Editing Defects Research Articles

Development of tRNA synthetases and connection to genetic code and disease

The genetic code is established by the aminoacylation reactions of aminoacyl tRNA synthetases, where amino acids are matched with triplet anticodons imbedded in the cognate tRNAs. The code established in this way is so robust that it gave birth to the entire tree of life. The tRNA synthetases are organized into two classes, based on their active site architectures. The details of this organization, and other considerations, suggest how the synthetases evolved by gene duplications, and how early proteins may have been statistical in nature, that is, products of a primitive code where one of several similar amino acids was used at a specific position in a polypeptide. The emergence of polypeptides with unique, defined sequences--true chemical entities--required extraordinary specificity of the aminoacylation reaction. This high specificity was achieved by editing activities that clear errors of aminoacylation and thereby prevent mistranslation. Defects in editing activities can be lethal and lead to pathologies in mammalian cells in culture. Even a mild defect in editing is casually associated with neurological disease in the mouse. Defects in editing are also mutagenic in an aging organism and suggest how mistranslation can lead to mutations that are fixed in the genome. Thus, clearance of mischarged tRNAs by the editing activities of tRNA synthetases was essential for development of the tree of life and has a role in the etiology of diseases that is just now being understood.

Natural Homolog of tRNA Synthetase Editing Domain Rescues Conditional Lethality Caused by Mistranslation

AlaXp is a widely distributed (from bacteria to humans) genome-encoded homolog of the editing domain of alanyl-tRNA synthetases. Editing repairs the confusion of serine and glycine for alanine through clearance of mischarged (with Ser or Gly) tRNA(Ala). Because genome-encoded fragments of editing domains of other synthetases are scarce, the AlaXp redundancy of the editing domain of alanyl-tRNA synthetase is thought to reflect an unusual sensitivity of cells to mistranslation at codons for Ala. Indeed, a small defect in the editing activity of alanyl-tRNA synthetase is causally linked to neurodegeneration in the mouse. Although limited earlier studies demonstrated that AlaXp deacylated mischarged tRNA(Ala) in vitro, the significance of this activity in vivo has not been clear. Here we describe a bacterial system specifically designed to investigate activity of AlaXp in vivo. Serine toxicity, experienced by a strain harboring an editing-defective alanyl-tRNA synthetase, was rescued by an AlaXp-encoding transgene. Rescue was dependent on amino acid residues in AlaXp that are needed for its in vitro catalytic activity. Thus, the editing activity per se of AlaXp was essential for suppressing mistranslation. The results support the idea that the unique widespread distribution of AlaXp arises from the singular difficulties, for translation, poised by alanine.

Evidence That tRNA Synthetase-Directed Proton Transfer Stops Mistranslation

To prevent mistranslation, aminoacyl-tRNA synthetases (AARSs) discriminate against noncognate amino acids and cellular metabolites. Defects in specificity produce statistical proteins which, in mammalian cells, lead to activation of the unfolded protein response and cell death. Because of inherent limitations in amino acid discrimination by a single active site, AARSs evolved a separate domain to clear mischarged amino acids. Although the structure of a widely distributed editing domain for ThrRS and AlaRS is known, the mechanism of amino acid clearance remains elusive. This domain has two motifs that together have four conserved residues in the pocket used to clear serine from mischarged tRNAs. Here, using ThrRS as an example, rapid single-turnover kinetics, mutagenesis, and solvent isotope analysis show that a strictly conserved histidine (between ThrRS and AlaRS) extracts a proton in the chemical step of the editing reaction. Three other conserved residues, and two additional residues in the editing pocket, are not directly implicated in the chemical step. These results are relevant to the previously reported mutagenesis of the homologous editing pocket of alanyl-tRNA synthetase, where even a mild defect in editing causes neurodegeneration in the mouse. Thus, a single proton-transfer event needed to prevent mistranslation can have profound implications for disease.

Phenylalanyl-tRNA synthetase editing defects result in efficient mistranslation of phenylalanine codons as tyrosine

Translational quality control is monitored at several steps, including substrate selection by aminoacyl-tRNA synthetases (aaRSs), and discrimination of aminoacyl-tRNAs by elongation factor Tu (EF-Tu) and the ribosome. Phenylalanyl-tRNA synthetase (PheRS) misactivates Tyr but is able to correct the mistake using a proofreading activity named editing. Previously we found that overproduction of editing-defective PheRS resulted in Tyr incorporation at Phe-encoded positions in vivo, although the misreading efficiency could not be estimated. This raised the question as to whether or not EF-Tu and the ribosome provide further proofreading mechanisms to prevent mistranslation of Phe codons by Tyr. Here we show that, after evading editing by PheRS, Tyr-tRNA(Phe) is recognized by EF-Tu as efficiently as the cognate Phe-tRNA(Phe). Kinetic decoding studies using full-length Tyr-tRNA(Phe) and Phe-tRNA(Phe), as well as a poly(U)-directed polyTyr/polyPhe synthesis assay, indicate that the ribosome lacks discrimination between Tyr-tRNA(Phe) and Phe-tRNA(Phe). Taken together, these data suggest that PheRS editing is the major proofreading step that prevents infiltration of Tyr into Phe codons during translation.

