production of reactive oxygen species. Our findings provide molecularlevel insights into the pathophysiology of maternally transmitted deafness arising from the synergy between tRNA Ser(UCN) and mitochondrial YARS mutations.
Defects of mitochondrial tRNA metabolisms have been associated with both syndromic deafness (hearing loss with other medical problems, such as diabetes) and nonsyndromic deafness (where hearing loss is the only obvious medical problem) (1)(2)(3)(4)(5). In humans, mitochondrial genomes (mtDNA) 3 encode 13 subunits of the oxidative phosphorylation system (OXPHOS), two rRNAs and 22 tRNAs required for translation (6,7). The formation of functional tRNA molecules used for protein synthesis requires the transcription, nucleolytic processing, posttranscriptional nucleotide modifications, and aminoacylation (4 -9). These proteins involved in the tRNA maturation processing, especially mitochondrial tRNA synthetases, encoded by nuclear genes, were synthesized in the cytosol and subsequently imported into mitochondria (7, 9 -11). These deafness-associated tRNA mutations have structural and functional consequences for corresponding tRNAs (1,12). These included the aberrant processing of 3Ј end tRNA Ser(UCN) precursor, caused by m.7445AϾG mutation (13,14), instability of the folded secondary structure of tRNA Glu due to m.14692AϾG mutation (15), deficient m 1 G37 modification of tRNA Asp caused by m.7551AϾG mutation (16), and defective aminoacylation of tRNA His resulting from m.12201TϾC muta-tion (17). Furthermore, alterations in the LARS2, KARS, IARS2, and NARS2 encoding mitochondrial leucyl-tRNA, lysyl-tRNA, isoleucyl-tRNA, and asparaginyl-tRNA synthetases have been associated with syndromic deafness, respectively (18 -21). Moreover, nonsyndromic deafness in some families was caused by the coexistence of the 12S rRNA m.1555AϾG mutation and p.Ala10Ser mutation in TRMU responsible for the biosynthesis of m 5 s 2 U at the wobble position of tRNA Gln , tRNA Glu , and tRNA Lys (22,23). However, the pathophysiology underlying deafness-linked aberrant tRNA metabolisms remains poorly understood.
As shown in Fig. 1A, the deafness-associated tRNA Ser(UCN) 7511AϾG mutation converted the A4-U69 base-pairing into a G4-U69 base-pairing at the aminoacyl acceptor stem of this tRNA (24 -27). This base-pairing may play an important role in the stability and identity of tRNA (24,25). We therefore hypothesized that the m.7511AϾG mutation perturbed both structure and function of tRNA Ser(UCN) . The m.7511AϾG mutation was identified in several families from different ethnic groups, with varying expressivity and penetrance of deafness (27)(28)(29)(30). In particular, 9 of 10 matrilineal relatives in a three-generation Chinese pedigree carrying the m.7511AϾG mutation exhibited hearing impairment, in contrast with only a small portion of hearing-impaired matrilineal relatives in two French pedigrees and one Japanese family carrying the same mtDNA mutation (27)(28)(29)(30). These findings suggest that the nuclear modifier genes, especially those involved in mitochondrial tRNA metabolism, contributed to the phenotypic expression of m.7511AϾG mutation. By target exome sequencing (genes encoding 20 mitochondrial tRNA synthetases and 25 tRNA modifying enzymes), we identified the known variant (c.572GϾT, p.191GlyϾVal) in the YARS2 gene encoding the mitochondrial tyrosyl-tRNA synthetase (31,32) that interacted with the m.7511AϾG mutation to cause hearing loss in a three-generation Chinese family with extremely high penetrance of hearing loss. In the present study, we further investigated the impact of the m.7511AϾG mutation on the structure and function of tRNA Ser(UCN) . The effects of YARS2 p.191GlyϾVal and m.7511AϾG mutations on mitochondrial functions were first assessed for the tRNA metabolism, including aminoacylation capacities and stability of tRNA, through the use of lymphoblastoid mutant cell lines derived from members of the Chinese family (individuals carrying only the m.7511AϾG mutation, only the YARS2 p.191GlyϾVal mutation or both m.7511AϾG and heterozygous or homozygous p.191GlyϾVal mutations), and genetically unrelated control subjects lacking these mutations. These cell lines were further evaluated for an effect on mitochondrial translation, respiration, production of ATP, mitochondrial membrane potential, and reactive oxygen species (ROS).

