Characterization of the Human Mitochondrial Methionyl-tRNA Synthetase

Spencer, Angela C.; Heck, Achim; Takeuchi, Naonobu; Watanabe, Kanako; Spremulli, Linda L.

doi:10.1021/bi049639w

Cited by 43 publications

(37 citation statements)

References 42 publications

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“…Aminoacylation of the bmtRNA Met transcript, individual half molecules, and half molecule complexes were conducted as described. 31 Yeast mitochondrial LeuRS (LeuRS mt ) was expressed in E. coli, isolated and purified. Cultures were inoculated with a single BL21 + (codon plus) colony harboring the plasmid pYM3-17, 74 which encodes the wildtype yeast LeuRS mt fused to an N-terminal six-histidine tag.…”

Section: Isolation Of Aminoacyl-trna Synthetases and Aminoacylation Omentioning

confidence: 99%

A Counterintuitive Mg2+-dependent and Modification-assisted Functional Folding of Mitochondrial tRNAs

Jones

Spencer

Hsu

et al. 2006

Journal of Molecular Biology

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Mitochondrial tRNAs (mtRNAs) often lack domains and posttranscriptional modifications that are found in cytoplasmic tRNAs. These structural and chemical elements normally stabilize the folding of cytoplasmic tRNAs into canonical structures that are competent for aminoacylation and translation. For example, the dihydrouridine (D) stem and loop domain is involved in the tertiary structure of cytoplasmic tRNAs through hydrogen bonds and a Mg 2+ bridge to the ribothymidine (T) stem and loop domain. These interactions are often absent in mtRNA because the D-domain is truncated or missing. Using gel mobility shift analyses, UV, circular dichroism and NMR spectroscopies and aminoacylation assays, we have investigated the functional folding interactions of chemically synthesized and site-specifically modified mitochondrial and cytoplasmic tRNAs. We found that Mg 2+ is critical for folding of the truncated D-domain of bovine mtRNA Met with the tRNA's T-domain. Contrary to the expectation that Mg 2+ stabilizes RNA folding, the mtRNA Met Ddomain structure was unfolded and relaxed, rather than stabilized in the presence of Mg 2+ . Because the D-domain is transcribed prior to the T-domain, we conclude that Mg 2+ prevents misfolding of the 5′-half of bovine mtRNA Met facilitating its correct interaction with the T-domain. The interaction of the mtRNA Met D-domain with the T-domain was enhanced by a pseudouridine located in either the D-or T-domains compared to that of the unmodified RNAs (K d = 25.3, 24.6 and 44.4 μM, respectively). Mg 2+ also affected the folding interaction of a yeast mtRNA Leu1 , but had minimal effect on the folding of an E. coli cytoplasmic tRNA Leu . The D-domain modification, dihydrouridine, facilitated mtRNA Leu folding. These data indicate that conserved modifications assist and stabilize the formation of the functional mtRNA tertiary structure.

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Section: Isolation Of Aminoacyl-trna Synthetases and Aminoacylation Omentioning

confidence: 99%

A Counterintuitive Mg2+-dependent and Modification-assisted Functional Folding of Mitochondrial tRNAs

Jones

Spencer

Hsu

et al. 2006

Journal of Molecular Biology

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“…1C) suggesting that the mutated tRNA had a significantly altered structure. The hmMetRS is believed to be both structurally and functionally homologous to its prokaryotic counterpart (23). However, this work demonstrates that the hmMetRS is less discriminatory than E. coli MetRS for the structure of the tRNA.…”

Section: Aminoacylation Of the Normal And 8u3 C Mutated Trnamentioning

confidence: 70%

“…Met transcripts for aminoacylation experiments were prepared by in vitro transcription using the hammerhead ribozyme construct described previously (23). The hmtRNA Met was purified by denaturing (10%) PAGE (29:1 acrylamide:bisacrylamide prepared with 7 M urea, 90 mM Tris borate, 2 mM EDTA), visualized by UV shadowing, excised from the gel, and recovered by passive elution in water followed by ethanol precipitation.…”

Section: Rna Synthesis-human Mitochondrial Trnamentioning

confidence: 99%

“…Cells carrying a plasmid encoding the His 6 -tagged human mitochondrial MetRS were grown as described (23). The cells were lysed as described above, and the hmMetRS was further purified as described (23).…”

Section: Rna Synthesis-human Mitochondrial Trnamentioning

confidence: 99%

“…Met -The aminoacylation reactions for both the normal and 8U3 C mutated tRNA Met transcripts were performed essentially as described (23). Reaction mixtures (100 l) contained 50 mM Tris-HCl, pH 7.6, 2.5 mM MgCl 2 , 2.5 mM ATP, 0.2 mM spermine, 200 g/ml bovine serum albumin, 0.…”

