Aminoacylation Activity Research Articles

Coexisting bacterial aminoacyl-tRNA synthetase paralogs exhibit distinct phylogenetic backgrounds and functional compatibility with Escherichia coli.

Aminoacyl-tRNA synthetases (aaRSs) are universally essential enzymes that synthesize aminoacyl-tRNA substrates for protein synthesis. Although most organisms require a single aaRS gene for each proteinogenic amino acid to translate their genetic information, numerous species encode multiple gene copies of an aaRS. Growing evidence indicates that organisms acquire extra aaRS genes to sustain or adapt to their unique lifestyle. However, predicting and defining the function of repeated aaRS genes remains challenging due to their potentially unique physiological role in the host organism and the inconsistent annotation of repeated aaRS genes in the literature. Here, we carried out comparative, phylogenetic, and functional studies to determine the activity of coexisting paralogs of tryptophanyl-, tyrosyl-, seryl-, and prolyl-tRNA synthetases encoded in several human pathogenic bacteria. Our analyses revealed that, with a few exceptions, repeated aaRSs involve paralogous genes with distinct phylogenetic backgrounds. Using a collection of Escherichia coli strains that enabled the facile characterization of aaRS activity in vivo, we found that, in almost all cases, one aaRS displayed transfer RNA (tRNA) aminoacylation activity, whereas the other was not compatible with E. coli. Together, this work illustrates the challenges of identifying, classifying, and predicting the function of aaRS paralogs and highlights the complexity of aaRS evolution. Moreover, these results provide new insights into the potential role of aaRS paralogs in the biology of several human pathogens and foundational knowledge for the investigation of the physiological role of repeated aaRS paralogs across bacteria.

An anticodon-sensing T-boxzyme generates the elongator nonproteinogenic aminoacyl-tRNA in situ of a custom-made translation system for incorporation.

In the hypothetical RNA world, ribozymes could have acted as modern aminoacyl-tRNA synthetases (ARSs) to charge tRNAs, thus giving rise to the peptide synthesis along with the evolution of a primitive translation apparatus. We previously reported a T-boxzyme, Tx2.1, which selectively charges initiator tRNA with N-biotinyl-phenylalanine (BioPhe) in situ in a Flexible In-vitro Translation (FIT) system to produce BioPhe-initiating peptides. Here, we performed in vitro selection of elongation-capable T-boxzymes (elT-boxzymes), using para-azido-l-phenylalanine (PheAZ) as an acyl-donor. We implemented a new strategy to enrich elT-boxzyme-tRNA conjugates that self-aminoacylated on the 3'-terminus selectively. One of them, elT32, can charge PheAZ onto tRNA in trans in response to its cognate anticodon. Further evolution of elT32 resulted in elT49, with enhanced aminoacylation activity. We have demonstrated the translation of a PheAZ-containing peptide in an elT-boxzyme-integrated FIT system, revealing that elT-boxzymes are able to generate the PheAZ-tRNA in response to the cognate anticodon in situ of a custom-made translation system. This study, together with Tx2.1, illustrates a scenario where a series of ribozymes could have overseen aminoacylation and co-evolved with a primitive RNA-based translation system.

Toxic effects and molecular mechanisms of estuarian crustaceans (Scylla paramamosain) exposed to five commonly used benzophenones

Benzophenones (BPs) are commonly used in personal care products like sunscreens and are increasingly being released into the environment, raising concerns about their potential ecotoxic effects. BPs as emerging environmental contaminants, little is known about their toxic effects on estuarine organisms. This study firstly investigated the toxic effects of five commonly used BPs on mud crabs (Scylla paramamosain). The crabs were exposed to varying concentrations of BPs for 14 days. The results showed that BPs caused damage to antioxidant systems in crabs. Transcriptome sequencing revealed that BP-3 and BP-1 had a greater impact on the crabs compared to the other BPs. Specifically, BP-1 and BP-3 caused severe damage to organelles and ribosomes. BP affected catalytic activity and hydrolase activity, BP-2 affected phosphoenolpyruate carboxykinase activity, and BP-4 affected tRNA aminoacylation and hydrolase activity. These findings can enhance our understanding of the ecotoxicity of BPs and may help to protect estuarine ecosystems.

