Functional and molecular mitochondrial abnormalities associated with a C → T transition at position 3256 of the human mitochondrial genome

The effects of a pathogenic mitochondrial tRNA point mutation in organelle translation and RNA processing

Huiling Hao, Carlos T Moraes

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53 Citations (Scopus)

Abstract

We have previously identified, a mitochondrial DNA polymorphism (a C → T transition at position 3256, within the mitochondrial tRNALeu(UUR) gene) in a patient with a multisystem disorder. Although there were several indicators suggesting a pathogenetic role for this mtDNA polymorphism, its heteroplasmic nature made functional and molecular studies difficult to interpret. We have now fused enucleated fibroblasts from the patient with a mtDNA-less cell line to generate transmitochondrial cybrids harboring different proportions of mutated and wild-type mtDNA. Individual clones harboring essentially 100% wild-type or >99% mutated mtDNAs were characterized and studied for respiratory capacity, respiratory chain enzymes activity, mitochondrial protein synthesis, and RNA steady-state levels and processing. Our results showed that cell lines containing exclusively mutated mtDNAs respire poorly, over-produce lactic acid, and have significantly impaired activity of respiratory complexes I and IV. Molecular studies showed that mutant clones have a decrease in steady-state levels of mitochondrial tRNALeu(UUR), and a partial impairment of mitochondrial protein synthesis and steady-state levels, suggesting that these molecular abnormalities are involved in the pathogenetic mechanism of the mtDNA 3256 mutation.

Original languageEnglish
Pages (from-to)2347-2352
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number4
StatePublished - Jan 26 1996

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Mitochondrial Genome
Human Genome
Transfer RNA
Mitochondrial DNA
Point Mutation
Organelles
Genes
RNA
RNA, Transfer, Leu
Processing
Mitochondrial Proteins
Polymorphism
Clone Cells
Cells
Electron Transport Complex I
Cell Line
Enzyme activity
Fibroblasts
Electron Transport
Lactic Acid

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Functional and molecular mitochondrial abnormalities associated with a C → T transition at position 3256 of the human mitochondrial genome: The effects of a pathogenic mitochondrial tRNA point mutation in organelle translation and RNA processing",
abstract = "We have previously identified, a mitochondrial DNA polymorphism (a C → T transition at position 3256, within the mitochondrial tRNALeu(UUR) gene) in a patient with a multisystem disorder. Although there were several indicators suggesting a pathogenetic role for this mtDNA polymorphism, its heteroplasmic nature made functional and molecular studies difficult to interpret. We have now fused enucleated fibroblasts from the patient with a mtDNA-less cell line to generate transmitochondrial cybrids harboring different proportions of mutated and wild-type mtDNA. Individual clones harboring essentially 100{\%} wild-type or >99{\%} mutated mtDNAs were characterized and studied for respiratory capacity, respiratory chain enzymes activity, mitochondrial protein synthesis, and RNA steady-state levels and processing. Our results showed that cell lines containing exclusively mutated mtDNAs respire poorly, over-produce lactic acid, and have significantly impaired activity of respiratory complexes I and IV. Molecular studies showed that mutant clones have a decrease in steady-state levels of mitochondrial tRNALeu(UUR), and a partial impairment of mitochondrial protein synthesis and steady-state levels, suggesting that these molecular abnormalities are involved in the pathogenetic mechanism of the mtDNA 3256 mutation.",
author = "Huiling Hao and Moraes, {Carlos T}",
year = "1996",
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language = "English",
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AU - Moraes, Carlos T

PY - 1996/1/26

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N2 - We have previously identified, a mitochondrial DNA polymorphism (a C → T transition at position 3256, within the mitochondrial tRNALeu(UUR) gene) in a patient with a multisystem disorder. Although there were several indicators suggesting a pathogenetic role for this mtDNA polymorphism, its heteroplasmic nature made functional and molecular studies difficult to interpret. We have now fused enucleated fibroblasts from the patient with a mtDNA-less cell line to generate transmitochondrial cybrids harboring different proportions of mutated and wild-type mtDNA. Individual clones harboring essentially 100% wild-type or >99% mutated mtDNAs were characterized and studied for respiratory capacity, respiratory chain enzymes activity, mitochondrial protein synthesis, and RNA steady-state levels and processing. Our results showed that cell lines containing exclusively mutated mtDNAs respire poorly, over-produce lactic acid, and have significantly impaired activity of respiratory complexes I and IV. Molecular studies showed that mutant clones have a decrease in steady-state levels of mitochondrial tRNALeu(UUR), and a partial impairment of mitochondrial protein synthesis and steady-state levels, suggesting that these molecular abnormalities are involved in the pathogenetic mechanism of the mtDNA 3256 mutation.

AB - We have previously identified, a mitochondrial DNA polymorphism (a C → T transition at position 3256, within the mitochondrial tRNALeu(UUR) gene) in a patient with a multisystem disorder. Although there were several indicators suggesting a pathogenetic role for this mtDNA polymorphism, its heteroplasmic nature made functional and molecular studies difficult to interpret. We have now fused enucleated fibroblasts from the patient with a mtDNA-less cell line to generate transmitochondrial cybrids harboring different proportions of mutated and wild-type mtDNA. Individual clones harboring essentially 100% wild-type or >99% mutated mtDNAs were characterized and studied for respiratory capacity, respiratory chain enzymes activity, mitochondrial protein synthesis, and RNA steady-state levels and processing. Our results showed that cell lines containing exclusively mutated mtDNAs respire poorly, over-produce lactic acid, and have significantly impaired activity of respiratory complexes I and IV. Molecular studies showed that mutant clones have a decrease in steady-state levels of mitochondrial tRNALeu(UUR), and a partial impairment of mitochondrial protein synthesis and steady-state levels, suggesting that these molecular abnormalities are involved in the pathogenetic mechanism of the mtDNA 3256 mutation.

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