Queuosine salvage in Bartonella henselae Houston 1: a unique evolutionary path.

Queuosine (Q) stands out as the sole tRNA modification that can be synthesized via salvage pathways. Comparative genomic analyses identified specific bacteria that showed a discrepancy between the projected Q salvage route and the predicted substrate specificities of the two identified salvage proteins: (1) the distinctive enzyme tRNA guanine-34 transglycosylase (bacterial TGT, or bTGT), responsible for inserting precursor bases into target tRNAs; and (2) queuosine precursor transporter (QPTR), a transporter protein that imports Q precursors. Organisms such as the facultative intracellular pathogen Bartonella henselae, which possess only bTGT and QPTR but lack predicted enzymes for converting preQ1 to Q, would be expected to salvage the queuine (q) base, mirroring the scenario for the obligate intracellular pathogen Chlamydia trachomatis. However, sequence analyses indicate that the substrate-specificity residues of their bTGTs resemble those of enzymes inserting preQ1 rather than q. Intriguingly, MS analyses of tRNA modification profiles in B. henselae reveal trace amounts of preQ1, previously not observed in a natural context. Complementation analysis demonstrates that B. henselae bTGT and QPTR not only utilize preQ1, akin to their Escherichia coli counterparts, but can also process q when provided at elevated concentrations. The experimental and phylogenomic analyses suggest that the Q pathway in B. henselae could represent an evolutionary transition among intracellular pathogens - from ancestors that synthesized Q de novo to a state prioritizing the salvage of q. Another possibility that will require further investigations is that the insertion of preQ1 confers fitness advantages when B. henselae is growing outside a mammalian host.

Inducible auto-phosphorylation regulates a widespread family of nucleotidyltransferase toxins.

Nucleotidyltransferases (NTases) control diverse physiological processes, including RNA modification, DNA replication and repair, and antibiotic resistance. The Mycobacterium tuberculosis NTase toxin family, MenT, modifies tRNAs to block translation. MenT toxin activity can be stringently regulated by diverse MenA antitoxins. There has been no unifying mechanism linking antitoxicity across MenT homologues. Here we demonstrate through structural, biochemical, biophysical and computational studies that despite lacking kinase motifs, antitoxin MenA1 induces auto-phosphorylation of MenT1 by repositioning the MenT1 phosphoacceptor T39 active site residue towards bound nucleotide. Finally, we expand this predictive model to explain how unrelated antitoxin MenA3 is similarly able to induce auto-phosphorylation of cognate toxin MenT3. Our study reveals a conserved mechanism for the control of tuberculosis toxins, and demonstrates how active site auto-phosphorylation can regulate the activity of widespread NTases.

tRNA modifications and tRNA-derived small RNAs: new insights of tRNA in human disease.

tRNAs are codon decoders that convert the transcriptome into the proteome. The field of tRNA research is excited by the increasing discovery of specific tRNA modifications that are installed at specific, evolutionarily conserved positions by a set of specialized tRNA-modifying enzymes and the biogenesis of tRNA-derived regulatory fragments (tsRNAs) which exhibit copious activities through multiple mechanisms. Dysregulation of tRNA modification usually has pathological consequences, a phenomenon referred to as "tRNA modopathy". Current evidence suggests that certain tRNA-modifying enzymes and tsRNAs may serve as promising diagnostic biomarkers and therapeutic targets, particularly for chemoresistant cancers. In this review, we discuss the latest discoveries that elucidate the molecular mechanisms underlying the functions of clinically relevant tRNA modifications and tsRNAs, with a focus on malignancies. We also discuss the therapeutic potential of tRNA/tsRNA-based therapies, aiming to provide insights for the development of innovative therapeutic strategies. Further efforts to unravel the complexities inherent in tRNA biology hold the promise of yielding better biomarkers for the diagnosis and prognosis of diseases, thereby advancing the development of precision medicine for health improvement.

The subcellular distribution of miRNA isoforms, tRNA-derived fragments, and rRNA-derived fragments depends on nucleotide sequence and cell type.

