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Fungal Trl1 is an essential tRNA splicing enzyme composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that convert the 2',3'-cyclic-PO 4 and 5'-OH ends of tRNA exons into the 3'-OH,2'-PO 4 and 5'-PO 4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trifunctional Trl1 enzymes are present in most human fungal pathogens and are untapped targets for antifungal drug discovery. Mucorales species, deemed high-priority human pathogens by WHO, elaborate a noncanonical tRNA splicing apparatus in which a stand-alone monofunctional RNA ligase enzyme joins 3'-OH,2'-PO 4 and 5'-PO 4 termini. Here we identify a stand-alone Mucor circinelloides polynucleotide kinase (MciKIN) and affirm its biological activity in tRNA splicing by genetic complementation in yeast. Recombinant MciKIN catalyzes magnesium-dependent phosphorylation of 5'-OH RNA and DNA ends in vitro. MciKIN displays a strong preference for GTP as the phosphate donor in the kinase reaction, a trait shared with the stand-alone RNA kinase homologs from Mucorales species Rhizopus azygosporus (RazKIN) and Lichtheimia corymbifera (LcoKIN) and with the kinase domains of fungal Trl1 enzymes. We report a 1.65 Å crystal structure of RazKIN in complex with GDP•Mg 2+ that illuminates the basis for guanosine nucleotide specificity., (© 2024 Ghosh et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Uridine residues present at the wobble position of eukaryotic cytosolic tRNAs often carry a 5-carbamoylmethyl (ncm 5 ), 5-methoxycarbonylmethyl (mcm 5 ), or 5-methoxycarbonylhydroxymethyl (mchm 5 ) side-chain. The presence of these side-chains allows proper pairing with cognate codons, and they are particularly important in tRNA species where the U 34 residue is also modified with a 2-thio (s 2 ) group. The first step in the synthesis of the ncm 5 , mcm 5 , and mchm 5 side-chains is dependent on the six-subunit Elongator complex, whereas the thiolation of the 2-position is catalyzed by the Ncs6/Ncs2 complex. In both yeast and metazoans, allelic variants of Elongator subunit genes show genetic interactions with mutant alleles of SOD1 , which encodes the cytosolic Cu, Zn-superoxide dismutase. However, the cause of these genetic interactions remains unclear. Here, we show that yeast sod1 null mutants are impaired in the formation of 2-thio-modified U 34 residues. In addition, the lack of Sod1 induces a defect in the biosynthesis of wybutosine, which is a modified nucleoside found at position 37 of tRNA Phe Our results suggest that these tRNA modification defects are caused by superoxide-induced inhibition of the iron-sulfur cluster-containing Ncs6/Ncs2 and Tyw1 enzymes. Since mutations in Elongator subunit genes generate strong negative genetic interactions with mutant ncs6 and ncs2 alleles, our findings at least partially explain why the activity of Elongator can modulate the phenotypic consequences of SOD1/sod1 alleles. Collectively, our results imply that tRNA hypomodification may contribute to impaired proteostasis in Sod1-deficient cells., (© 2024 Xu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Translation-targeting toxic small alarmone synthetases (toxSAS) are effectors of bacterial toxin-antitoxin systems that pyrophosphorylate the 3'-CCA end of transfer RNA (tRNA) to prevent aminoacylation. toxSAS are implicated in antiphage immunity: Phage detection triggers the toxSAS activity to shut down viral production. We show that the toxSAS FaRel2 inspects the tRNA acceptor stem to specifically select tRNA Gly and tRNA Thr . The first, second, fourth, and fifth base pairs of the stem act as the specificity determinants. We show that the toxSASs PhRel2 and CapRel SJ46 differ in tRNA specificity from FaRel2 and rationalize this through structural modeling: While the universal 3'-CCA end slots into a highly conserved CCA recognition groove, the acceptor stem recognition region is variable across toxSAS diversity. As phages use tRNA isoacceptors to overcome tRNA-targeting defenses, we hypothesize that highly evolvable modular tRNA recognition allows for the escape of viral countermeasures through tRNA substrate specificity switching.

Pancreatic adenocarcinoma (PAAD) is a life-threatening cancer. Exploring new diagnosis and treatment targets helps improve its prognosis. tRNA-derived small non-coding RNAs (tsRNAs) are a novel type of gene expression regulators and their dysregulation is closely related to many human cancers. Yet the expression and functions of tsRNAs in PAAD are not well understood. Our study used RNA sequencing to identify tsRNA expression profiles in PAAD cells cultured in no or high glucose media and found tRF-18-8R6546D2 was an uncharacterized tsRNA, which has significantly high expression in PAAD cells and tissues. Clinically, tRF-18-8R6546D2 is linked to poor prognosis in PAAD patients and can be used to distinguish them from healthy populations. Functionally, in vitro and vivo, tRF-18-8R6546D2 over-expression promoted PAAD cell proliferation, migration and invasion, inhibited apoptosis, whereas tRF-18-8R6546D2 knock-down showed opposite effects. Mechanistically, tRF-18-8R6546D2 promoted PAAD malignancy partly by directly silencing ASCL2 and further regulating its downstream genes such as MYC and CASP3. These findings show that tRF-18-8R6546D2 is a novel oncogenic factor and can be a promising diagnostic or prognostic biomarker and therapeutic target for PAAD., 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.)

