A coronary artery disease-associated tRNAThr mutation altered mitochondrial function, apoptosis and angiogenesis.

The tissue specificity of mitochondrial tRNA mutations remains largely elusive. In this study, we demonstrated the deleterious effects of tRNAThr 15927G>A mutation that contributed to pathogenesis of coronary artery disease. The m.15927G>A mutation abolished the highly conserved base-pairing (28C-42G) of anticodon stem of tRNAThr. Using molecular dynamics simulations, we showed that the m.15927G>A mutation caused unstable tRNAThr structure, supported by decreased melting temperature and slower electrophoretic mobility of mutated tRNA. Using cybrids constructed by transferring mitochondria from a Chinese family carrying the m.15927G>A mutation and a control into mitochondrial DNA (mtDNA)-less human umbilical vein endothelial cells, we demonstrated that the m.15927G>A mutation caused significantly decreased efficiency in aminoacylation and steady-state levels of tRNAThr. The aberrant tRNAThr metabolism yielded variable decreases in mtDNA-encoded polypeptides, respiratory deficiency, diminished membrane potential and increased the production of reactive oxygen species. The m.15927G>A mutation promoted the apoptosis, evidenced by elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 3, 7, 9 and PARP. Moreover, the lower wound healing cells and perturbed tube formation were observed in mutant cybrids, indicating altered angiogenesis. Our findings provide new insights into the pathophysiology of coronary artery disease, which is manifested by tRNAThr mutation-induced alterations.

Sequence-specific and Shape-selective RNA Recognition by the Human RNA 5-Methylcytosine Methyltransferase NSun6.

Human NSun6 is an RNA methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-l-methionine (SAM) to C72 of tRNAThr and tRNACys In the current study, we used mass spectrometry to demonstrate that human NSun6 indeed introduces 5-methylcytosine (m5C) into tRNA, as expected. To further reveal the tRNA recognition mechanism of human NSun6, we measured the methylation activity of human NSun6 and its kinetic parameters for different tRNA substrates and their mutants. We showed that human NSun6 requires a well folded, full-length tRNA as its substrate. In the acceptor region, the CCA terminus, the target site C72, the discriminator base U73, and the second and third base pairs (2:71 and 3:70) of the acceptor stem are all important RNA recognition elements for human NSun6. In addition, two specific base pairs (11:24 and 12:23) in the D-stem of the tRNA substrate are involved in interacting with human NSun6. Together, our findings suggest that human NSun6 relies on a delicate network for RNA recognition, which involves both the primary sequence and tertiary structure of tRNA substrates.

Human polypyrimidine tract-binding protein interacts with mitochondrial tRNA(Thr) in the cytosol.

Human polypyrimidine tract-binding protein PTB is a multifunctional RNA-binding protein with four RNA recognition motifs (RRM1 to RRM4). PTB is a nucleocytoplasmic shuttle protein that functions as a key regulator of alternative pre-mRNA splicing in the nucleoplasm and promotes internal ribosome entry site-mediated translation initiation of viral and cellular mRNAs in the cytoplasm. Here, we demonstrate that PTB and its paralogs, nPTB and ROD1, specifically interact with mitochondrial (mt) tRNA(Thr) both in human and mouse cells. In vivo and in vitro RNA-binding experiments demonstrate that PTB forms a direct interaction with the T-loop and the D-stem-loop of mt tRNA(Thr) using its N-terminal RRM1 and RRM2 motifs. RNA sequencing and cell fractionation experiments show that PTB associates with correctly processed and internally modified, mature mt tRNA(Thr) in the cytoplasm outside of mitochondria. Consistent with this, PTB activity is not required for mt tRNA(Thr) biogenesis or for correct mitochondrial protein synthesis. PTB association with mt tRNA(Thr) is largely increased upon induction of apoptosis, arguing for a potential role of the mt tRNA(Thr)/PTB complex in apoptosis. Our results lend strong support to the recently emerging conception that human mt tRNAs can participate in novel cytoplasmic processes independent from mitochondrial protein synthesis.

Discovery of the ß-barrel-type RNA methyltransferase responsible for N6-methylation of N6-threonylcarbamoyladenosine in tRNAs.

Methylation is a versatile reaction involved in the synthesis and modification of biologically active molecules, including RNAs. N(6)-methyl-threonylcarbamoyl adenosine (m(6)t(6)A) is a post-transcriptional modification found at position 37 of tRNAs from bacteria, insect, plants, and mammals. Here, we report that in Escherichia coli, yaeB (renamed as trmO) encodes a tRNA methyltransferase responsible for the N(6)-methyl group of m(6)t(6)A in tRNA(Thr) specific for ACY codons. TrmO has a unique single-sheeted ß-barrel structure and does not belong to any known classes of methyltransferases. Recombinant TrmO employs S-adenosyl-L-methionine (AdoMet) as a methyl donor to methylate t(6)A to form m(6)t(6)A in tRNA(Thr). Therefore, TrmO/YaeB represents a novel category of AdoMet-dependent methyltransferase (Class VIII). In a ΔtrmO strain, m(6)t(6)A was converted to cyclic t(6)A (ct(6)A), suggesting that t(6)A is a common precursor for both m(6)t(6)A and ct(6)A. Furthermore, N(6)-methylation of t(6)A enhanced the attenuation activity of the thr operon, suggesting that TrmO ensures efficient decoding of ACY. We also identified a human homolog, TRMO, indicating that m(6)t(6)A plays a general role in fine-tuning of decoding in organisms from bacteria to mammals.