Domain collapse and active site ablation generate a widespread animal mitochondrial seryl-tRNA synthetase.

Through their aminoacylation reactions, aminoacyl tRNA-synthetases (aaRS) establish the rules of the genetic code throughout all of nature. During their long evolution in eukaryotes, additional domains and splice variants were added to what is commonly a homodimeric or monomeric structure. These changes confer orthogonal functions in cellular activities that have recently been uncovered. An unusual exception to the familiar architecture of aaRSs is the heterodimeric metazoan mitochondrial SerRS. In contrast to domain additions or alternative splicing, here we show that heterodimeric metazoan mitochondrial SerRS arose from its homodimeric ancestor not by domain additions, but rather by collapse of an entire domain (in one subunit) and an active site ablation (in the other). The collapse/ablation retains aminoacylation activity while creating a new surface, which is necessary for its orthogonal function. The results highlight a new paradigm for repurposing a member of the ancient tRNA synthetase family.

Mitochondrial RNA m3C methyltransferase METTL8 relies on an isoform-specific N-terminal extension and modifies multiple heterogenous tRNAs

Methyltransferase-like 8 (METTL8) encodes a mitochondria-localized METTL8-Iso1 and a nucleolus-distributed METTL8-Iso4 isoform, which differ only in their N-terminal extension (N-extension), by mRNA alternative splicing. METTL8-Iso1 generates 3-methylcytidine at position 32 (m3C32) of mitochondrial tRNAThr and tRNASer(UCN). Whether METTL8-Iso4 is an active m3C32 methyltransferase and the role of the N-extension in mitochondrial tRNA m3C32 formation remain unclear. Here, we revealed that METTL8-Iso4 was inactive in m3C32 generation due to the lack of N-extension, which contains several absolutely conserved modification-critical residues; the counterparts were likewise essential in cytoplasmic m3C32 biogenesis by methyltransferase-like 2A (METTL2A) or budding yeasts tRNA N3-methylcytidine methyltransferase (Trm140), in vitro and in vivo. Cross-compartment/species tRNA modification assays unexpectedly found that METTL8-Iso1 efficiently introduced m3C32 to several cytoplasmic or even bacterial tRNAs in vitro. m3C32 did not influence tRNAThrN6-threonylcarbamoyladenosine (t6A) modification or aminoacylation. In addition to its interaction with mitochondrial seryl-tRNA synthetase (SARS2), we further discovered an interaction between mitochondrial threonyl-tRNA synthetase (TARS2) and METTL8-Iso1. METTL8-Iso1 substantially stimulated the aminoacylation activities of SARS2 and TARS2 in vitro, suggesting a functional connection between mitochondrial tRNA modification and charging. Altogether, our results deepen the mechanistic insights into mitochondrial m3C32 biogenesis and provide a valuable route to prepare cytoplasmic/bacterial tRNAs with only a m3C32 moiety, aiding in future efforts to investigate its effects on tRNA structure and function.

Loss of threonyl-tRNA synthetase-like protein Tarsl2 has little impact on protein synthesis but affects mouse development

Aminoacyl-tRNA synthetases (aaRSs) are essential components for mRNA translation. Two sets of aaRSs are required for cytoplasmic and mitochondrial translation in vertebrates. Interestingly, TARSL2 is a recently evolved duplicated gene of TARS1 (encoding cytoplasmic threonyl-tRNA synthetase) and represents the only duplicated aaRS gene in vertebrates. Although TARSL2 retains the canonical aminoacylation and editing activities in vitro, whether it is a true tRNA synthetase for mRNA translation in vivo is unclear. In this study, we showed that Tars1 is an essential gene since homozygous Tars1 knockout mice were lethal. In contrast, when Tarsl2 was deleted in mice and zebrafish, neither the abundance nor the charging levels of tRNAThrs were changed, indicating that cells relied on Tars1 but not on Tarsl2 for mRNA translation. Furthermore, Tarsl2 deletion did not influence the integrity of the multiple tRNA synthetase complex (MSC), suggesting that Tarsl2 is a peripheral member of the MSC. Finally, we observed that Tarsl2-deleted mice exhibited severe developmental retardation, elevated metabolic capacity, and abnormal bone and muscle development after 3 weeks. Collectively, these data suggest that, despite its intrinsic activity, loss of Tarsl2 has little influence on protein synthesis but does affect mouse development.

