alanyl-tRNA Synthetase Research Articles

RNA-seq analysis of mitochondria-related genes regulated by AMPK in the human trophoblast cell line BeWo.

How AMP activated protein kinase (AMPK) signaling regulates mitochondrial functions and mitophagy in human trophoblast cells remains unclear. This study was designed to investigate potential players mediating the regulation of AMPK on mitochondrial functions and mitophagy by next generation RNA-seq. We compared ATP production in protein kinase AMP-activated catalytic subunit alpha 1/2 (PRKAA1/2) knockdown (AKD) and control BeWo cells using the Seahorse real-time ATP rate test, then analyzed gene expression profiling by RNA-seq. Differentially expressed genes (DEG) were examined by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Then protein-protein interactions (PPI) among mitochondria related genes were further analyzed using Metascape and Ingenuity Pathway Analysis (IPA) software. Both mitochondrial and glycolytic ATP production in AKD cells were lower than in the control BeWo cells (CT), with a greater reduction of mitochondrial ATP production. A total of 1092 DEGs were identified, with 405 upregulated and 687 downregulated. GO analysis identified 60 genes associated with the term 'mitochondrion' in the cellular component domain. PPI analysis identified three clusters of mitochondria related genes, including aldo-keto reductase family 1 member B10 and B15 (AKR1B10, AKR1B15), alanyl-tRNA synthetase 1 (AARS1), mitochondrial ribosomal protein S6 (MRPS6), mitochondrial calcium uniporter dominant negative subunit beta (MCUB) and dihydrolipoamide branched chain transacylase E2 (DBT). In summary, this study identified multiple mitochondria related genes regulated by AMPK in BeWo cells, and among them, three clusters of genes may potentially contribute to altered mitochondrial functions in response to reduced AMPK signaling.

Evaluation of the Role of Tanshinone I in an In Vitro System of Charcot-Marie-Tooth Disease Type 2N.

Charcot-Marie-Tooth disease type 2N (CMT2N) is an inherited nerve disorder caused by mutations in the alanyl-tRNA synthetase (AlaRS) gene, resulting in muscle weakness and sensory issues. Currently, there is no cure for CMT2N. Here, we found that all five AlaRS mutations in the aminoacylation domain can interact with neuropilin-1 (Nrp1), which is consistent with our previous findings. Interestingly, three of these mutations did not affect alanine activation activity. We then performed a high-throughput screen of 2000 small molecules targeting the prevalent R329H mutant. Using thermal stability assays (TSA), biolayer interferometry (BLI), ATP consumption, and proteolysis assays, we identified Tanshinone I as a compound that binds to and modifies the conformation of the R329H mutant and other CMT-related AlaRS mutants interacting with Nrp1. Molecular docking and dynamic simulation studies further clarified Tanshinone I's binding mode, indicating its potential against various AlaRS mutants. Furthermore, co-immunoprecipitation (Co-IP) and pull-down assays showed that Tanshinone I significantly reduces the binding of AlaRS mutants to Nrp1. Collectively, these findings suggest that Tanshinone I, by altering the conformation of mutant proteins, disrupts the pathological interaction between AlaRS CMT mutants and Nrp1, potentially restoring normal Nrp1 function. This makes Tanshinone I a promising therapeutic candidate for CMT2N.

AARS1 and AARS2 sense L-lactate to regulate cGAS as global lysine lactyltransferases.

L-lactate modifies proteins through lactylation1, but how this process occurs is unclear. Here we identify the alanyl-tRNA synthetases AARS1 and AARS2 (AARS1/2) as intracellular L-lactate sensors required for L-lactate to stimulate the lysine lactylome in cells. AARS1/2 and the evolutionarily conserved Escherichia coli orthologue AlaRS bind to L-lactate with micromolar affinity and they directly catalyse L-lactate for ATP-dependent lactylation on the lysine acceptor end. In response to L-lactate, AARS2 associates with cyclic GMP-AMP synthase (cGAS) and mediates its lactylation and inactivation in cells and in mice. By establishing a genetic code expansion orthogonal system for lactyl-lysine incorporation, we demonstrate that the presence of a lactyl moiety at a specific cGAS amino-terminal site abolishes cGAS liquid-like phase separation and DNA sensing in vitro and in vivo. A lactyl mimetic knock-in inhibits cGAS, whereas a lactyl-resistant knock-in protects mice against innate immune evasion induced through high levels of L-lactate. MCT1 blockade inhibits cGAS lactylation in stressed mice and restores innate immune surveillance, which in turn antagonizes viral replication. Thus, AARS1/2 are conserved intracellular L-lactate sensors and have an essential role as lactyltransferases. Moreover, a chemical reaction process of lactylation targets and inactivates cGAS.

