Mistranslating the genetic code with leucine in yeast and mammalian cells.

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.

Targeting immune cell glutamyl-prolyl-transfer RNA synthetase 1 (EPRS1) to prevent fibrosis after tubulointerstitial nephritis.

Glutamyl-prolyl-transfer RNA synthetase 1 is an enzyme that connects glutamic acid and proline to transfer RNA during protein synthesis. In this issue, a study by Kang et al. examined the role of the immune cell glutamyl-prolyl-transfer RNA synthetase 1 in toxin-induced tubulointerstitial nephritis mice. The study demonstrated that blocking glutamyl-prolyl-transfer RNA synthetase 1 may be a therapeutic target to attenuate fibrosis after toxin-induced tubulointerstitial nephritis.

Practical Approaches to Genetic Code Expansion with Aminoacyl-tRNA Synthetase/tRNA Pairs.

Genetic code expansion (GCE) can enable the site-selective incorporation of non-canonical amino acids (ncAAs) into proteins. GCE has advanced tremendously in the last decade and can be used to create biorthogonal handles, monitor and control proteins inside cells, study post-translational modifications, and engineer new protein functions. Since establishing our laboratory, our research has focused on applications of GCE in protein and enzyme engineering using aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs. This topic has been reviewed extensively, leaving little doubt that GCE is a powerful tool for engineering proteins and enzymes. Therefore, for this young faculty issue, we wanted to provide a more technical look into the methods we use and the challenges we think about in our laboratory. Since starting the laboratory, we have successfully engineered over a dozen novel aaRS/tRNA pairs tailored for various GCE applications. However, we acknowledge that the field can pose challenges even for experts. Thus, herein, we provide a review of methodologies in ncAA incorporation with some practical commentary and a focus on challenges, emerging solutions, and exciting developments.

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.

Allosteric inhibition of tRNA synthetase Gln4 by N-pyrimidinyl-ß-thiophenylacrylamides exerts highly selective antifungal activity.

Candida species are among the most prevalent causes of systemic fungal infections, which account for ∼1.5 million annual fatalities. Here, we build on a compound screen that identified the molecule N-pyrimidinyl-ß-thiophenylacrylamide (NP-BTA), which strongly inhibits Candida albicans growth. NP-BTA was hypothesized to target C. albicans glutaminyl-tRNA synthetase, Gln4. Here, we confirmed through in vitro amino-acylation assays NP-BTA is a potent inhibitor of Gln4, and we defined how NP-BTA arrests Gln4's transferase activity using co-crystallography. This analysis also uncovered Met496 as a critical residue for the compound's species-selective target engagement and potency. Structure-activity relationship (SAR) studies demonstrated the NP-BTA scaffold is subject to oxidative and non-oxidative metabolism, making it unsuitable for systemic administration. In a mouse dermatomycosis model, however, topical application of the compound provided significant therapeutic benefit. This work expands the repertoire of antifungal protein synthesis target mechanisms and provides a path to develop Gln4 inhibitors.

Pathophysiology of human mitochondrial tRNA metabolism.

Mitochondria play multiple critical roles in cellular activity. In particular, mitochondrial translation is pivotal in the regulation of mitochondrial and cellular homeostasis. In this forum article, we discuss human mitochondrial tRNA metabolism and highlight its tight connection with various mitochondrial diseases caused by mutations in aminoacyl-tRNA synthetases, tRNAs, and tRNA-modifying enzymes.

Adding α,α-disubstituted and ß-linked monomers to the genetic code of an organism.

The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1-3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4-6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs-this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-L-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several ß-amino acids, α,α-disubstituted-amino acids and ß-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of ß-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers.

Enhanced Directed Evolution in Mammalian Cells Yields a Hyperefficient Pyrrolysyl tRNA for Noncanonical Amino Acid Mutagenesis.

Heterologous tRNAs used for noncanonical amino acid (ncAA) mutagenesis in mammalian cells typically show poor activity. We recently introduced a virus-assisted directed evolution strategy (VADER) that can enrich improved tRNA mutants from naïve libraries in mammalian cells. However, VADER was limited to processing only a few thousand mutants; the inability to screen a larger sequence space precluded the identification of highly active variants with distal synergistic mutations. Here, we report VADER2.0, which can process significantly larger mutant libraries. It also employs a novel library design, which maintains base-pairing between distant residues in the stem regions, allowing us to pack a higher density of functional mutants within a fixed sequence space. VADER2.0 enabled simultaneous engineering of the entire acceptor stem of M. mazei pyrrolysyl tRNA (tRNAPyl ), leading to a remarkably improved variant, which facilitates more efficient incorporation of a wider range of ncAAs, and enables facile development of viral vectors and stable cell-lines for ncAA mutagenesis.