The m.7511A>G mutation altered the stability and conformation of tRNA Ser(UCN)
As shown in Fig. 1A, the m.7511AϾG mutation changed the typical A4-U69 base-pairing into a noncanonical G4-U69 basepairing at the acceptor stems. To experimentally test the effect of m.7511AϾG mutation on the stability of tRNA Ser(UCN) , we examined the melting temperatures (T m ) of WT (A4) and mutant (G4) tRNA Ser(UCN) transcripts. These T m values were determined by calculating the derivatives of the absorbance against a temperature curve. As shown in Fig. 1B, the T m values for WT (A4) and mutant (G4) tRNA Ser(UCN) transcripts were 41.7 and 51°C, respectively. These data suggested that the tRNA Ser(UCN) with a G4:U69 bp may be more stable than the tRNA Ser(UCN) with an A4:U69 bp.
As shown in Fig. 1C, electrophoretic patterns showed that the mutant (G4) tRNA Ser(UCN) transcript migrated faster than the WT (A4) tRNA Ser(UCN) transcript under native conditions. However, there was no difference of migration pattern between WT (A4) and mutant (G4) tRNA Ser(UCN) transcripts under denaturing conditions. These data indicated that the m.7511AϾG mutation resulted in the conformational change of tRNA Ser(UCN) .

Clinical presentation of a hearing-impaired Han Chinese pedigree
One Han Chinese hearing-impaired proband carrying the m.7511AϾG mutation was identified among 2651 Chinese hearing-impaired probands but absent in 574 Chinese hearing-normal controls (28). As shown in Fig. S1A, the Chinese family exhibited extremely high penetrance of hearing loss. As shown in Fig. S2 and Table S1, 9 of 10 matrilineal relatives exhibited the variable degree of hearing impairment (two with mild hearing loss, six with moderate hearing loss, and one with severe hearing loss), whereas none of other members in this family had hearing loss. The age-at-onset of hearing loss ranged from 5 to 55 years old, with an average of 25 years old. There was no evidence that any of the other members of this family had any other causes to account for hearing loss. These matrilineal relatives showed no other clinical abnormalities, including cardiac failure, muscular diseases, visual failure, and neurological disorders. Further analysis showed that the m.7511AϾG mutation was present in homoplasmy in all matrilineal relatives but not in other members of this family (Fig. S1B).

Targeting exome sequence analysis
The higher penetrance of hearing loss in this Chinese family implied that nuclear modifier genes, especially for genes involved in mitochondrial tRNA metabolism, influence the phenotypic manifestation of m.7511AϾG mutation. To test this hypothesis, we performed targeting exome-sequencing analyses of 45 genes encoding 20 mitochondrial tRNA synthetases and 25 tRNA-modifying enzymes (Table S2) among seven matrilineal relatives (II-5, II-7, III-3, III-4, III-5, III-6, and III-7) and two married-in controls (II-4 and II-5) of WZD200 pedigree carrying the m.7511AϾG mutation. As a result, we identified the known (c.572GϾT, p.191GlyϾVal) mutation in the YARS2 gene encoding the mitochondrial tyrosyl-tRNA synthetase in six hearing-impaired matrilineal relatives but not in the hearing-normal matrilineal relative (III-7). We further analyzed the presence of the c.572GϾT mutation in three symptomatic members and six asymptomatic subjects of this Chinese family and 13 symptomatic members and five asymptomatic A nuclear modifier for deafness expression of tRNA mutation subjects of a Japanese family (30), by restriction fragment length polymorphism analysis, because the c.572GϾT mutation disrupted a Tsp45I site (32). In the Chinese family, the symptomatic subjects (II-1 and II-5) and married-in control (I-1) carried the homozygous c.572GϾT mutation, the symptomatic subjects (I-2, II-2, II-7, III-3, III-4, III-5 and III-6) harbored the heterozygous c.572GϾT mutation, and the asymptomatic individual (III-7) and three married controls lacked the c.572GϾT mutation ( Fig. S1A and Table S1). However, this mutation was absent in the members of the Japanese family (30). These suggested that the c.572GϾT mutation may increase the penetrance of hearing loss in the Chinese family.

