Richard Giegé

Mutation of the Mitochondrial Tyrosyl-tRNA Synthetase Gene, YARS2, Causes Myopathy, Lactic Acidosis, and Sideroblastic Anemia—MLASA Syndrome

Mitochondrial respiratory chain disorders are a heterogeneous group of disorders in which the underlying genetic defect is often unknown. We have identified a pathogenic mutation (c.156C>G [p.F52L]) in YARS2, located at chromosome 12p11.21, by using genome-wide SNP-based homozygosity analysis of a family with affected members displaying myopathy, lactic acidosis, and sideroblastic anemia (MLASA). We subsequently identified the same mutation in another unrelated MLASA patient. The YARS2 gene product, mitochondrial tyrosyl-tRNA synthetase (YARS2), was present at lower levels in skeletal muscle whereas fibroblasts were relatively normal. Complex I, III, and IV were dysfunctional as indicated by enzyme analysis, immunoblotting, and immunohistochemistry. A mitochondrial protein-synthesis assay showed reduced levels of respiratory chain subunits in myotubes generated from patient cell lines. A tRNA aminoacylation assay revealed that mutant YARS2 was still active; however, enzyme kinetics were abnormal compared to the wild-type protein. We propose that the reduced aminoacylation activity of mutant YARS2 enzyme leads to decreased mitochondrial protein synthesis, resulting in mitochondrial respiratory chain dysfunction. MLASA has previously been associated with PUS1 mutations; hence, the YARS2 mutation reported here is an alternative cause of MLASA.

tRNA structure and aminoacylation efficiency.

Publisher Summary This chapter discusses the role of tRNA structure in the recognition process with synthetases and on the implications for aminoacylation efficiency. Many examples are taken from our own research on several specific aminoacylation systems, for example aspartate, histidine, valine, but concepts are presented more globally with reference to the complete set of aminoacylation systems. It emphasizes on the importance of tRNA-like structures for understanding the interaction of canonical tRNAs with synthetase. Although tRNA-like molecules found in some plant viral RNAs do not participate in protein synthesis, they represent interesting natural mutants to be compared to canonical tRNAs. This is also the case of tRNAlike structures found in some messenger RNAs as well as of bizarre tRNAs from mitochondria . In addition, competition and kinetic effects may also contribute to the overall specificity of the various aminoacylation systems; the balance between the concentration of tRNAs and synthetases would be essential for ensuring optimal specificity. According to this view, individual aminoacylation systems do not work at their optimal chemical efficiency, but work instead to assure optimal discrimination among the different aminoacylation systems. Such a balance may be perturbed under certain physiological or pathological conditions. Finally, this chapter discusses a comparison of recent results with previous observations, and show how old concepts established phenomenologically can now be tested more explicitly.

Factors determining the specificity of the tRNA aminoacylation reaction: Non-absolute specificity of tRNA-aminoacyl-tRNA synthetase recognition and particular importance of the maximal velocity

Summary It is generally believed that the specificity of tRNA aminoacylation results solely from a specific recognition between the aminoacyl-tRNA synthetase and the cognate tRNA. In fact, this specificity is not absolute: this is supported by the following observations (1) the existence of tRNA mischarging in homologous systems under usual aminoacylation conditions, (2) the existence of inhibitions produced by « non-cognatetRNA species in correct aminoacylation reactions, (3) the lack of specificity of AMP- and PPi- independent aminoacyl-tRNA synthetase catalysed deacylation of aminoacyl-tRNA species, (4) the isolation of complexes between aminoacyl-tRNA synthetases and non-cognate tRNA species. The affinities between aminoacyl-tRNA synthetases and non-cognate tRNA species, estimated by the Km measurements in mischarging reactions, have been found only diminished by 1 or 2 orders of magnitude as compared to the values found in specific systems, whereas the Vmax values for mischarging have been found diminished by 3 or 4 orders of magnitude. This suggests that tRNA aminoacylation depends more upon the maximal velocity of the reaction than upon the recognition between aminoacyl-tRNA synthetase and tRNA. Furthermore, we found that the recognition of a tRNA by an aminoacyl-tRNA synthetase does not seem to require the 3′ terminal part of the amino acid acceptor stem. As the importance of this part of the tRNA molecule during the aminoacylation process has been well established, it is possible that it is involved in determining the Vmax of the aminoacylation reaction, probably by positioning the 3′ terminal adenosine in the catalytic site of the enzyme. In conclusion, it appears that the specificity of the tRNA aminoacylation reaction proceeds through two discrimination mechanisms: the first one, measured by the Km, acts at the recognition level; the second one, which is more effective, is measured by the Vmax values. Competition phenomena have been observed between cognate and non-cognate tRNA species. They enhance the specificity of the tRNA aminoacylation, but their contribution to the specificity is low compared to that brought by Km and Vmax. Finally we found that a more rapid enzymatic deacylation of mischarged tRNA species (as compared to correctly charged ones) cannot be considered as a general mechanism for correction of misaminoacylation.

