Transfer RNAs (tRNAs) are the most highly modified cellular RNAs, both with respect to the proportion of nucleotides that are modified within the tRNA sequence and with respect to the extraordinary diversity in tRNA modification chemistry. However, the functions of many different tRNA modifications are only beginning to emerge. tRNAs have two general clusters of modifications. The first cluster is within the anticodon stem-loop including several modifications essential for protein translation. The second cluster of modifications is within the tRNA elbow, and roles for these modifications are less clear. In general, tRNA elbow modifications are typically not essential for cell growth, but nonetheless several tRNA elbow modifications have been highly conserved throughout all domains of life. In addition to forming modifications, many tRNA modifying enzymes have been demonstrated or hypothesized to also play an important role in folding tRNA acting as tRNA chaperones. In this review, we summarize the known functions of tRNA modifying enzymes throughout the lifecycle of a tRNA molecule, from transcription to degradation. Thereby, we describe how tRNA modification and folding by tRNA modifying enzymes enhance tRNA maturation, tRNA aminoacylation, and tRNA function during protein synthesis, ultimately impacting cellular phenotypes and disease.

RNA profiling studies have revealed that ∼75% of the human genome is transcribed to RNA but only a meagre fraction of it is translated to proteins. Majority of transcribed RNA constitute a specialized pool of non-coding RNAs. Human genome contains approximately 506 genes encoding a set of 51 different tRNAs, constituting a unique class of non-coding RNAs that not only have essential housekeeping functions as translator molecules during protein synthesis, but have numerous uncharted regulatory functions. Intriguing findings regarding a variety of non-canonical functions of tRNAs, tRNA derived fragments (tRFs), esoteric epitranscriptomic modifications of tRNAs, along with aminoacyl-tRNA synthetases (AARSs) and ARS-interacting multifunctional proteins (AIMPs), envision a \'peripheral dogma\' controlling the flow of genetic information in the backdrop of qualitative information wrung out of the long-live central dogma of molecular biology, to drive cells towards either proliferation or differentiation programs. Our review will substantiate intriguing peculiarities of tRNA gene clusters, atypical tRNA-transcription from internal promoters catalysed by another distinct RNA polymerase enzyme, dynamically diverse tRNA epitranscriptome, intricate mechanism of tRNA-charging by AARSs governing translation fidelity, epigenetic regulation of gene expression by tRNA fragments, and the role of tRNAs and tRNA derived/associated molecules as quantitative determinants of the functional proteome, covertly orchestrating the process of tumorigenesis, through a deregulated tRNA-ome mediating selective codon-biased translation of cancer related gene transcripts.

Starvation of Schizosaccharomyces pombe for inorganic phosphate elicits adaptive transcriptome changes in which mRNAs driving ribosome biogenesis, tRNA biogenesis, and translation are globally downregulated, while those for autophagy and phosphate mobilization are upregulated. Here, we interrogated three components of the starvation response: upregulated autophagy; the role of transcription factor Pho7 (an activator of the PHO regulon); and upregulated expression of ecl3, one of three paralogous genes (ecl1, ecl2, and ecl3) collectively implicated in cell survival during other nutrient stresses. Ablation of autophagy factor Atg1 resulted in early demise of phosphate-starved fission yeast, as did ablation of Pho7. Transcriptome profiling of phosphate-starved pho7Δ cells highlighted Pho7 as an activator of genes involved in phosphate acquisition and mobilization, not limited to the original three-gene PHO regulon, and additional starvation-induced genes (including ecl3) not connected to phosphate dynamics. Pho7-dependent gene induction during phosphate starvation tracked with the presence of Pho7 DNA-binding elements in the gene promoter regions. Fewer ribosome protein genes were downregulated in phosphate-starved pho7Δ cells versus WT, which might contribute to their shortened lifespan. An ecl3Δ mutant elicited no gene expression changes in phosphate-replete cells and had no impact on survival during phosphate starvation. By contrast, pan-ecl deletion (ecl123Δ) curtailed lifespan during chronic phosphate starvation. Phosphate-starved ecl123Δ cells experienced a more widespread downregulation of mRNAs encoding aminoacyl tRNA synthetases vis-à-vis WT or pho7Δ cells. Collectively, these results enhance our understanding of fission yeast phosphate homeostasis and survival during nutrient deprivation.