An editing-defective aminoacyl-tRNA synthetase is mutagenic in aging bacteria via the SOS response

Mistranslation in bacterial and mammalian cells leads to production of statistical proteins that are, in turn, associated with specific cell or animal pathologies, including death of bacterial cells, apoptosis of mammalian cells in culture, and neurodegeneration in the mouse. A major source of mistranslation comes from heritable defects in the editing activities of aminoacyl-tRNA synthetases. These activities clear errors of aminoacylation by deacylation of mischarged tRNAs. We hypothesized that, in addition to previously reported phenotypes in bacterial and mammalian systems, errors of aminoacylation could be mutagenic and lead to disease. As a first step in testing this hypothesis, the effect of an editing defect in a single tRNA synthetase on the accumulation of mutations in aging bacteria was investigated. A striking, statistically significant, enhancement of the mutation rate in aging bacteria was found. This enhancement comes from an increase in error-prone DNA repair through induction of the bacterial SOS response. Thus, mistranslation, as caused by an editing-defective tRNA synthetase, can lead to heritable genetic changes that could, in principle, be linked to disease.

Global Effects of Mistranslation from an Editing Defect in Mammalian Cells

Aminoacyl-tRNA synthetases prevent mistranslation, or genetic code ambiguity, through specialized editing reactions. Mutations that disrupt editing in bacteria adversely affect cell growth and viability, and recent work in the mouse supports the idea that translational errors caused by an editing defect lead to a neurological disease-like phenotype. To further investigate the connection of mistranslation to cell pathology, we introduced an inducible transgene expressing an editing-deficient valyl-tRNA synthetase into mammalian cells. Introducing mistranslation precipitated a disruption of cell morphology and membrane blebbing, accompanied by activation of caspase-3, consistent with an apoptotic response. Addition of a noncanonical amino acid that is misactivated, but not cleared, by the editing-defective enzyme exacerbated these effects. A special ambiguity-detecting sensor provided direct readout of mistranslation in vivo, supporting the possibility that decreased translational fidelity could be associated with disease.

Pigment deficiency in nightshade/tobacco cybrids is caused by the failure to edit the plastid ATPase alpha-subunit mRNA.

The subgenomes of the plant cell, the nuclear genome, the plastome, and the chondriome are known to interact through various types of coevolving macromolecules. The combination of the organellar genome from one species with the nuclear genome of another species often leads to plants with deleterious phenotypes, demonstrating that plant subgenomes coevolve. The molecular mechanisms behind this nuclear-organellar incompatibility have been elusive, even though the phenomenon is widespread and has been known for >70 years. Here, we show by direct and reverse genetic approaches that the albino phenotype of a flowering plant with the nuclear genome of Atropa belladonna (deadly nightshade) and the plastome of Nicotiana tabacum (tobacco) develops as a result of a defect in RNA editing of a tobacco-specific editing site in the plastid ATPase alpha-subunit transcript. A plastome-wide analysis of RNA editing in these cytoplasmic hybrids and in plants with a tobacco nucleus and nightshade chloroplasts revealed additional defects in the editing of species-specific editing sites, suggesting that differences in RNA editing patterns in general contribute to the pigment deficiencies observed in interspecific nuclear-plastidial incompatibilities.

Elucidation of tRNA-dependent editing by a class II tRNA synthetase and significance for cell viability.

Editing of misactivated amino acids by class I tRNA synthetases is encoded by a specialized internal domain specific to class I enzymes. In contrast, little is known about editing activities of the structurally distinct class II enzymes. Here we show that the class II alanyl-tRNA synthetase (AlaRS) has a specialized internal domain that appears weakly related to an appended domain of threonyl-tRNA synthetase (ThrRS), but is unrelated to that found in class I enzymes. Editing of misactivated glycine or serine was shown to require a tRNA cofactor. Specific mutations in the aforementioned domain disrupt editing and lead to production of mischarged tRNA. This class-specific editing domain was found to be essential for cell growth, in the presence of elevated concentrations of glycine or serine. In contrast to ThrRS, where the editing domain is not found in all three kingdoms of living organisms, it was incorporated early into AlaRSs and is present throughout evolution. Thus, tRNA-dependent editing by AlaRS may have been critical for making the genetic code sufficiently accurate to generate the tree of life.