The ribosome, together with its tRNA substrates, links genotype to phenotype by translating the genetic information carried by mRNA into protein. During the past half-century, the structure and mechanisms of action of the ribosome have emerged from mystery and confusion. It is now evident that the ribosome is an ancient RNA-based molecular machine of staggering structural complexity and that it is fundamentally similar in all living organisms. The three central functions of protein synthesis-decoding, catalysis of peptide bond formation, and translocation of mRNA and tRNA-are based on elegant mechanisms that evolved from the properties of RNA, the founding macromolecule of life. Moreover, all three of these functions (and even life itself) seem to proceed in defiance of entropy. Protein synthesis thus appears to exploit both the energy of GTP hydrolysis and peptide bond formation to constrain the directionality and accuracy of events taking place on the ribosome., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

The chemical and physical properties of proteins are limited by the 20 canonical amino acids. Genetic code manipulation allows for the incorporation of noncanonical amino acids (ncAAs) that enhance or alter protein functionality. This review explores advances in the three main strategies for introducing ncAAs into biosynthesized proteins, focusing on the role of high throughput screening in these advancements. The first section discusses engineering aminoacyl-tRNA synthetases (aaRSs) and tRNAs, emphasizing how novel selection methods improve characteristics including ncAA incorporation efficiency and selectivity. The second section examines high-throughput techniques for improving protein translation machinery, enabling accommodation of alternative genetic codes. This includes opportunities to enhance ncAA incorporation through engineering cellular components unrelated to translation. The final section highlights various discovery platforms for high-throughput screening of ncAA-containing proteins, showcasing innovative binding ligands and enzymes that are challenging to create with only canonical amino acids. These advances have led to promising drug leads and biocatalysts. Overall, the ability to discover unexpected functionalities through high-throughput methods significantly influences ncAA incorporation and its applications. Future innovations in experimental techniques, along with advancements in computational protein design and machine learning, are poised to further elevate this field.

Over the course of evolution, land plant mitochondrial genomes have lost many transfer RNA (tRNA) genes and the import of nucleus-encoded tRNAs is essential for mitochondrial protein synthesis. By contrast, plastidial genomes of photosynthetic land plants generally possess a complete set of tRNA genes and the existence of plastidial tRNA import remains a long-standing question. The early vascular plants of the Selaginella genus show an extensive loss of plastidial tRNA genes while retaining photosynthetic capacity, and represent an ideal model for answering this question. Using purification, northern blot hybridization, and high-throughput tRNA sequencing, a global analysis of total and plastidial tRNA populations was undertaken in Selaginella kraussiana. We confirmed the expression of all plastidial tRNA genes and, conversely, observed that nucleus-encoded tRNAs corresponding to these plastidial tRNAs were generally excluded from the chloroplasts. We then demonstrated a selective and differential plastidial import of around forty nucleus-encoded tRNA species, likely compensating for the insufficient coding capacity of plastidial-encoded tRNAs. In-depth analysis revealed differential import of tRNA isodecoders, leading to the identification of specific situations. This includes the expression and import of nucleus-encoded tRNAs expressed from plastidial or bacterial-like genes inserted into the nuclear genome. Overall, our results confirm the existence of molecular processes that enable tRNAs to be selectively imported not only into mitochondria, as previously described, but also into chloroplasts, when necessary., Competing Interests: Competing interests statement:The authors declare no competing interest.

There has been recent interest in trying to understand the connection between transfer RNA (tRNA) posttranscriptional modifications and changes in-cellular environmental conditions. Here, we report on the identification of the modified nucleoside 5-methylcytidine (m 5 C) in Escherichia coli tRNAs. This modification was determined to be present at position 49 of tRNA Tyr-QUA-II. Moreover, m 5 C levels in this tRNA are significantly elevated under high reactive oxygen specieis (ROS) conditions in E. coli cells. We identified the known ribosomal RNA methyltransferase rsmF as the enzyme responsible for m 5 C synthesis in tRNA and enzyme transcript levels are responsive to elevated levels of ROS in the cell. We further find that changes in m 5 C levels in this tRNA are not specific to Fenton-like reaction conditions elevating ROS, but heat shock can also induce increased modification of tRNA Tyr-QUA-II. Altogether, this work illustrates how cells adapt to changing environmental conditions through variations in tRNA modification profiles., Competing Interests: Competing interests statement:The authors declare no competing interest.