Reductions in the steady-state levels of mitochondrial tRNAs
To test if the m.7511AϾG mutation affected the conformation of tRNA Ser(UCN) ex vivo, total RNAs from mitochondria isolated from various cell lines were electrophoresed through 15% polyacrylamide gel (native condition) and then electroblotted onto a positively charged nylon membrane (Roche  (12). An arrow denotes the location of the m.7511AϾG mutation. B, thermal stability of WT (A4) and mutant (G4) tRNA Ser(UCN) . Absorbance (Abs) of WT and mutant (MT) was measured at 260 nm with a heating rate of 1°C/min from 25 to 95°C (red curves). First derivative, generated with the expression dA/dT, showed the rate of absorbance change (blue curves). The calculations were based on three independent experiments. C, in vitro analysis of the conformation of tRNA Ser(UCN) . WT and mutant tRNA Ser(UCN) transcripts were electrophoresed through native polyacrylamide gel, electroblotted, and hybridized with the DIG-labeled oligonucleotide probes specific for tRNA Ser(UCN) . D, Northern blot analysis of tRNA under native conditions. Two micrograms of total mitochondrial RNA from mutant and control cell lines were electrophoresed through native polyacrylamide gel, electroblotted, and hybridized with DIG-labeled oligonucleotide probes specific for the tRNA Ser(UCN) and tRNA Leu(CUN) , respectively.
Applied Science) for hybridization analysis with digoxigenin (DIG)-labeled oligodeoxynucleotide probes for tRNA Ser(UCN) and tRNA Leu(CUN) , respectively. As shown in Fig. 1D, the electrophoretic patterns showed that the tRNA Ser(UCN) in the mutant cell lines (III-7) carrying the m.7511AϾG mutation migrated faster than those of one cell line (A61) lacking this mutation. However, there were no differences in the migration of tRNA Leu(CUN) between WT and mutant cell lines.
To further examine the effect of m.7511AϾG and c.572GϾT mutations on the stability of tRNA, mitochondrial RNAs from various cell lines were subjected to Northern blotting and hybridized with DIG-labeled oligodeoxynucleotide probes specific for tRNA Ser(UCN) , tRNA Tyr , tRNA Glu , tRNA Asp , tRNA Met , tRNA Lys , tRNA Leu(UUR) , and 5S rRNA, respectively. For comparison, the average levels of each tRNA in various cell lines were normalized according to the level of the 5S rRNA. As shown in Fig. 2, the steady-state levels of tRNA Ser(UCN) in the cell line III-7, bearing only the m.7511AϾG mutation, and tRNA Tyr in the cell line I-1, carrying the homozygous c.572GϾT mutation, were decreased 49.5 and 33.7%, respectively, as compared with those in the control cell line (A61) lacking these mutations. Strikingly, cells harboring both m.7511AϾG and c.572GϾT mutations exhibited drastic decreases in levels of tRNA Ser(UCN) and tRNA Tyr as well as various reductions in the other tRNAs (Fig. 2B). In particular, the average steady-state levels of tRNA Ser(UCN) , tRNA Tyr , tRNA Glu , tRNA Asp , tRNA Met , tRNA Lys , and tRNA Leu(UUR) in mutant cell lines carrying both m.7511AϾG and homozygous c.572GϾT mutations were decreased by 72.2, 58.9, 33.5, 69.6, 86.1, 13.5, and 21.2%, as compared with the average values in the control cell line (A61), respectively. Furthermore, the average steadystate levels of tRNA Ser(UCN) , tRNA Tyr , tRNA Glu , tRNA Asp , tRNA Met , tRNA Lys , and tRNA Leu(UUR) in mutant cell lines carrying both m.7511AϾG and heterozygous c.572GϾT mutations were decreased by 75.8, 51.1, 35.5, 64.4, 71.1, 11.2, and 26.1%, as compared with the average values in the control cell line (A61), respectively.

Defects in tRNA aminoacylation
The aminoacylation capacities of tRNA Ser(UCN) , tRNA Tyr , tRNA Thr , tRNA Leu(UUR) , tRNA Lys , and tRNA Ser(AGY) in various control and mutant cell lines were examined by using electrophoresis in an acid polyacrylamide/urea gel system to separate uncharged tRNA species from the corresponding charged tRNA, electroblotting and hybridizing with the above tRNA probes. As shown in Fig. 3A, the slower-migrating band (top band) represents the charged tRNA, and the faster-migrating band (bottom band) represents uncharged tRNA. The electrophoretic patterns revealed two stacked bands present for the WT tRNA Ser(UCN) and two well-separated bands for the mutant tRNA Ser(UCN) . Furthermore, either charged or uncharged tRNA Ser(UCN) migrated faster in all mutant cell lines carrying the m.7511AϾG mutation than those in other cell lines lacking the mutation. To further distinguish nonaminoacylated tRNA from aminoacylated tRNA, samples of tRNAs were deacylated after heating for 10 min at 60°C (pH 9.0) and then run in parallel. As shown in Fig. 3, the deacylated samples gave only one band (uncharged tRNA) in both mutant and control cell lines.
As shown in Fig

