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Translational fidelity relies critically on correct aminoacyl-tRNA supply. The trans-editing factor AlaX predominantly hydrolyzes Ser-tRNAAla, functioning as a third sieve of alanyl-tRNA synthetase (AlaRS). Despite extensive studies in bacteria and archaea, the mechanism of trans-editing in mammals remains largely unknown. Here, we show that human AlaX (hAlaX), which is exclusively distributed in the cytoplasm, is an active trans-editing factor with strict Ser-specificity. In vitro, both hAlaX and yeast AlaX (ScAlaX) were capable of hydrolyzing nearly all Ser-mischarged cytoplasmic and mitochondrial tRNAs; and robustly edited cognate Ser-charged cytoplasmic and mitochondrial tRNASers. In vivo or cell-based studies revealed that loss of ScAlaX or hAlaX readily induced Ala- and Thr-to-Ser misincorporation. Overexpression of hAlaX impeded the decoding efficiency of consecutive Ser codons, implying its regulatory role in Ser codon decoding. Remarkably, yeast cells with ScAlaX deletion responded differently to translation inhibitor treatment, with a gain in geneticin resistance, but sensitivity to cycloheximide, both of which were rescued by editing-capable ScAlaX, alanyl- or threonyl-tRNA synthetase. Altogether, our results demonstrated the previously undescribed editing peculiarities of eukaryotic AlaXs, which provide multiple checkpoints to maintain the speed and fidelity of genetic decoding., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Lactylation has been recently identified as a new type of posttranslational modification occurring widely on lysine residues of both histone and nonhistone proteins. The acetyltransferase p300 is thought to mediate protein lactylation, yet the cellular concentration of the proposed lactyl-donor, lactyl-coenzyme A, is about 1,000 times lower than that of acetyl-CoA, raising the question of whether p300 is a genuine lactyltransferase. Here, we report that alanyl-tRNA synthetase 1 (AARS1) moonlights as a bona fide lactyltransferase that directly uses lactate and ATP to catalyze protein lactylation. Among the candidate substrates, we focused on the Hippo pathway, which has a well-established role in tumorigenesis. Specifically, AARS1 was found to sense intracellular lactate and translocate into the nucleus to lactylate and activate the YAP-TEAD complex; and AARS1 itself was identified as a Hippo target gene that forms a positive-feedback loop with YAP-TEAD to promote gastric cancer (GC) cell proliferation. Consistently, the expression of AARS1 was found to be upregulated in GC, and elevated AARS1 expression was found to be associated with poor prognosis for patients with GC. Collectively, this work found AARS1 with lactyltransferase activity in vitro and in vivo and revealed how the metabolite lactate is translated into a signal of cell proliferation.