Trypanosoma brucei multi-aminoacyl-tRNA synthetase complex formation limits promiscuous tRNA proofreading.

Faithful mRNA decoding depends on the accuracy of aminoacyl-tRNA synthetases (ARSs). Aminoacyl-tRNA proofreading mechanisms have been well-described in bacteria, humans, and plants. However, our knowledge of translational fidelity in protozoans is limited. Trypanosoma brucei (Tb) is a eukaryotic, protozoan pathogen that causes Human African Trypanosomiasis, a fatal disease if untreated. Tb undergoes many physiological changes that are dictated by nutrient availability throughout its insect-mammal lifecycle. In the glucose-deprived insect vector, the tsetse fly, Tb use proline to make ATP via mitochondrial respiration. Alanine is one of the major by-products of proline consumption. We hypothesize that the elevated alanine pool challenges Tb prolyl-tRNA synthetase (ProRS), an ARS known to misactivate alanine in all three domains of life, resulting in high levels of misaminoacylated Ala-tRNAPro. Tb encodes two domains that are members of the INS superfamily of aminoacyl-tRNA deacylases. One homolog is appended to the N-terminus of Tb ProRS, and a second is the major domain of multi-aminoacyl-tRNA synthetase complex (MSC)-associated protein 3 (MCP3). Both ProRS and MCP3 are housed in the Tb MSC. Here, we purified Tb ProRS and MCP3 and observed robust Ala-tRNAPro deacylation activity from both enzymes in vitro. Size-exclusion chromatography multi-angle light scattering used to probe the oligomerization state of MCP3 revealed that although its unique N-terminal extension confers homodimerization in the absence of tRNA, the protein binds to tRNA as a monomer. Kinetic assays showed MCP3 alone has relaxed tRNA specificity and promiscuously hydrolyzes cognate Ala-tRNAAla; this activity is significantly reduced in the presence of Tb alanyl-tRNA synthetase, also housed in the MSC. Taken together, our results provide insight into translational fidelity mechanisms in Tb and lay the foundation for exploring MSC-associated proteins as novel drug targets.

Comprehensive assessment of recessive, pathogenic AARS1 alleles in a humanized yeast model reveals loss-of-function and dominant-negative effects.

Alanyl-tRNA synthetase 1 (AARS1) encodes the enzyme that ligates tRNA molecules to alanine in the cytoplasm, which is required for protein translation. Variants in AARS1 have been implicated in early-onset, multi-system recessive phenotypes and in later-onset dominant peripheral neuropathy; to date, no single variant has been associated with both dominant and recessive diseases raising questions about shared mechanisms between the two inheritance patterns. AARS1 variants associated with recessive disease are predicted to result in null or hypomorphic alleles and this has been demonstrated, in part, via yeast complementation assays. However, pathogenic alleles have not been assessed in a side-by-side manner to carefully scrutinize the strengths and limitations of this model system. To address this, we employed a humanized yeast model to evaluate the functional consequences of all AARS1 missense variants reported in recessive disease. The majority of variants showed variable loss-of-function effects, ranging from no growth to significantly reduced growth. These data deem yeast a reliable model to test the functional consequences of human AARS1 variants; however, our data indicate that this model is prone to false-negative results and is not informative for genotype-phenotype studies. We next tested missense variants associated with no growth for dominant-negative effects. Interestingly, K81T AARS1, a variant implicated in recessive disease, demonstrated loss-of-function and dominant-negative effects, indicating that certain AARS1 variants may be capable of causing both dominant and recessive disease phenotypes.

Eukaryotic AlaX provides multiple checkpoints for quality and quantity of aminoacyl-tRNAs in translation.

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.

Alanyl-tRNA synthetase, AARS1, is a lactate sensor and lactyltransferase that lactylates p53 and contributes to tumorigenesis

Lysine lactylation is a post-translational modification that links cellular metabolism to protein function. Here, we find that AARS1 functions as a lactate sensor that mediates global lysine lacylation in tumor cells. AARS1 binds to lactate and catalyzes the formation of lactate-AMP, followed by transfer of lactate to the lysince acceptor residue. Proteomics studies reveal a large number of AARS1 targets, including p53 where lysine 120 and lysine 139 in the DNA binding domain are lactylated. Generation and utilization of p53 variants carrying constitutively lactylated lysine residues revealed that AARS1 lactylation of p53 hinders its liquid-liquid phase separation, DNA binding, and transcriptional activation. AARS1 expression and p53 lacylation correlate with poor prognosis among cancer patients carrying wild type p53. β-alanine disrupts lactate binding to AARS1, reduces p53 lacylation, and mitigates tumorigenesis in animal models. We propose that AARS1 contributes to tumorigenesis by coupling tumor cell metabolism to proteome alteration.