Study of the Allosteric Mechanism of Human Mitochondrial Phenylalanyl-tRNA Synthetase by Transfer Entropy via an Improved Gaussian Network Model and Co-evolution Analyses.

We propose an improved transfer entropy approach called the dynamic version of the force constant fitted Gaussian network model based on molecular dynamics ensemble (dfcfGNMMD) to explore the allosteric mechanism of human mitochondrial phenylalanyl-tRNA synthetase (hmPheRS), one of the aminoacyl-tRNA synthetases that play a crucial role in translation of the genetic code. The dfcfGNMMD method can provide reliable estimates of the transfer entropy and give new insights into the role of the anticodon binding domain in driving the catalytic domain in aminoacylation activity and into the effects of tRNA binding and residue mutation on the enzyme activity, revealing the causal mechanism of the allosteric communication in hmPheRS. In addition, we incorporate the residue dynamic and co-evolutionary information to further investigate the key residues in hmPheRS allostery. This study sheds light on the mechanisms of hmPheRS allostery and can provide important information for related drug design.

RNA granule-clustered mitochondrial aminoacyl-tRNA synthetases form multiple complexes with the potential to fine-tune tRNA aminoacylation.

Mitochondrial RNA metabolism is suggested to occur in identified compartmentalized foci, i.e. mitochondrial RNA granules (MRGs). Mitochondrial aminoacyl-tRNA synthetases (mito aaRSs) catalyze tRNA charging and are key components in mitochondrial gene expression. Mutations of mito aaRSs are associated with various human disorders. However, the suborganelle distribution, interaction network and regulatory mechanism of mito aaRSs remain largely unknown. Here, we found that all mito aaRSs partly colocalize with MRG, and this colocalization is likely facilitated by tRNA-binding capacity. A fraction of human mitochondrial AlaRS (hmtAlaRS) and hmtSerRS formed a direct complex via interaction between catalytic domains in vivo. Aminoacylation activities of both hmtAlaRS and hmtSerRS were fine-tuned upon complex formation in vitro. We further established a full spectrum of interaction networks via immunoprecipitation and mass spectrometry for all mito aaRSs and discovered interactions between hmtSerRS and hmtAsnRS, between hmtSerRS and hmtTyrRS and between hmtThrRS and hmtArgRS. The activity of hmtTyrRS was also influenced by the presence of hmtSerRS. Notably, hmtSerRS utilized the same catalytic domain in mediating several interactions. Altogether, our results systematically analyzed the suborganelle localization and interaction network of mito aaRSs and discovered several mito aaRS-containing complexes, deepening our understanding of the functional and regulatory mechanisms of mito aaRSs.

Heterozygous Seryl-tRNA Synthetase 1 Variants Cause Charcot-Marie-Tooth Disease.

Despite the increasing number of genes associated with Charcot-Marie-Tooth (CMT) disease, many patients currently still lack appropriate genetic diagnosis for this disease. Autosomal dominant mutations in aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT. Here, we describe causal missense mutations in the gene encoding seryl-tRNA synthetase 1 (SerRS) for 3 families affected with CMT. Whole-exome sequencing was performed in 16 patients and 14 unaffected members of 3 unrelated families. The functional impact of the genetic variants identified was investigated using bioinformatic prediction tools and confirmed using cellular and biochemical assays. Combined linkage analysis for the 3 families revealed significant linkage (Zmax LOD=6.9) between the genomic co-ordinates on chromosome 1: 108681600-110300504. Within the linkage region, heterozygous SerRS missense variants segregated with the clinical phenotype in the 3 families. The mutant SerRS proteins exhibited reduced aminoacylation activity and abnormal SerRS dimerization, which suggests the impairment of total protein synthesis and induction of eIF2α phosphorylation. Our findings suggest the heterozygous SerRS variants identified represent a novel cause for autosomal dominant CMT. Mutant SerRS proteins are known to impact various molecular and cellular functions. Our findings provide significant advances on the current understanding of the molecular mechanisms associated with ARS-related CMT. ANN NEUROL 2023;93:244-256.