Post-transcriptional modification of RNA regulates gene expression at multiple levels. ALKBH8 is a tRNA-modifying enzyme that methylates wobble uridines in a subset of tRNAs to modulate translation. Through methylation of tRNA-selenocysteine, ALKBH8 promotes selenoprotein synthesis and regulates redox homeostasis. Pathogenic variants in ALKBH8 have been linked to intellectual disability disorders in the human population, but the role of ALKBH8 in the nervous system is unknown. Through in vivo studies in Drosophila , we show that ALKBH8 controls oxidative stress in the brain to restrain synaptic growth and support learning and memory. ALKBH8 null animals lack wobble uridine methylation and exhibit reduced protein synthesis in the nervous system, including a specific decrease in selenoprotein levels. Either loss of ALKBH8 or independent disruption of selenoprotein synthesis results in ectopic synapse formation. Genetic expression of antioxidant enzymes fully suppresses synaptic overgrowth in ALKBH8 null animals, confirming oxidative stress as the underlying cause of dysregulation. ALKBH8 null animals also exhibit associative memory impairments that are reversed by pharmacological antioxidant treatment. Together, these findings demonstrate the critical role of tRNA wobble uridine modification in redox homeostasis in the developing nervous system and reveal antioxidants as a potential therapy for ALKBH8-associated intellectual disability., Competing Interests: Competing interests statement:The authors declare no competing interest.

Retrons are bacterial genetic elements that encode a reverse transcriptase and, in combination with toxic effector proteins, can serve as antiphage defense systems. However, the mechanisms of action of most retron effectors, and how phages evade retrons, are not well understood. Here, we show that some phages can evade retrons and other defense systems by producing specific tRNAs. We find that expression of retron-Eco7 effector proteins (PtuA and PtuB) leads to degradation of tRNA Tyr and abortive infection. The genomes of T5 phages that evade retron-Eco7 include a tRNA-rich region, including a highly expressed tRNA Tyr gene, which confers protection against retron-Eco7. Furthermore, we show that other phages (T1, T7) can use a similar strategy, expressing a tRNA Lys , to counteract a tRNA anticodon defense system (PrrC170)., Competing Interests: Competing interests A.H.A., Y.T., K.W., and K.Kiga are co-inventors on a pending patent submitted by the National Institute of Infectious Diseases, which is based on the results reported in this paper. The remaining authors declare no competing interests., (© 2024. The Author(s).)

All known bacterial tRNAs adopt the canonical cloverleaf 2D and L-shaped 3D structures. We aimed to explore whether alternative tRNA structures could be introduced in bacterial translation. To this end, we crafted a vitamin-based genetic system to evolve Escherichia coli toward activity of structurally non-canonical tRNAs. The system reliably couples (escape frequency <10-12) growth with the activities of a novel orthogonal histidine suppressor tRNA (HisTUAC) and of the cognate ARS (HisS) via suppression of a GTA valine codon in the mRNA of an enzyme in thiamine biosynthesis (ThiN). Suppression results in the introduction of an essential histidine and thereby confers thiamine prototrophy. We then replaced HisTUAC in the system with non-canonical suppressor tRNAs and selected for growth. A strain evolved to utilize mini HisT, a tRNA lacking the D-arm, and we identified the responsible mutation in an RNase gene (pnp) involved in tRNA degradation. This indicated that HisS, the ribosome, and EF-Tu accept mini HisT ab initio, which we confirmed genetically and through in vitro translation experiments. Our results reveal a previously unknown flexibility of the bacterial translation machinery for the accepted fold of the adaptor of the genetic code and demonstrate the power of the vitamin-based suppression system., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Toxins of toxin-antitoxin systems use diverse mechanisms to inhibit bacterial growth. In this study, we characterize the translation inhibitor toxin MenT3 of Mycobacterium tuberculosis, the bacterium responsible for tuberculosis in humans. We show that MenT3 is a robust cytidine specific tRNA nucleotidyltransferase in vitro, capable of modifying the aminoacyl acceptor ends of most tRNA but with a marked preference for tRNA Ser , to which long stretches of cytidines are added. Furthermore, transcriptomic-wide analysis of MenT3 targets in M. tuberculosis identifies tRNA Ser as the sole target of MenT3 and reveals significant detoxification attempts by the essential CCA-adding enzyme PcnA in response to MenT3. Finally, under physiological conditions, only in the presence the native menAT3 operon, an active pool of endogenous MenT3 targeting tRNA Ser in M. tuberculosis is detected, likely reflecting the importance of MenT3 during infection., (© 2024. The Author(s).)