The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA.

The ribosome selects a correct transfer RNA (tRNA) for each amino acid added to the polypeptide chain, as directed by messenger RNA. Aminoacyl-tRNA is delivered to the ribosome by elongation factor Tu (EF-Tu), which hydrolyzes guanosine triphosphate (GTP) and releases tRNA in response to codon recognition. The signaling pathway that leads to GTP hydrolysis upon codon recognition is critical to accurate decoding. Here we present the crystal structure of the ribosome complexed with EF-Tu and aminoacyl-tRNA, refined to 3.6 angstrom resolution. The structure reveals details of the tRNA distortion that allows aminoacyl-tRNA to interact simultaneously with the decoding center of the 30S subunit and EF-Tu at the factor binding site. A series of conformational changes in EF-Tu and aminoacyl-tRNA suggests a communication pathway between the decoding center and the guanosine triphosphatase center of EF-Tu.

The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA.

Threonylcarbamoyladenosine (t(6)A) is a universal modification found at position 37 of ANN decoding tRNAs, which imparts a unique structure to the anticodon loop enhancing its binding to ribosomes in vitro. Using a combination of bioinformatic, genetic, structural and biochemical approaches, the universal protein family YrdC/Sua5 (COG0009) was shown to be involved in the biosynthesis of this hypermodified base. Contradictory reports on the essentiality of both the yrdC wild-type gene of Escherichia coli and the SUA5 wild-type gene of Saccharomyces cerevisiae led us to reconstruct null alleles for both genes and prove that yrdC is essential in E. coli, whereas SUA5 is dispensable in yeast but results in severe growth phenotypes. Structural and biochemical analyses revealed that the E. coli YrdC protein binds ATP and preferentially binds RNA(Thr) lacking only the t(6)A modification. This work lays the foundation for elucidating the function of a protein family found in every sequenced genome to date and understanding the role of t(6)A in vivo.

Mitochondrial ND5 T12338C, tRNA(Cys) T5802C, and tRNA(Thr) G15927A variants may have a modifying role in the phenotypic manifestation of deafness-associated 12S rRNA A1555G mutation in three Han Chinese pedigrees.

We report here on the clinical, genetic, and molecular characterization of three Han Chinese pedigrees with aminoglycoside-induced and nonsyndromic hearing loss. Clinical evaluation revealed the variable phenotype of hearing impairment including severity, age-at-onset, audiometric configuration in these subjects. The penetrance of hearing loss in WZD8, WZD9, and WZD10 pedigrees were 46%, 46%, and 50%, respectively, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrance of hearing loss in these pedigrees were 23%, 31%, and 37.5%, respectively. Mutational analysis of the complete mitochondrial genomes showed the homoplasmic A1555G mutation and distinct sets of mitochondrial DNA variants belonging to haplogroups D4b2b, B5b1, and F2, respectively. Of these, the tRNA(Cys) T5802C, tRNA(Thr) A15924C, and ND5 T12338C variants are of special interest as these variants occur at positions which are highly evolutionarily conserved nucleotides of tRNAs or amino acid of polypeptide. These homoplasmic mtDNA variants were absent among 156 unrelated Chinese controls. The T5802C and G15927A variants disrupted a highly conserved A-U or C-G base-pairing at the anticodon-stem of tRNA(Cys) or tRNA(Thr), while the ND5 T12338C mutation resulted in the replacement of the translation-initiating methionine with a threonine, and also located in two nucleotides adjacent to the 3' end of the tRNA(Leu(CUN)). Thus, mitochondrial dysfunctions, caused by the A1555G mutation, would be worsened by these mtDNA variants. Therefore, these mtDNA mutations may have a potential modifier role in increasing the penetrance and expressivity of the deafness-associated 12S rRNA A1555G mutation in those Chinese pedigrees.

Achieving error-free translation; the mechanism of proofreading of threonyl-tRNA synthetase at atomic resolution.

The fidelity of aminoacylation of tRNA(Thr) by the threonyl-tRNA synthetase (ThrRS) requires the discrimination of the cognate substrate threonine from the noncognate serine. Misacylation by serine is corrected in a proofreading or editing step. An editing site has been located 39 A away from the aminoacylation site. We report the crystal structures of this editing domain in its apo form and in complex with the serine product, and with two nonhydrolyzable analogs of potential substrates: the terminal tRNA adenosine charged with serine, and seryl adenylate. The structures show how serine is recognized, and threonine rejected, and provide the structural basis for the editing mechanism, a water-mediated hydrolysis of the mischarged tRNA. When the adenylate analog binds in the editing site, a phosphate oxygen takes the place of one of the catalytic water molecules, thereby blocking the reaction. This rules out a correction mechanism that would occur before the binding of the amino acid on the tRNA.