Neuropathy-associated Fars2 deficiency affects neuronal development and potentiates neuronal apoptosis by impairing mitochondrial function

BackgroundNeurodegenerative diseases encompass an extensive and heterogeneous group of nervous system disorders which are characterized by progressive degeneration and death of neurons. Many lines of evidence suggest the participation of mitochondria dysfunction in these diseases. Mitochondrial phenylalanyl-tRNA synthetase, encoded by FARS2, catalyzes the transfer of phenylalanine to its cognate tRNA for protein synthesis. As a member of mt-aaRSs genes, FARS2 missense homozygous mutation c.424G > T (p.D142Y) found in a Chinese consanguineous family first built the relationship between pure hereditary spastic paraplegia (HSP) and FARS2 gene. More FARS2 variations were subsequently found to cause heterogeneous group of neurologic disorders presenting three main phenotypic manifestations: infantile-onset epileptic mitochondrial encephalopathy, later-onset spastic paraplegia and juvenile onset refractory epilepsy. Studies showed that aminoacylation activity is frequently disrupt in cases with FARS2 mutations, indicating a loss-of-function mechanism. However, the underlying pathogenesis of neuropathy-associated Fars2 deficiency is still largely unknown.ResultsEarly gestation lethality of global Fars2 knockout mice was observed prior to neurogenesis. The conditional Fars2 knockout-mouse model delayed lethality to late-gestation, resulting in a thinner cortex and an enlarged ventricle which is consist with the MRI results revealing cortical atrophy and reduced cerebral white matter volume in FARS2-deficient patients. Delayed development of neurite outgrowth followed by neuronal apoptosis was confirmed in Fars2-knockdown mouse primary cultured neurons. Zebrafish, in which fars2 was knocked down, exhibited aberrant motor neuron function including reduced locomotor capacity which well restored the spastic paraplegia phenotype of FARS2-deficient patients. Altered mitochondrial protein synthesis and reduced levels of oxidative phosphorylation complexes were detected in Fars2-deficient samples. And thus, reduced ATP, total NAD levels and mitochondrial membrane potential, together with increased ROS production, revealed mitochondrial dysfunction both in vitro and in vivo. Dctn3 is a potential downstream molecule in responds to Fars2 deficient in neurons, which may provide some evidence for the development of pathogenesis study and therapeutic schedule.ConclusionsThe Fars2 deficiency genetic models developed in this study cover the typical clinical manifestations in FARS2 patients, and help clarify how neuropathy-associated Fars2 deficiency, by damaging the mitochondrial respiratory chain and impairing mitochondrial function, affects neuronal development and potentiates neuronal cell apoptosis.

Systemic inflammatory syndrome in children with FARSA deficiency.

Variants in aminoacyl‐tRNA synthetases (ARSs) genes are associated to a broad spectrum of human inherited diseases. Patients with defective PheRS, encoded by FARSA and FARSB, display brain abnormalities, interstitial lung disease and facial dysmorphism. We investigated four children from two unrelated consanguineous families carrying two missense homozygous variants in FARSA with significantly reduced PheRS‐mediated aminoacylation activity. In addition to the core ARS‐phenotype, all patients showed an inflammatory profile associated with autoimmunity and interferon score, a clinical feature not ascribed to PheRS‐deficient patients to date. JAK inhibition improved lung disease in one patient. Our findings expand the genetic and clinical spectrum of FARSA‐related disease.

Recognition of tRNAIle with a UAU anticodon by isoleucyl-tRNA synthetase in lactic acid bacteria.