Pathogenic variants in subunits of RNA polymerase (Pol) III cause a spectrum of Polr3 -related neurodegenerative diseases including 4H leukodystrophy. Disease onset occurs from infancy to early adulthood and is associated with a variable range and severity of neurological and non-neurological features. The molecular basis of Polr3 -related disease pathogenesis is unknown. We developed a postnatal whole-body mouse model expressing pathogenic Polr3a mutations to examine the molecular mechanisms by which reduced Pol III transcription results primarily in central nervous system phenotypes. Polr3a mutant mice exhibit behavioral deficits, cerebral pathology and exocrine pancreatic atrophy. Transcriptome and immunohistochemistry analyses of cerebra during disease progression show a reduction in most Pol III transcripts, induction of innate immune and integrated stress responses and cell-type-specific gene expression changes reflecting neuron and oligodendrocyte loss and microglial activation. Earlier in the disease when integrated stress and innate immune responses are minimally induced, mature tRNA sequencing revealed a global reduction in tRNA levels and an altered tRNA profile but no changes in other Pol III transcripts. Thus, changes in the size and/or composition of the tRNA pool have a causal role in disease initiation. Our findings reveal different tissue- and brain region-specific sensitivities to a defect in Pol III transcription., Competing Interests: RM, EM, VC, ES, IW No competing interests declared, (© 2024, Moir, Merheb et al.)

Mutations modification enzymes including the tRNA N 7 -methylguanosine (m 7 G) methyltransferase complex component WDR4 were frequently found in patients with neural disorders, while the pathogenic mechanism and therapeutic intervention strategies are poorly explored. In this study, we revealed that patient-derived WDR4 mutation leads to temporal and cell-type-specific neural degeneration, and directly causes neural developmental disorders in mice. Mechanistically, WDR4 point mutation disrupts the interaction between WDR4 and METTL1 and accelerates METTL1 protein degradation. We further uncovered that impaired tRNA m 7 G modification caused by Wdr4 mutation decreases the mRNA translation of genes involved in mTOR pathway, leading to elevated endoplasmic reticulum stress markers, and increases neural cell apoptosis. Importantly, treatment with stress-attenuating drug Tauroursodeoxycholate (TUDCA) significantly decreases neural cell death and improves neural functions of the Wdr4 mutated mice. Moreover, adeno-associated virus mediated transduction of wild-type WDR4 restores METTL1 protein level and tRNA m 7 G modification in the mouse brain, and achieves long-lasting therapeutic effect in Wdr4 mutated mice. Most importantly, we further demonstrated that both TUDCA treatment and WDR4 restoration significantly improve the survival and functions of human iPSCs-derived neuron stem cells that harbor the patient's WDR4 mutation. Overall, our study uncovers molecular insights underlying WDR4 mutation in the pathogenesis of neural diseases and develops two promising therapeutic strategies for treatment of neural diseases caused by impaired tRNA modifications., Competing Interests: Competing interests statement:The authors declare no competing interest.

Transfer RNA-derived small RNAs (tsRNAs) - categorized as tRNA-derived fragments (tRFs), tRNA-derived stress-induced RNAs (tiRNAs) and internal tRF (itRF) - are small non-coding RNAs that participate in various cellular processes such as translation inhibition and responses to cellular stress. We here identified tsRNA profiles within susceptible tissues in animal models of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Parkinson's disease (PD) to pinpoint disease-specific tsRNAs and those shared across neurodegenerative diseases. We performed small RNA sequencing in the SOD1G93A and TDP43A315T mouse models of ALS (spinal cord), the TauP301S model of FTD (hippocampus), and the parkin/POLG model of PD (substantia nigra). Bioinformatic analysis showed higher expression of 5' tiRNAs selectively in the two ALS models, lower expression of 3' tRFs in both the ALS and FTD mouse models, and lower expression of itRF Arg in the PD model. Experimental validation confirmed the expression of tsRNAs. Gene Ontology analysis of targets associated with validated 3' tRFs indicated functions in the regulation of synaptic and neuronal pathways. Our profiling of tsRNAs indicates disease-specific fingerprints in animal models of neurodegeneration, which require validation in human disease., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Embryogenesis entails dramatic shifts in mRNA translation and turnover that reprogram gene expression during cellular proliferation and differentiation. Codon identity modulates mRNA stability during early vertebrate embryogenesis, but how the composition of tRNA pools is matched to translational demand is unknown. By quantitative profiling of tRNA repertoires in zebrafish embryos during the maternal-to-zygotic transition, we show that zygotic tRNA repertoires are established after the onset of gastrulation, succeeding the major wave of zygotic mRNA transcription. Maternal and zygotic tRNA pools are distinct, but their reprogramming does not result in a better match to the codon content of the zygotic transcriptome. Instead, we find that an increase in global translation at gastrulation sensitizes decoding rates to tRNA supply, thus destabilizing maternal mRNAs enriched in slowly translated codons. Translational activation and zygotic tRNA expression temporally coincide with an increase of TORC1 activity at gastrulation, which phosphorylates and inactivates the RNA polymerase III repressor Maf1a/b. Our data indicate that a switch in global translation, rather than tRNA reprogramming, determines the onset of codon-dependent maternal mRNA decay during zebrafish embryogenesis., Competing Interests: Disclosure and competing interests statement AB and DDN are inventors on a patent application filed by the Max Planck Society pertaining to the mim-tRNAseq technology. The other authors declare no competing interests., (© 2024. The Author(s).)