BACKGROUND: MicroRNA isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) represent most of the small non-coding RNAs (sncRNAs) found in cells. Members of these three classes modulate messenger RNA (mRNA) and protein abundance and are dysregulated in diseases. Experimental studies to date have assumed that the subcellular distribution of these molecules is well-understood, independent of cell type, and the same for all isoforms of a sncRNA. RESULTS: We tested these assumptions by investigating the subcellular distribution of isomiRs, tRFs, and rRFs in biological replicates from three cell lines from the same tissue and same-sex donors that model the same cancer subtype. In each cell line, we profiled the isomiRs, tRFs, and rRFs in the nucleus, cytoplasm, whole mitochondrion (MT), mitoplast (MP), and whole cell. Using a rigorous mathematical model we developed, we accounted for cross-fraction contamination and technical errors and adjusted the measured abundances accordingly. Analyses of the adjusted abundances show that isomiRs, tRFs, and rRFs exhibit complex patterns of subcellular distributions. These patterns depend on each sncRNA's exact sequence and the cell type. Even in the same cell line, isoforms of the same sncRNA whose sequences differ by a few nucleotides (nts) can have different subcellular distributions. CONCLUSIONS: SncRNAs with similar sequences have different subcellular distributions within and across cell lines, suggesting that each isoform could have a different function. Future computational and experimental studies of isomiRs, tRFs, and rRFs will need to distinguish among each molecule's various isoforms and account for differences in each isoform's subcellular distribution in the cell line at hand. While the findings add to a growing body of evidence that isomiRs, tRFs, rRFs, tRNAs, and rRNAs follow complex intracellular trafficking rules, further investigation is needed to exclude alternative explanations for the observed subcellular distribution of sncRNAs.

Maximal Genetic Code Symmetry Is a Physicochemical Purine-Pyrimidine Symmetry Language for Transcription and Translation in the Flow of Genetic Information from DNA to Proteins.

Until now, research has not taken into consideration the physicochemical purine-pyrimidine symmetries of the genetic code in the transcription and translation processes of proteinogenesis. Our Supersymmetry Genetic Code table, developed in 2022, is common and unique for all RNA and DNA living species. Its basic structure is a purine-pyrimidine symmetry net with double mirror symmetry. Accordingly, the symmetry of the genetic code directly shows its organisation based on the principle of nucleotide Watson-Crick and codon-anticodon pairing. The maximal purine-pyrimidine symmetries of codons show that each codon has a strictly defined and unchangeable position within the genetic code. We discovered that the physicochemical symmetries of the genetic code play a fundamental role in recognising and differentiating codons from mRNA and the anticodon tRNA and aminoacyl-tRNA synthetases in the transcription and translation processes. These symmetries also support the wobble hypothesis with non-Watson-Crick pairing interactions between the translation process from mRNA to tRNA. The Supersymmetry Genetic Code table shows a specific arrangement of the second base of codons, according to which it is possible that an anticodon from tRNA recognises whether a codon from mRNA belongs to an amino acid with two or four codons, which is very important in the purposeful use of the wobble pairing process. Therefore, we show that canonical and wobble pairings essentially do not lead to misreading and errors during translation, and we point out the role of physicochemical purine-pyrimidine symmetries in decreasing disorder according to error minimisation and preserving the integrity of biological processes during proteinogenesis.

N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation.

The ribosome utilizes hydrogen bonding between mRNA codons and aminoacyl-tRNAs to ensure rapid and accurate protein production. Chemical modification of mRNA nucleobases can adjust the strength and pattern of this hydrogen bonding to alter protein synthesis. We investigate how the N1-methylpseudouridine (m1Ψ) modification, commonly incorporated into therapeutic and vaccine mRNA sequences, influences the speed and fidelity of translation. We find that m1Ψ does not substantially change the rate constants for amino acid addition by cognate tRNAs or termination by release factors. However, we also find that m1Ψ can subtly modulate the fidelity of amino acid incorporation in a codon-position and tRNA dependent manner in vitro and in human cells. Our computational modeling shows that altered energetics of mRNA:tRNA interactions largely account for the context dependence of the low levels of miscoding we observe on Ψ and m1Ψ containing codons. The outcome of translation on modified mRNA bases is thus governed by the sequence context in which they occur.

Oncogenic tRNA-derived fragment tRF-Leu-CAG promotes tumorigenesis of lung cancer via targeting TCEA3 and increasing autophagy.

