A Hypertension-Associated tRNA^Ala Mutation Alters tRNA Metabolism and Mitochondrial Function

Abstract

In this report, we investigated the pathophysiology of a novel hypertension-associated mitochondrial tRNAAla 5655A → G (m.5655A → G) mutation. The destabilization of a highly conserved base pairing (A1-U72) at the aminoacyl acceptor stem by an m.5655A → G mutation altered the tRNAAla function. An in vitro processing analysis showed that the m.5655A → G mutation reduced the efficiency of tRNAAla precursor 5′ end cleavage catalyzed by RNase P. By using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mitochondrial DNA (mtDNA)-less (ρo) cells, we showed a 41% reduction in the steady-state level of tRNAAla in mutant cybrids. The mutation caused an improperly aminoacylated tRNAAla, as suggested by aberrantly aminoacylated tRNAAla and slower electrophoretic mobility of mutated tRNA. A failure in tRNAAla metabolism contributed to variable reductions in six mtDNA-encoded polypeptides in mutant cells, ranging from 21% to 37.5%, with an average of a 29.1% reduction, compared to levels of the controls. The impaired translation caused reduced activities of mitochondrial respiration chains. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These caused increases in the production of reactive oxygen species in the mutant cybrids. The data provide evidence for the association of the tRNAAla 5655A → G mutation with hypertension.

A deafness-associated tRNAAspmutation alters the m1G37 modification, aminoacylation and stability of tRNAAspand mitochondrial function

“…The bromodeoxyuridine (BrdU) resistant 143B.TK − cell line was grown in Dulbecco's Modified Eagle Medium (Life Technologies) (containing 4.5 mg of glucose and 0.11 mg pyruvate/ml), supplemented with 100 μg of BrdU/ml and 5% fetal bovine serum. The mtDNA-less ρ°206 cell line, derived from 143B.TK − (34,35) was grown under the same conditions as the parental line, except for the addition of 50 μg of uridine/ml. Transformation by cytoplasts of mtDNA-less ρ°206 cells using one affected subject (III-6) and one control individual (A4) was performed as described elsewhere (34,35,37).…”

“…The mtDNA-less ρ°206 cell line, derived from 143B.TK − (34,35) was grown under the same conditions as the parental line, except for the addition of 50 μg of uridine/ml. Transformation by cytoplasts of mtDNA-less ρ°206 cells using one affected subject (III-6) and one control individual (A4) was performed as described elsewhere (34,35,37). All cybrid cell lines constructed with enucleated lymphoblastoid cell lines were maintained in the same medium as the 143B.TK − cell line.…”

“…For the aminoacylation assays, total mitochondrial RNAs were isolated under acid conditions, and 2 μg of total mitochondrial RNAs was electrophoresed at 4°C through an acid (pH 3.0) 10% polyacrylamide–7 M urea gel to separate the charged and uncharged tRNA as detailed elsewhere (43,44). To further distinguish non-aminoacylated tRNA from aminoacylated tRNA, samples of tRNAs were deacylated by being heated for 10 min at 60°C at pH 8.3 and then run in parallel (35,44). The gels were then electroblotted onto a positively charged nylon membrane (Roche) for the hybridization analysis with oligodeoxynucleotide probes as described above.…”

“…Mitochondrial membrane potential was assessed with JC-10 Assay Kit-Microplate (Abcam) following general manufacturer's recommendations with some modifications, as detailed elsewhere (20,35). …”

“…It was also proposed that an impairment of mitochondrial translation caused by the mt–tRNA mutation alters the respiration, production of adenosine triphoshate (ATP) and reactive oxygen species (ROS). To further investigate the pathogenic mechanism of the m.7551A > G mutation, cybrid cell lines were constructed by transferring mitochondria from lymphoblastoid cell lines derived from an affected matrilineal relative in a Chinese family carrying the mtDNA mutation and from a control individual lacking the mtDNA mutation, into human mtDNA-less (ρ°) cells (34–35). First, we examined if the m.7551A > G mutation created the m 1 G37 modification of mt–tRNA Asp by using primer extension.…”