Leber's hereditary optic neuropathy (LHON), a maternally inherited ocular disease, is predominantly caused by mitochondrial DNA (mtDNA) mutations. Mitochondrial tRNA variants are hypothesized to amplify the pathogenic impact of three primary mutations. However, the exact mechanisms remained unclear. In the present study, the synergistic effect of the tRNA Glu 14693A > G and ND6 14484T > C mutations in three Chinese families affected by LHON is investigated. The m.14693A > G mutation nearly abolishes the pseudouridinylation at position 55 of tRNA Glu , leading to structural abnormalities, decreased stability, aberrant mitochondrial protein synthesis, and increased autophagy. In contrast, the ND6 14484T > C mutation predominantly impairs complex I function, resulting in heightened apoptosis and virtually no induction of mitochondrial autophagy compared to control cell lines. The presence of dual mutations in the same cell lines exhibited a coexistence of both upregulated cellular stress responses to mitochondrial damage, indicating a scenario of autophagy and mutation dysregulation within these dual-mutant cell lines. The data proposes a novel hypothesis that mitochondrial tRNA gene mutations generally lead to increased mitochondrial autophagy, while mutations in genes encoding mitochondrial proteins typically induce apoptosis, shedding light on the intricate interplay between different genetic factors in the manifestation of LHON., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Hybrid rice, widely planted in Asia, is pathogen resistant and has superior yields, making it a major contributor to global food security. The two-line hybrid rice system, which utilizes mutants exhibiting photo-/thermo-sensitive genic male sterility (P/TGMS), is the leading hybrid rice breeding technology. Mutations in THERMO-SENSITIVE GENIC MALE STERILE 5 (TMS5) accounts for over 95% of current TGMS lines. We previously found that tms5 carries a mutation in ribonuclease Z S1 . Despite its importance for breeding robust rice lines, the mechanism underlying tms5-mediated TGMS remains elusive. Here, we demonstrate that TMS5 is a tRNA 2',3'-cyclic phosphatase. The tms5 mutation leads to accumulation of 2',3'-cyclic phosphate (cP)-ΔCCA-tRNAs (tRNAs without 3' CCA ended with cP), which is exacerbated by high temperatures, and reduction in the abundance of mature tRNAs, particularly alanine tRNAs (tRNA-Alas). Overexpression of tRNA-Alas in the tms5 mutant restores male fertility to 70%. Remarkably, male fertility of tms5 mutant is completely restored at high temperatures by knocking out OsVms1 which encodes the enzyme for cP-ΔCCA-tRNA generation. Our study reveals the mechanism underlying tms5-mediated TGMS in rice and provides mechanistic insight into the further improvement of TGMS in hybrid crop development., (© 2024. The Author(s) under exclusive licence to Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences.)

The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) long noncoding RNA (lncRNA) has key roles in regulating transcription, splicing, tumorigenesis, etc. Its maturation and stabilization require precise processing by RNase P, which simultaneously initiates the biogenesis of a 3' cytoplasmic MALAT1-associated small cytoplasmic RNA (mascRNA). mascRNA was proposed to fold into a transfer RNA (tRNA)-like secondary structure but lacks eight conserved linking residues required by the canonical tRNA fold. Here we report crystal structures of human mascRNA before and after processing, which reveal an ultracompact, quasi-tRNA-like structure. Despite lacking all linker residues, mascRNA faithfully recreates the characteristic 'elbow' feature of tRNAs to recruit RNase P and ElaC homolog protein 2 (ELAC2) for processing, which exhibit distinct substrate specificities. Rotation and repositioning of the D-stem and anticodon regions preclude mascRNA from aminoacylation, avoiding interference with translation. Therefore, a class of metazoan lncRNA loci uses a previously unrecognized, unusually streamlined quasi-tRNA architecture to recruit select tRNA-processing enzymes while excluding others to drive bespoke RNA biogenesis, processing and maturation., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