Enzymic recognition of amino acids drove the evolution of primordial genetic codes.

How genetic information gained its exquisite control over chemical processes needed to build living cells remains an enigma. Today, the aminoacyl-tRNA synthetases (AARS) execute the genetic codes in all living systems. But how did the AARS that emerged over three billion years ago as low-specificity, protozymic forms then spawn the full range of highly-specific enzymes that distinguish between 22 diverse amino acids? A phylogenetic reconstruction of extant AARS genes, enhanced by analysing modular acquisitions, reveals six AARS with distinct bacterial, archaeal, eukaryotic, or organellar clades, resulting in a total of 36 families of AARS catalytic domains. Small structural modules that differentiate one AARS family from another played pivotal roles in discriminating between amino acid side chains, thereby expanding the genetic code and refining its precision. The resulting model shows a tendency for less elaborate enzymes, with simpler catalytic domains, to activate amino acids that were not synthesised until later in the evolution of the code. The most probable evolutionary route for an emergent amino acid type to establish a place in the code was by recruiting older, less specific AARS, rather than adapting contemporary lineages. This process, retrofunctionalisation, differs from previously described mechanisms through which amino acids would enter the code.

Mitochondrial aminoacyl-tRNA synthetases trigger unique compensatory mechanisms in neurons.

Mitochondrial aminoacyl-tRNA synthetase (mt-ARS) mutations cause severe, progressive, and often lethal diseases with highly heterogeneous and tissue-specific clinical manifestations. This study investigates the molecular mechanisms triggered by three different mt-ARS defects caused by biallelic mutations in AARS2, EARS2, and RARS2, using an in vitro model of human neuronal cells. We report distinct molecular mechanisms of mitochondrial dysfunction among the mt-ARS defects studied. Our findings highlight the ability of proliferating neuronal progenitor cells (iNPCs) to compensate for mitochondrial translation defects and maintain balanced levels of oxidative phosphorylation (OXPHOS) components, which becomes more challenging in mature neurons. Mutant iNPCs exhibit unique compensatory mechanisms, involving specific branches of the integrated stress response, which may be gene-specific or related to the severity of the mitochondrial translation defect. RNA sequencing revealed distinct transcriptomic profiles showing dysregulation of neuronal differentiation and protein translation. This study provides valuable insights into the tissue-specific compensatory mechanisms potentially underlying the phenotypes of patients with mt-ARS defects. Our novel in vitro model may more accurately represent the neurological presentation of patients and offer an improved platform for future investigations and therapeutic development.

Disease association and therapeutic routes of aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (ARSs) are enzymes that catalyze the ligation of amino acids to tRNAs for translation. Beyond their traditional role in translation, ARSs have acquired regulatory functions in various biological processes (epi-translational functions). With their dual-edged activities, aberrant expression, secretion, and mutations of ARSs are associated with human diseases, including cancer, autoimmune diseases, and neurological diseases. The increasing numbers of newly unveiled activities and disease associations of ARSs have spurred interest in novel drug development, targeting disease-related catalytic and noncatalytic activities of ARSs as well as harnessing ARSs as sources for biological therapeutics. This review speculates how the translational and epi-translational activities of ARSs can be related and describes how their activities can be linked to diseases and drug discovery.

Truncation-Free Genetic Code Expansion with Tetrazine Amino Acids for Quantitative Protein Ligations.

Quantitative labeling of biomolecules is necessary to advance areas of antibody-drug conjugation, super-resolution microscopy imaging of molecules in live cells, and determination of the stoichiometry of protein complexes. Bio-orthogonal labeling to genetically encodable noncanonical amino acids (ncAAs) offers an elegant solution; however, their suboptimal reactivity and stability hinder the utility of this method. Previously, we showed that encoding stable 1,2,4,5-tetrazine (Tet)-containing ncAAs enables rapid, complete conjugation, yet some expression conditions greatly limited the quantitative reactivity of the Tet-protein. Here, we demonstrate that reduction of on-protein Tet ncAAs impacts their reactivity, while the leading cause of the unreactive protein is near-cognate suppression (NCS) of UAG codons by endogenous aminoacylated tRNAs. To overcome incomplete conjugation due to NCS, we developed a more catalytically efficient tRNA synthetase and developed a series of new machinery plasmids harboring the aminoacyl tRNA synthetase/tRNA pair (aaRS/tRNA pair). These plasmids enable robust production of homogeneously reactive Tet-protein in truncation-free cell lines, eliminating the contamination caused by NCS and protein truncation. Furthermore, these plasmid systems utilize orthogonal synthetic origins, which render these machinery vectors compatible with any common expression system. Through developing these new machinery plasmids, we established that the aaRS/tRNA pair plasmid copy-number greatly affects the yields and quality of the protein produced. We then produced quantitatively reactive soluble Tet-Fabs, demonstrating the utility of this system for rapid, homogeneous conjugations of biomedically relevant proteins.

