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Cardio-metabolic diseases, including a cluster of metabolic disorders and their secondary affections on cardiovascular physiology, are gradually brought to the forefront by researchers due to their high prevalence and mortality, as well as an unidentified pathogenesis. tRNA-derived small RNAs (tsRNAs), cleaved by several specific enzymes and once considered as some "metabolic junks" in the past, have been proved to possess numerous functions in human bodies. More interestingly, such a potential also seems to influence the progression of cardio-metabolic diseases to some extent. In this review, the biogenesis, classification and mechanisms of tsRNAs will be discussed based on some latest studies, and their relations with several cardio-metabolic diseases will be highlighted in sequence. Lastly, some future prospects, such as their clinical applications as biomarkers and therapeutic targets will also be mentioned, in order to provide researchers with a comprehensive understanding of the research status of tsRNAs as well as its association with cardio-metabolic diseases, thus presenting as a beacon to indicate directions for the next stage of study., Competing Interests: Declaration of competing interest All the authors declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The success of bacterial pathogens depends on the coordinated expression of virulence determinants. Regulatory circuits that drive pathogenesis are complex, multilayered, and incompletely understood. Here, we reveal that alterations in tRNA modifications define pathogenic phenotypes in the opportunistic pathogen Pseudomonas aeruginosa . We demonstrate that the enzymatic activity of GidA leads to the introduction of a carboxymethylaminomethyl modification in selected tRNAs. Modifications at the wobble uridine base (cmnm 5 U 34 ) of the anticodon drives translation of transcripts containing rare codons. Specifically, in P. aeruginosa the presence of GidA-dependent tRNA modifications modulates expression of genes encoding virulence regulators, leading to a cellular proteomic shift toward pathogenic and well-adapted physiological states. Our approach of profiling the consequences of chemical tRNA modifications is general in concept. It provides a paradigm of how environmentally driven tRNA modifications govern gene expression programs and regulate phenotypic outcomes responsible for bacterial adaption to challenging habitats prevailing in the host niche., Competing Interests: Competing interests statement:The authors declare no competing interest.

Transfer RNAs (tRNAs) are small non-coding RNAs playing a central role during protein synthesis. Besides translation, growing evidence suggests that in many contexts, precursor or mature tRNAs can also be processed into smaller fragments playing many non-canonical regulatory roles in different biological pathways with oncogenic relevance. Depending on the source, these molecules can be classified as tRNA halves (also known as tiRNAs) or tRNA-derived fragments (tRFs), and furtherly divided into 5'-tRNA and 3'-tRNA halves, or tRF-1, tRF-2, tRF-3, tRF-5, and i-tRF, respectively. Unlike DNA and mRNA, high-throughput sequencing of tRNAs is challenging, because of technical limitations of currently developed sequencing methods. In recent years, different sequencing approaches have been proposed allowing the quantification and identification of an increasing number of tRNA fragments with critical functions in distinct physiological and pathophysiological processes. In the present review, we discussed pros and cons of recent advances in different sequencing methods, also introducing the expanding repertoire of bioinformatics tool and resources specifically focused on tRNA research and discussing current issues in the study of these small RNA molecules. Furthermore, we discussed the potential value of tRNA fragments as diagnostic and prognostic biomarkers for different types of cancers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The tsRNAs (tRNA-derived small RNAs) are a novel class of small non-coding RNAs derived from transfer-RNAs. Colon adenocarcinoma (COAD) is the most malignant intestinal tumor. This study focused on the identification and characterization of tsRNA biomarkers in colon adenocarcinomas. Data processing and bioinformatic analyses were performed with the packages of R and Python software. The cell proliferation, migration and invasion abilities were determined by CCK-8 and transwell assays. Luciferase reporter assay was used to test the binding of tsRNA with its target genes. With computational methods, we identified the tRNA fragments profiles within COAD datasets, and discriminated forty-two differentially expressed tsRNAs between paired colon adenocarcinomas and non-tumor controls. Among the fragments derived from the 3' end of tRNA-His-GUG (a histidyl-transfer-RNA), tRFdb-3013a and tRFdb-3013b (tRFdb-3013a/b) were notably decreased in colon and rectum adenocarcinomas, especially, tRFdb-3013a/b might tend to be down-regulated in patients with lymphatic or vascular invasion present. The clinical survival of colorectal adenocarcinoma patients with low tRFdb-3013a/b expression was significantly worse than that of high expression patients. In colon adenocarcinoma cells, tRFdb-3013a could have inhibited cell proliferations, and reduced cell migration and invasion abilities. The enrichment analyses showed that most of tRFdb-3013a correlated-genes were enriched in the extracellular matrix associated GO terms, phagosome pathway, and a GSEA molecular signature pathway. Additionally, the 3'UTR of ST3GAL1 mRNA was predicted to contain the binding site of tRFdb-3013a/b, tRFdb-3013a/b might directly target and regulate ST3GAL1 expression in colon adenocarcinomas. These results suggested that tRFdb-3013a/b might serve as novel biomarkers for diagnosis and prognosis of colon adenocarcinomas, and act a key player in the progression of colon adenocarcinomas.

Metastasis is a significant clinical challenge responsible for cancer mortality and non-response to treatment. However, the molecular mechanisms driving metastasis remain unclear, limiting the development of efficient diagnostic and therapeutic approaches. Recent breakthroughs in cancer biology have discovered a group of small non-coding RNAs called tRNA-derived fragments (tRFs), which play a critical role in the metastatic behavior of various tumors. tRFs are produced from cleavage modifications of tRNAs and have different functional classes based on the pattern of these modifications. They perform post-transcriptional regulation through microRNA-like functions, displacing RNA-binding proteins, and play a role in translational regulation by inducing ribosome synthesis, translation initiation, and epigenetic regulation. Tumor cells manipulate tRFs to develop and survive the tumor mass, primarily by inducing metastasis. Multiple studies have demonstrated the potential of tRFs as therapeutic, diagnostic, and prognostic targets for tumor metastasis. This review discusses the production and function of tRFs in cells, their aberrant molecular contributions to the metastatic environment, and their potential as promising targets for anti-metastasis treatment strategies., Competing Interests: Declaration of competing interest The authors confirm that there are no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Accurate pairing of amino acids and tRNAs is a prerequisite for faithful translation of genetic information during protein biosynthesis. Here we present the effects of proteome-wide mistranslation of isoleucine (Ile) by canonical valine (Val) or non-proteinogenic norvaline (Nva) in a genetically engineered Escherichia coli strain with an editing-defective isoleucyl-tRNA synthetase (IleRS). Editing-defective IleRS efficiently mischarges both Val and Nva to tRNA Ile and impairs the translational accuracy of Ile decoding. When mistranslation was induced by the addition of Val or Nva to the growth medium, an Ile-to-Val or Ile-to-Nva substitution of up to 20 % was measured by high-resolution mass spectrometry. This mistranslation level impaired bacterial growth, promoted the SOS response and filamentation during stationary phase, caused global proteome dysregulation and upregulation of the cellular apparatus for maintaining proteostasis, including the major chaperones (GroES/EL, DnaK/DnaJ/GrpE and HtpG), the disaggregase ClpB and the proteases (Lon, HslV/HslU, ClpA, ClpS). The most important consequence of mistranslation appears to be non-specific protein aggregation, which is effectively counteracted by the disaggregase ClpB. Our data show that E. coli can sustain high isoleucine mistranslation levels and remain viable despite excessive protein aggregation and severely impaired translational fidelity. However, we show that inaccurate translation lowers bacterial resilience to heat stress and decreases bacterial survival at elevated temperatures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein-only RNase P (PRORP) is an essential enzyme responsible for the 5' maturation of precursor tRNAs (pre-tRNAs). PRORPs are classified into three categories with unique molecular architectures, although all three classes of PRORPs share a mechanism and have similar active sites. Single subunit PRORPs, like those found in plants, have multiple isoforms with different localizations, substrate specificities, and temperature sensitivities. Most recently, Arabidopsis thaliana PRORP2 was shown to interact with TRM1A and B, highlighting a new potential role between these enzymes. Work with At PRORPs led to the development of a ribonuclease that is being used to protect against plant viruses. The mitochondrial RNase P complex, found in metazoans, consists of PRORP, TRMT10C, and SDR5C1, and has also been shown to have substrate specificity, although the cause is unknown. Mutations in mitochondrial tRNA and mitochondrial RNase P have been linked to human disease, highlighting the need to continue understanding this complex. The last class of PRORPs, homologs of Aquifex RNase P (HARPs), is found in thermophilic archaea and bacteria. This most recently discovered type of PRORP forms a large homo-oligomer complex. Although numerous structures of HARPs have been published, it is still unclear how HARPs bind pre-tRNAs and in what ratio. There is also little investigation into the substrate specificity and ideal conditions for HARPs. Moving forward, further work is required to fully characterize each of the three classes of PRORP, the pre-tRNA binding recognition mechanism, the rules of substrate specificity, and how these three distinct classes of PRORP evolved. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems., (© 2024 The Authors. WIREs RNA published by Wiley Periodicals LLC.)

Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit protein RPL26 (also known as uL24) 1,2 . This modification, which is known as UFMylation, is orchestrated by the UFM1 ribosome E3 ligase (UREL) complex, comprising UFL1, UFBP1 and CDK5RAP3 (ref. 3 ). However, the catalytic mechanism of UREL and the functional consequences of UFMylation are unclear. Here we present cryo-electron microscopy structures of UREL bound to 60S ribosomes, revealing the basis of its substrate specificity. UREL wraps around the 60S subunit to form a C-shaped clamp architecture that blocks the tRNA-binding sites at one end, and the peptide exit tunnel at the other. A UFL1 loop inserts into and remodels the peptidyl transferase centre. These features of UREL suggest a crucial function for UFMylation in the release and recycling of stalled or terminated ribosomes from the ER membrane. In the absence of functional UREL, 60S-SEC61 translocon complexes accumulate at the ER membrane, demonstrating that UFMylation is necessary for releasing SEC61 from 60S subunits. Notably, this release is facilitated by a functional switch of UREL from a 'writer' to a 'reader' module that recognizes its product-UFMylated 60S ribosomes. Collectively, we identify a fundamental role for UREL in dissociating 60S subunits from the SEC61 translocon and the basis for UFMylation in regulating protein homeostasis at the ER., (© 2024. The Author(s).)

Serine-proline (Ser-Pro) backbone-modified dipeptide analogues are powerful tools to investigate the role of cis-trans isomerization in the regulation of the cell cycle and transcription. These studies have previously been limited to synthetic peptides, whose synthesis is a challenge for larger peptides due to the compounding yield loss incurred in each step. We now introduce a method for the aminoacylation of tRNA with dipeptides and dipeptide analogs to permit the installation of cis- and trans-locked Ser-Pro analogues into full-length proteins. To that end, we synthesized the 3,5-dinitrobenzyl (DNB)-activated esters of a native Ser-Pro dipeptide and its cis- and trans-locked alkene analogs. Murakami et al. created the DNB flexizyme (dFx), a ribozyme that acylates tRNA with DNB esters of amino acids to permit unnatural amino acids to be incorporated into proteins. A tRNA from yeast that recognizes the amber stop codon, along with the dFx flexizyme, were generated by in vitro transcription with T7 RNA polymerase. dFx was used to successfully catalyze the chemical misacylation of truncated amber tRNA with the Ser-Pro-DNB activated dipeptide. This method allows the introduction of non-native Ser-Pro dipeptide mimics into full-length proteins by in vitro transcription-translation. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 3,5-dinitrobenzyl activated esters of Ser-Pro Basic Protocol 2: Preparation of truncated amber tRNA Basic Protocol 3: Acylation of amber-tRNA by the dFx flexizyme Basic Protocol 4: PAGE electrophoresis of tRNA SerPro ., (© 2024 Wiley Periodicals LLC.)