NEAT1 long noncoding RNA orchestrates paraspeckle assembly and impacts tumorigenesis, fertility and immunity. Its maturation requires RNase P cleavage yielding an unstable transfer RNA-like multiple endocrine neoplasia-β tRNA-like transcript (menRNA) due to CCACCA addition. Here we report the crystal structure of human menRNA, which partially mimics tRNAs to drive RNase P and ELAC2 processing. Biophysical analyses uncover an RNA-centric, riboswitch-like mechanism whereby the nascent CCA reshapes the RNA folding landscape and propels a spontaneous conformational isomerization that directs repeat CCA addition, marking the menRNA and defective tRNAs for degradation., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Site-specific incorporation of noncanonical amino acids (ncAAs) can be realized by genetic code expansion (GCE) technology. Different orthogonal tRNA synthetase/tRNA (RS/tRNA) pairs have been developed to introduce a ncAA at the desired site, delivering a wide variety of functionalities that can be installed into selected proteins. Cytoplasmic expression of RS/tRNA pairs can cause a problem with background ncAA incorporation into host proteins. The application of orthogonally translating organelles (OTOs), inspired by the concept of phase separation, provides a solution for this issue in mammalian cells, allowing site-specific and protein-selective ncAA incorporation. So far, only Methanosarcina mazei (Mm) pyrrolysyl-tRNA synthetase (PylRS) has been used within OTOs, limiting the method's potential. Here, we explored the implementation of four other widely used orthogonal RS/tRNA pairs with OTOs, which, to our surprise, were unsuccessful in generating mRNA-selective GCE. Next, we tested several experimental solutions and developed a new chimeric phenylalanyl-RS/tRNA pair that enables ncAA incorporation in OTOs in a site-specific and protein-selective manner. Our work reveals unaccounted design constraints in the spatial engineering of enzyme functions using designer organelles and presents a strategy to overcome those in vivo. We then discuss current limitations and future directions of in-cell engineering in general and protein engineering using GCE specifically., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: “E.A.L. holds patents related to OTOs. M.E.S. and M.J. declare no competing interests.”., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The expansion of VapBC TA systems in M. tuberculosis has been linked with its fitness and survival upon exposure to stress conditions. Here, we have functionally characterized VapBC13 and VapBC26 TA modules of M. tuberculosis. We report that overexpression of VapC13 and VapC26 toxins in M. tuberculosis results in growth inhibition and transcriptional reprogramming. We have also identified various regulatory proteins as hub nodes in the top response network of VapC13 and VapC26 overexpression strains. Further, analysis of RNA protection ratios revealed potential tRNA targets for VapC13 and VapC26. Using in vitro ribonuclease assays, we demonstrate that VapC13 and VapC26 degrade serT and leuW tRNA, respectively. However, no significant changes in rRNA cleavage profiles were observed upon overexpression of VapC13 and VapC26 in M. tuberculosis. In order to delineate the role of these TA systems in M. tuberculosis physiology, various mutant strains were constructed. We show that in comparison to the parental strain, ΔvapBC13 and ΔvapBC26 strains were mildly susceptible to oxidative stress. Surprisingly, the growth patterns of parental and mutant strains were comparable in aerosol-infected guinea pigs. These observations imply that significant functional redundancy exists for some TA systems from M. tuberculosis., (© 2024. The Author(s).)

Codon bias analysis of SARS-CoV-2 reveals suboptimal adaptation for translation in human cells it infects. The detailed examination of the codons preferentially used by SARS-CoV-2 shows a strong preference for Lys AAA , Gln CAA , Glu GAA , and Arg AGA , which are infrequently used in human genes. In the absence of an adapted tRNA pool, efficient decoding of these codons requires a 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 ) modification at the U 34 wobble position of the corresponding tRNAs (tLys UUU ; tGln UUG ; tGlu UUC ; tArg UCU ). The optimal translation of SARS-CoV-2 open reading frames (ORFs) may therefore require several adjustments to the host's translation machinery, enabling the highly biased viral genome to achieve a more favorable "Ready-to-Translate" state in human cells. Experimental approaches based on LC-MS/MS quantification of tRNA modifications and on alteration of enzymatic tRNA modification pathways provide strong evidence to support the hypothesis that SARS-CoV-2 induces U 34 tRNA modifications and relies on these modifications for its lifecycle. The conclusions emphasize the need for future studies on the evolution of SARS-CoV-2 codon bias and its ability to alter the host tRNA pool through the manipulation of RNA modifications.

Transfer RNAs (tRNAs) contain dozens of chemical modifications. These modifications are critical for maintaining tRNA tertiary structure and optimizing protein synthesis. Here we advance the use of Nanopore direct RNA-sequencing (DRS) to investigate the synergy between modifications that are known to stabilize tRNA structure. We sequenced the 42 cytosolic tRNA isoacceptors from wild-type yeast and five tRNA-modifying enzyme knockout mutants. These data permitted comprehensive analysis of three neighboring and conserved modifications in T-loops: 5-methyluridine (m5U54), pseudouridine (Ψ55), and 1-methyladenosine (m1A58). Our results were validated using direct measurements of chemical modifications by mass spectrometry. We observed concerted T-loop modification circuits-the potent influence of Ψ55 for subsequent m1A58 modification on more tRNA isoacceptors than previously observed. Growing cells under nutrient depleted conditions also revealed a novel condition-specific increase in m1A58 modification on some tRNAs. A global and isoacceptor-specific classification strategy was developed to predict the status of T-loop modifications from a user-input tRNA DRS dataset, applicable to other conditions and tRNAs in other organisms. These advancements demonstrate how orthogonal technologies combined with genetics enable precise detection of modification landscapes of individual, full-length tRNAs, at transcriptome-scale., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