BACKGROUND: Lung cancer is a prevalent and severe form of malignant tumors worldwide. tRF-Leu-CAG, a recently discovered non-coding single-stranded small RNA derived from transfer RNA, has sparked interest in exploring its biological functions and potential molecular mechanisms in lung cancer. METHODS: The abundance of tRF-Leu-CAG was measured via quantitative real-time polymerase chain reaction (qRT-PCR) in 96 sets of lung cancer tissue samples obtained from clinical patients. Subsequently, both in vivo and in vitro experiments were conducted to validate the biological functions of tRF-Leu-CAG in lung cancer. Furthermore, an exploration of the potential target genes of tRF-Leu-CAG and its association with autophagy and drug resistance in lung cancer was undertaken. RESULTS: Our analysis revealed a significant upregulation of tRF-Leu-CAG in non-small cell lung cancer (NSCLC) tissues. Additionally, we observed that heightened expression of tRF-Leu-CAG significantly augmented the proliferation and migration of NSCLC cells, facilitated cell cycle progression, and suppressed apoptosis. Furthermore, we identified transcription elongation factor A3 (TCEA3) as a direct target gene of tRF-Leu-CAG. TCEA3 inhibited the proliferation and migration of NSCLC, and tRF-Leu-CAG promoted the proliferation and migration of NSCLC by mediating the silencing of TCEA3. Moreover, we demonstrated that the augmentation of paclitaxel resistance by tRF-Leu-CAG was contingent on autophagy. Finally, tRF-Leu-CAG notably accelerated tumor growth and promoted the process of epithelial-mesenchymal transition (EMT) in vivo. CONCLUSIONS: tRF-Leu-CAG promotes NSCLC tumor growth and metastasis by targeting TCEA3 and promotes paclitaxel resistance by enhancing cellular autophagy. These results provide potentially effective targets and therapeutic options for the clinical treatment of NSCLC.

Decoding the role of tRNA modifications in cancer progression.

Epitranscriptomic modification of tRNA has recently gained traction in the field of cancer biology. The presence of such modifications on tRNA appears to allow for translational control of processes central to progression and malignant transformation. Methyltransferase Like 1 protein (METTL1), along with other epitranscriptomic writers (e.g. NSUN3, NAT10, ELP3, etc.), has recently been investigated in multiple cancer types. Here, we review the impact of such tRNA modifications in tumorigenesis and the progression of cancer toward drug resistance and metastasis. Regulation of central cellular processes relied upon by malignant cancer cells through modulation of the tRNA epitranscriptome represents an area with great potential to bring novel first-in-class therapies to the clinic.

Alternate conformational trajectories in ribosome translocation.

Translocation in protein synthesis entails the efficient and accurate movement of the mRNA-[tRNA]2 substrate through the ribosome after peptide bond formation. An essential conformational change during this process is the swiveling of the small subunit head domain about two rRNA 'hinge' elements. Using iterative selection and molecular dynamics simulations, we derive alternate hinge elements capable of translocation in vitro and in vivo and describe their effects on the conformational trajectory of the EF-G-bound, translocating ribosome. In these alternate conformational pathways, we observe a diversity of swivel kinetics, hinge motions, three-dimensional head domain trajectories and tRNA dynamics. By finding alternate conformational pathways of translocation, we identify motions and intermediates that are essential or malleable in this process. These findings highlight the plasticity of protein synthesis and provide a more thorough understanding of the available sequence and conformational landscape of a central biological process.

Temperature-Dependent tRNA Modifications in Bacillales.

Transfer RNA (tRNA) modifications are essential for the temperature adaptation of thermophilic and psychrophilic organisms as they control the rigidity and flexibility of transcripts. To further understand how specific tRNA modifications are adjusted to maintain functionality in response to temperature fluctuations, we investigated whether tRNA modifications represent an adaptation of bacteria to different growth temperatures (minimal, optimal, and maximal), focusing on closely related psychrophilic (P. halocryophilus and E. sibiricum), mesophilic (B. subtilis), and thermophilic (G. stearothermophilus) Bacillales. Utilizing an RNA sequencing approach combined with chemical pre-treatment of tRNA samples, we systematically profiled dihydrouridine (D), 4-thiouridine (s4U), 7-methyl-guanosine (m7G), and pseudouridine (Ψ) modifications at single-nucleotide resolution. Despite their close relationship, each bacterium exhibited a unique tRNA modification profile. Our findings revealed increased tRNA modifications in the thermophilic bacterium at its optimal growth temperature, particularly showing elevated levels of s4U8 and Ψ55 modifications compared to non-thermophilic bacteria, indicating a temperature-dependent regulation that may contribute to thermotolerance. Furthermore, we observed higher levels of D modifications in psychrophilic and mesophilic bacteria, indicating an adaptive strategy for cold environments by enhancing local flexibility in tRNAs. Our method demonstrated high effectiveness in identifying tRNA modifications compared to an established tool, highlighting its potential for precise tRNA profiling studies.