RNase P and RNase mitochondrial RNA processing (MRP) are ribonucleoproteins (RNPs) that consist of a catalytic RNA and a varying number of protein cofactors. RNase P is responsible for precursor tRNA maturation in all three domains of life, while RNase MRP, exclusive to eukaryotes, primarily functions in rRNA biogenesis. While eukaryotic RNase P is associated with more protein cofactors and has an RNA subunit with fewer auxiliary structural elements compared to its bacterial cousin, the double-anchor precursor tRNA recognition mechanism has remarkably been preserved during evolution. RNase MRP shares evolutionary and structural similarities with RNase P, preserving the catalytic core within the RNA moiety inherited from their common ancestor. By incorporating new protein cofactors and RNA elements, RNase MRP has established itself as a distinct RNP capable of processing ssRNA substrates. The structural information on RNase P and MRP helps build an evolutionary trajectory, depicting how emerging protein cofactors harmonize with the evolution of RNA to shape different functions for RNase P and MRP. Here, we outline the structural and functional relationship between RNase P and MRP to illustrate the coevolution of RNA and protein cofactors, a key driver for the extant, diverse RNP world., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Reversible modification of target proteins by ubiquitin and ubiquitin-like proteins (UBLs) is widely used by eukaryotic cells to control protein fate and cell behaviour 1 . UFM1 is a UBL that predominantly modifies a single lysine residue on a single ribosomal protein, uL24 (also called RPL26), on ribosomes at the cytoplasmic surface of the endoplasmic reticulum (ER) 2,3 . UFM1 conjugation (UFMylation) facilitates the rescue of 60S ribosomal subunits (60S) that are released after ribosome-associated quality-control-mediated splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER 3,4 . Neither the molecular mechanism by which the UFMylation machinery achieves such precise target selection nor how this ribosomal modification promotes 60S rescue is known. Here we show that ribosome UFMylation in vivo occurs on free 60S and we present sequential cryo-electron microscopy snapshots of the heterotrimeric UFM1 E3 ligase (E3(UFM1)) engaging its substrate uL24. E3(UFM1) binds the L1 stalk, empty transfer RNA-binding sites and the peptidyl transferase centre through carboxy-terminal domains of UFL1, which results in uL24 modification more than 150 Å away. After catalysing UFM1 transfer, E3(UFM1) remains stably bound to its product, UFMylated 60S, forming a C-shaped clamp that extends all the way around the 60S from the transfer RNA-binding sites to the polypeptide tunnel exit. Our structural and biochemical analyses suggest a role for E3(UFM1) in post-termination release and recycling of the large ribosomal subunit from the ER membrane., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Errors in protein translation can lead to non-genetic, phenotypic mutations, including amino acid misincorporations. While phenotypic mutations can increase protein diversity, the systematic characterization of their proteome-wide frequencies and their evolutionary impact has been lacking. Here, we developed a mechanistic model of translation errors to investigate how selection acts on protein populations produced by amino acid misincorporations. We fitted the model to empirical observations of misincorporations obtained from over a hundred mass spectrometry datasets of E. coli and S. cerevisiae. We found that on average 20% to 23% of proteins synthesized in the cell are expected to harbor at least one amino acid misincorporation, and that deleterious misincorporations are less likely to occur. Combining misincorporation probabilities and the estimated fitness effects of amino acid substitutions in a population genetics framework, we found 74% of mistranslation events in E. coli and 94% in S. cerevisiae to be neutral. We further show that the set of available synonymous tRNAs is subject to evolutionary pressure, as the presence of missing tRNAs would increase codon-anticodon cross-reactivity and misincorporation error rates. Overall, we find that the translation machinery is likely optimal in E. coli and S. cerevisiae and that both local solutions at the level of codons and a global solution such as the tRNA pool can mitigate the impact of translation errors. We provide a framework to study the evolutionary impact of codon-specific translation errors and a method for their proteome-wide detection across organisms and conditions., Competing Interests: Conflict of Interest The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Ribosomal protein eL42 (formerly known as L36A), a small protein of the large (60S) subunit of the eukaryotic ribosome, is a component of its exit (E) site. The residue K53 of this protein resides within the motif QSGYGGQTK mainly conserved in eukaryotes, and it is located in the immediate vicinity of the CCA-terminus of the ribosome-bound tRNA in the hybrid P/E state. To examine the role of this eL42 motif in translation, we obtained HEK293T cells producing the wild-type FLAG-tagged protein or its mutant forms with either single substitutions of conserved amino acid residues in the above motif, or simultaneous replacements in positions 45 and 51 or 45 and 53. Examination of the level of exogenous eL42 in fractions of polysome profiles from the target protein-producing cells by the Western blotting revealed that neither single substitution affects the assembly of 60S ribosomal subunits and 80S ribosomes or critically decreases the level of polysomes, but the latter was observed with the double replacements. Analysis of tRNAs bound to 80S ribosomes containing eL42 with double substitutions and examination their peptidyl transferase activity enabled estimation the stage of the elongation cycle, in which amino acid residues of the conserved eL42 motif are involved. We clearly show that cooperative interactions implicating the eL42 residues Q45, Q51, and K53 play a critical role in the ability of the human ribosome to perform properly elongation cycle at the step of deacylated tRNA dissociation from the E site in the human cell., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Diverse environmental insults induce the integrated stress response (ISR), which features eIF2 phosphorylation and translational control that serves to restore protein homeostasis. The eIF2 kinase GCN2 is a first responder in the ISR that is activated by amino acid depletion and other stresses not directly related to nutrients. Two mechanisms are suggested to trigger an ordered process of GCN2 activation during stress: GCN2 monitoring stress via accumulating uncharged tRNAs or by stalled and colliding ribosomes. Our results suggest that while ribosomal collisions are indeed essential for GCN2 activation in response to translational elongation inhibitors, conditions that trigger deacylation of tRNAs activate GCN2 via its direct association with affected tRNAs. Both mechanisms require the GCN2 regulatory domain related to histidyl tRNA synthetases. GCN2 activation by UV irradiation features lowered amino acids and increased uncharged tRNAs and UV-induced ribosome collisions are suggested to be dispensable. We conclude that there are multiple mechanisms that activate GCN2 during diverse stresses., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 (tRNA Pyl ), 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., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

As cellular engineering progresses from simply overexpressing proteins to imparting complex phenotypes through multigene expression, judicious appropriation of cellular resources is essential. Since codon use is degenerate and biased, codons may control cellular resources at a translational level. We investigate how partitioning transfer RNA (tRNA) resources by incorporating dissimilar codon usage can drastically alter interdependence of expression level and burden on the host. By isolating the effect of individual codons' use during translation elongation while eliminating confounding factors, we show that codon choice can trans-regulate fitness of the host and expression of other heterologous or native genes. We correlate specific codon usage patterns with host fitness and derive a coding scheme for multigene expression called the Codon Health Index (CHI, χ). This empirically derived coding scheme (χ) enables the design of multigene expression systems that avoid catastrophic cellular burden and is robust across several proteins and conditions.

Trillions of microorganisms, collectively known as the microbiome, inhabit our bodies with the gut microbiome being of particular interest in biomedical research. Bacteriophages, the dominant virome constituents, can utilize suppressor tRNAs to switch to alternative genetic codes (e.g., the UAG stop-codon is reassigned to glutamine) while infecting hosts with the standard bacterial code. However, what triggers this switch and how the bacteriophage manipulates its host is poorly understood. Here, we report the discovery of a subgroup of minimal hepatitis delta virus (HDV)-like ribozymes - theta ribozymes - potentially involved in the code switch leading to the expression of recoded lysis and structural phage genes. We demonstrate their HDV-like self-scission behavior in vitro and find them in an unreported context often located with their cleavage site adjacent to tRNAs, indicating a role in viral tRNA maturation and/or regulation. Every fifth associated tRNA is a suppressor tRNA, further strengthening our hypothesis. The vast abundance of tRNA-associated theta ribozymes - we provide 1753 unique examples - highlights the importance of small ribozymes as an alternative to large enzymes that usually process tRNA 3'-ends. Our discovery expands the short list of biological functions of small HDV-like ribozymes and introduces a previously unknown player likely involved in the code switch of certain recoded gut bacteriophages., (© 2024. The Author(s).)

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., (© The Author(s) 2023. Published by Oxford University Press.)

Several methods are available to visualize and assess the kinetics and efficiency of elemental steps of protein biosynthesis. However, each of these methods has its own limitations. Here, we present a novel, simple and convenient tool for monitoring stepwise in vitro translation initiated by BODIPY-Met-tRNA. Synthesis and release of very short, 1-7 amino acids, BODIPY-labeled peptides, can be monitored using urea-polyacrylamide gel electrophoresis. Very short BODIPY-labeled oligopeptides might be resolved this way, in contrast to widely used Tris-tricine gel electrophoresis, which is suitable to separate peptides larger than 1 kDa. The method described in this manuscript allows one to monitor the steps of translation initiation, peptide transfer, translocation, and termination as well as their inhibition at an unprecedented single amino acid resolution., (© 2024 Marina et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The ribosome is a ribonucleoprotein complex found in all domains of life. Its role is to catalyze protein synthesis, the messenger RNA (mRNA)-templated formation of amide bonds between α-amino acid monomers. Amide bond formation occurs within a highly conserved region of the large ribosomal subunit known as the peptidyl transferase center (PTC). Here we describe the step-wise design and characterization of mini-PTC 1.1, a 284-nucleotide RNA that recapitulates many essential features of the Escherichia coli PTC. Mini-PTC 1.1 folds into a PTC-like structure under physiological conditions, even in the absence of r-proteins, and engages small molecule analogs of A- and P-site tRNAs. The sequence of mini-PTC 1.1 differs from the wild type E. coli ribosome at 12 nucleotides that were installed by a cohort of citizen scientists using the on-line video game Eterna. These base changes improve both the secondary structure and tertiary folding of mini-PTC 1.1 as well as its ability to bind small molecule substrate analogs. Here, the combined input from Eterna citizen-scientists and RNA structural analysis provides a robust workflow for the design of a minimal PTC that recapitulates many features of an intact ribosome., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

MTU1 controls intramitochondrial protein synthesis by catalyzing the 2-thiouridine modification of mitochondrial transfer RNAs (mt-tRNAs). Missense mutations in the MTU1 gene are associated with life-threatening reversible infantile hepatic failure. However, the molecular pathogenesis is not well understood. Here, we investigated 17 mutations associated with this disease, and our results showed that most disease-related mutations are partial loss-of-function mutations, with three mutations being particularly severe. Mutant MTU1 is rapidly degraded by mitochondrial caseinolytic peptidase (CLPP) through a direct interaction with its chaperone protein CLPX. Notably, knockdown of CLPP significantly increased mutant MTU1 protein expression and mt-tRNA 2-thiolation, suggesting that accelerated proteolysis of mutant MTU1 plays a role in disease pathogenesis. In addition, molecular dynamics simulations demonstrated that disease-associated mutations may lead to abnormal intermolecular interactions, thereby impairing MTU1 enzyme activity. Finally, clinical data analysis underscores a significant correlation between patient prognosis and residual 2-thiolation levels, which is partially consistent with the AlphaMissense predictions. These findings provide a comprehensive understanding of MTU1-related diseases, offering prospects for modification-based diagnostics and novel therapeutic strategies centered on targeting CLPP., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Even though Bacillus subtilis is one of the most studied organisms, no function has been identified for about 20% of its proteins. Among these unknown proteins are several RNA- and ribosome-binding proteins suggesting that they exert functions in cellular information processing. In this work, we have investigated the RNA-binding protein YlxR. This protein is widely conserved in bacteria and strongly constitutively expressed in B. subtilis suggesting an important function. We have identified the RNA subunit of the essential RNase P as the binding partner of YlxR. The main activity of RNase P is the processing of 5' ends of pre-tRNAs. In vitro processing assays demonstrated that the presence of YlxR results in reduced RNase P activity. Chemical cross-linking studies followed by in silico docking analysis and experiments with site-directed mutant proteins suggest that YlxR binds to the region of the RNase P RNA that is important for binding and cleavage of the pre-tRNA substrate. We conclude that the YlxR protein is a novel interaction partner of the RNA subunit of RNase P that serves to finetune RNase P activity to ensure appropriate amounts of mature tRNAs for translation. We rename the YlxR protein RnpM for RNase P modulator., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Extensive research has focused on genetic code reprogramming using flexizymes (Fxs), ribozymes enabling diverse tRNA acylation. Here we describe a nucleoside-modification strategy for the preparation of flexizyme variants derived from 2'-OMe, 2'-F, and 2'-MOE modifications with unique and versatile activities, enabling the charging of tRNAs with a broad range of substrates. This innovative strategy holds promise for synthetic biology applications, offering a robust pathway to expand the genetic code for diverse substrate incorporation.

Acetylation plays a critical role in regulating eukaryotic transcription via the modification of histones. Beyond this well-documented function, a less explored biological frontier is the potential for acetylation to modify and regulate the function of RNA molecules themselves. N 4 -Acetylcytdine (ac 4 C) is a minor RNA nucleobase conserved across all three domains of life (archaea, bacteria, and eukarya), a conservation that suggests a fundamental role in biological processes. Unlike many RNA modifications that are controlled by large enzyme families, almost all organisms catalyze ac 4 C using a homologue of human Nat10, an essential disease-associated acetyltransferase enzyme.A critical step in defining the fundamental functions of RNA modifications has been the development of methods for their sensitive and specific detection. This Account describes recent progress enabling the use of chemical sequencing reactions to map and quantify ac 4 C with single-nucleotide resolution in RNA. To orient readers, we first provide historical background of the discovery of ac 4 C and the enzymes that catalyze its formation. Next, we describe mechanistic experiments that led to the development of first- and second-generation sequencing reactions able to determine ac 4 C's position in a polynucleotide by exploiting the nucleobase's selective susceptibility to reduction by hydride donors. A notable feature of this chemistry, which may serve as a prototype for nucleotide resolution RNA modification sequencing reactions more broadly, is its ability to drive a penetrant and detectable gain of signal specifically at ac 4 C sites. Emphasizing practical applications, we present how this optimized chemistry can be integrated into experimental workflows capable of sensitive, transcriptome-wide analysis. Such readouts can be applied to quantitatively define the ac 4 C landscape across the tree of life. For example, in human cell lines and yeast, this method has uncovered that ac 4 C is highly selective, predominantly occupying dominant sites within rRNA (rRNA) and tRNA (tRNA). By contrast, when we extend these analyses to thermophilic archaea they identify the potential for much more prevalent patterns of cytidine acetylation, leading to the discovery of a role for this modification in adaptation to environmental stress. Nucleotide resolution analyses of ac 4 C have also allowed for the determination of structure-activity relationships required for short nucleolar RNA (snoRNA)-catalyzed ac 4 C deposition and the discovery of organisms with unexpectedly divergent tRNA and rRNA acetylation signatures. Finally, we share how these studies have shaped our approach to evaluating novel ac 4 C sites reported in the literature and highlight unanswered questions and new directions that set the stage for future research in the field.

Cancer onset and progression are known to be regulated by genetic and epigenetic events, including RNA modifications (a.k.a. epitranscriptomics). So far, more than 150 chemical modifications have been described in all RNA subtypes, including messenger, ribosomal, and transfer RNAs. RNA modifications and their regulators are known to be implicated in all steps of post-transcriptional regulation. The dysregulation of this complex yet delicate balance can contribute to disease evolution, particularly in the context of carcinogenesis, where cells are subjected to various stresses. We sought to discover RNA modifications involved in cancer cell adaptation to inhospitable environments, a peculiar feature of cancer stem cells (CSCs). We were particularly interested in the RNA marks that help the adaptation of cancer cells to suspension culture, which is often used as a surrogate to evaluate the tumorigenic potential. For this purpose, we designed an experimental pipeline consisting of four steps: (1) cell culture in different growth conditions to favor CSC survival; (2) simultaneous RNA subtype (mRNA, rRNA, tRNA) enrichment and RNA hydrolysis; (3) the multiplex analysis of nucleosides by LC-MS/MS followed by statistical/bioinformatic analysis; and (4) the functional validation of identified RNA marks. This study demonstrates that the RNA modification landscape evolves along with the cancer cell phenotype under growth constraints. Remarkably, we discovered a short epitranscriptomic signature, conserved across colorectal cancer cell lines and associated with enrichment in CSCs. Functional tests confirmed the importance of selected marks in the process of adaptation to suspension culture, confirming the validity of our approach and opening up interesting prospects in the field.

RNA modification C 2 -methyladenosine (m 2 A) exists in both rRNA and tRNA of Escherichia coli (E. coli), installed by the methyltransferase RlmN using a radical-S-adenosylmethionine (SAM) mechanism. However, the precise function of m 2 A in tRNA and its ubiquity in plants have remained unclear. Here we discover the presence of m 2 A in chloroplast rRNA and tRNA, as well as cytosolic tRNA, in multiple plant species. We identify six m 2 A-modified chloroplast tRNAs and two m 2 A-modified cytosolic tRNAs across different plants. Furthermore, we characterize three Arabidopsis m 2 A methyltransferases-RLMNL1, RLMNL2, and RLMNL3-which methylate chloroplast rRNA, chloroplast tRNA, and cytosolic tRNA, respectively. Our findings demonstrate that m 2 A37 promotes a relaxed conformation of tRNA, enhancing translation efficiency in chloroplast and cytosol by facilitating decoding of tandem m 2 A-tRNA-dependent codons. This study provides insights into the molecular function and biological significance of m 2 A, uncovering a layer of translation regulation in plants., (© 2024. The Author(s).)

Lung cancer, being the most widespread and lethal form of cancer globally, has a high incidence and mortality rate primarily attributed to challenges associated with early detection, extensive metastasis, and frequent recurrence. In the context of lung cancer development, noncoding RNA molecules have a crucial role in governing gene expression and protein synthesis. Specifically, tRNA-derived fragments (tRFs), a subset of noncoding RNAs, exert significant biological influences on cancer progression, encompassing transcription and translation processes as well as epigenetic regulation. This article primarily examines the mechanisms by which tRFs modulate gene expression and contribute to tumorigenesis in lung cancer. Furthermore, we provide a comprehensive overview of the current bioinformatics analysis of tRFs in lung cancer, with the objective of offering a systematic and efficient approach for studying the expression profiling, functional enrichment, and molecular mechanisms of tRFs in this disease. Finally, we discuss the clinical significance and potential avenues for future research on tRFs in lung cancer. This paper presents a comprehensive systematic review of the existing research findings on tRFs in lung cancer, aiming to offer improved biomarkers and drug targets for clinical management of lung cancer., (© 2023. Springer Nature Switzerland AG.)

Human tRNA (uracil-5-)-methyltransferase 2 homolog A (TRMT2A) is the dedicated enzyme for the methylation of uridine 54 in transfer RNA (tRNA). Human TRMT2A has also been described as a modifier of polyglutamine (polyQ)-derived neuronal toxicity. The corresponding human polyQ pathologies include Huntington's disease and constitute a family of devastating neurodegenerative diseases. A polyQ tract in the corresponding disease-linked protein causes neuronal death and symptoms such as impaired motor function, as well as cognitive impairment. In polyQ disease models, silencing of TRMT2A reduced polyQ-associated cell death and polyQ protein aggregation, suggesting this protein as a valid drug target against this class of disorders. In this paper, the 1.6 Å resolution crystal structure of the RNA-recognition motif (RRM) from Drosophila melanogaster, which is a homolog of human TRMT2A, is described and analysed., (open access.)

Nucleobase-specific noncovalent interactions play a crucial role in translation. Herein, we provide a comprehensive analysis of the stacks between different RNA components in the crystal structures of the bacterial ribosome caught at different translation stages. Analysis of tRNA||rRNA stacks reveals distinct behaviour; both the A-and E-site tRNAs exhibit unique stacking patterns with 23S rRNA bases, while P-site tRNAs stack with 16S rRNA bases. Furthermore, E-site stacks exhibit diverse face orientations and ring topologies-rare for inter-chain RNA interactions-with higher average interaction energies than A or P-site stacks. This suggests that stacking may be essential for stabilizing tRNA progression through the E-site. Additionally, mRNA||rRNA stacks reveal other geometries, which depend on the tRNA binding site, whereas 16S rRNA||23S rRNA stacks highlight the importance of specific bases in maintaining the integrity of the translational complex by linking the two rRNAs. Furthermore, tRNA||mRNA stacks exhibit distinct geometries and energetics at the E-site, indicating their significance during tRNA translocation and elimination. Overall, both A and E-sites display a more diverse distribution of inter-RNA stacks compared to the P-site. Stacking interactions in the active ribosome are not simply accidental byproducts of biochemistry but are likely invoked to compensate and support the integrity and dynamics of translation., Competing Interests: Declaration of Competing Interest JFT is the President and CEO of Binary Star Research Services. BSRS had no input into the methodology or conclusions of this study and has no business interests in the specific subject of this study. The other authors claim no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Transfer RNAs (tRNAs) have a crucial role in protein synthesis, and in recent years, their therapeutic potential for the treatment of genetic diseases - primarily those associated with a mutation altering mRNA translation - has gained significant attention. Engineering tRNAs to readthrough nonsense mutation-associated premature termination of mRNA translation can restore protein synthesis and function. In addition, supplementation of natural tRNAs can counteract effects of missense mutations in proteins crucial for tRNA biogenesis and function in translation. This Review will present advances in the development of tRNA therapeutics with high activity and safety in vivo and discuss different formulation approaches for single or chronic treatment modalities. The field of tRNA therapeutics is still in its early stages, and a series of challenges related to tRNA efficacy and stability in vivo, delivery systems with tissue-specific tropism, and safe and efficient manufacturing need to be addressed., (© 2023. Springer Nature Limited.)

tRNA modifications are crucial in all organisms to ensure tRNA folding and stability, and accurate translation. In both the yeast Saccharomyces cerevisiae and the evolutionarily distant yeast Schizosaccharomyces pombe, mutants lacking certain tRNA body modifications (outside the anticodon loop) are temperature sensitive due to rapid tRNA decay (RTD) of a subset of hypomodified tRNAs. Here we show that for each of two S. pombe mutants subject to RTD, mutations in ribosomal protein genes suppress the temperature sensitivity without altering tRNA levels. Prior work showed that S. pombe trm8Δ mutants, lacking 7-methylguanosine, were temperature sensitive due to RTD, and that one class of suppressors had mutations in the general amino acid control (GAAC) pathway, which was activated concomitant with RTD, resulting in further tRNA loss. We now find that another class of S. pombe trm8Δ suppressors have mutations in rpl genes, encoding 60S subunit proteins, and that suppression occurs with minimal restoration of tRNA levels and reduced GAAC activation. Furthermore, trm8Δ suppression extends to other mutations in the large or small ribosomal subunit. We also find that S. pombe tan1Δ mutants, lacking 4-acetylcytidine, are temperature sensitive due to RTD, that one class of suppressors have rpl mutations, associated with minimal restoration of tRNA levels, and that suppression extends to other rpl and rps mutations. However, although S. pombe tan1Δ temperature sensitivity is associated with some GAAC activation, suppression by an rpl mutation only modestly inhibits GAAC activation. We propose a model in which ribosomal protein mutations result in reduced ribosome concentrations, leading to both reduced ribosome collisions and a reduced requirement for tRNA, with these effects having different relative importance in trm8Δ and tan1Δ mutants. This model is consistent with our results in S. cerevisiae trm8Δ trm4Δ mutants, known to undergo RTD, fueling speculation that this model applies across eukaryotes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 De Zoysa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Early metastasis is the primary factor in the very poor prognosis of pancreatic ductal adenocarcinoma (PDAC), with liver metastasis being the most common form of distant metastasis in PDAC. To investigate the mechanism of PDAC liver metastasis, we found that PDAC cells can promote the formation of pre-metastatic niches (PMNs) through exosomes to facilitate liver metastasis in the early stage. In our study, hepatic stellate cells (HSCs) were treated with PDAC-derived exosomes (PDAC-exo), and the activation of HSCs was detected. A novel transfer RNA-derived fragment, the tRF-GluCTC-0005 was obtained by small RNA sequencing from serum exosomes of PDAC patients. Bioinformatics analysis and RNA pull-down assays revealed the interaction between WDR1 and tRF-GluCTC-0005. A KPC transgenic mouse model and an AAV-mediated sh-WDR1 mouse model were used to detect the mechanism of liver metastasis in vivo. Finally, the dual luciferase reporter assay, protein mutation truncation assay, Co-IP assay, and flow cytometry assay were used to explore the molecular mechanism in HSCs activation and PMNs formation. We found that the tRF-GluCTC-0005 in exosomes binds to the 3' untranslated region of the mRNA of the WDRl in HSCs and increases mRNA stability. The N-terminals of WDR1 bind to the YAP protein directly, inhibit YAP phosphorylation, and promote the expression of YAP transcription factors. The tRF-GluCTC-0005 in PDAC-exo significantly recruits myeloid-derived suppressor cells (MDSCs) in the liver, creating a PMNs immunosuppressive microenvironment and further advancing liver metastasis from PDAC. Our results suggest that the key of PDAC liver metastasis is the activation of HSCs through upregulation of WDR1 by tRF-GluCTC-0005 in exosomes, which mediates the infiltration of MDSCs to form PMNs., (© 2024. The Author(s).)

The N 6 -threonylcarbamoyl adenosine (t 6 A) tRNA modification is critical for ensuring translation fidelity across three domains of life. Our prior work highlighted the KEOPS complex, organized in a Pcc1-Kae1-Bud32-Cgi121 linear arrangement, not only serves an evolutionarily conserved role in t 6 A tRNA modification but also exerts diverse functional impacts on pathobiological characteristics in Cryptococcus neoformans , a leading cause of fungal meningitis worldwide. However, the extent to which the pleiotropic functions of the KEOPS complex are specifically tied to tRNA modification remains uncertain. To address this, we undertook a functional characterization of Sua5, responsible for generating the precursor threonylcarbamoyl-adenylate (TC-AMP) for t 6 A tRNA modification, using a reverse genetics approach. Comparative phenotypic analyses with KEOPS mutants revealed that Sua5 plays a vital role in multiple cellular processes, such as t 6 A tRNA modification, growth, sexual development, stress response, and virulence factor production, thus reflecting the multifaceted functions of the KEOPS complex. In support of this, sua5 Δ bud32 Δ double mutants showed phenotypes comparable to those of the corresponding single mutants. Intriguingly, a SUA5 allele lacking a mitochondria targeting sequence ( SUA5 MTS Δ ) was sufficient to restore the wild-type phenotypes in the sua5 Δ mutant, suggesting that Sua5's primary functional locus may be cytosolic, akin to the KEOPS complex. Further supporting this, the deletion of Qri7, a mitochondrial paralog of Kae1, had no discernible phenotypic impact on C. neoformans . We concluded that cytosolic t 6 A tRNA modifications, orchestrated by Sua5 and the KEOPS complex, are central to the regulation of diverse pathobiological functions in C. neoformans .IMPORTANCEUnderstanding cellular functions at the molecular level is crucial for advancing disease treatments. Our research reveals a critical connection between the KEOPS complex and Sua5 in Cryptococcus neoformans , a significant cause of fungal meningitis. While the KEOPS complex is known for its versatile roles in cellular processes, Sua5 is specialized in t 6 A tRNA modification. Our key finding is that the diverse roles of the KEOPS complex, ranging from cell growth and stress response to virulence, are fundamentally linked to its function in t 6 A tRNA modification. This conclusion is supported by the remarkable similarities between the impacts of Sua5 and KEOPS on these processes, despite their roles in different steps of the t 6 A modification pathway. This newfound understanding deepens our insight into fungal biology and opens new avenues for developing potential therapies against dangerous fungal diseases., Competing Interests: The authors declare no conflict of interest.

Plants have evolved sophisticated mechanisms to regulate gene expression to activate immune responses against pathogen infections. However, how the translation system contributes to plant immunity is largely unknown. The evolutionarily conserved thiolation modification of transfer RNA (tRNA) ensures efficient decoding during translation. Here, we show that tRNA thiolation is required for plant immunity in Arabidopsis . We identify a cgb mutant that is hyper-susceptible to the pathogen Pseudomonas syringae. CGB encodes ROL5, a homolog of yeast NCS6 required for tRNA thiolation. ROL5 physically interacts with CTU2, a homolog of yeast NCS2. Mutations in either ROL5 or CTU2 result in loss of tRNA thiolation. Further analyses reveal that both transcriptome and proteome reprogramming during immune responses are compromised in cgb . Notably, the translation of salicylic acid receptor NPR1 is reduced in cgb , resulting in compromised salicylic acid signaling. Our study not only reveals a regulatory mechanism for plant immunity but also uncovers an additional biological function of tRNA thiolation., Competing Interests: XZ, HC, ZD, YW, LZ, CW, XY, QC, SY No competing interests declared, (© 2024, Zheng, Chen et al.)

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68)., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nitrofurantoin is a commonly used chemotherapeutic agent in the treatment of uncomplicated urinary tract infections caused by the problematic multidrug resistant Gram-negative pathogen Klebsiella pneumoniae . The present study aims to elucidate the mechanism of nitrofurantoin action and high-level resistance in K. pneumoniae using whole-genome sequencing (WGS), qPCR analysis, mutation structural modeling and untargeted metabolomic analysis. WGS profiling of evolved highly resistant mutants (nitrofurantoin minimum inhibitory concentrations > 256 mg/L) revealed modified expression of several genes related to membrane transport (porin ompK36 and efflux pump regulator oqxR ) and nitroreductase activity ( ribC and nfsB , involved in nitrofurantoin reduction). Untargeted metabolomics analysis of total metabolites extracted at 1 and 4 h post-nitrofurantoin treatment revealed that exposure to the drug caused a delayed effect on the metabolome which was most pronounced after 4 h. Pathway enrichment analysis illustrated that several complex interrelated metabolic pathways related to nitrofurantoin bacterial killing (aminoacyl-tRNA biosynthesis, purine metabolism, central carbohydrate metabolism, and pantothenate and CoA biosynthesis) and the development of nitrofurantoin resistance (riboflavin metabolism) were significantly perturbed. This study highlights for the first time the key role of efflux pump regulator oqxR in nitrofurantoin resistance and reveals global metabolome perturbations in response to nitrofurantoin, in K. pneumoniae .IMPORTANCEA quest for novel antibiotics and revitalizing older ones (such as nitrofurantoin) for treatment of difficult-to-treat Gram-negative bacterial infections has become increasingly popular. The precise antibacterial activity of nitrofurantoin is still not fully understood. Furthermore, although the prevalence of nitrofurantoin resistance remains low currently, the drug's fast-growing consumption worldwide highlights the need to comprehend the emerging resistance mechanisms. Here, we used multidisciplinary techniques to discern the exact mechanism of nitrofurantoin action and high-level resistance in Klebsiella pneumoniae , a common cause of urinary tract infections for which nitrofurantoin is the recommended treatment. We found that the expression of multiple genes related to membrane transport (including active efflux and passive diffusion of drug molecules) and nitroreductase activity was modified in nitrofurantoin-resistant strains, including oqxR, the transcriptional regulator of the oqxAB efflux pump. Furthermore, complex interconnected metabolic pathways that potentially govern the nitrofurantoin-killing mechanisms (e.g., aminoacyl-tRNA biosynthesis) and nitrofurantoin resistance (riboflavin metabolism) were significantly inhibited following nitrofurantoin treatment. Our study could help inform the improvement of nitrofuran derivatives, the development of new pharmacophores, or drug combinations to support the resurgence of nitrofurantoin in the management of multidrug resistant K. pneumouniae infection., Competing Interests: The authors declare no conflict of interest.

Background: In recent years, many studies have confirmed that hypoxia and hypoxia inducible factor (HIF)-1α drive the development of colorectal cancer (CRC). HIF-1α also modulates epitranscriptomic remodeling to regulate cancer development. However, the mechanism by which RNA methylation is altered under hypoxic conditions and the underlying regulatory mechanisms in CRC remain unclear., Methods: Here, seven common types of modifications of mRNA and tRNA were quantitated using liquid chromatography-tandem mass spectrometry. To validate the robustness of the profiling data, modifications that were consistently altered across the three CRC cell lines under hypoxia were validated via dot blot analysis. Then, 10 enzymes that could regulate the abundance of three RNA modifications in tRNA were measured in CRC cells after hypoxia treatment using quantitative real-time polymerase chain reaction. Furthermore, the regulatory role of HIF-1α in the expression of methyltransferase 1 (METTL1) under hypoxic conditions was confirmed using METTL1 promoter activity assays and HIF-1α small interfering RNA (siRNA). The binding capacity of HIF-1α to each hypoxia response element (HRE) in the promoter of METTL1 was investigated by performing Chromatin immunoprecipitation assay (ChIP)., Results: Abundance of RNA modifications was altered more consistently and significantly in tRNA than in mRNA under hypoxic conditions. In addition, the abundance of N7-methyleguanosine (m 7 G) modification in tRNA decreased significantly under hypoxic conditions. As a methyltransferase of the m 7 G modification in tRNA, the expression of METTL1 mRNA was drastically downregulated under hypoxic conditions. Mechanistically, suppression of HIF-1α by siRNA upregulated the METTL1 promoter activity. Furthermore, ChIP showed that HIF-1α could bind with an HRE in the promoter region of METTL1, indicating that METTL1 is a direct target of HIF-1α in CRC cells under hypoxic conditions., Conclusions: Our study revealed that the abundance of the m 7 G modification in tRNA was drastically reduced in CRC cells dependent on the HIF-1α-mediated inhibition of METTL1 transcription under hypoxic conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The tolerance of the translation apparatus toward noncanonical amino acids (ncAAs) has enabled the creation of diverse natural-product-like peptide libraries using mRNA display for use in drug discovery. Typical experiments testing for ribosomal ncAA incorporation involve radioactive end point assays to measure yield alongside mass spectrometry experiments to validate incorporation. These end point assays require significant postexperimental manipulation for analysis and prevent higher throughput analysis and optimization experiments. Continuous assays for in vitro translation involve the synthesis of fluorescent proteins which require the full complement of canonical AAs for function and are therefore of limited utility for testing of ncAAs. Here, we describe a new, continuous fluorescence assay for in vitro translation based on detection of a short peptide tag using an affinity clamp protein, which exhibits changes in its fluorescent properties upon binding. Using this assay in a 384-well format, we were able to validate the incorporation of a variety of ncAAs and also quickly test for the codon reading specificities of a variety of Escherichia coli tRNAs. This assay enables rapid assessment of ncAAs and optimization of translation components and is therefore expected to advance the engineering of the translation apparatus for drug discovery and synthetic biology.

tRNAs are among the most abundant and essential biomolecules in cells. These spontaneously folding, extensively structured yet conformationally flexible anionic polymers literally bridge the worlds of RNAs and proteins, and serve as Rosetta stones that decipher and interpret the genetic code. Their ubiquitous presence, functional irreplaceability, and privileged access to cellular compartments and ribosomes render them prime targets for both endogenous regulation and exogenous manipulation. There is essentially no part of the tRNA that is not touched by another interaction partner, either as programmed or imposed by an external adversary. Recent progresses in genetic, biochemical, and structural analyses of the tRNA interactome produced a wealth of new knowledge into their interaction networks, regulatory functions, and molecular interfaces. In this review, I describe and illustrate the general principles of tRNA recognition by proteins and other RNAs, and discuss the underlying molecular mechanisms that deliver affinity, specificity, and functional competency., Competing Interests: Declaration of interests The author declares no competing interests., (Published by Elsevier Ltd.)

In Saccharomyces cerevisiae , a single homolog of the tRNA methyltransferase Trm10 performs m 1 G9 modification on 13 different tRNAs. Here we provide evidence that the m 1 G9 modification catalyzed by S. cerevisiae Trm10 plays a biologically important role for one of these tRNA substrates, tRNA Trp Overexpression of tRNA Trp (and not any of 38 other elongator tRNAs) rescues growth hypersensitivity of the trm10Δ strain in the presence of the antitumor drug 5-fluorouracil (5FU). Mature tRNA Trp is depleted in trm10Δ cells, and its levels are further decreased upon growth in 5FU, while another Trm10 substrate (tRNA Gly ) is not affected under these conditions. Thus, m 1 G9 in S. cerevisiae is another example of a tRNA modification that is present on multiple tRNAs but is only essential for the biological function of one of those species. In addition to the effects of m 1 G9 on mature tRNA Trp , precursor tRNA Trp species accumulate in the same strains, an effect that is due to at least two distinct mechanisms. The levels of mature tRNA Trp are rescued in the trm10Δmet22Δ strain, consistent with the known role of Met22 in tRNA quality control, where deletion of met22 causes inhibition of 5'-3' exonucleases that catalyze tRNA decay. However, none of the known Met22-associated exonucleases appear to be responsible for the decay of hypomodified tRNA Trp , based on the inability of mutants of each enzyme to rescue the growth of the trm10Δ strain in the presence of 5FU. Thus, the surveillance of tRNA Trp appears to constitute a distinct tRNA quality control pathway in S. cerevisiae ., (© 2024 Bowles and Jackman; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Optimized transfer RNA (tRNA) codon use can speed up antibody generation.

tRFtarget 1.0 (http://trftarget.net/) is a platform consolidating both computationally predicted and experimentally validated binding sites between transfer RNA-derived fragments (tRFs) and target genes (or transcripts) across multiple organisms. Here, we introduce a newly released version of tRFtarget 2.0, in which we integrated 6 additional tRF sources, resulting in a comprehensive collection of 2614 high-quality tRF sequences spanning across 9 species, including 1944 Homo sapiens tRFs and one newly incorporated species Rattus norvegicus. We also expanded target genes by including ribosomal RNAs, long non-coding RNAs, and coding genes >50 kb in length. The predicted binding sites have surged up to approximately 6 billion, a 20.5-fold increase than that in tRFtarget 1.0. The manually curated publications relevant to tRF targets have increased to 400 and the gene-level experimental evidence has risen to 232. tRFtarget 2.0 introduces several new features, including a web-based tool that identifies potential binding sites of tRFs in user's own datasets, integration of standardized tRF IDs, and inclusion of external links to contents within the database. Additionally, we enhanced website framework and user interface. With these improvements, tRFtarget 2.0 is more user-friendly, providing researchers a streamlined and comprehensive platform to accelerate their research progress., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The incorporation of non-canonical amino acids (ncAAs) into proteins is a powerful technique used in various research fields. Genetic code expansion (GCE) is the most common way to achieve this: a specific codon is selected to be decoded by a dedicated tRNA orthogonal to the endogenous ones. In the past 30 years, great progress has been made to obtain novel tRNA synthetases (aaRSs) accepting a variety of ncAAs with distinct physicochemical properties, to develop robust in vitro assays or approaches for codon reassignment. This sparked the use of the technique, leading to the accumulation of publications, from which gathering all relevant information can appear daunting. Here we present iNClusive (https://non-canonical-aas.biologie.uni-freiburg.de/), a manually curated, extensive repository using standardized nomenclature that provides organized information on ncAAs successfully incorporated into target proteins as verified by mass spectrometry. Since we focused on tRNA synthetase-based tRNA loading, we provide the sequence of the tRNA and aaRS used for the incorporation. Derived from more than 687 peer-reviewed publications, it currently contains 2432 entries about 466 ncAAs, 569 protein targets, 500 aaRSs and 144 tRNAs. We foresee iNClusive will encourage more researchers to experiment with ncAA incorporation thus contributing to the further development of this exciting technique., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Neointimal hyperplasia is a pathological vascular remodeling caused by abnormal proliferation and migration of subintimal vascular smooth muscle cells (VSMCs) following intimal injury. There is increasing evidence that tRNA-derived small RNA (tsRNA) plays an important role in vascular remodeling. The purpose of this study is to search for tsRNAs signature of neointima formation and to explore their potential functions. The balloon injury model of rat common carotid artery was replicated to induce intimal hyperplasia, and the differentially expressed tsRNAs (DE-tsRNAs) in arteries with intimal hyperplasia were screened by small RNA sequencing and tsRNA library. A total of 24 DE-tsRNAs were found in the vessels with intimal hyperplasia by small RNA sequencing. In vitro, tRF-Glu-CTC inhibited the expression of fibromodulin (FMOD) in VSMCs, which is a negative modulator of TGF-β1 activity. tRF-Glu-CTC also increased VSMC proliferation and migration. In vivo experiments showed that inhibition of tRF-Glu-CTC expression after balloon injury of rat carotid artery can reduce the neointimal area. In conclusion, tRF-Glu-CTC expression is increased after vascular injury and inhibits FMOD expression in VSMCs, which influences neointima formation. On the other hand, reducing the expression of tRF-Glu-CTC after vascular injury may be a potential approach to prevent vascular stenosis., (© 2023. The Author(s).)

RNA molecules are not merely a combination of four bases of A, C, G, and U. Chemical modifications occur in almost all RNA species and play diverse roles in gene expression regulation. The abundant cellular RNAs, such as ribosomal RNA (rRNA) and transfer RNA (tRNA), are known to have the highest density of RNA modifications, which exert critical functions in rRNA and tRNA biogenesis, stability, and subsequent translation. In recent years, modifications on low-abundance RNA species in mammalian cells, such as messenger RNA (mRNA), regulatory noncoding RNA (ncRNA), and chromatin-associated RNA (caRNA), have been shown to contain multiple different chemical modifications with functional significance. As the most abundant mRNA modification in mammals, N 6 -methyladenosine (m 6 A) affects nearly every stage of mRNA processing and metabolism, with the antibody-based m 6 A-MeRIP-seq (methylated RNA immunoprecipitation sequencing) followed by high-throughput sequencing widely employed in mapping m 6 A distribution transcriptome-wide in diverse biological systems. In addition to m 6 A, other chemical modifications such as pseudouridine (Ψ), 2'- O -methylation (N m ), 5-methylcytidine (m 5 C), internal N 7 -methylguanosine (m 7 G), N 1 -methyladenosine (m 1 A), N 4 -acetylcytidine (ac 4 C), etc. also exist in polyA-tailed RNA in mammalian cells, requiring effective mapping approaches for whole-transcriptome profiling of these non-m 6 A mRNA modifications. Like m 6 A, the antibody-based enrichment followed by sequencing has been the primary method to study distributions of these modifications. Methods to more quantitatively map these modifications would dramatically improve our understanding of distributions and modification density of these chemical marks on RNA, thereby bettering informing functional implications. In this Account, aimed at both single-base resolution and modification fraction quantification, we summarize our recent advances in developing a series of chemistry- or biochemistry-based methods to quantitatively map RNA modifications, including m 6 A, Ψ, m 5 C, m 1 A, 2'- O -methylation (N m ), and internal m 7 G, in mammalian mRNA at base resolution. These new methods, including m 6 A-SAC-seq, eTAM-seq, BID-seq, UBS-seq, DAMM-seq, m 1 A-quant-seq, Nm-Mut-seq, and m 7 G-quant-seq, promise to conduct base-resolution mapping of most major mRNA modifications with low RNA input and uncover dynamic changes in modification stoichiometry during biological and physiological processes, facilitating future investigations on these RNA modifications in regulating cellular gene expression and as potential biomarkers for clinical diagnosis and prognosis. These quantitative sequencing methods allow the mapping of most mRNA modifications with limited input sample requirements. The same modifications on diverse RNA species, such as caRNA, ncRNA, nuclear nascent RNA, mitochondrial RNA, cell-free RNA (cfRNA), etc., could be sequenced using the same methods.

2'- O -ribose methylation (2'- O -Me) is one of the most common RNA modifications detected in ribosomal RNAs (rRNA) from bacteria to eukaryotic cells. 2'- O -Me favours a specific RNA conformation and protects RNA from hydrolysis. Moreover, rRNA 2'- O -Me might stabilize its interactions with messenger RNA (mRNA), transfer RNA (tRNA) or proteins. The extent of rRNA 2'- O -Me fluctuates between species from 3-4 sites in bacteria to tens of sites in archaea, yeast, algae, plants and human. Depending on the organism as well as the rRNA targeting site and position, the 2'- O -Me reaction can be carried out by several site-specific RNA methyltransferases (RMTase) or by a single RMTase associated to specific RNA guides. Here, we review current progresses in rRNA 2'- O -Me (sites/Nm and RMTases) in plants and compare the results with molecular clues from unicellular (bacteria, archaea, algae and yeast) as well as multicellular (human and plants) organisms.

Emerging microorganism Pseudomonas putida KT2440 is utilized for the synthesis of biobased chemicals from renewable feedstocks and for bioremediation. However, the methods for analyzing, engineering, and regulating the biosynthetic enzymes and protein complexes in this organism remain underdeveloped.Such attempts can be advanced by the genetic code expansion-enabled incorporation of noncanonical amino acids (ncAAs) into proteins, which also enables further controls over the strain's biological processes. Here, we give a step-by-step account of the incorporation of two ncAAs into any protein of interest (POI) in response to a UAG stop codon by two commonly used orthogonal archaeal tRNA synthetase and tRNA pairs. Using superfolder green fluorescent protein (sfGFP) as an example, this method lays down a solid foundation for future work to study and enhance the biological functions of KT2440., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The identification of a wide variety of RNA molecules using high-throughput sequencing techniques in the transcriptome pool of living organisms has revealed hidden regulatory insights in the cell. The class of non-coding RNA fragments produced from transfer RNA, or tRFs, is one such example. They are heterogeneously sized molecules with lengths ranging between 15 and 50 nt. They have a history of being dysregulated in human malignancies and other illnesses. The detection of these molecules has been made easier by a variety of bioinformatics techniques. The various types of tRFs and how they relate to cancer are covered in this chapter. It also provides a summary of the biological significance of tRFs reported in human cancer. Additionally, it emphasizes the utilities of databases and computational tools that have been created by different research teams for the investigation of tRFs. This will further aid the exploration and analysis of tRFs in cancer research and will support future advancement and a better comprehension of these molecules., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Nucleotide modifications are occurrent in all types of RNA and play an important role in RNA structure formation and stability. Modified bases not only possess the ability to shift the RNA structure ensemble towards desired functional confirmations. By changes in the base pairing partner preference, they may even enlarge or reduce the conformational space, i.e., the number and types of structures the RNA molecule can adopt. However, most methods to predict RNA secondary structure do not provide the means to include the effect of modifications on the result. With the help of a heavily modified transfer RNA (tRNA) molecule, this chapter demonstrates how to include the effect of different base modifications into secondary structure prediction using the ViennaRNA Package. The constructive approach demonstrated here allows for the calculation of minimum free energy structure and suboptimal structures at different levels of modified base support. In particular we, show how to incorporate the isomerization of uridine to pseudouridine ( Ψ ) and the reduction of uridine to dihydrouridine (D)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)