In almost all eubacteria, the AUA codon is translated by tRNAIle2 bearing lysidine at the wobble position. Lysidine is introduced by tRNAIle lysidine synthetase (TilS) via post-transcriptional modification of the cytidine of tRNAIle2 (CAU). Lactobacillus casei and Lactobacillus plantarum have tilS homologues and tRNAIle2 (CAU) genes. In addition, L. casei also has another tRNAIle2 gene with an UAU anticodon. L. plantarum hasa tRNAIle (UAU)-like RNA. Here, we demonstrate that L. casei tRNAIle2 (UAU) is charged with isoleucine by L. casei isoleucyl-tRNA synthetase (IleRS) but not by L. plantarum IleRS, even though the amino acid identity of these two enzymes is over 60%. It has been reported that, in Mycoplasma mobile, which has its tRNAIle2 (UAU) but no tilS homologue, an Arg residue at position 865 of the IleRS is required for recognition of the UAU anticodon. This position is occupied by an Arg also in the IleRSs from both of the Lactobacillus species. Thus, other residues in L. casei, IleRS should also contribute to the recognition of tRNAIle2 (UAU). We found that a chimeric L. casei IleRS in which the N-terminal domain was replaced by the corresponding region of L. plantatarum IleRS has very low aminoacylation activity towards both tRNAIle2 (UAU) and tRNAIle1 (GAU). The A18G mutant had barely detectable aminoacylation activity towards either of the tRNAsIle . However, a double point mutant of A18G and G19N aminoacylated tRNAIle1 (GAU), but not tRNAIle2 (UAU). Our results suggest that, for L. casei IleRS, Ala18 and Gly19 also play a critical role in recognition of tRNAIle2 (UAU).

Human lysyl-tRNA synthetase evolves a dynamic structure that can be stabilized by forming complex.

The evolutionary necessity of aminoacyl-tRNA synthetases being associated into complex is unknown. Human lysyl-tRNA synthetase (LysRS) is one component of the multi-tRNA synthetase complex (MSC), which is not only critical for protein translation but also involved in multiple cellular pathways such as immune response, cell migration, etc. Here, combined with crystallography, CRISPR/Cas9-based genome editing, biochemistry, and cell biology analyses, we show that the structures of LysRSs from metazoan are more dynamic than those from single-celled organisms. Without the presence of MSC scaffold proteins, such as aminoacyl-tRNA synthetase complex-interacting multifunctional protein 2 (AIMP2), human LysRS is free from the MSC. The interaction with AIMP2 stabilizes the closed conformation of LysRS, thereby protects the essential aminoacylation activity under stressed conditions. Deleting AIMP2 from the human embryonic kidney 293 cells leads to retardation in cell growth in nutrient deficient mediums. Together, these results suggest that the evolutionary emergence of the MSC in metazoan might be to protect the aminoacyl-tRNA synthetase components from being modified or recruited for use in other cellular pathways.

Gain of C-Ala enables AlaRS to target the L-shaped tRNAAla.

Unlike many other aminoacyl-tRNA synthetases, alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout biology. While Caenorhabditis elegans cytoplasmic AlaRS (CeAlaRSc) retains the prototype structure, its mitochondrial counterpart (CeAlaRSm) contains only a residual C-terminal domain (C-Ala). We demonstrated herein that the C-Ala domain from CeAlaRSc robustly binds both tRNA and DNA. It bound different tRNAs but preferred tRNAAla. Deletion of this domain from CeAlaRSc sharply reduced its aminoacylation activity, while fusion of this domain to CeAlaRSm selectively and distinctly enhanced its aminoacylation activity toward the elbow-containing (or L-shaped) tRNAAla. Phylogenetic analysis showed that CeAlaRSm once possessed the C-Ala domain but later lost most of it during evolution, perhaps in response to the deletion of the T-arm (part of the elbow) from its cognate tRNA. This study underscores the evolutionary gain of C-Ala for docking AlaRS to the L-shaped tRNAAla.

TRNA Synthetases Are Recruited to Yeast Ribosomes by rRNA Expansion Segment 7L but Do Not Require Association for Functionality.