Aminoacyl-tRNA synthetase interactions in SARS-CoV-2 infection.

Aminoacyl-tRNA synthetases (aaRSs) are ancient enzymes that serve a foundational role in the efficient and accurate translation of genetic information from messenger RNA to proteins. These proteins play critical, non-canonical functions in a multitude of cellular processes. Multiple viruses are known to hijack the functions of aaRSs for proviral outcomes, while cells modify antiviral responses through non-canonical functions of certain synthetases. Recent findings have revealed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronaviral disease 19 (COVID-19), utilizes canonical and non-canonical functions of aaRSs, establishing a complex interplay of viral proteins, cellular factors and host aaRSs. In a striking example, an unconventional multi-aaRS complex consisting of glutamyl-prolyl-, lysyl-, arginyl- and methionyl-tRNA synthetases interact with a previously unknown RNA-element in the 3'-end of SARS-CoV-2 genomic and subgenomic RNAs. This review aims to highlight the aaRS-SARS-CoV-2 interactions identified to date, with possible implications for the biology of host aaRSs in SARS-CoV-2 infection.

Multiomics of GCN4-Dependent Replicative Lifespan Extension Models Reveals Gcn4 as a Regulator of Protein Turnover in Yeast.

We have shown that multiple tRNA synthetase inhibitors can increase lifespan in both the nematode C. elegans and the budding yeast S. cerevisiae by acting through the conserved transcription factor Gcn4 (yeast)/ATF-4 (worms). To further understand the biology downstream from this conserved transcription factor in the yeast model system, we looked at two different yeast models known to have upregulated Gcn4 and GCN4-dependent increased replicative lifespan. These two models were rpl31aΔ yeast and yeast treated with the tRNA synthetase inhibitor borrelidin. We used both proteomic and RNAseq analysis of a block experimental design that included both of these models to identify GCN4-dependent changes in these two long-lived strains of yeast. Proteomic analysis of these yeast indicate that the long-lived yeast have increased abundances of proteins involved in amino acid biosynthesis. The RNAseq of these same yeast uncovered further regulation of protein degradation, identifying the differential expression of genes associated with autophagy and the ubiquitin-proteasome system (UPS). The data presented here further underscore the important role that GCN4 plays in the maintenance of protein homeostasis, which itself is an important hallmark of aging. In particular, the changes in autophagy and UPS-related gene expression that we have observed could also have wide-ranging implications for the understanding and treatment of diseases of aging that are associated with protein aggregation.

Axonal polyneuropathy and ataxia in children: consider Perrault Syndrome, a case report.

BACKGROUND: Perrault Syndrome (PRLTS) is a rare, autosomal recessive disorder that presents with bilateral sensorineural hearing loss in all patients and gonadal dysfunction in females. It has been linked to variants in CLPP, ERAL1, HARS2, HSD17B4, LARS2, and TWNK genes. All reported cases due to TWNK variants have included neurologic features, such as ataxia and axonal sensorimotor neuropathy. CASE PRESENTATION: A 4.5-year-old female presented to neuromuscular clinic due to ataxia. Neurological examination revealed truncal ataxia and steppage gait, reduced deep tendon reflexes, and axonal sensorimotor polyneuropathy. Auditory brainstem response testing revealed an uncommon type of sensorineural hearing loss known as auditory neuropathy/auditory synaptopathy (AN/AS) affecting both ears. Magnetic Resonance Imaging (MRI) revealed subtle cauda equina enhancement. Nerve conduction studies led to a provisional diagnosis of chronic inflammatory demyelinating polyneuropathy (CIDP), and intravenous immune globulin (IVIG) was initiated. The patient was unresponsive to treatment, thus whole exome testing (WES) was conducted in tandem with IVIG weaning. WES revealed a compound heterozygous state with two variants in the TWNK gene and a diagnosis of Perrault Syndrome was made. CONCLUSIONS: Perrault Syndrome should be considered in the differential for children who present with bilateral sensorineural hearing loss, axonal polyneuropathy, and ataxia. Further examination includes testing for ovarian dysgenesis and known PRLTS genetic variants.

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery.

Stop codon suppression using dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs allows for genetically encoded, site-specific incorporation of non-canonical amino acids (ncAAs) as chemical handles for protein labeling and modification. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal pyrrolysine tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells.