Designing a Novel Temperature- and Noncanonical Amino Acid-Controlled Biological Logic Gate in Escherichia coli.

Genetic code expansion (GCE) is a powerful strategy that expands the genetic code of an organism for incorporating noncanonical amino acids into proteins using engineered tRNAs and aminoacyl-tRNA synthetases (aaRSs). While GCE has opened up new possibilities for synthetic biology, little is known about the potential side effects of exogenous aaRS/tRNA pairs. In this study, we investigated the impact of exogenous aaRS and amber suppressor tRNA on gene expression in Escherichia coli. We discovered that in DH10ß ΔcyaA, transformed with the F1RP/F2P two-hybrid system, the high consumption rate of cellular adenosine triphosphate by exogenous aaRS/tRNA at elevated temperatures induces temperature sensitivity in the expression of genes regulated by the cyclic AMP receptor protein (CRP). We harnessed this temperature sensitivity to create a novel biological AND gate in E. coli, responsive to both p-benzoylphenylalanine (BzF) and low temperature, using a BzF-dependent variant of E. coli chorismate mutase and split subunits of Bordetella pertussis adenylate cyclase. Our study provides new insights into the unexpected effects of exogenous aaRS/tRNA pairs and offers a new approach for constructing a biological logic gate.

[4Fe-4S]-dependent enzymes in non-redox tRNA thiolation.

Post-transcriptional modification of nucleosides in transfer RNAs (tRNAs) is an important process for accurate and efficient translation of the genetic information during protein synthesis in all domains of life. In particular, specific enzymes catalyze the biosynthesis of sulfur-containing nucleosides, such as the derivatives of 2-thiouridine (s2U), 4-thiouridine (s4U), 2-thiocytidine (s2C), and 2-methylthioadenosine (ms2A), within tRNAs. Whereas the mechanism that has prevailed for decades involved persulfide chemistry, more and more tRNA thiolation enzymes have now been shown to contain a [4Fe-4S] cluster. This review summarizes the information over the last ten years concerning the biochemical, spectroscopic and structural characterization of [4Fe-4S]-dependent non-redox tRNA thiolation enzymes.

RudS: bacterial desulfidase responsible for tRNA 4-thiouridine de-modification.

In this study, we present an extensive analysis of a widespread group of bacterial tRNA de-modifying enzymes, dubbed RudS, which consist of a TudS desulfidase fused to a Domain of Unknown Function 1722 (DUF1722). RudS enzymes exhibit specific de-modification activity towards the 4-thiouridine modification (s4U) in tRNA molecules, as indicated by our experimental findings. The heterologous overexpression of RudS genes in Escherichia coli significantly reduces the tRNA 4-thiouridine content and diminishes UVA-induced growth delay, indicating the enzyme's role in regulating photosensitive tRNA s4U modification. Through a combination of protein modeling, docking studies, and molecular dynamics simulations, we have identified amino acid residues involved in catalysis and tRNA binding. Experimental validation through targeted mutagenesis confirms the TudS domain as the catalytic core of RudS, with the DUF1722 domain facilitating tRNA binding in the anticodon region. Our results suggest that RudS tRNA modification eraser proteins may play a role in regulating tRNA during prokaryotic stress responses.

Translational T-box riboswitches bind tRNA by modulating conformational flexibility.

T-box riboswitches are noncoding RNA elements involved in genetic regulation of most Gram-positive bacteria. They regulate amino acid metabolism by assessing the aminoacylation status of tRNA, subsequently affecting the transcription or translation of downstream amino acid metabolism-related genes. Here we present single-molecule FRET studies of the Mycobacterium tuberculosis IleS T-box riboswitch, a paradigmatic translational T-box. Results support a two-step binding model, where the tRNA anticodon is recognized first, followed by interactions with the NCCA sequence. Furthermore, after anticodon recognition, tRNA can transiently dock into the discriminator domain even in the absence of the tRNA NCCA-discriminator interactions. Establishment of the NCCA-discriminator interactions significantly stabilizes the fully bound state. Collectively, the data suggest high conformational flexibility in translational T-box riboswitches; and supports a conformational selection model for NCCA recognition. These findings provide a kinetic framework to understand how specific RNA elements underpin the binding affinity and specificity required for gene regulation.