Protein biosynthesis is essential for any organism, yet how this process is regulated is not fully understood at the molecular level. During evolution, ribosomal RNA expanded in specific regions, referred to as rRNA expansion segments (ES). First functional roles of these expansions have only recently been discovered. Here we address the role of ES7La located in the large ribosomal subunit for factor recruitment to the yeast ribosome and the potential consequences for translation. Truncation of ES7La has only minor effects on ribosome biogenesis, translation efficiency and cell doubling. Using yeast rRNA deletion strains coupled with ribosome-specific mass spectrometry we analyzed the interactome of ribosomes lacking ES7La. Three aminoacyl-tRNA synthetases showed reduced ribosome association. Synthetase activities however remained unaltered suggesting that the pool of aminoacylated tRNAs is unaffected by the ES deletion. These results demonstrated that aminoacylation activities of tRNA synthetases per se do not rely on ribosome association. These findings suggest a role of ribosome-associated aminoacyl-tRNA synthetase beyond their core enzymatic functions.

Treatment of ARS deficiencies with specific amino acids

PurposeRecessive cytosolic aminoacyl-tRNA synthetase (ARS) deficiencies are severe multiorgan diseases, with limited treatment options. By loading transfer RNAs (tRNAs) with their cognate amino acids, ARS are essential for protein translation. However, it remains unknown why ARS deficiencies lead to specific symptoms, especially early life and during infections. We set out to increase pathophysiological insight and improve therapeutic possibilities. MethodsIn fibroblasts from patients with isoleucyl-RS (IARS), leucyl-RS (LARS), phenylalanyl-RS-beta-subunit (FARSB), and seryl-RS (SARS) deficiencies, we investigated aminoacylation activity, thermostability, and sensitivity to ARS-specific amino acid concentrations, and developed personalized treatments. ResultsAminoacylation activity was reduced in all patients, and further diminished at 38.5/40°C (PLARS and PFARSB), consistent with infectious deteriorations. With lower cognate amino acid concentrations, patient fibroblast growth was severely affected. To prevent local and/or temporal deficiencies, we treated patients with corresponding amino acids (follow-up: 1/2–2 2/3rd years), and intensified treatment during infections. All patients showed beneficial treatment effects, most strikingly in growth (without tube feeding), head circumference, development, coping with infections, and oxygen dependency. ConclusionFor these four ARS deficiencies, we observed a common disease mechanism of episodic insufficient aminoacylation to meet translational demands and illustrate the power of amino acid supplementation for the expanding ARS patient group. Moreover, we provide a strategy for personalized preclinical functional evaluation. [Display omitted]

Selective and competitive functions of the AAR and UPR pathways in stress-induced angiogenesis

The amino acid response (AAR) and unfolded protein response (UPR) pathways converge on eIF2α phosphorylation, which is catalyzed by Gcn2 and Perk, respectively, under different stresses. This close interconnection makes it difficult to specify different functions of AAR and UPR. Here, we generated a zebrafish model in which loss of threonyl-tRNA synthetase (Tars) induces angiogenesis dependent on Tars aminoacylation activity. Comparative transcriptome analysis of the tars-mutant and wild-type embryos with/without Gcn2- or Perk-inhibition reveals that only Gcn2-mediated AAR is activated in the tars-mutants, whereas Perk functions predominantly in normal development. Mechanistic analysis shows that, while a considerable amount of eIF2α is normally phosphorylated by Perk, the loss of Tars causes an accumulation of uncharged tRNAThr, which in turn activates Gcn2, leading to phosphorylation of an extra amount of eIF2α. The partial switchover of kinases for eIF2α largely overwhelms the functions of Perk in normal development. Interestingly, although inhibition of Gcn2 and Perk in this stress condition both can reduce the eIF2α phosphorylation levels, their functional consequences in the regulation of target genes and in the rescue of the angiogenic phenotypes are dramatically different. Indeed, genetic and pharmacological manipulations of these pathways validate that the Gcn2-mediated AAR, but not the Perk-mediated UPR, is required for tars-deficiency induced angiogenesis. Thus, the interconnected AAR and UPR pathways differentially regulate angiogenesis through selective functions and mutual competitions, reflecting the specificity and efficiency of multiple stress response pathways that evolve integrally to enable an organism to sense/respond precisely to various types of stresses.