A nuclear modifier for deafness expression of tRNA mutation Decreases in the levels of mitochondrial proteins
To assess whether the c.572GϾT mutation enhanced the defects in mitochondrial translation associated with m.7511AϾG mutation, a Western blot analysis was carried out to examine the levels of seven mtDNA encoding polypeptides (of respiratory complex) in various cell lines with VDAC as a loading control. As shown in

Reduced activities of respiratory complexes I, III, and IV
To examine whether the c.572GϾT mutation worsened the respiratorydeficiencycausedbym.7511AϾGmutation,wemeasured the activities of respiratory complexes by isolating mitochondria from mutant and control cell lines (33,34). As shown in

Respiration defects in mutant cells
To further assess whether the m.7511AϾG and c.572GϾT mutations altered cellular bioenergetics, we examined the oxygen consumption rates (OCR) of various mutant and control A nuclear modifier for deafness expression of tRNA mutation cell lines using a Seahorse Bioscience XF-96 extracellular flux analyzer (35,36). In this system, a single experiment can measure all major aspects of mitochondrial coupling and respiratory control, including basal respiration, O 2 consumption attributed to ATP production, proton leak, maximum respiratory rate, reserve capacity, and nonmitochondrial respiration (Fig. 6B). As shown in Fig. 6C, the basal OCR in the mutant cell lines carrying only the c.572GϾT mutation, the m.7511AϾG mutation, or both the m.7511AϾG and heterozygous or homozygous c.572GϾT mutations were 92.5, 68.6, 47.0, and 36.9% of the mean values measured in the control cell lines (p Ͻ 0.05), respectively. To investigate which of the enzyme complexes of the respiratory chain was affected in the mutant cell lines, OCR was measured after the sequential addition of oligomycin (to inhibit the ATP synthase), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (to uncouple the mitochondrial inner membrane and allow for maximum electron flux through the electron transfer chain), rotenone (to inhibit complex I), and antimycin A (to inhibit complex III) (56). The differences between the basal OCR and the drug-insensitive

A nuclear modifier for deafness expression of tRNA mutation
OCR yielded the amount of ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity, and nonmitochondrial OCR. As illustrated in Fig. 6C

Reduced levels in mitochondrial ATP production
To examine the capacity of oxidative phosphorylation, we measured the levels of cellular and mitochondrial ATP produc-tion using a luciferin/luciferase assay. Populations of cells from various mutant and control cell lines were incubated in the medium in the presence of glucose (total cellular ATP production) or 2-deoxy-D-glucose with pyruvate (mitochondrial ATP production). As shown in Fig. 7, the levels of mitochondrial ATP production in mutant cell lines carrying only the c.572GϾT mutation, the m.7511AϾG mutation, or both the m.7511AϾG and heterozygous or homozygous c.572GϾT mutations were 77.6, 58.9, 62.9, and 44.8% of the control cell lines. Moreover, the levels of total cellular ATP production in the above mutant cell lines were 97.5, 84.3, 79.5, and 67.1%, relative to the mean value measured in the control cell lines, respectively.

Decreases in mitochondrial membrane potentials
The mitochondrial membrane potential (⌬⌿ m ) generated by proton pumps (complexes I, III, and IV) is an essential component in the process of energy storage during oxidative phosphorylation (37). We examined the levels of ⌬⌿ m in the mutant and control cell lines using a fluorescence probe JC-10 assay system. The ratios of fluorescence intensity excitation/emission ϭ 490/590 and 490/530 nm (FL590/FL530) were recorded to reflect the ⌬⌿ m level of each sample. As shown in Fig. 8, the ⌬⌿ m levels of mutant cell lines harboring only the c.572GϾT mutation, the m.7511AϾG mutation, or both the m.7511AϾG and heterozygous or homozygous c.572GϾT mutations were 97.5, 71.1, 67.7, and 64.1% of the mean values measured in the control cell lines, respectively. In contrast, the ⌬⌿ m levels in mutant cell lines in the presence of FCCP were comparable with those measured in the control cell lines.