Enzyme redesign and genetic code expansion.

Enzyme design is an important application of computational protein design (CPD). It can benefit enormously from the additional chemistries provided by noncanonical amino acids (ncAAs). These can be incorporated into an 'expanded' genetic code, and introduced in vivo into target proteins. The key step for genetic code expansion is to engineer an aminoacyl-transfer RNA (tRNA) synthetase (aaRS) and an associated tRNA that handles the ncAA. Experimental directed evolution has been successfully used to engineer aaRSs and incorporate over 200 ncAAs into expanded codes. But directed evolution has severe limits, and is not yet applicable to noncanonical AA backbones. CPD can help address several of its limitations, and has begun to be applied to this problem. We review efforts to redesign aaRSs, studies that designed new proteins and functionalities with the help of ncAAs, and some of the method developments that have been used, such as adaptive landscape flattening Monte Carlo, which allows an enzyme to be redesigned with substrate or transition state binding as the design target.

Elucidating the structure-function attributes of a trypanosomal arginyl-tRNA synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are fundamental components of the protein translation machinery. In light of their pivotal role in protein synthesis and structural divergence among species, they have always been considered potential targets for the development of antimicrobial compounds. Arginyl-tRNA synthetase from Trypanosoma cruzi (TcArgRS), the parasite responsible for causing Chagas Disease, contains a 100-amino acid insertion that was found to be completely absent in the human counterpart of similar length, as ascertained from multiple sequence alignment results. Thus, we were prompted to perform a preliminary characterization of TcArgRS using biophysical, biochemical, and bioinformatics tools. We expressed the protein in E. coli and validated its in-vitro enzymatic activity. Additionally, analysis of DTNB kinetics, Circular dichroism (CD) spectra, and ligand-binding studies using intrinsic tryptophan fluorescence measurements aided us to understand some structural features in the absence of available crystal structures. Our study indicates that TcArgRS can discriminate between L-arginine and its analogues. Among the many tested substrates, only L-canavanine and L-thioarginine, a synthetic arginine analogue exhibited notable activation. The binding of various substrates was also determined using in silico methods. This study may provide a viable foundation for studying small compounds that can be targeted against TcArgRS.

Phosphocode-dependent glutamyl-prolyl-tRNA synthetase 1 signaling in immunity, metabolism, and disease.

Ubiquitously expressed aminoacyl-tRNA synthetases play essential roles in decoding genetic information required for protein synthesis in every living species. Growing evidence suggests that they also function as crossover mediators of multiple biological processes required for homeostasis. In humans, eight cytoplasmic tRNA synthetases form a central machinery called the multi-tRNA synthetase complex (MSC). The formation of MSCs appears to be essential for life, although the role of MSCs remains unclear. Glutamyl-prolyl-tRNA synthetase 1 (EPRS1) is the most evolutionarily derived component within the MSC that plays a critical role in immunity and metabolism (beyond its catalytic role in translation) via stimulus-dependent phosphorylation events. This review focuses on the role of EPRS1 signaling in inflammation resolution and metabolic modulation. The involvement of EPRS1 in diseases such as cancer is also discussed.

Phenylalanyl-tRNA synthetase deficiency caused by biallelic variants in FARSA gene and literature review.

BACKGROUND: Aminoacyl-tRNA synthetases (ARSs) are indispensable enzymes for protein biosynthesis in cells. The phenylalanyl-tRNA synthetase (FARS1) located in cytoplasm which consists of two FARS alpha subunits (FARSA) and two FARS beta subunits (FARSB). Autosomal recessive inheritance of pathogenic variants of FARSA or FARSB can result in defective FARS1 which are characterized by interstitial lung disease, liver disease, brain abnormalities, facial dysmorphism and growth restriction. METHODS: Exome sequencing was used to detect the candidate variants. The in silico prediction and expressional level analysis were performed to evaluate the pathogenicity of the variations. Additionally, we presented the patient's detailed clinical information and compared the clinical feature with other previously reported patients with FARSA-deficiency. RESULTS: We identified compound heterozygous rare missense variants (c.1172 T > C/ p.Leu391Pro and c.1211G > A/ p.Arg404His) in FARSA gene in a Chinese male patient. The protein structure prediction and the analysis of levels of FARSA and FARSB subunits indicated both variants pathogenic. Clinical feature review indicated inflammatory symptoms in young infants may be an additional key feature. Thyroid dysfunction should be considered as a phenotype with variable penetrance. CONCLUSIONS: Our results expanded the current phenotypic and genetic spectrum of FARSA-deficiency.