The alanyl-tRNA synthetase AARS1 moonlights as a lactyltransferase to promote YAP signaling in gastric cancer.

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.

Abstract 1804 Extending the scope of a coupled assay to measure alanyl-tRNA synthetase activity

Abstract 1804 Extending the scope of a coupled assay to measure alanyl-tRNA synthetase activity

Common evolutionary origins of the bacterial glycyl tRNA synthetase and alanyl tRNA synthetase.

Aminoacyl-tRNA synthetases (aaRSs) establish the genetic code. Each aaRS covalently links a given canonical amino acid to a cognate set of tRNA isoacceptors. Glycyl tRNA aminoacylation is unusual in that it is catalyzed by different aaRSs in different lineages of the Tree of Life. We have investigated the phylogenetic distribution and evolutionary history of bacterial glycyl tRNA synthetase (bacGlyRS). This enzyme is found in early diverging bacterial phyla such as Firmicutes, Acidobacteria, and Proteobacteria, but not in archaea or eukarya. We observe relationships between each of six domains of bacGlyRS and six domains of four different RNA-modifying proteins. Component domains of bacGlyRS show common ancestry with (i) the catalytic domain of class II tRNA synthetases; (ii) the HD domain of the bacterial RNase Y; (iii) the body and tail domains of the archaeal CCA-adding enzyme; (iv) the anti-codon binding domain of the arginyl tRNA synthetase; and (v) a previously unrecognized domain that we call ATL (Ancient tRNA latch). The ATL domain has been found thus far only in bacGlyRS and in the universal alanyl tRNA synthetase (uniAlaRS). Further, the catalytic domain of bacGlyRS is more closely related to the catalytic domain of uniAlaRS than to any other aminoacyl tRNA synthetase. The combined results suggest that the ATL and catalytic domains of these two enzymes are ancestral to bacGlyRS and uniAlaRS, which emerged from common protein ancestors by bricolage, stepwise accumulation of protein domains, before the last universal common ancestor of life.

Elucidation of productive alanine recognition mechanism by Escherichia coli alanyl-tRNA synthetase

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.

Anticodon sequence determines the impact of mistranslating tRNAAla variants

ABSTRACT Transfer RNAs (tRNAs) maintain translation fidelity through accurate charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic message is incorporated into proteins and has applications in biotechnology, therapeutics and is relevant to disease. Since the alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in tRNAAla and the anticodon plays no role in charging, tRNAAla variants with anticodon mutations have the potential to mis-incorporate alanine. Here, we characterize the impact of the 60 non-alanine tRNAAla anticodon variants on the growth of Saccharomyces cerevisiae. Overall, 36 tRNAAla anticodon variants decreased growth in single- or multi-copy. Mass spectrometry analysis of the cellular proteome revealed that 52 of 57 anticodon variants, not decoding alanine or stop codons, induced mistranslation when on single-copy plasmids. Variants with G/C-rich anticodons resulted in larger growth deficits than A/U-rich variants. In most instances, synonymous anticodon variants impact growth differently, with anticodons containing U at base 34 being the least impactful. For anticodons generating the same amino acid substitution, reduced growth generally correlated with the abundance of detected mistranslation events. Differences in decoding specificity, even between synonymous anticodons, resulted in each tRNAAla variant mistranslating unique sets of peptides and proteins. We suggest that these differences in decoding specificity are also important in determining the impact of tRNAAla anticodon variants.

Sequence-specific targeting of Caenorhabditis elegans C-Ala to the D-loop of tRNAAla

Alanyl-tRNA synthetase retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Alac) robustly binds both ligands. How Ce-C-Alac targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Alac are responsible for DNA and tRNA binding, respectively. Ce-C-Alac specifically recognized the conserved invariant base G18 in the D-loop of tRNAAla through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C.elegans mitochondrial C-Ala robustly bound both tRNAAla and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C.elegans C-Ala specifically targets the D-loop of tRNAAla.

