The RNA sequence context defines the mechanistic routes by which yeast arginyl-tRNA synthetase charges tRNA.

Arginylation of tRNA transcripts by yeast arginyl-tRNA synthetase can be triggered by two alternate recognition sets in anticodon loops: C35 and U36 or G36 in tRNA(Arg) and C36 and G37 in tRNA(Asp) (Sissler M, Giegé R, Florentz C, 1996, EMBO J 15:5069-5076). Kinetic studies on tRNA variants were done to explore the mechanisms by which these sets are expressed. Although the synthetase interacts in a similar manner with tRNA(Arg) and tRNA(Asp), the details of the interaction patterns are idiosyncratic, especially in anticodon loops (Sissler M, Eriani G, Martin F, Giegé R, Florentz C, 1997, Nucleic Acids Res 25:4899-4906). Exchange of individual recognition elements between arginine and aspartate tRNA frameworks strongly blocks arginylation of the mutated tRNAs, whereas full exchange of the recognition sets leads to efficient arginine acceptance of the transplanted tRNAs. Unpredictably, the similar catalytic efficiencies of native and transplanted tRNAs originate from different k(cat) and Km combinations. A closer analysis reveals that efficient arginylation results from strong anticooperative effects between individual recognition elements. Nonrecognition nucleotides as well as the tRNA architecture are additional factors that tune efficiency. Altogether, arginyl-tRNA synthetase is able to utilize different context-dependent mechanistic routes to be activated. This confers biological advantages to the arginine aminoacylation system and sheds light on its evolutionary relationship with the aspartate system.comparative studyjournal articleresearch support, non-u.s. gov't1998 Junimporte

Pathology-related mutation A7526G (A9G) helps in the understanding of the 3D structural core of human mitochondrial tRNA(Asp).

More than 130 mutations in human mitochondrial tRNA (mt-tRNA) genes have been correlated with a variety of neurodegenerative and neuromuscular disorders. Their molecular impacts are of mosaic type, affecting various stages of tRNA biogenesis, structure, and/or functions in mt-translation. Knowledge of mammalian mt-tRNA structures per se remains scarce however. Primary and secondary structures deviate from classical tRNAs, while rules for three-dimensional (3D) folding are almost unknown. Here, we take advantage of a myopathy-related mutation A7526G (A9G) in mt-tRNA(Asp) to investigate both the primary molecular impact underlying the pathology and the role of nucleotide 9 in the network of 3D tertiary interactions. Experimental evidence is presented for existence of a 9-12-23 triple in human mt-tRNA(Asp) with a strongly conserved interaction scheme in mammalian mt-tRNAs. Mutation A7526G disrupts the triple interaction and in turn reduces aspartylation efficiency.letterresearch support, non-u.s. gov't2009 Aug2009 06 17importe

Major identity determinants for enzymatic formation of ribothymidine and pseudouridine in the T psi-loop of yeast tRNAs.

Almost all transfer RNA molecules sequenced so far contain two universal modified nucleosides at positions 54 and 55, respectively: ribothymidine (T54) and pseudouridine (psi 55). To identify the tRNA elements recognized by tRNA:m5uridine-54 methyltransferase and tRNA:pseudouridine-55 synthase from the yeast Saccharomyces cerevisiae, a set of 43 yeast tRNA(Asp) mutants were used. Some variants contained point mutations, while the others included progressive reductions in size down to a tRNA minisubstrate consisting of the T psi-loop with only one G.C base-pair as stem (9-mer). All substrates (full-sized tRNA(Asp) and various minihelices) were produced in vitro by T7 transcription and tested using yeast extract (S100) as a source of enzymatic activities and S-adenosyl-L-methionine as a methyl donor. The results indicate that the minimal substrate for enzymatic formation of psi 55 is a stem/loop structure with only four G.C base-pairs in the stem, while a longer stem is required for efficient T54 formation. None of the conserved nucleotides (G53, C56, A58 and C61) and U54 for psi 55 or U55 for T54 formation can be replaced by any of the other three canonical nucleotides. Yeast tRNA:m5uridine-54 methyltransferase additionally requires the presence of a pyrimidine-60 in the loop. Interestingly, in a tRNA(Asp) variant in which the T psi-loop was permuted with the anticodon-loop, the new U32 and U33 residues derived from the T psi-loop were quantitatively converted to T32 and psi 33, respectively. Structural mapping of this variant with ethylnitrosourea confirmed that the intrinsic characteristic structure of the T psi-loop was conserved upon permutation and that the displaced anticodon-loop did not acquire a T psi-loop structure. These results demonstrate that a local conformation rather than the exact location of the U-U sequence within the tRNA architecture is the important identity determinant for recognition by yeast tRNA:m5uridine-54 methyltransferase and tRNA:pseudouridine-55 synthase.journal articleresearch support, non-u.s. gov't1997 Dec 12importe

Loss of a primordial identity element for a mammalian mitochondrial aminoacylation system.