Increase of ROS production
Respiratory deficiency can increase the production of ROS (38,39). In this study, we measured the levels of ROS generation in mutant and control cell lines with flow cytometry under normal and H 2 O 2 -stimulated conditions. To detect the capacity of reaction upon increasing levels of ROS under oxidative stress, we calculated the ratio of geometric mean intensity between unstimulated and stimulated with H 2 O 2 in each cell line. As shown in Fig. 9, the levels of ROS generation in the mutant cell lines harboring only the c.572GϾT mutation, the m.7511AϾG mutation, or both the m.7511AϾG and heterozygous or homozygous c.572GϾT mutations were 119.3, 109.6, 127.3, and 140.6% of the control cell lines.

The pathogenicity of tRNA Ser(UCN) 7511A>G mutation
In the present study, we further investigated the molecular mechanism of the deafness-associated m.7511AϾG mutation. Indeed, the occurrence of the m.7511AϾG mutation in several hearing-impaired families from different ethic backgrounds strongly indicated that this mutation is involved in the pathogenesis of deafness (26 -30). The m.7511AϾG mutation caused the substitution of the A4:U69 base-pairing with G4:U69 basepairing at the aminoacyl acceptor stem of tRNA Ser(UCN) (12,26,27). In fact, this A4:U69 base-pairing may play an important role in the stability and identity of tRNA (12, 24, 25, 39 -42). Therefore, it was hypothesized that m.7511AϾG mutation led to structural and functional consequences for tRNA Ser(UCN) , including the processing of RNA precursors, stability, and aminoacylation of tRNA Ser(UCN) . In particular, the substitution A4:U69 base-pairing with G4:U69 base-pairing caused by the m.7511AϾG mutation may restrict the accessible conformation space of tRNA Ser(UCN) (43)(44)(45). Here, the altered structure of tRNA Ser(UCN) caused by the m.7511AϾG mutation was evidenced by the increased melting temperature and electrophoretic mobility of mutated tRNA with respect to the WT molecule in vitro or ex vivo. The instability of mutant tRNA was further supported by marked reductions in the steady-state level of tRNA Ser(UCN) in the cybrid mutant cell lines (26) and lymphoblastoid cell lines carrying the m.7511AϾG mutation in the present study.
Furthermore, the substitution A4:U69 base-pairing with G4:U69 base-pairing induced by the m.7511AϾG mutation may result in the faulty interaction of tRNA Ser(UCN) with mitochondrial seryl-tRNA synthetase, thereby altering the aminoacylation properties of tRNA Ser(UCN) (39,(43)(44)(45)(46). Indeed, all human AlaRS mischarged to noncognate tRNAs, such as tRNA Cys and tRNA Asp , with the G4:U69 bp (45,46). Therefore, mutant tRNA Ser(UCN) with G4:U69 bp can be mischarged with other amino acids. In this study, the possible mischarging to noncognate tRNAs of mutant tRNA Ser(UCN) may account for the improperly aminoacylated tRNA Ser(UCN) , as suggested by the aberrantly aminoacylated tRNA Ser(UCN) in the mutant cell lines and faster electrophoretic mobility of mutated tRNA with re- presented the ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity, and nonmitochondrial OCR in mutant and control cell lines. Nonmitochondrial OCR was determined as the OCR after rotenone/antimycin A treatment. Basal OCR was determined as OCR before oligomycin minus OCR after rotenone/ antimycin A. ATP-linked OCR was determined as OCR before oligomycin minus OCR after oligomycin. Proton leak was determined as basal OCR minus ATP-linked OCR. Maximal OCR was determined as the OCR after FCCP minus nonmitochondrial OCR. Reserve capacity was defined as the difference between maximal OCR after FCCP minus basal OCR. OCR values were expressed in pmol of oxygen/min/g of protein.
The average values of three determinations for each cell line were shown. Graph details and symbols are explained in the legend to Fig. 2.

A nuclear modifier for deafness expression of tRNA mutation
spect to the WT molecules. Alternatively, the mutant tRNA Ser(UCN) may be charged to a lesser extent by the mitochondrial serylsynthetase. In this study, only mildly reduced efficiencies of aminoacylated tRNA Ser(UCN) were observed in a mutant cell line carrying only the m.7511AϾG mutation, in contrast to marked decreases of aminoacylation in the tRNA Leu(UUR) with the 14AϾG substitution or tRNA Lys with the 55AϾG mutation (47)(48)(49). Improper aminoacylation and instability of tRNA Ser(UCN) were responsible for marked reductions in the level of tRNA Ser(UCN) observed in a cell line carrying the m.7511AϾG mutation, as in the cases of other pathogenic tRNA mutations (47)(48)(49)(50)(51). The aberrant tRNA Ser(UCN) metabolism resulted in the impairment of mitochondrial translation, defective oxidative phosphorylation, and increasing production of oxidative reactive species (15,16,17,26). The resultant mitochondrial dysfunctions would lead to the dysfunction or death of cochlear cells, thereby contributing to the development of hearing loss.