High expression of VARS promotes the growth of multiple myeloma cells by causing imbalance in valine metabolism.

Multiple myeloma (MM) is a plasma cell malignancy occurring in middle and old age. MM is still an incurable disease due to its frequent recurrence and drug resistance. However, its pathogenesis is still unclear. Abnormal amino acid metabolism is one of the important characteristics of MM, and the important metabolic pathway of amino acids participates in protein synthesis as basic raw materials. Aminoacyl transfer ribonucleic acid synthetase (ARS) gene is a key regulatory gene in protein synthesis. This study aims to explore the molecular mechanism for ARS, a key factor of amino acid metabolism, in regulating amino acid metabolism in MM and affecting MM growth. The corresponding gene number was combined with the gene expression profile GSE5900 dataset and GSE2658 dataset in Gene Expression Omnibus (GEO) database to standardize the gene expression data of ARS. GSEA_4.2.0 software was used to analyze the difference of gene enrichment between healthy donors (HD) and MM patients in GEO database. GraphPad Prism 7 was used to draw heat maps and perform data analysis. Kaplan-Meier and Cox regression model were used to analyze the expression of ARS gene and the prognosis of MM patients, respectively. Bone marrow samples from 7 newly diagnosed MM patients were collected, CD138+ and CD138- cells were obtained by using CD138 antibody magnetic beads, and the expression of ARS in MM clinical samples was analyzed by real-time RT-PCR. Human B lymphocyte GM12878 cells and human MM cell lines ARP1, NCI-H929, OCI-MY5, U266, RPMI 8266, OPM-2, JJN-3, KMS11, MM1.s cells were selected as the study objects. The expression of ARS in MM cell lines was analyzed by real-time RT-PCR and Western blotting. Short hairpin RNA (shRNA) lentiviruses were used to construct gene knock-out plasmids (VARS-sh group). No-load plasmids (scramble group) and gene knock-out plasmids (VARS-sh group) were transfected into HEK 293T cells with for virus packaging, respectively. Stable expression cell lines were established by infecting ARP1 and OCI-MY5 cells, and the effects of knockout valyl-tRNA synthetase (VARS) gene on proliferation and apoptosis of MM cells were detected by cell counting and flow cytometry, respectively. GEO data were divided into a high expression group and a low expression group according to the expression of VARS. Bioinformatics analysis was performed to explore the downstream pathways affected by VARS. Gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) and high performance liquid chromatography (HPLC) were used to detect the valine content in CD138+ cells and ARP1, OCI-MY5 cells and supernatant of knockdown VARS gene in bone marrow samples from patients, respectively. Gene enrichment analysis showed that tRNA processing related genes were significantly enriched in MM compared with HD (P<0.0001). Further screening of tRNA processing-pathway related subsets revealed that cytoplasmic aminoacyl tRNA synthetase family genes were significantly enriched in MM (P<0.0001). The results of gene expression heat map showed that the ARS family genes except alanyl-tRNA synthetase (AARS), arginyl-tRNA synthetase (RARS), seryl-tRNA synthetase (SARS) in GEO data were highly expressed in MM (all P<0.01). With the development of monoclonal gammopathy of undetermined significance (MGUS) to MM, the gene expression level was increased gradually. Kaplan-Meier univariate analysis of survival results showed that there were significant differences in the prognosis of MM patients in methionyl-tRNA synthetase (MARS), asparaginyl-tRNA synthetase (NARS) and VARS between the high expression group and the low expression group (all P<0.05). Cox regression model multivariate analysis showed that the high expression of VARS was associated with abnormal overall survival time of MM (HR=1.83, 95% CI 1.10 to 3.06, P=0.021). The high expression of NARS (HR=0.90, 95% CI 0.34 to 2.38) and MARS (HR=1.59, 95% CI 0.73 to 3.50) had no effect on the overall survival time of MM patients (both P>0.05). Real-time RT-PCR and Western blotting showed that VARS, MARS and NARS were highly expressed in CD138+ MM cells and MM cell lines of clinical patients (all P<0.05). Cell counting and flow cytometry results showed that the proliferation of MM cells by knockout VARS was significantly inhibited (P<0.01), the proportion of apoptosis was significantly increased (P<0.05). Bioinformatics analysis showed that in addition to several pathways including the cell cycle regulated by VARS, the valine, leucine and isoleucine catabolic pathways were upregulated. Non-targeted metabolomics data showed reduced valine content in CD138+ tumor cells in MM patients compared to HD (P<0.05). HPLC results showed that compared with the scramble group, the intracellular and medium supernatant content of ARP1 cells and the medium supernatant of OCI-MY5 in the VARS-shRNA group was increased (all P<0.05). MM patients with abnormal high expression of VARS have a poor prognosis. VARS promotes the malignant growth of MM cells by affecting the regulation of valine metabolism.