Section: Assay For the Aminoacylation Of Human Mitochondrial Trnamentioning

confidence: 99%

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A Disease-causing Point Mutation in Human Mitochondrial tRNAMet Results in tRNA Misfolding Leading to Defects in Translational Initiation and Elongation

Jones

Graham

et al. 2008

Journal of Biological Chemistry

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The mitochondrial tRNA genes are hot spots for mutations that lead to human disease. A single point mutation (T4409C) in the gene for human mitochondrial tRNA Met (hmtRNA Met ) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNA Met with a C8. The hmtRNA Met serves both in translational initiation and elongation in human mitochondria making this tRNA of particular interest in mitochondrial protein synthesis. Here we show that the single 8U3 C mutation leads to a failure of the tRNA to respond conformationally to Mg 2؉ . This mutation results in a drastic disruption of the structure of the hmtRNA Met , which significantly reduces its aminoacylation. The small fraction of hmtRNA Met that can be aminoacylated is not formylated by the mitochondrial Met-tRNA transformylase preventing its function in initiation, and it is unable to form a stable ternary complex with elongation factor EF-Tu preventing any participation in chain elongation. We have used structural probing and molecular reconstitution experiments to examine the structures formed by the normal and mutated tRNAs. In the presence of Mg 2؉ , the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg 2؉ , the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg 2؉ -binding site on the tRNA required for formation of the biologically active structure. This work establishes a foundation for understanding the physiological consequences of the numerous mitochondrial tRNA mutations that result in disease in humans.Human mitochondria are subcellular organelles that produce more than 90% of the energy required by the cell. The mitochondrial genome encodes 13 proteins necessary for energy production, two rRNAs and all of the 22 tRNAs required for the synthesis of these proteins (1, 2). Mammalian mitochondrial tRNAs have several unusual features that distinguish them from canonical tRNAs. In many cases, they lack a number of the conserved or semi-conserved nucleotides that play important roles in creating the L-shaped tertiary structure of prokaryotic and eukaryotic cytoplasmic tRNAs (3). There is little detailed structural information on these tRNAs. No data are currently available that examine the structure of mammalian mitochondrial tRNAs with single nucleotide resolution. However, chemical and enzymatic probing has lead to the idea that these tRNAs have retained the basic cloverleaf structure of canonical tRNAs but that they lack several conserved tertiary interactions leading to a weaker three-dimensional structure (4 -8). In particular, a number of the long range interactions between the Dand T-arms of the tRNAs appear to be missing.All 22 tRNAs that function in mammalian mitochondria are encoded in the mitochondrial DNA. Considerable interest in mitochondrial tRNAs centers on the occurrence of diseases arising from mutations in their genes that lead to maternally inherited genetic diso...

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Novel, Compound Heterozygous, Single-Nucleotide Variants inMARS2Associated with Developmental Delay, Poor Growth, and Sensorineural Hearing Loss

et al. 2015

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Novel, single nucleotide mutations were identified in the mitochondrial methionyl amino-acyl-tRNA synthetase gene (MARS2) via whole exome sequencing in two affected siblings with developmental delay, poor growth, and sensorineural hearing loss. We show that compound heterozygous mutations c.550C>T:p.Gln184* and c.424C>T:p.Arg142Trp in MARS2 lead to decreased MARS2 protein levels in patient lymphoblasts. Analysis of respiratory complex (RC) enzyme activities in patient fibroblasts revealed decreased Complex I and IV activities. Immunoblotting of patient fibroblast and lymphoblast samples revealed reduced protein levels of NDUFB8 and COXII, representing Complex I and IV respectively. Additionally, overexpression of wild-type MARS2 in patient fibroblasts increased NDUFB8 and COXII protein levels. These findings suggest that recessive single nucleotide mutations in MARS2 are causative for a new mitochondrial translation deficiency disorder with a primary phenotype including developmental delay and hypotonia. Identification of additional patients with single nucleotide mutations in MARS2 is necessary to determine if pectus carinatum is also a consistent feature of this syndrome.

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Characterization of the Human Mitochondrial Methionyl-tRNA Synthetase

Cited by 43 publications

References 42 publications

A Counterintuitive Mg2+-dependent and Modification-assisted Functional Folding of Mitochondrial tRNAs

A Counterintuitive Mg2+-dependent and Modification-assisted Functional Folding of Mitochondrial tRNAs

A Disease-causing Point Mutation in Human Mitochondrial tRNAMet Results in tRNA Misfolding Leading to Defects in Translational Initiation and Elongation

Novel, Compound Heterozygous, Single-Nucleotide Variants inMARS2Associated with Developmental Delay, Poor Growth, and Sensorineural Hearing Loss

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