In mammalian mitochondria the translational machinery is of dual origin with tRNAs encoded by a simplified and rapidly evolving mitochondrial (mt) genome and aminoacyl-tRNA synthetases (aaRS) coded by the nuclear genome, and imported. Mt-tRNAs are atypical with biased sequences, size variations in loops and stems, and absence of residues forming classical tertiary interactions, whereas synthetases appear typical. This raises questions about identity elements in mt-tRNAs and adaptation of their cognate mt-aaRSs. We have explored here the human mt-aspartate system in which a prokaryotic-type AspRS, highly similar to the Escherichia coli enzyme, recognizes a bizarre tRNA(Asp). Analysis of human mt-tRNA(Asp) transcripts confirms the identity role of the GUC anticodon as in other aspartylation systems but reveals the non-involvement of position 73. This position is otherwise known as the site of a universally conserved major aspartate identity element, G73, also known as a primordial identity signal. In mt-tRNA(Asp), position 73 can be occupied by any of the four nucleotides without affecting aspartylation. Sequence alignments of various AspRSs allowed placing Gly-269 at a position occupied by Asp-220, the residue contacting G73 in the crystallographic structure of E. coli AspRS-tRNA(Asp) complex. Replacing this glycine by an aspartate renders human mt-AspRS more discriminative to G73. Restriction in the aspartylation identity set, driven by a rapid mutagenic rate of the mt-genome, suggests a reverse evolution of the mt-tRNA(Asp) identity elements in regard to its bacterial ancestor.journal articleresearch support, non-u.s. gov't2006 Jun 092006 04 05importe

The concept of RNA-assisted protein folding: the role of tRNA

We suggest that tRNA actively participates in the transfer of 3D information from mRNA to peptides - in addition to its well-known, "classical" role of translating the 3-letter RNA codes into the one letter protein code. The tRNA molecule displays a series of thermodynamically favored configurations during translation, a movement which places the codon and coded amino acids in proximity to each other and make physical contact between some amino acids and their codons possible. This specific codon-amino acid interaction of some selected amino acids is necessary for the transfer of spatial information from mRNA to coded proteins, and is known as RNA-assisted protein folding

Clinical, neuroradiological, and molecular characterization of mitochondrial threonyl-tRNA-synthetase (TARS2)-related disorder

PURPOSE: Biallelic variants in TARS2, encoding the mitochondrial threonyl-tRNA-synthetase, have been reported in a small group of individuals displaying a neurodevelopmental phenotype, but with limited neuroradiological data and insufficient evidence for causality of the variants. METHODS: Exome or genome sequencing was carried out in 15 families. Clinical and neuroradiological evaluation was performed for all affected individuals, including review of 10 previously reported individuals. The pathogenicity of TARS2 variants was evaluated using in vitro assays, and a zebrafish model. RESULTS: We report 18 new individuals harboring biallelic TARS2 variants. Phenotypically, these individuals show developmental delay/intellectual disability, regression, cerebellar and cerebral atrophy, basal ganglia signal alterations, hypotonia, cerebellar signs and increased blood lactate. In vitro studies showed that variants within the TARS2301-381 region had decreased binding to Rag GTPases, likely impairing mTORC1 activity. The zebrafish model recapitulated key features of the human phenotype and unraveled dysregulation of downstream targets of mTORC1 signaling. Functional testing of the variants confirmed the pathogenicity in a zebrafish model. CONCLUSION: We define the clinico-radiological spectrum of TARS2-related mitochondrial disease, unveil the likely involvement of the mTORC1 signaling pathway as a distinct molecular mechanism, and establish a TARS2 zebrafish model as an important tool to study variant pathogenicity

Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome.

Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome