tRNA Modification Enzymes GidA and MnmE: Potential Role in Virulence of Bacterial Pathogens

Transfer RNA (tRNA) is an RNA molecule that carries amino acids to the ribosomes for protein synthesis. These tRNAs function at the peptidyl (P) and aminoacyl (A) binding sites of the ribosome during translation, with each codon being recognized by a specific tRNA. Due to this specificity, tRNA modification is essential for translational efficiency. Many enzymes have been implicated in the modification of bacterial tRNAs, and these enzymes may complex with one another or interact individually with the tRNA. Approximately, 100 tRNA modification enzymes have been identified with glucose-inhibited division (GidA) protein and MnmE being two of the enzymes studied. In Escherichia coli and Salmonella, GidA and MnmE bind together to form a functional complex responsible for the proper biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm⁵s²U34) of tRNAs. Studies have implicated this pathway in a major pathogenic regulatory mechanism as deletion of gidA and/or mnmE has attenuated several bacterial pathogens like Salmonella enterica serovar Typhimurium, Pseudomonas syringae, Aeromonas hydrophila, and many others. In this review, we summarize the potential role of the GidA/MnmE tRNA modification pathway in bacterial virulence, interactions with the host, and potential therapeutic strategies resulting from a greater understanding of this regulatory mechanism.     

Making Sense of “Nonsense” and More: Challenges and Opportunities in the Genetic Code Expansion,in the World of tRNA Modifications

The evolutional development of the RNA translation process that leads to protein synthesis based on naturally occurring amino acids has its continuation via synthetic biology, the so-called rational bioengineering. Genetic code expansion (GCE) explores beyond the natural translational processes to further enhance the structural properties and augment the functionality of a wide range of proteins. Prokaryotic and eukaryotic ribosomal machinery have been proven to accept engineered tRNAs from orthogonal organisms to efficiently incorporate noncanonical amino acids (ncAAs) with rationally designed side chains. These side chains can be reactive or functional groups, which can be extensively utilized in biochemical, biophysical, and cellular studies. Genetic code extension offers the contingency of introducing more than one ncAA into protein through frameshift suppression, multi-site-specific incorporation of ncAAs, thereby increasing the vast number of possible applications. However, different mediating factors reduce the yield and efficiency of ncAA incorporation into synthetic proteins. In this review, we comment on the recent advancements in genetic code expansion to signify the relevance of systems biology in improving ncAA incorporation efficiency. We discuss the emerging impact of tRNA modifications and metabolism in protein design. We also provide examples of the latest successful accomplishments in synthetic protein therapeutics and show how codon expansion has been employed in various scientific and biotechnological applications.     

Downregulation of eRF1 by RNA interference increases mis-acylated tRNA suppression efficiency in human cells

The site-specific incorporation of non-natural amino acids into proteins by nonsense suppression has been widely used to investigate protein structure and function. Usually this technique exhibits low incorporation efficiencies of non-natural amino acids into proteins. We describe for the first time an approach for achieving an increased level of nonsense codon suppression with synthetic suppressor tRNAs in cultured human cells. We find that the intracellular concentration of the eukaryotic release factor 1 (eRF1) is a critical parameter influencing the efficiency of amino acid incorporation by nonsense suppression. Using RNA interference we were able to lower eRF1 gene expression significantly. We achieved a five times higher level of amino acid incorporation as compared with non-treated control cells, as demonstrated by enhanced green fluorescent protein (EGFP) fluorescence recovery after importing a mutated reporter mRNA together with an artificial amber suppressor tRNA.     

RTCB Complex Regulates Stress-Induced tRNA Cleavage

Under stress conditions, transfer RNAs (tRNAs) are cleaved by stress-responsive RNases such as angiogenin, generating tRNA-derived RNAs called tiRNAs. As tiRNAs contribute to cytoprotection through inhibition of translation and prevention of apoptosis, the regulation of tiRNA production is critical for cellular stress response. Here, we show that RTCB ligase complex (RTCB-LC), an RNA ligase complex involved in endoplasmic reticulum (ER) stress response and precursor tRNA splicing, negatively regulates stress-induced tiRNA production. Knockdown of RTCB significantly increased stress-induced tiRNA production, suggesting that RTCB-LC negatively regulates tiRNA production. Gel-purified tiRNAs were repaired to full-length tRNAs by RtcB in vitro, suggesting that RTCB-LC can generate full length tRNAs from tiRNAs. As RTCB-LC is inhibited under oxidative stress, we further investigated whether tiRNA production is promoted through the inhibition of RTCB-LC under oxidative stress. Although hydrogen peroxide (H₂O₂) itself did not induce tiRNA production, it rapidly boosted tiRNA production under the condition where stress-responsive RNases are activated. We propose a model of stress-induced tiRNA production consisting of two factors, a trigger and booster. This RTCB-LC-mediated boosting mechanism may contribute to the effective stress response in the cell.     

The Clinical Significance of Transfer RNAs Present in Extracellular Vesicles

Extracellular vesicles (EVs) are important for intercellular signalling in multi-cellular organisms. However, the role of mature transfer RNAs (tRNAs) and tRNA fragments in EVs has yet to be characterised. This systematic review aimed to identify up-to-date literature on tRNAs present within human EVs and explores their potential clinical significance in health and disease. A comprehensive and systematic literature search was performed, and the study was conducted in accordance with PRISMA guidelines. Electronic databases MEDLINE and EMBASE were searched up until 1 January 2022. From 685 papers, 60 studies were identified for analysis. The majority of papers reviewed focussed on the role of EV tRNAs in cancers (31.7%), with numerous other conditions represented. Blood and cell lines were the most common EV sources, representing 85.9% of protocols used. EV isolation methods included most known methods, precipitation being the most common (49.3%). The proportion of EV tRNAs was highly variable, ranging between 0.04% to >95% depending on tissue source. EV tRNAs are present in a multitude of sources and show promise as disease markers in breast cancer, gastrointestinal cancers, and other diseases. EV tRNA research is an emerging field, with increasing numbers of papers highlighting novel methodologies for tRNA and tRNA fragment discovery.     

Adaptation of the Romanomermis culicivorax CCA-Adding Enzyme to Miniaturized Armless tRNA Substrates

The mitochondrial genome of the nematode Romanomermis culicivorax encodes for miniaturized hairpin-like tRNA molecules that lack D- as well as T-arms, strongly deviating from the consensus cloverleaf. The single tRNA nucleotidyltransferase of this organism is fully active on armless tRNAs, while the human counterpart is not able to add a complete CCA-end. Transplanting single regions of the Romanomermis enzyme into the human counterpart, we identified a beta-turn element of the catalytic core that—when inserted into the human enzyme—confers full CCA-adding activity on armless tRNAs. This region, originally identified to position the 3′-end of the tRNA primer in the catalytic core, dramatically increases the enzyme’s substrate affinity. While conventional tRNA substrates bind to the enzyme by interactions with the T-arm, this is not possible in the case of armless tRNAs, and the strong contribution of the beta-turn compensates for an otherwise too weak interaction required for the addition of a complete CCA-terminus. This compensation demonstrates the remarkable evolutionary plasticity of the catalytic core elements of this enzyme to adapt to unconventional tRNA substrates.     

tRNA Biology in the Pathogenesis of Diabetes: Role of Genetic and Environmental Factors

The global rise in type 2 diabetes results from a combination of genetic predisposition with environmental assaults that negatively affect insulin action in peripheral tissues and impair pancreatic β-cell function and survival. Nongenetic heritability of metabolic traits may be an important contributor to the diabetes epidemic. Transfer RNAs (tRNAs) are noncoding RNA molecules that play a crucial role in protein synthesis. tRNAs also have noncanonical functions through which they control a variety of biological processes. Genetic and environmental effects on tRNAs have emerged as novel contributors to the pathogenesis of diabetes. Indeed, altered tRNA aminoacylation, modification, and fragmentation are associated with β-cell failure, obesity, and insulin resistance. Moreover, diet-induced tRNA fragments have been linked with intergenerational inheritance of metabolic traits. Here, we provide a comprehensive review of how perturbations in tRNA biology play a role in the pathogenesis of monogenic and type 2 diabetes.     

A Pyrimido-Quinoxaline Fused Heterocycle Lights Up Transfer RNA upon Binding at the Mg2+ Binding Site

Transfer RNAs (tRNAs) are fundamental molecules in cellular translation. In this study we have highlighted a fluorescence-based perceptive approach for tRNAs by using a quinoxaline small molecule. We have synthesised a water-soluble fluorescent pyrimido-quinoxaline-fused heterocycle containing a mandatory piperazine tail ( DS1 ) with a large Stokes shift (∼160 nm). The interaction between DS1 and tRNA results in significant fluorescence enhancement of the molecule with K_d∼5 μM and multiple binding sites. Our work reveals that the DS1 binding site overlaps with the specific Mg²⁺ ion binding site in the D loop of tRNA. As a proof-of-concept, the molecule inhibited Pb²⁺-induced cleavage of yeast tRNA^Phe in the D loop. In competitive binding assays, the fluorescence of DS1 -tRNA complex is quenched by a known tRNA-binder, tobramycin. This indicates the displacement of DS1 and, indeed, a substantiation of specific binding at the site of tertiary interaction in the central region of tRNA. The ability of compound DS1 to bind tRNA with a higher affinity compared to DNA and single-stranded RNA offers a promising approach to developing tRNA-based biomarker diagnostics in the future.     

Probing the Substrate Requirements of the In Vitro Geranylation Activity of Selenouridine Synthase (SelU)

Natural RNA modifications diversify the structures and functions of existing nucleic acid building blocks. Geranyl is one of the most hydrophobic groups recently identified in bacterial tRNAs. Selenouridine synthase (SelU, also called mnmH) is an enzyme with a dual activity which catalyzes selenation and geranylation in tRNAs containing 2-thiouridine using selenophosphate or geranyl-pyrophosphate as cofactors. In this study, we explored the in vitro geranylation process of tRNA anticodon stem loops (ASL) mediated by SelU and showed that the geranylation activity was abolished when U₃₅ was mutated to A₃₅ (ASL-tRNA^Lys_(s2U)UU to ASL-tRNA^Ile_(s2U)AU). By examining the SelU cofactor geranyl-pyrophosphate (gePP) and its analogues, we found that only the geranyl group, but not dimethylallyl- and farnesyl-pyrophosphate with either shorter or longer terpene chains, could be incorporated into ASL. The degree of tRNA geranylation in the end-point analysis for SelU follows the order of ASL^Lys_(s2UUU) ASL^Gln_(s2UUG)>ASL^Glu_(s2UUC). These findings suggest a putative mechanism for substrate discrimination by SelU and reveal key factors that might influence its enzymatic activity. Given that SelU plays an important role in bacterial translation systems, inhibiting this enzyme and targeting its geranylation and selenation pathways could be exploited as a promising strategy to develop SelU-based antibiotics.     

Stress Granules and Acute Ischemic Stroke: Beyond mRNA Translation

Ischemic stroke is a leading cause of death and disability worldwide. Following an ischemic insult, cells undergo endoplasmic reticulum (ER) stress, which increases the ER’s protein-folding and degradative capacities and blocks the global synthesis of proteins by phosphorylating the eukaryotic translation initiation factor 2-alpha (eIF2α). Phosphorylation of eIF2α is directly related to the dynamics of stress granules (SGs), which are membraneless organelles composed of RNA-binding proteins and mRNA. SGs play a critical role in mRNA metabolism and translational control. Other translation factors are also linked to cellular pathways, including SG dynamics following a stroke. Because the formation of SGs is closely connected to mRNA translation, it is interesting to study the relationship between SG dynamics and cellular outcome in cases of ischemic damage. Therefore, in this review, we focus on the role of SG dynamics during cerebral ischemia.     

Phosphorylation Stoichiometries of Human Eukaryotic Initiation Factors

Eukaryotic translation initiation factors are the principal molecular effectors regulating the process converting nucleic acid to functional protein. Commonly referred to as eIFs (eukaryotic initiation factors), this suite of proteins is comprised of at least 25 individual subunits that function in a coordinated, regulated, manner during mRNA translation. Multiple facets of eIF regulation have yet to be elucidated; however, many of the necessary protein factors are phosphorylated. Herein, we have isolated, identified and quantified phosphosites from eIF2, eIF3, and eIF4G generated from log phase grown HeLa cell lysates. Our investigation is the first study to globally quantify eIF phosphosites and illustrates differences in abundance of phosphorylation between the residues of each factor. Thus, identification of those phosphosites that exhibit either high or low levels of phosphorylation under log phase growing conditions may aid researchers to concentrate their investigative efforts to specific phosphosites that potentially harbor important regulatory mechanisms germane to mRNA translation.     

Loss of Individual Mitochondrial Ribonuclease P Complex Proteins Differentially Affects Mitochondrial tRNA Processing In Vivo

Over a thousand nucleus-encoded mitochondrial proteins are imported from the cytoplasm; however, mitochondrial (mt) DNA encodes for a small number of critical proteins and the entire suite of mt:tRNAs responsible for translating these proteins. Mitochondrial RNase P (mtRNase P) is a three-protein complex responsible for cleaving and processing the 5′-end of mt:tRNAs. Mutations in any of the three proteins can cause mitochondrial disease, as well as mutations in mitochondrial DNA. Great strides have been made in understanding the enzymology of mtRNase P; however, how the loss of each protein causes mitochondrial dysfunction and abnormal mt:tRNA processing in vivo has not been examined in detail. Here, we used Drosophila genetics to selectively remove each member of the complex in order to assess their specific contributions to mt:tRNA cleavage. Using this powerful model, we find differential effects on cleavage depending on which complex member is lost and which mt:tRNA is being processed. These data revealed in vivo subtleties of mtRNase P function that could improve understanding of human diseases.     

Exploring the Minimal RNA Substrate of Flexizymes

Flexizymes are tRNA acylation ribozymes that have been successfully used to facilitate genetic code reprogramming. They are capable of charging acid substrates onto various tRNAs and tRNA analogues. However, their minimal RNA substrate has not been investigated. Here we have designed fluorescently labeled short RNAs corresponding to the four, three, and two bases (4bRNA, 3bRNA, 2bRNA) at the tRNA 3′-end and explored the minimal RNA substrate of flexizymes, dFx and eFx. 3bRNA was the observed minimal RNA substrate of the flexizymes, but the efficiency of acylation of this short RNA was two to three times lower than that of 4bRNA. The efficiency of acylation of 4bRNA was comparable with that of the microhelix, a 22-base RNA conventionally used as a tRNA analogue for analyzing acylation efficiency. We also compared the efficiencies of acylation of the microhelix and 4bRNA with various acid substrates. Thanks to the short length of 4bRNA, its acyl-4bRNA products exhibited larger mobility shifts in gel electrophoresis than those exhibited by acyl-microhelix products with every substrate tested. This indicated that 4bRNA was an ideal RNA substrate for analyzing the efficiency of acylation by flexizymes.     

Ribosomal Protein S5e is Implicated in Translation Initiation through its Interaction with the N‐Terminal Domain of Initiation Factor eIF2α

A key step of translation initiation in eukaryotes is formation of the 48S preinitiation complex (PIC) containing the 40S ribosome, a set of eukaryotic initiation factors (eIFs), mRNA, and initiator Met‐tRNA interacting with mRNA start codon; however, the PIC structure remains substantially unknown. Here, we apply formaldehyde‐induced protein–protein crosslinks to identify contacts between ribosomal protein S5e (rpS5e, “e” stands for “eukaryotic”) and eIFs within the mammalian PIC, assembled on either model canonical or IRES‐containing mRNA. Using immunoblotting and mass spectrometry, we show that with both types of mRNA, rpS5e crosslinks to eIF2α. Comparative analysis of peptides resulting from trypsinolysis of the crosslinked proteins before and after crosslink reversal reveals crosslinked peptides in the N‐terminal parts of rpS5e and eIF2α. Application of these data to a model PIC structure obtained with the use of available structures indicates that eIF2α undergoes major conformation rearrangements to enable contacts of the factor with rpS5e. These contacts are suggested to maintain the correct positioning of eIF2α relative to other PIC components; this could be essential for start‐codon selection by the PIC.