Alanyl-tRNA synthetase (AlaRS) incorrectly recognizes both a slightly smaller glycine and a slightly larger serine in addition to alanine, and the probability of incorrect identification is extremely low at 1/300 and 1/170, respectively. Alanine is the second smallest amino acid after glycine; however, the mechanism by which AlaRS specifically identifies small differences in side chains with high accuracy remains unknown. In this study, using a malachite green assay, we aimed to elucidate the alanine recognition mechanism of a fragment (AlaRS368N) containing only the amino acid activation domain of Escherichia coli AlaRS. This method quantifies monophosphate by decomposing pyrophosphate generated during aminoacyl-AMP production. AlaRS368N produced far more pyrophosphate when glycine or serine was used as a substrate than when alanine was used. Among several mutants tested, an AlaRS mutant in which the widely conserved aspartic acid at the 235th position (D235) near the active center was replaced with glutamic acid (D235E) increased pyrophosphate release for the alanine substrate, compared to that from glycine and serine. These results suggested that D235 is optimal for AlaRS to specifically recognize alanine. Alanylation activities of an RNA minihelix by the mutants of valine at the 214th position (V214) of another fragment (AlaRS442N), which is the smallest AlaRS with alanine charging activity, suggest the existence of the van der Waals-like interaction between the side chain of V214 and the methyl group of the alanine substrate., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: The mitochondrial alanyl-tRNA synthetase 2 (AARS2) as one of aminoacyl-tRNA synthases (ARSs) performs amino acid transportation and involves protein synthesis. However, its role in cancer remains largely unexplored., Methods: In this study, more than 10,000 samples were enrolled to explore genomic alterations, biological function, prognosis, and clinical treatment based on AARS2 across pan-cancer. The molecular characterization of AARS2 was confirmed in hepatocellular carcinoma (HCC) using proteomics analysis, quantitative real-time PCR, western blotting, immunohistochemical staining, and cell experiments., Results: For genomic landscape, the AARS2 was dramatically upregulated in multiple cancers, which might be mainly caused by copy number alteration rather than mutation and methylation. The abnormal expression of AARS2 was prominently associated with activity of cancer pathways and performed oncogenic roles in most cancers. Systematic experiments in vitro substantiated the elevated expression of AARS2, and the deficiency of it inhibited cell proliferation and cell migration in HCC. Meanwhile, our findings suggested that AARS2 could serve as a novel promising and stable biomarker for assessing prognosis and immunotherapy. Moreover, a variety of therapeutic drugs and targeted pathways were proposed for cancer treatment, which might enhance clinical efficacy., Conclusion: The AARS2 could serve as a new oncogenic gene that promotes cell proliferation and migration in HCC. The comprehensive investigations increased the understanding of AARS2 across human cancers and generated beginning insights of AARS2 in genomic landscape, molecular biological function, prognosis, and clinical treatment., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that ligate tRNA molecules to cognate amino acids. Heterozygosity for missense variants or small in-frame deletions in six ARS genes causes dominant axonal peripheral neuropathy. These pathogenic variants reduce enzyme activity without significantly decreasing protein levels and reside in genes encoding homo-dimeric enzymes. These observations raise the possibility that neuropathy-associated ARS variants exert a dominant-negative effect, reducing overall ARS activity below a threshold required for peripheral nerve function. To test such variants for dominant-negative properties, we developed a humanized yeast assay to co-express pathogenic human alanyl-tRNA synthetase (AARS1) mutations with wild-type human AARS1. We show that multiple loss-of-function AARS1 mutations impair yeast growth through an interaction with wild-type AARS1, but that reducing this interaction rescues yeast growth. This suggests that neuropathy-associated AARS1 variants exert a dominant-negative effect, which supports a common, loss-of-function mechanism for ARS-mediated dominant peripheral neuropathy., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Homochirality of the cellular proteome is attributed to the L-chiral bias of the translation apparatus. The chiral specificity of enzymes was elegantly explained using the 'four-location' model by Koshland two decades ago. In accordance with the model, it was envisaged and noted that some aminoacyl-tRNA synthetases (aaRS) that charge larger amino acids are porous to D-amino acids. However, a recent study showed that alanyl-tRNA synthetase (AlaRS) can mischarge D-alanine and that its editing domain, but not the universally present D-aminoacyl-tRNA deacylase (DTD), is responsible for correcting the chirality-based error. Here, using in vitro and in vivo data coupled with structural analysis, we show that AlaRS catalytic site is a strict D-chiral rejection system and therefore does not activate D-alanine. It obviates the need for AlaRS editing domain to be active against D-Ala-tRNAAla and we show that it is indeed the case as it only corrects L-serine and glycine mischarging. We further provide direct biochemical evidence showing activity of DTD on smaller D-aa-tRNAs that corroborates with the L-chiral rejection mode of action proposed earlier. Overall, while removing anomalies in the fundamental recognition mechanisms, the current study further substantiates how chiral fidelity is perpetuated during protein biosynthesis., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout its biology, consisting of catalytic, tRNA-recognition, editing, and C-Ala domains. The catalytic and tRNA-recognition domains catalyze aminoacylation, the editing domain hydrolyzes mischarged tRNA Ala , and C-Ala-the major tRNA-binding module-targets the elbow of the L-shaped tRNA Ala . Interestingly, a mini-AlaRS lacking the editing and C-Ala domains is recovered from the Tupanvirus of the amoeba Acanthamoeba castellanii. Here we show that Tupanvirus AlaRS (TuAlaRS) is phylogenetically related to its host's AlaRS. Despite lacking the conserved amino acid residues responsible for recognition of the identity element of tRNA Ala (G3:U70), TuAlaRS still specifically recognized G3:U70-containing tRNA Ala . In addition, despite lacking C-Ala, TuAlaRS robustly binds and charges micro Ala (an RNA substrate corresponding to the acceptor stem of tRNA Ala ) as well as tRNA Ala , indicating that TuAlaRS exclusively targets the acceptor stem. Moreover, this mini-AlaRS could functionally substitute for yeast AlaRS in vivo. This study suggests that TuAlaRS has developed a new tRNA-binding mode to compensate for the loss of C-Ala., (© 2023. The Author(s).)