Drosophila Models for Charcot-Marie-Tooth Neuropathy Related to Aminoacyl-tRNA Synthetases.

Aminoacyl-tRNA synthetases (aaRS) represent the largest cluster of proteins implicated in Charcot–Marie–Tooth neuropathy (CMT), the most common neuromuscular disorder. Dominant mutations in six aaRS cause different axonal CMT subtypes with common clinical characteristics, including progressive distal muscle weakness and wasting, impaired sensory modalities, gait problems and skeletal deformities. These clinical manifestations are caused by “dying back” axonal degeneration of the longest peripheral sensory and motor neurons. Surprisingly, loss of aminoacylation activity is not a prerequisite for CMT to occur, suggesting a gain-of-function disease mechanism. Here, we present the Drosophila melanogaster disease models that have been developed to understand the molecular pathway(s) underlying GARS1- and YARS1-associated CMT etiology. Expression of dominant CMT mutations in these aaRSs induced comparable neurodegenerative phenotypes, both in larvae and adult animals. Interestingly, recent data suggests that shared molecular pathways, such as dysregulation of global protein synthesis, might play a role in disease pathology. In addition, it has been demonstrated that the important function of nuclear YARS1 in transcriptional regulation and the binding properties of mutant GARS1 are also conserved and can be studied in D. melanogaster in the context of CMT. Taken together, the fly has emerged as a faithful companion model for cellular and molecular studies of aaRS-CMT that also enables in vivo investigation of candidate CMT drugs.

A non-translational role of threonyl-tRNA synthetase in regulating JNK signaling during myogenic differentiation.

Aminoacyl-tRNA synthetases (aaRSs) are house-keeping enzymes that are essential for protein synthesis. However, it has become increasingly evident that some aaRSs also have non-translational functions. Here we report the identification of a non-translational function of threonyl-tRNA synthetase (ThrRS) in myogenic differentiation. We find that ThrRS negatively regulates myoblast differentiation in vitro and injury-induced skeletal muscle regeneration in vivo. This function is independent of amino acid binding or aminoacylation activity of ThrRS, and knockdown of ThrRS leads to enhanced differentiation without affecting the global protein synthesis rate. Furthermore, we show that the non-catalytic new domains (UNE-T and TGS) of ThrRS are both necessary and sufficient for the myogenic function. In searching for a molecular mechanism of this new function, we find the kinase JNK to be a downstream target of ThrRS. Our data further reveal MEKK4 and MKK4 as upstream regulators of JNK in myogenesis and the MEKK4-MKK4-JNK pathway to be a mediator of the myogenic function of ThrRS. Finally, we show that ThrRS physically interacts with Axin1, disrupts Axin1-MEKK4 interaction and consequently inhibits JNK signaling. In conclusion, we uncover a non-translational function for ThrRS in the maintenance of homeostasis of skeletal myogenesis and identify the Axin1-MEKK4-MKK4-JNK signaling axis to be an immediate target of ThrRS action.

Crystal structure of Nanoarchaeum equitans tyrosyl-tRNA synthetase and its aminoacylation activity toward tRNATyr with an extra guanosine residue at the 5ʹ-terminus

tRNATyr of Nanoarchaeum equitans has a remarkable feature with an extra guanosine residue at the 5ʹ-terminus. However, the N. equitans tRNATyr mutant without extra guanosine at the 5ʹ-end was tyrosylated by tyrosyl-tRNA synthase (TyrRS). We solved the crystal structure of N. equitans TyrRS at 2.80 Å resolution. By comparing the present solved structure with the complex structures TyrRS with tRNATyr of Thermus thermophilus and Methanocaldococcus jannaschii, an arginine substitution mutant of N. equitans TyrRS at Ile200 (I200R), which is the putative closest candidate to the 5ʹ-phosphate of C1 of N. equitans tRNATyr, was prepared. The I200R mutant tyrosylated not only wild-type tRNATyr but also the tRNA without the G-1 residue. Further tyrosylation analysis revealed that the second base of the anticodon (U35), discriminator base (A73), and C1:G72 base pair are strong recognition sites.