Aminoacyl-tRNA synthetases (AaRSs) are valuable \"housekeeping\" enzymes that ensure the accurate transmission of genetic information in living cells, where they aminoacylated tRNA molecules with their cognate amino acid and provide substrates for protein biosynthesis. In addition to their translational or canonical function, they contribute to nontranslational/moonlighting functions, which are mediated by the presence of other domains on the proteins. This was supported by several reports which claim that AaRS has a significant role in gene transcription, apoptosis, translation, and RNA splicing regulation. Noncanonical/ nontranslational functions of AaRSs also include their roles in regulating angiogenesis, inflammation, cancer, and other major physio-pathological processes. Multiple AaRSs are also associated with a broad range of physiological and pathological processes; a few even serve as cytokines. Therefore, the multifunctional nature of AaRSs suggests their potential as viable therapeutic targets as well. Here, our discussion will encompass a range of noncanonical functions attributed to Aminoacyl-tRNA Synthetases (AaRSs), highlighting their links with a diverse array of human diseases.

Tuberculosis caused by Mycobacterium tuberculosis (M.tb) has killed millions worldwide. Antibiotic resistance leads to the ineffectiveness of the current therapies. Aminoacyl tRNA synthetase (aaRS) class of proteins involved in protein synthesis are promising bacterial targets for developing new therapies. Here, we carried out a systematic comparative study on the aaRS sequences from M.tb and human. We listed important M.tb aaRS that could be explored as potential M.tb targets alongside the detailed conformational space analysis of methionyl-tRNA synthetase (MetRS) in apo- and substrate-bound form, which is among the proposed targets. Understanding the conformational dynamics is central to the mechanistic understanding of MetRS, as the substrate binding leads to the conformational changes causing the reaction to proceed. We performed the most complete simulation study of M.tb MetRS for 6 microseconds (2 systems × 3 runs × 1 microsecond) in the apo and substrate-bound states. Interestingly, we observed differential features, showing comparatively large dynamics for the holo simulations, whereas the apo structures became slightly compact with reduced solvent exposed area. In contrast, the ligand size decreased significantly in holo structures possibly to relax ligand conformation. Our findings correlate with experimental studies, thus validating our protocol. Adenosine monophosphate moiety of the substrate exhibited quite higher fluctuations than the methionine. His21 and Lys54 were found to be the important residues forming prominent hydrogen bond and salt-bridge interactions with the ligand. The ligand-protein affinity decreased during simulations as computed by MMGBSA analysis over the last 500 ns trajectories, which indicates the conformational changes upon ligand binding. These differential features could be further explored for designing new M.tb inhibitors.

Transfer RNAs (tRNAs) decode messenger RNA codons to peptides at the ribosome. The nuclear genome contains many tRNA genes for each amino acid and even each anticodon. Recent evidence indicates that expression of these tRNAs in neurons is regulated, and they are not functionally redundant. When specific tRNA genes are nonfunctional, this results in an imbalance between codon demand and tRNA availability. Furthermore, tRNAs are spliced, processed, and posttranscriptionally modified. Defects in these processes lead to neurological disorders. Finally, mutations in the aminoacyl tRNA synthetases (aaRSs) also lead to disease. Recessive mutations in several aaRSs cause syndromic disorders, while dominant mutations in a subset of aaRSs lead to peripheral neuropathy, again due to an imbalance between tRNA supply and codon demand. While it is clear that disrupting tRNA biology often leads to neurological disease, additional research is needed to understand the sensitivity of neurons to these changes.

Glycyl-tRNA synthetases (GlyRSs) have different oligomeric structures depending on the organisms. While a dimeric α2 GlyRS species is present in archaea, eukaryotes and some eubacteria, a heterotetrameric α2β2 GlyRS species is found in most eubacteria. Here, we present the crystal structure of heterotetrameric α2β2 GlyRS, consisting of the full-length α and β subunits, from Lactobacillus plantarum (LpGlyRS), gram-positive lactic bacteria. The α2β2LpGlyRS adopts the same X-shaped structure as the recently reported Escherichia coli α2β2 GlyRS. A tRNA docking model onto LpGlyRS suggests that the α and β subunits of LpGlyRS together recognize the L-shaped tRNA structure. The α and β subunits of LpGlyRS together interact with the 3\'-end and the acceptor region of tRNAGly, and the C-terminal domain of the β subunit interacts with the anticodon region of tRNAGly. The biochemical analysis using tRNA variants showed that in addition to the previously defined determinants G1C72 and C2G71 base pairs, C35, C36 and U73 in eubacterial tRNAGly, the identification of bases at positions 4 and 69 in tRNAGly is required for efficient glycylation by LpGlyRS. In this case, the combination of a purine base at Position 4 and a pyrimidine base at Position 69 in tRNAGly is preferred.