By linking amino acids to their codon assignments, transfer RNAs (tRNAs) are essential for protein synthesis and translation fidelity. Some human tRNA variants cause amino acid mis-incorporation at a codon or set of codons. We recently found that a naturally occurring tRNA Ser variant decodes phenylalanine codons with serine and inhibits protein synthesis. Here, we hypothesized that human tRNA variants that misread glycine (Gly) codons with alanine (Ala) will also disrupt protein homeostasis. The A3G mutation occurs naturally in tRNA Gly variants (tRNA Gly CCC , tRNA Gly GCC ) and creates an alanyl-tRNA synthetase (AlaRS) identity element (G3 : U70). Because AlaRS does not recognize the anticodon, the human tRNA Ala AGC G35C (tRNA Ala ACC ) variant may function similarly to mis-incorporate Ala at Gly codons. The tRNA Gly and tRNA Ala variants had no effect on protein synthesis in mammalian cells under normal growth conditions; however, tRNA Gly GCC A3G depressed protein synthesis in the context of proteasome inhibition. Mass spectrometry confirmed Ala mistranslation at multiple Gly codons caused by the tRNA Gly GCC A3G and tRNA Ala AGC G35C mutants, and in some cases, we observed multiple mistranslation events in the same peptide. The data reveal mistranslation of Ala at Gly codons and defects in protein homeostasis generated by natural human tRNA variants that are tolerated under normal conditions. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

To report a new genetic cause of distal hereditary motor neuropathy (dHMN), which is likely associated with worsening during pregnancy. We collected the clinical data of a patient with severe weakness of the lower limbs induced by repeated pregnancy and performed relevant experimental examinations, including neuromuscular electrophysiological examination, neuromuscular biopsy, and genetic testing. The patient reported weakness of the right lower extremity after delivery of the first child. Initially, the right foot was weak during lifting, and symptoms gradually progressed to weakness when landing on the toe during walking. She then developed weakness of the right lower extremity and thinning of the right leg. After an interval of 2.5 years, after delivery of the second child, her left lower extremity developed asthenia, with the same symptoms as previously reported for the right lower extremity. Subsequently, weakness of both lower extremities became progressively worse, and she developed difficulty sitting up, getting out of bed, and walking. Physical examination showed that both upper limb vertebral tracts were damaged and both lower extremity motor nerves were damaged. Electrophysiology suggested motor axonal neurogenic damage. Brain magnetic resonance imaging demonstrated leukodystrophy. Sural nerve biopsy suggested mild axonal damage. Skeletal muscle biopsy suggested neurogenic skeletal muscle damage. Genetic testing suggested that there was a heterozygous mutation at the shear site of the AARS gene. An AARS mutation may cause dHMN associated with pyramidal tract signs.

Background: Aminoacyl tRNA-synthetases are ubiquitously-expressed enzymes that attach amino acids to their cognate tRNA molecules. Mutations in several genes encoding aminoacyl tRNA-synthetases, have been associated with peripheral neuropathy, i.e. AARS1, GARS1, HARS1, YARS1 and WARS1. The pathogenic mechanism underlying AARS1-related neuropathy is not known., Methods: From 2012 onward, all probands presenting at Telemark Hospital (Skien, Norway) with peripheral neuropathy were screened for variants in AARS1 using an "in-house" next-generation sequencing panel. DNA from patient's family members was examined by Sanger sequencing. Blood from affected family members and healthy controls were used for quantification of AARS1 mRNA and alanine. Proteomic analyses were conducted in peripheral blood mononuclear cells (PBMC) from four affected family members and five healthy controls., Results: Seventeen individuals in two Norwegian families affected by Charcot-Marie-Tooth disease (CMT) were characterized in this study. The heterozygous NM_001605.2:c.976C > T p.(Arg326Trp) AARS1 mutation was identified in ten affected family members. All living carriers had a mild to severe length-dependent sensorimotor neuropathy. Three deceased obligate carriers aged 74-98 were reported to be unaffected, but were not examined in the clinic. Proteomic studies in PBMC from four affected individuals suggest an effect on the immune system mediated by components of a systemic response to chronic injury and inflammation. Furthermore, altered expression of proteins linked to mitochondrial function/dysfunction was observed. Proteomic data are available via ProteomeXchange using identifier PXD023842., Conclusion: This study describes clinical and neurophysiological features linked to the p.(Arg326Trp) variant of AARS1 in CMT-affected members of two Norwegian families. Proteomic analyses based on of PBMC from four CMT-affected individuals suggest that involvement of inflammation and mitochondrial dysfunction might contribute to AARS1 variant-associated peripheral neuropathy., (© 2022. The Author(s).)

Background: Missense mutations in the mitochondrial alanyl-tRNA synthetase 2 (AARS2) gene are clinically associated with infantile mitochondrial cardiomyopathy or adult-onset leukoencephalopathy with early ovarian failure. To date, approximately 40 cases have been reported related to AARS2 mutations, while its genetic and phenotypic spectrum remains to be defined., Case Presentation: We identified a 24-year-old Chinese female patient with adult-onset leukoencephalopathy carrying novel compound heterozygous pathogenic mutations in the AARS2 gene (c.718C > T and c.1040 + 1G > A) using a whole-exome sequencing approach., Conclusions: Our findings further extend the mutational spectrum of AARS2-related leukoencephalopathy and highlight the importance of the whole-exome sequencing in precisely diagnosing adult-onset leukoencephalopathies., (© 2022. The Author(s).)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)