EF-P is a translation factor that facilitates the formation of peptide bonds between consecutive prolines. Using FRET between EF-P and ribosomal protein bL33, we studied dynamics and specificity of EF-P binding to the ribosome. Our findings reveal that EF-P rapidly scans for a free E site and can bind to any ribosome containing a P-site tRNA, regardless of the ribosome's functional state. The interaction with uL1 is essential for EF-P binding, while the β-Lys modification of EF-P doubles the association rate. Specific interactions with the D-loop of tRNAPro or tRNAfMet and via the β-Lys group with the tRNA in the peptidyl transferase center reduce the rate of EF-P dissociation from the ribosome, providing the specificity for complexes that need help in catalyzing peptide bond formation. The nature of the E-site codon has little effect on EF-P binding kinetics. Although EF-P dissociation is reduced upon recognizing its correct tRNA substrate, it remains sufficiently rapid compared to tRNA translocation and does not affect the translocation rate. These results highlight the importance of EF-P's scanning-engagement mechanism for dynamic substrate recognition during rapid translation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ribosomal translation at the origin of life requires controlled aminoacylation to produce mono-aminoacyl esters of tRNAs. Herein, we show that transient annealing of short RNA oligo:amino acid mixed anhydrides to an acceptor strand enables the sequential transfer of aminoacyl residues to the diol of an overhang, first forming aminoacyl esters then peptidyl esters. Using N-protected aminoacyl esters prevents unwanted peptidyl ester formation in this manner. However, N-acyl-aminoacyl transfer is not stereoselective., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

tRNAs are evolutionarily ancient molecular decoders essential for protein translation. In eukaryotes, tRNAs and other short, noncoding RNAs are transcribed by RNA polymerase (Pol) III, an enzyme that promotes ageing in yeast, worms, and flies. Here, we show that a partial reduction in Pol III activity specifically disrupts tRNA levels. This effect is conserved across worms, flies, and mice, where computational models indicate that it impacts mRNA decoding. In all 3 species, reduced Pol III activity increases proteostatic resilience. In worms, it activates the unfolded protein response (UPR) and direct disruption of tRNA metabolism is sufficient to recapitulate this. In flies, decreasing Pol III's transcriptional initiation on tRNA genes by a loss-of-function in the TFIIIC transcription factor robustly extends lifespan, improves proteostatic resilience and recapitulates the broad-spectrum benefits to late-life health seen following partial Pol III inhibition. We provide evidence that a partial reduction in Pol III activity impacts translation, quantitatively or qualitatively, in both worms and flies, indicating a potential mode of action. Our work demonstrates a conserved and previously unappreciated role of tRNAs in animal ageing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Malik 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.)

Ribonuclease P (RNase P) plays a vital role in the maturation of tRNA across bacteria, archaea, and eukaryotes. However, how RNase P assembles various components to achieve specific cleavage of precursor tRNA (pre-tRNA) in different organisms remains elusive. In this study, we employed single-molecule fluorescence resonance energy transfer to probe the dynamics of RNase P from E. coli ( Escherichia coli ) and Mja ( Methanocaldococcus jannaschii ) during pre-tRNA cleavage by incorporating five Cy3-Cy5 pairs into pre-tRNA and RNase P. Our results revealed significant differences in the assembly and catalytic mechanisms of RNase P between E. coli and Mja at both the RNA and protein levels. Specifically, the RNA of E. coli RNase P ( Eco RPR) can adopt an active conformation that is capable of binding and cleaving pre-tRNA with high specificity independently. The addition of the protein component of E. coli RNase P (RnpA) enhances and accelerates pre-tRNA cleavage efficiency by increasing and stabilizing the active conformation. In contrast, Mja RPR is unable to form the catalytically active conformation on its own, and at least four proteins are required to induce the correct folding of Mja RPR. Mutation experiments suggest that the functional deficiency of Mja RPR arises from the absence of the second structural layer, and proper intermolecular assembly is essential for Mja RNase P to be functional over a broad temperature range. We propose models to illustrate the distinct catalytic patterns and RNA-protein interactions of RNase P in these two organisms., Competing Interests: Competing interests statement:The authors declare no competing interest.