Crucial role of conserved lysine 277 in the fidelity of tRNA aminoacylation by Escherichia coli valyl-tRNA synthetase.

Valyl-tRNA synthetase (ValRS) from Escherichia coli undergoes covalent valylation by a donor valyl adenylate synthesized by the enzyme itself. ValRS could also be modified, although to a lesser extent, by the noncognate isosteric substrate L-threonine from a donor threonyl adenylate synthesized by the synthetase itself, or by the nonsubstrate methionine from methionyl adenylate produced by catalytic amounts of methionyl-tRNA synthetase. MALDI mass spectrometry analysis designated lysines 154, 162, 170, 533, 554, 593, 894, 930, and 940 of ValRS as the target residues for the attachment of valine. Following autothreonylation, lysines 162, 170, 178, 277, 291, 554, 580, 593, 861, 894, and 930 were found to be modified. Finally, L-Met-labeled residues were lysines 118, 162, 170, 178, 277, and 938. Alignment of the available ValRS amino acid sequences showed that lysines 277 and 554 are strictly conserved (with the exception concerning replacement of Lys-277 with a methionine or a tyrosine in archaebacteria), suggesting that these residues might be functionally significant. Indeed, lysine 554 of ValRS is the first lysine of the Lys-Met-Ser-Lys-Ser signature of the catalytic site of class I aminoacyl-tRNA synthetases. Lys-277 which is labeled by L-threonine or L-methionine, and not by L-valine, is located at or near the editing site, in the three-dimensional structure of ValRS. The role of lysine 277 was evaluated by site-directed mutagenesis. The Lys277Ala mutant (K277A) exhibited a posttransfer Thr-tRNA(Val) editing rate that was significantly lower than that observed for the wild-type enzyme. In addition, the K277A substitution altered amino acid discrimination in the editing site, resulting in hydrolysis of the correctly charged cognate Val-tRNA(Val). Finally, significant amounts of mischarged Thr-tRNA(Val) were produced by the K277A mutant, and not by wild-type ValRS. Altogether, our results designate Lys-277 as a likely candidate for nucleophilic attack of misacylated tRNA in the editing site of ValRS.

The methyl group of the N6-methyl-N6-threonylcarbamoyladenosine in tRNA of Escherichia coli modestly improves the efficiency of the tRNA.

tRNA species that read codons starting with adenosine (A) contain N6-threonylcarbamoyladenosine (t6A) derivatives adjacent to and 3' of the anticodons from all organisms. In Escherichia coli there are 12 such tRNA species of which two (tRNA(Thr1)GGU and tRNA(Thr3)GGU) have the t6A derivative N6-methyl-N6-threonylcarbamoyladenosine (m6t6A37). We have isolated a mutant of E. coli that lacks the m6t6A37 in these two tRNA(Thr)GGU species. These tRNA species in the mutant are likely to have t6A37 instead of m6t6A37. We show that the methyl group of m6t6A37 originates from S-adenosyl-L-methionine and that the gene (tsaA) which most likely encodes tRNA(m6t6A37)methyltransferase is located at min 4.6 on the E. coli chromosomal map. The growth rate of the cell, the polypeptide chain elongation rate, and the selection of Thr-tRNA(Thr)GGU to the ribosomal A site programmed with either of the cognate codons ACC and ACU were the same for the tsaA1 mutant as for the congenic wild-type strain. The expression of the threonine operon is regulated by an attenuator which contains in its leader mRNA seven ACC codons that are read by these two m6t6A37-containing tRNA(Thr)GGU species. We show that the tsaA1 mutation resulted in a twofold derepression of this operon, suggesting that the lack of the methyl group of m6t6A37 in tRNA(Thr)GGU slightly reduces the efficiency of this tRNA to read cognate codon ACC.

A novel mutation in the mitochondrial tRNA(Thr) gene associated with a mitochondrial encephalomyopathy.

A novel G-to-A transition at nucleotide 15915 in mtDNA is described. The patient showed a combination of muscle weakness, hearing loss, mental retardation, and seizures. Muscle biopsy showed RRFs and focal COX deficiency. We sequenced all mtDNA, and found 5 novel nucleotide substitutions. Three of them were synonymous mutations, one was a missense mutation in cytochrome b gene (A-->G at nt 15422), and the last one was the 15915 mutation in tRNA(Thr) gene. We screened for the 15422 and the 15915 mutations with mismatch primers and found that one of 104 normal individuals carried the former one and none of 175 had the latter one. The 15422 mutation existed in homoplasmic states both in the patient and the normal individual, suggesting that this is a polymorphism. In contrast the 15915 mutation resided in heteroplasmic states in muscle, skin fibroblast and blood. The nucleotide substitution at nt 15915 disrupts a highly conserved base pair in anticodon stem of the tRNA(Thr). Our data suggest that the 15915 mutation is an additional mtDNA mutation responsible for mitochondrial encephalomyopathies.