The YARS2 p.191Gly>Val mutation enhanced the phenotypic manifestation of the m.7511A>G mutation
Genetic modifiers involved in mitochondrial tRNA metabolism modulate the phenotypic manifestation of the deafnessassociated 12S rRNA mutations (22,23,52). In this study, the penetrances of hearing loss in this Chinese family harboring both the m.7511AϾG and YARS2 p.191GlyϾVal mutations were significantly higher than those in the French and Japanese families carrying only the m.7511AϾG mutation (29,30). Furthermore, cell lines bearing both p.191GlyϾVal and m.7511AϾG mutations exhibited greater mitochondrial dysfunctions than those carrying only p.191GlyϾVal or m.7511AϾG mutation. Strikingly, mutant cell lines harboring both m.7511AϾG and p.191GlyϾVal mutations exhibited not only more decreases in the aminoacylation efficiencies of tRNA Ser(UCN) and tRNA Tyr but also deficient aminoacylation of tRNA Thr , tRNA Lys , tRNA Leu(UUR) , and tRNA Ser(AGY) , as compared with those in the cell lines carrying only the p.191GlyϾVal or m.7511AϾG mutation. The aberrantly aminoacylated tRNA makes the mutant tRNA metabolically less stable and more subject to degradation, thereby lowering the level of the tRNA in mutant cell lines (17,26,40). In the present study, mutant cell lines bearing only the m.7511AϾG mutation exhibited 48% reductions in the level of tRNA Ser(UCN) , and mutant cell lines harboring only the p.191GlyϾVal mutation displayed 33.7% decreases in the level of tRNA Tyr. , respectively. By contrast, mutant cell lines harboring both m.7511AϾG and homozygous p.191GlyϾVal mutations reveled 70% decreases in the level of tRNA Ser(UCN) and 59% reductions in the level of tRNA Tyr as well as various decreases in the levels of other tRNAs, including tRNA Glu , tRNA Asp , tRNA Met , tRNA Lys , and tRNA Leu(UUR) . Notably, ϳ70% reductions in the steady-state levels of tRNA Ser(UCN) in the cells carrying both p.191GlyϾVal and m.7511AϾG mutations were in good agreement with the 75% decrease in the levels of tRNA Ser(UCN) in those in the mutant cybrid cells bearing the m.7511AϾG, tRNA Ala 5655AϾG, and ND1 3308TϾC mutations (26). These data strongly suggested that the synergic interaction between the YARS2 p.191GlyϾVal and m.7511AϾG mutations mediated mitochondrial tRNA metabolisms, especially exacertbating the defects of tRNA Ser(UCN) and tRNA Tyr metabolisms. Notably, mutations in the TRMU involved in biosynthesis of m 5 s 2 U at the wobble position of tRNA Gln , tRNA Glu , and tRNA Lys affected the metabolism of not only tRNA Lys , tRNA Glu , tRNA-Gln , but also other mitochondrial tRNA (22,53).
Both shortage of and aberrant aminoacylation of tRNAs led to impairments of mitochondrial translation. In this investigation, 50% decreases in the levels of mtDNA encoding proteins observed in the mutant cells carrying both the m.7511AϾG and p.191GlyϾVal mutations are below the proposed threshold level (50%) to produce a clinical phenotype associated with a mtDNA mutation (23,47,48,54). The defects of mitochondrial translation were responsible for the respiratory deficiency, uncoupling of the oxidative pathway for ATP synthesis, diminished mitochondrial membrane potentials, and overproduction of ROS (7,48,52,55). In particular, more drastic decreases of oxygen consumption rates, mitochondrial ATP production, and mitochondrial membrane potentials and increases of ROS production were observed in the cell lines carrying both the p.191GlyϾVal and m.7511AϾG mutations than those in cell lines carrying only the p.191GlyϾVal or m.7511AϾG mutation. These mitochondrial dysfunctions yielded a preferential effect on the hair cells and neurons in the cochlea, because cochlear functions depend on a very high rate of ATP production (56 -58). This would result in the dysfunction or death of hair cells and neurons in the cochlea carrying both the p.191GlyϾVal and m.7511AϾG mutations, thereby producing a phenotype of hearing loss.
In summary, we demonstrated that the pathophysiology of maternally inherited deafness was manifested by aberrant