Identity elements for specific aminoacylation of yeast tRNA(Asp) by cognate aspartyl-tRNA synthetase.

The nucleotides crucial for the specific aminoacylation of yeast tRNA(Asp) by its cognate synthetase have been identified. Steady-state aminoacylation kinetics of unmodified tRNA transcripts indicate that G34, U35, C36, and G73 are important determinants of tRNA(Asp) identity. Mutations at these positions result in a large decrease (19- to 530-fold) of the kinetic specificity constant (ratio of the catalytic rate constant kcat and the Michaelis constant Km) for aspartylation relative to wild-type tRNA(Asp). Mutation to G10-C25 within the D-stem reduced kcat/Km eightfold. This fifth mutation probably indirectly affects the presentation of the highly conserved G10 nucleotide to the synthetase. A yeast tRNA(Phe) was converted into an efficient substrate for aspartyl-tRNA synthetase through introduction of the five identity elements. The identity nucleotides are located in regions of tight interaction between tRNA and synthetase as shown in the crystal structure of the complex and suggest sites of base-specific contacts.

The crystallization of biological macromolecules from precipitates: evidence for Ostwald ripening

Abstract Crystals were obtained by different methods under conditions where nucleation and growth occur from precipitated macromolecular material. The phenomenon was observed with compounds of different size and nature, such as thaumatin, concanavalin A, an α-amylase, a thermostable aspartyl-tRNA synthetase, the nucleo-protein complex between a tRNA Asp transcript and its cognate yeast aspartyl-tRNA synthetase, and tomato bushy stunt virus. In each system, after a rather rapid precipitation step at high supersaturation lasting one to several days, a few microcrystals appear after prolonged equilibration at constant temperature. With α-amylase, the virus and the thermostable synthetase, crystallization is accompanied by appearance of depletion zones around the growing crystals and growth of the largest crystals at the expense of the smaller ones. These features are evidences for crystal growth by Ostwald ripening. In the case of thaumatin, concanavalin A and the nucleo-protein complex, crystallization occurs by a phase transition mechanism since it is never accompanied by the disappearance of the smallest crystals. A careful analysis with thermostable aspartyl-tRNA synthetase indicates that its crystallization at 4°C under high supersaturation starts by a phase transition mechanism with the formation of small crystals within an amorphous protein precipitate. Ostwald ripening follows over a period of up to three/four months with a growth rate of about 0.8 A/s that is 13 times slower than that of crystals growing at 20°C in the absence of precipitate without ripening. At the end of the ripening process at 4°C, only one unique synthetase crystal remains per microassay with dimensions as large as 1 mm.

Diagnostic of precipitant for biomacromolecule crystallization by quasi-elastic light-scattering

The translational diffusion coefficient D25,w of hen egg-white lysozyme and concanavalin A from the jack bean is measured in various precipitating agent solutions as a function of salt and protein concentration using quasi-elastic light-scattering. With some precipitants, in undersaturated protein solutions, a protein or salt concentration dependence of the diffusion coefficient of the scatters is observed. It can be correlated with the inability of the protein to crystallize in this precipitant once the solution is supersaturated. These variations of D25,w are interpreted in terms of non-specific interactions and/or aggregation that prevent the protein from making appropriate contacts to form a crystal. With other precipitants known to lead to crystallization, no significant variation of the diffusion coefficient with increasing concentration was observed, indicating that under such conditions up to saturation the proteins remain essentially monodisperse. Application of this technique to find crystallization conditions of other proteins is discussed.