Viral hijacking of hnRNPH1 unveils a G-quadruplex-driven mechanism of stress control.

Viral genomes are enriched with G-quadruplexes (G4s), non-canonical structures formed in DNA or RNA upon assembly of four guanine stretches into stacked quartets. Because of their critical roles, G4s are potential antiviral targets, yet their function remains largely unknown. Here, we characterize the formation and functions of a conserved G4 within the polymerase coding region of orthoflaviviruses of the Flaviviridae family. Using yellow fever virus, we determine that this G4 promotes viral replication and suppresses host stress responses via interactions with hnRNPH1, a host nuclear protein involved in RNA processing. G4 binding to hnRNPH1 causes its cytoplasmic retention with subsequent impacts on G4-containing tRNA fragments (tiRNAs) involved in stress-mediated reductions in translation. As a result, these host stress responses and associated antiviral effects are impaired. These data reveal that the interplay between hnRNPH1 and both host and viral G4 targets controls the integrated stress response and viral replication.

The role of tRF-Val-CAC-010 in lung adenocarcinoma: implications for tumorigenesis and metastasis.

OBJECTIVE: Transfer RNA-derived fragments (tRFs) are short non-coding RNA (ncRNA) sequences, ranging from 14 to 30 nucleotides, produced through the precise cleavage of precursor and mature tRNAs. While tRFs have been implicated in various diseases, including cancer, their role in lung adenocarcinoma (LUAD) remains underexplored. This study aims to investigate the impact of tRF-Val-CAC-010, a specific tRF molecule, on the phenotype of LUAD cells and its role in tumorigenesis and progression in vivo. METHODS: The expression level of tRF-Val-CAC-010 was quantified using quantitative real-time polymerase chain reaction (qRT-PCR). Specific inhibitors and mimics of tRF-Val-CAC-010 were synthesized for transient transfection. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8), while cell invasion and migration were evaluated through Transwell invasion and scratch assays. Flow cytometry was utilized to analyze cell cycle and apoptosis. The in vivo effects of tRF-Val-CAC-010 on tumor growth and metastasis were determined through tumor formation and metastasis imaging experiments in nude mice. RESULTS: The expression level of tRF-Val-CAC-010 was upregulated in A549 and PC9 LUAD cells (P < 0.01). Suppression of tRF-Val-CAC-010 expression resulted in decreased proliferation of A549 and PC9 cells (P < 0.001), reduced invasion and migration of A549 (P < 0.05, P < 0.001) and PC9 cells (P < 0.05, P < 0.01), enhanced apoptosis in both A549 (P < 0.05) and PC9 cells (P < 0.05), and increased G2 phase cell cycle arrest in A549 cells (P < 0.05). In vivo, the tumor formation volume in the tRF-inhibitor group was significantly smaller than that in the model and tRF-NC groups (P < 0.05). The metastatic tumor flux value in the tRF-inhibitor group was also significantly lower than that in the model and tRF-NC groups (P < 0.05). CONCLUSION: This study demonstrates that tRF-Val-CAC-010 promotes proliferation, migration, and invasion of LUAD cells and induces apoptosis in vitro, however, its specific effects on the cell cycle require further elucidation. Additionally, tRF-Val-CAC-010 enhances tumor formation and metastasis in vivo. Therefore, tRF-Val-CAC-010 may serve as a novel diagnostic biomarker and potential therapeutic target for LUAD.

tRF-27 competitively Binds to G3BPs and Activates MTORC1 to Enhance HER2 Positive Breast Cancer Trastuzumab Tolerance.