Genetic Code Ambiguity: CELL VIABILITY RELATED TO SEVERITY OF EDITING DEFECTS IN MUTANT tRNA SYNTHETASES*

The rules of the genetic code are established in reactions that aminoacylate tRNAs with specific amino acids. Ambiguity in the code is prevented by editing activities whereby incorrect aminoacylations are cleared by specialized hydrolytic reactions of aminoacyl tRNA synthetases. Whereas editing reactions have long been known, their significance for cell viability is still poorly understood. Here we investigated in vitro and in vivo four different mutations in the center for editing that diminish the proofreading activity of valyl-tRNA synthetase (ValRS). The four mutant enzymes were shown to differ quantitatively in the severity of the defect in their ability to clear mischarged tRNA in vitro. Strikingly, in the presence of excess concentrations of alpha-aminobutyrate, one of the amino acids that is misactivated by ValRS, growth of bacterial strains bearing these mutant alleles is arrested. The concentration of misactivated amino acid required for growth arrest correlates inversely in a rank order with the degree of the editing defect seen in vitro. Thus, cell viability depends directly on the suppression of genetic code ambiguity by these specific editing reactions and is finely tuned to any perturbation of these reactions.

The human anti-thyroid peroxidase autoantibody repertoire in Graves' and Hashimoto's autoimmune thyroid diseases.

Human anti-thyroid peroxidase (TPO) autoantibodies (aAb) are generated during autoimmune thyroid diseases (AITD). Within recent years, increasing knowledge of the TPO-specific aAb repertoire, gained mainly by the use of combinatorial library methodology, has led to the cloning and sequencing of around 180 human anti-TPO aAb. Analysis of the immunoglobulin (Ig) variable (V) genes encoding the TPO aAb in the ImMunoGeneTics database (IMGT) (http://imgt.cines.fr) reveals major features of the TPO-directed aAb repertoire during AITD. Heavy chain VH domains of TPO-specific aAb from Graves' disease patients preferentially use D proximal IGHV1 genes, whereas those from Hashimoto's thyroiditis are characterized more frequently by IGHV3 genes, mainly located in the middle of the IGH locus. A large proportion of the anti-TPO heavy chain VH domains is obtained following a VDJ recombination process that uses inverted D genes. J distal IGKV1 and IGLV1 genes are predominantly used in TPO aAb. In contrast to the numerous somatic hypermutations in the TPO-specific heavy chains, there is only limited amino acid replacement in most of the TPO-specific light chains, particularly in those encoded by J proximal IGLV or IGKV genes, suggesting that a defect in receptor editing can occur during aAb generation in AITD. Among the predominant IGHV1 or IGKV1 TPO aAb, conserved somatic mutations are the hallmark of the TPO aAb repertoire. The aim of this review is to provide new insights into aAb generation against TPO, a major autoantigen involved in AITD.

Receptor revision and systemic lupus.

Studies over the past 10 years have shown that B cells can undergo secondary heavy- or light-chain immunoglobulin (Ig) rearrangements at various stages of their normal development, a process termed receptor editing. In the bone marrow, this mechanism is important to maintain tolerance because it can extinguish a self-reactive specificity without having to physically eliminate a potentially autoreactive B cell. In the periphery, secondary rearrangements may also play a role in the diversification and maturation of an immune response, although conclusive evidence for this process is still required. Individuals with systemic autoimmune diseases, such as lupus, show evidence of intricate abnormalities in receptor editing. On the one hand, decreased editing may not eliminate the self-reactive specificities that emerge during B-cell development in the bone marrow. Conversely, excessive secondary rearrangements, especially in the periphery where tolerance mechanisms are less effective, can result in the production of autoantibodies by edited B cells. It will be important to assess whether the complex editing defects observed during lupus are a primary susceptibility factor to this disease or if they are secondary to other abnormalities of lymphocyte development in these autoimmune patients.

A new mutator strain of Bacillus subtilis.

Bacillus subtilis strain SB1207, widely used in our laboratory, was found to be highly temperature-sensitive and to exhibit a strong SOS-independent mutator phenotype at elevated temperatures. Both chromosomal and plasmid-borne genes were affected by the mutator. Lethality and mutator phenotype could not be attributed to a replication shut off or to thymine starvation. Due to the high frequency of base misincorporation, the mutator phenotype probably results from an editing defect rather than from a post-replication defect (mismatch repair).

D. The Line Spectrum of Pulsating Variable Stars

(Ed. note:Encouraged by the success of the more informal approach in Christy's presentation, we tried an even more extreme experiment in this session, I-D. In essence, Kraft held the floor continuously all morning, and for the hour and a half afternoon session, serving as a combined Summary-Introductory speaker and a marathon-moderator of a running discussion on the line spectrum of cepheids. There was almost continuous interruption of his presentation; and most points raised from the floor were followed through in detail, no matter how digressive to the main presentation. This approach turned out to be much too extreme. It is wearing on the speaker, and the other members of the symposium feel more like an audience and less like participants in a dissective discussion. Because Kraft presented a compendious collection of empirical information, and, based on it, an exceedingly novel series of suggestions on the cepheid problem, these defects were probably aggravated by the first and alleviated by the second. I am much indebted to Kraft for working with me on a preliminary editing, to try to delete the side-excursions and to retain coherence about the main points. As usual, however, all responsibility for defects in final editing is wholly my own.)