Developing an understanding of the interactions between an antibiotic and its binding site in a pathogen cell is the key to antibiotic design-an important cost-saving methodology compared to the costly and time-consuming random trial-and-error approach. The rapid development of antibiotic resistance provides an impetus for such studies. Recent years have witnessed the beginning of the use of combined computational techniques, including computer simulations and quantum mechanical computations, to understand how antibiotics bind at the active site of aminoacyl tRNA synthetases (aaRSs) from pathogens. Such computational protocols assist the knowledge-based design of antibiotics targeting aaRSs, which are their validated targets. After the ideas behind the protocols and their strategic planning are discussed, the protocols are described along with their major outcomes. This is followed by an integration of results from the different basic protocols. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Analysis of active-site residues from primary sequence of synthetase and transfer RNAs Basic Protocol 2: Molecular dynamics simulation-based protocol to study the structure and dynamics of the aaRS active site:antibiotic complex Basic Protocol 3: Quantum mechanical method-based protocol to study the structure and dynamics of the aaRS active site:antibiotic complex.

Aminoacyl tRNA synthetases (aaRSs) are a well-studied family of enzymes with a canonical role in charging tRNAs with a specific amino acid. These proteins appear to also have non-canonical roles, including post-transcriptional regulation of mRNA expression. Many aaRSs were found to bind mRNAs and regulate their translation into proteins. However, the mRNA targets, mechanism of interaction, and regulatory consequences of this binding are not fully resolved. Here, we focused on yeast cytosolic threonine tRNA synthetase (ThrRS) to decipher its impact on mRNA binding. Affinity purification of ThrRS with its associated mRNAs followed by transcriptome analysis revealed a preference for mRNAs encoding RNA polymerase subunits. An mRNA that was significantly bound compared to all others was the mRNA encoding RPC10, a small subunit of RNA polymerase III. Structural modeling suggested that this mRNA includes a stem-loop element that is similar to the anti-codon stem loop (ASL) structure of ThrRS cognate tRNA (tRNAThr). We introduced random mutations within this element and found that almost every change from the normal sequence leads to reduced binding by ThrRS. Furthermore, point mutations at six key positions that abolish the predicted ASL-like structure showed a significant decrease in ThrRS binding with a decrease in RPC10 protein levels. Concomitantly, tRNAThr levels were reduced in the mutated strain. These data suggest a novel regulatory mechanism in which cellular tRNA levels are regulated through a mimicking element within an RNA polymerase III subunit in a manner that involves the tRNA cognate aaRS.

Mitochondrial diseases (MDs) may result from mutations affecting nuclear or mitochondrial genes, encoding mitochondrial proteins, or non-protein-coding mitochondrial RNA. Despite the great variability of affected genes, in the most severe cases, a neuromuscular and neurodegenerative phenotype is observed, and no specific therapy exists for a complete recovery from the disease. The most used treatments are symptomatic and based on the administration of antioxidant cocktails combined with antiepileptic/antipsychotic drugs and supportive therapy for multiorgan involvement. Nevertheless, the real utility of antioxidant cocktail treatments for patients affected by MDs still needs to be scientifically demonstrated. Unfortunately, clinical trials for antioxidant therapies using α-tocopherol, ascorbate, glutathione, riboflavin, niacin, acetyl-carnitine and coenzyme Q have met a limited success. Indeed, it would be expected that the employed antioxidants can only be effective if they are able to target the specific mechanism, i.e., involving the central and peripheral nervous system, responsible for the clinical manifestations of the disease. Noteworthily, very often the phenotypes characterizing MD patients are associated with mutations in proteins whose function does not depend on specific cofactors. Conversely, the administration of the antioxidant cocktails might determine the suppression of endogenous oxidants resulting in deleterious effects on cell viability and/or toxicity for patients. In order to avoid toxicity effects and before administering the antioxidant therapy, it might be useful to ascertain the blood serum levels of antioxidants and cofactors to be administered in MD patients. It would be also worthwhile to check the localization of mutations affecting proteins whose function should depend (less or more directly) on the cofactors to be administered, for estimating the real need and predicting the success of the proposed cofactor/antioxidant-based therapy.