About 20% of breast cancer patients are positive for HER2. The efficacy of current treatments is limited by primary and secondary resistance to trastuzumab. tRNA-derived fragments (tRFs) have shown crucial regulatory roles in various cancers. This study aimed to evaluate the role of tRF-27 in regulating the resistance of HER2-positive breast cancer against trastuzumab. tRF-27 was highly expressed in trastuzumab-resistant cells, and its expression level could predict the resistance to trastuzumab. High expression of tRF-27 promoted the growth and proliferation of trastuzumab-exposed cells. RNA-pulldown assay and mass spectrometry were performed to identify Ras GTPase-activating protein-binding proteins 1 and 2 (G3BPs) (two proteins targeted by tRF-27); RNA-immunoprecipitation (RIP) to confirm their bindings; co-immunoprecipitation (co-IP) and RNA-pulldown assay to determine the binding domains between G3BPs and tRF-27.tRF-27 bound to the nuclear transport factor 2 like domain(NTF2 domain) of G3BPs through a specific sequence. tRF-27 relied on G3BPs and NTF2 domain to increase trastuzumab tolerance. tRF-27 competed with lysosomal associated membrane protein 1(LAMP1) for NTF2 domain, thereby inhibiting lysosomal localization of G3BPs and tuberous sclerosis complex (TSC). Overexpression of tRF-27 inhibited phosphorylation of TSCs and promoted the activation of mechanistic target of rapamycin complex 1(MTORC1) to enhance cell proliferation and entice the resistance of HER2-positive breast cancer against trastuzumab.

tsRNA-GlyGCC promotes colorectal cancer progression and 5-FU resistance by regulating SPIB.

BACKGROUND: tRNA-derived small RNAs (tsRNAs) are newly discovered non-coding RNA, which are generated from tRNAs and are reported to participate in several biological processes in diseases, especially cancer; however, the mechanism of tsRNA involvement in colorectal cancer (CRC) and 5-fluorouracil (5-FU) is still unclear. METHODS: RNA sequencing was performed to identify differential expression of tsRNAs in CRC tissues. CCK8, colony formation, transwell assays, and tumor sphere assays were used to investigate the role of tsRNA-GlyGCC in 5-FU resistance in CRC. TargetScan and miRanda were used to identify the target genes of tsRNA-GlyGCC. Biotin pull-down, RNA pull-down, luciferase assay, ChIP, and western blotting were used to explore the underlying molecular mechanisms of action of tsRNA-GlyGCC. The MeRIP assay was used to investigate the N(7)-methylguanosine RNA modification of tsRNA-GlyGCC. RESULTS: In this study, we uncovered the feature of tsRNAs in human CRC tissues and confirmed a specific 5' half tRNA, 5'tiRNA-Gly-GCC (tsRNA-GlyGCC), which is upregulated in CRC tissues and modulated by METTL1-mediated N(7)-methylguanosine tRNA modification. In vitro and in vivo experiments revealed the oncogenic role of tsRNA-GlyGCC in 5-FU drug resistance in CRC. Remarkably, our results showed that tsRNA-GlyGCC modulated the JAK1/STAT6 signaling pathway by targeting SPIB. Poly (ß-amino esters) were synthesized to assist the delivery of 5-FU and tsRNA-GlyGCC inhibitor, which effectively inhibited tumor growth and enhanced CRC sensitive to 5-FU without obvious adverse effects in subcutaneous tumor. CONCLUSIONS: Our study revealed a specific tsRNA-GlyGCC-engaged pathway in CRC progression. Targeting tsRNA-GlyGCC in combination with 5-FU may provide a promising nanotherapeutic strategy for the treatment of 5-FU-resistance CRC.

Unravelling tRNA fragments in DENV pathogenesis: Insights from RNA sequencing.

Small non-coding RNAs (sncRNAs) derived from tRNAs are known as tRNA-derived small RNAs (tsRNAs). These tsRNAs are further categorized into tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs), which play significant roles in the various molecular mechanisms underlying certain human diseases. However, the generation of tsRNAs and their potential roles during Dengue virus (DENV) infection is not yet known. Here, we performed small RNA sequencing to identify the generation and alterations in tsRNAs expression profiles of DENV-infected Huh7 cells. Upon DENV infection, tRNA fragmentation was found to be increased. We identified a significant number of differentially expressed tsRNAs during DENV infection. Interestingly, the 3'tRF population showed upregulation, while the i-tRF population exhibited downregulation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to analyze the impact of differentially expressed tsRNAs on DENV pathogenesis. Our results suggest that differentially expressed tsRNAs are involved in transcriptional regulation via RNA polymerase II promoter and metabolic pathways. Overall, our study contributes significantly to our understanding of the roles played by tsRNAs in the complex dynamics of DENV infection.

Conserved 5-methyluridine tRNA modification modulates ribosome translocation.

While the centrality of posttranscriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one of the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA in Escherichia coli, Trm2 in Saccharomyces cerevisiae) exhibit altered tRNA modification patterns. Furthermore, m5U54-deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that relative to wild-type cells, trm2Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.