Aminoacylated tmRNA from Escherichia coli interacts with prokaryotic elongation factor Tu

Eubacterial tmRNAs (10Sa RNAs) are unique because they function, at least in Escherichia coli , both as tRNA and mRNA (for a review, see Muto et al., 1998). These ∼360 ± 40-nt-long RNAs are charged with alanine at their 3′ ends by alanyl-tRNA synthetases or AlaRS (Komine et al., 1994; Ushida et al., 1994). Alanylation occurs thanks to the presence of the equivalent of the G 3 -U 70 pair, the major identity element for the alanylation of canonical tRNAs (Hou & Schimmel, 1988; McClain & Foss, 1988). Bacterial tmRNAs also have a short reading frame coding for 9 to 27 amino acids, depending on the species. E. coli tmRNA mediates recycling of ribosomes stalled at the end of terminatorless mRNAs, via a trans -translation process (Tu et al., 1995; Keiler et al., 1996; Himeno et al., 1997; Withey & Friedman, 1999). In E. coli , this amino acid tag is cotranslationally added to polypeptides synthesized from mRNAs lacking a termination codon, and the added 11-amino-acid C-terminal tag makes the protein a target for specific proteolysis (Keiler et al., 1996).

Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity

A limitation for a universal use of T7 RNA polymerase for in vitro tRNA transcription lies in the nature of the often unfavorable 5′‐terminal sequence of the gene to be transcribed. To overcome this drawback, a hammerhead ribozyme sequence was introduced between a strong T7 RNA polymerase promoter and the tDNA sequence. Transcription of this construct gives rise to a ‘transzyme’ molecule, the autocatalytic activity of which liberates a 5′‐OH tRNA transcript starting with the proper nucleotide. The method was optimized for transcription of yeast tRNATyr, starting with 5′‐C1, and operates as well for yeast tRNAAsp with 5′‐U1. Although the tRNAs produced by the transzyme method are not phosphorylated, they are fully active in aminoacylation with k cat and K m parameters quasi identical to those of their phosphorylated counterparts.

Conformation in solution of yeast tRNAAsp transcripts deprived of modified nucleotides

A synthetic gene of yeast aspartic acid tRNA with a promoter for phage T7 RNA polymerase was cloned in Escherichia coli. The in vitro transcribed tRNA(Asp) molecules are deprived of modified nucleotides and retain their aspartylation capacity. The solution conformation of these molecules was mapped with chemical structural probes and compared to that of fully modified molecules. Significant differences in reactivities were observed in Pb2+ cleavage of the RNAs and in modification of the bases with dimethyl sulphate. The most striking result concerns C56, which becomes reactive in unmodified tRNA(Asp), indicating the disruption of the C56-G19 base pair involved in the D- and T-loop interaction. The chemical data indicate that unmodified tRNA(Asp) transcripts possess a relaxed conformation compared to that of the native tRNA. This conclusion is confirmed by thermal melting experiments. Thus it can be proposed that post-transcriptional modifications of nucleotides in tRNA stabilize the biologically active conformations in these molecules.

Toward a more complete view of tRNA biology

Transfer RNAs are ancient molecules present in all domains of life. In addition to translating the genetic code into protein and defining the second genetic code together with aminoacyl-tRNA synthetases, tRNAs act in many other cellular functions. Robust phenomenological observations on the role of tRNAs in translation, together with massive sequence and crystallographic data, have led to a deeper physicochemical understanding of tRNA architecture, dynamics and identity. In vitro studies complemented by cell biology data already indicate how tRNA behaves in cellular environments, in particular in higher Eukarya. From an opposite approach, reverse evolution considerations suggest how tRNAs emerged as simplified structures from the RNA world. This perspective discusses what basic questions remain unanswered, how these answers can be obtained and how a more rational understanding of the function and dysfunction of tRNA can have applications in medicine and biotechnology.