A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

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.

Comprehensive proteogenomic characterization of early duodenal cancer reveals the carcinogenesis tracks of different subtypes

The subtypes of duodenal cancer (DC) are complicated and the carcinogenesis process is not well characterized. We present comprehensive characterization of 438 samples from 156 DC patients, covering 2 major and 5 rare subtypes. Proteogenomics reveals LYN amplification at the chromosome 8q gain functioned in the transmit from intraepithelial neoplasia phase to infiltration tumor phase via MAPK signaling, and illustrates the DST mutation improves mTOR signaling in the duodenal adenocarcinoma stage. Proteome-based analysis elucidates stage-specific molecular characterizations and carcinogenesis tracks, and defines the cancer-driving waves of the adenocarcinoma and Brunner’s gland subtypes. The drug-targetable alanyl-tRNA synthetase (AARS1) in the high tumor mutation burden/immune infiltration is significantly enhanced in DC progression, and catalyzes the lysine-alanylation of poly-ADP-ribose polymerases (PARP1), which decreases the apoptosis of cancer cells, eventually promoting cell proliferation and tumorigenesis. We assess the proteogenomic landscape of early DC, and provide insights into the molecular features corresponding therapeutic targets.

Design principles and functional basis of enantioselectivity of alanyl-tRNA synthetase and a chiral proofreader during protein biosynthesis.

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.

A naturally occurring mini-alanyl-tRNA synthetase

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 tRNAAla, and C-Ala―the major tRNA-binding module―targets the elbow of the L-shaped tRNAAla. 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 tRNAAla (G3:U70), TuAlaRS still specifically recognized G3:U70-containing tRNAAla. In addition, despite lacking C-Ala, TuAlaRS robustly binds and charges microAla (an RNA substrate corresponding to the acceptor stem of tRNAAla) as well as tRNAAla, 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.

Perseverance of protein homeostasis despite mistranslation of glycine codons with alanine.

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 tRNASer 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 tRNAGly variants (tRNAGlyCCC, tRNAGlyGCC) and creates an alanyl-tRNA synthetase (AlaRS) identity element (G3 : U70). Because AlaRS does not recognize the anticodon, the human tRNAAlaAGC G35C (tRNAAlaACC) variant may function similarly to mis-incorporate Ala at Gly codons. The tRNAGly and tRNAAla variants had no effect on protein synthesis in mammalian cells under normal growth conditions; however, tRNAGlyGCC A3G depressed protein synthesis in the context of proteasome inhibition. Mass spectrometry confirmed Ala mistranslation at multiple Gly codons caused by the tRNAGlyGCC A3G and tRNAAlaAGC 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'.

Alanyl-tRNA Synthetase 1 Gene Variants in Hereditary Neuropathy: Genotype and Phenotype Overview.

Background and ObjectivesOur objective was to report 2 novel variants and to reclassify previously reported alanyl-tRNA synthetase 1 (AARS1) variants associated with hereditary neuropathy and to summarize the clinical features of a previously published cohort of patients.MethodsWe performed detailed neurologic and electrophysiologic assessments and segregation analysis of 2 unrelated families with Charcot-Marie-Tooth (CMT) disease with novel variants in the AARS1 gene. Via literature search, we found studies that included neuropathy cases with AARS1 variants; we then reviewed and reclassified these variants.ResultsWe identified 2 CMT families harboring previously unreported likely pathogenic AARS1 variants: c.1823C>A p.(Thr608Lys) and c.1815C>G p.(His605Gln). In addition, we reinterpreted a total of 35 different AARS1 variants reported in cases with neuropathy from the literature: 9 variants fulfilled the current criteria for being (likely) pathogenic. We compiled and summarized standardized clinical and genotypic information for 90 affected individuals from 32 families with (likely) pathogenic AARS1 variants. Most experienced motor weakness and sensory loss in the lower limbs.DiscussionIn total, 11 AARS1 variants can currently be classified as pathogenic or likely pathogenic and are associated with sensorimotor axonal or intermediate, slowly progressive polyneuropathy with common asymmetry and variable age of symptom onset with no apparent involvement of other organ systems.