A nuclear modifier for deafness expression of tRNA mutation
tRNA metabolisms due to the combination of YARS2 p.191GlyϾVal with tRNA Ser(UCN) 7511AϾG mutations. The m.7511AϾG mutation altered both the structure and function of tRNA Ser(UCN) . The p.191GlyϾVal mutation deteriorated the aberrant tRNA metabolisms associated with the m.7511AϾG mutation. The aberrant tRNA metabolisms resulted in defective mitochondrial translation, respiratory deficiency, decreasing ATP production, and increasing ROS production. These biochemical defects led to the high penetrance and occurrence of deafness in the Chinese family carrying both the m.7511AϾG and p.191GlyϾVal mutations. Our findings provide new insights into the pathophysiology of maternally inherited deafness, manifested by the synergetic interaction between mitochondrial and nuclear gene products underlying aberrant tRNA metabolism.

Subjects
One Han Chinese family (WZD200), as shown in Fig. S1A, was recruited from the Otology Clinics of Wenzhou Medical University (Zhejiang, China), as described previously (28). Comprehensive history-taking, physical examination, and audiological examination were performed to identify any syndromic findings, history of exposure to aminoglycosides, and genetic factors related to hearing impairment in all available members of this Chinese pedigree, as detailed previously (59,60). The 574 control subjects were from a panel of unaffected subjects of Han Chinese ancestry from the same region. This study followed the principles of the Declaration of Helsinki. Informed consent was obtained from the participants prior to their participation in the study, under protocols approved by the Ethics Committees of Zhejiang University and the Wenzhou Medical University.

Mitochondrial DNA-sequencing analysis
Genomic DNA was isolated from whole blood of participants using the QIAamp DNA Blood Mini Kit (Qiagen, catalog no. 51104). The entire mtDNAs of the family members of WZD200 (I-1, II-1, II-2, III-5, and III-7) and one Chinese control subject (A61) were PCR-amplified in 24 overlapping fragments using sets of the light (L) and heavy (H) strand oligonucleotide primers, as described previously (61). These sequence results were compared with the updated consensus Cambridge sequence (GenBank TM accession number NC_012920) (6). For the analysis for the presence and level of the m.7511AϾG mutation, the PCR DNA fragments (117 bp) spanning the tRNA Ser(UCN) gene were amplified using genomic DNA as the template and the oligodeoxynucleotides 5Ј-CCCCATGGCCTCCATGACTTT-TTAAA-3Ј and 5Ј-CTACTTGCGCTGCATGTGCCATTAA-GAT-3Ј. The resultant 117-bp segments were digested with the restriction enzyme DraI and analyzed by electrophoresis through a 14% polyacrylamide gel. After ethidium bromide staining, the ImageQuant program was used to determine the proportions of digested and undigested PCR product to ascertain whether the m.7511AϾG mutation was present in homoplasmy in these subjects (Fig. S1B).

Target exome sequencing
A panel of exome sequencings (genes encoding 20 mitochondrial tRNA synthetases and 25 tRNA-modifying enzymes, Table S1) of seven matrilineal relatives (II-5, II-7, III-3, III-4,  III-5, III-6, and III-7) carrying the m.7511AϾG mutation and two married-in controls (II-4 and II-6) of WZD200 pedigree were performed by BGI (Shenzhen, China). High-quality genomic DNA (3 g) was captured by hybridization using the SureSelect XT Human All Exon 50Mb kit (Agilent Technologies). Samples were prepared according to the manufacturer's instructions. Each captured library was run on a HiSeq 2000 instrument, and sequences were generated as 90-bp pair-end reads. An average of 82 million paired reads were generated per sample, the mean duplication rate was 6.37%, and 98% of the targeted region was covered by at least 50 ϫ mean depth. All sequencing reads were mapped to the human reference genome (GRCh37) at UCSC. The software SOAPsnp was used to assemble the consensus sequence and call genotypes in target regions. GATK (Indel Genotyper version 1.0) was used for indel detection. The threshold for filtering SNPs included the following criteria. SNP quality score should be м20; sequencing depth should be between 4 and 200; estimated copy number should be no more than 2; and the distance between two SNPs should be larger than 5.

Mutation analysis of YARS2 gene
Five pairs of primers for PCR-amplifying exons and their flanking sequences, including splicing-donor and acceptorconsensus sequences of YARS2, were used for this analysis, as described previously (32). Fragments spanning five exons and flanking sequences from seven matrilineal relatives (II-5, II-7, III-3, III-4, III-5, III-6, and III-7) and three married-in controls (I-1, II-4, and II-6) carrying the m.7511AϾG mutation in the Chinese family and two genetically unrelated Chinese controls were PCR-amplified, purified, and subsequently analyzed by Sanger sequencing. These sequence results were compared with the YARS2 genomic sequence (RefSeq NC_000012.12). Genotyping for the c.572GϾT mutation in other subjects was PCR-amplified for exon 1 and followed by digestion of the 626-bp segment with the restriction enzyme Tsp45I. The forward and reverse primers for exon 1 are 5Ј-GACTCGCTT-CATGTGGGTCAT-3Ј and 5Ј-CGAAGGGCAGCAACT-ACAATC-3Ј, respectively. The Tsp45I-digested products were analyzed on 10% polyacrylamide gel (Fig. S1C).

Cell lines and culture conditions
Lymphoblastoid cell lines were immortalized by transformation with the Epstein-Barr virus, as described elsewhere (62). Cell lines derived from five members of the Chinese family (hearing-impaired subjects II-2 and III-4 harboring both m.7511AϾG and heterozygous c.572GϾT mutations, II-1 and II-5 carrying both m.7511AϾG and homozygous c.572GϾT mutations, a hearing-normal individual (I-1) bearing only the homozygous c.572GϾT mutation, and one hearing-normal subject (III-7) carrying only the m.7511AϾG mutation) and two genetically unrelated control individuals (A61 and A62) lacking these mutations (Table S3) were grown in the RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum.

UV melting assays
UV melting assays were carried out as described previously (50,63). The WT and mutant tRNA Ser(UCN) transcripts were generated as detailed elsewhere (64). The transcripts were diluted in buffer including 50 mM sodium phosphate (pH 7.0), 50 mM NaCl, 5 mM MgCl 2 , and 0.1 mM EDTA. Absorbance against temperature melting curves were measured at 260 nm with a heating rate of 0.5°C/min from 25 to 95°C through an Agilent Cary 100 UV spectrophotometer.
The aminoacylation assays were carried out as detailed previously (63,66). To further distinguish nonaminoacylated tRNA from aminoacylated tRNA, total RNAs were treated by heat shock for 10 min at 60°C at pH 9.0 and then run in parallel (63,66). DIG-labeled oligodeoxynucleotide probes for tRNA Ser(UCN) , tRNA Tyr , tRNA Ser(AGY) , tRNA Leu(UUR) , tRNA Lys , and tRNA Thr were as described above. Quantification of density in each band was performed as detailed previously (63,66).
For the tRNA mobility shift assay, 2 g of total mitochondrial RNAs were electrophoresed through a 10% polyacrylamide native gel at room temperature in 50 mM Tris-glycine buffer. After electrophoresis, the gels were treated according to the procedure for the tRNA Northern blot analysis described above.

Assays of activities of respiratory complexes
The enzymatic activities of complex I, II, III, and IV were assayed as detailed elsewhere (33,67,68). Briefly, complex I (NADH ubiquinone oxidoreductase) activity was determined by following the oxidation of NADH with ubiquinone as the electron acceptor. complex III (ubiquinone cytochrome c oxi-doreductase) activity was measured as the reduction of cytochrome c (III) using D-ubiquinol-2 as the electron donor. The activity of complex IV (cytochrome c oxidase) was monitored by following the oxidation of cytochrome c (II).

Measurements of oxygen consumption
OCR in lymphoblastoid cell lines were measured with a Seahorse Bioscience XF-96 extracellular flux analyzer (Seahorse Bioscience), as detailed previously (17,35,36).

ATP measurements
The Cell Titer-Glo luminescent cell viability assay kit (Promega) was used for the measurement of cellular and mitochondrial ATP levels, according to the modified manufacturer's instructions (17,68).

Assessment of mitochondrial membrane potential
The JC-10 Assay Kit-Microplate (Abcam) was used to assess the mitochondrial membrane potential, according to a modification of the manufacturer's instructions (37).

Measurement of ROS production
ROS measurements were performed following the procedures detailed previously (40,50,69).

Computer analysis
Statistical analysis was performed using the unpaired, twotailed Student's t test contained in the Microsoft Excel program (version 2017). Differences were considered significant at p Ͻ 0.05.