As the most ancient RNA, transfer RNAs (tRNAs) play a more complex role than their constitutive function as amino acid transporters in the protein synthesis process. The transcription and maturation of tRNA in cells are subject to stringent regulation, resulting in the formation of tissue- and cell-specific tRNA pools with variations in tRNA overall abundance, composition, modification, and charging levels. The heterogeneity of tRNA pools contributes to facilitating the formation of histocyte-specific protein expression patterns and is involved in diverse biological processes. Moreover, tRNAs can be recognized by various RNase under physiological and pathological conditions to generate tRNA-derived small RNAs (tsRNAs) and serve as small regulatory RNAs in various biological processes. Here, we summarize these recent insights into the heterogeneity of tRNA and highlight the advances in the regulation of tRNA function and tsRNA biogenesis by tRNA modifications. We synthesize diverse mechanisms of tRNA and tsRNA in embryonic development, cell fate determination, and epigenetic inheritance regulation. We also discuss the potential clinical applications based on the new knowledge of tRNA and tsRNA as diagnostic and prognostic biomarkers and new therapeutic strategies for multiple diseases.

Human mitochondrial tRNAs (mt-tRNAs), critical for mitochondrial biogenesis, are frequently associated with pathogenic mutations. These mt-tRNAs have unusual sequence motifs and require post-transcriptional modifications to stabilize their fragile structures. However, whether a modification that stabilizes a wild-type (WT) mt-tRNA would also stabilize its pathogenic variants is unknown. Here we show that the N 1 -methylation of guanosine at position 9 (m 1 G9) of mt-Leu(UAA), while stabilizing the WT tRNA, has a destabilizing effect on variants associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). This differential effect is further demonstrated, as removal of the m 1 G9 methylation, while damaging to the WT tRNA, is beneficial to the major pathogenic variant, improving the structure and activity of the variant. These results have therapeutic implications, suggesting that the N 1 -methylation of mt-tRNAs at position 9 is a determinant of pathogenicity and that controlling the methylation level is an important modulator of mt-tRNA-associated diseases., (© 2024. The Author(s).)

Nonuniform local translation speed dictates diverse protein biogenesis outcomes. To unify known and uncover unknown principles governing eukaryotic elongation rate, we developed a machine learning pipeline to analyze RiboSeq datasets. We find that the chemical nature of the incoming amino acid determines how codon optimality influences elongation rate, with hydrophobic residues more dependent on transfer RNA (tRNA) levels than charged residues. Unexpectedly, we find that wobble interactions exert a widespread effect on elongation pausing, with wobble-mediated decoding being slower than Watson-Crick decoding, irrespective of tRNA levels. Applying our ribosome pausing principles to ribosome collisions reveals that disomes arise upon apposition of fast-decoding and slow-decoding signatures. We conclude that codon choice and tRNA pools are evolutionarily constrained to harmonize elongation rate with cotranslational folding while minimizing wobble pairing and deleterious stalling.

5-Methylcytosine (5mC) is an established epigenetic mark in vertebrate genomic DNA, but whether its oxidation intermediates formed during TET-mediated DNA demethylation possess an instructive role of their own that is also physiologically relevant remains unresolved. Here, we reveal a 5-formylcytosine (5fC) nuclear chromocenter, which transiently forms during zygotic genome activation (ZGA) in Xenopus and mouse embryos. We identify this chromocenter as the perinucleolar compartment, a structure associated with RNA Pol III transcription. In Xenopus embryos, 5fC is highly enriched on Pol III target genes activated at ZGA, notably at oocyte-type tandem arrayed tRNA genes. By manipulating Tet and Tdg enzymes, we show that 5fC is required as a regulatory mark to promote Pol III recruitment as well as tRNA expression. Concordantly, 5fC modification of a tRNA transgene enhances its expression in vivo. The results establish 5fC as an activating epigenetic mark during zygotic reprogramming of Pol III gene expression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here, we identify C12orf29 (RLIG1) as an atypical ATP-grasp enzyme that ligates RNA. Human RLIG1 and its homologs autoadenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5'-phosphate and a 3'-hydroxyl. RLIG1 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro. Transcriptomic analyses of Rlig1 knockout mice revealed significant alterations in global tRNA levels in the brains of female mice, but not in those of male mice. Furthermore, crystal structures of a RLIG1 homolog from Yasminevirus bound to nucleotides revealed a minimal and atypical RNA ligase fold with a conserved active site architecture that participates in catalysis. Collectively, our results identify RLIG1 as an RNA ligase and suggest its involvement in tRNA biology., Competing Interests: Competing interests statement:The authors declare no competing interest.

tRNA-derived fragments (tRFs) are novel small noncoding RNAs (sncRNAs) that range from approximately 14 to 50 nt. They are generated by the cleavage of mature tRNAs or precursor tRNAs (pre-tRNAs) at specific sites. Based on their origin and length, tRFs can be classified into three categories: (1) tRF-1 s; (2) tRF-3 s, tRF-5 s, and internal tRFs (i-tRFs); and (3) tRNA halves. They play important roles in stress response, signal transduction, and gene expression processes. Recent studies have identified differential expression of tRFs in various tumors. Aberrantly expressed tRFs have critical clinical value and show promise as new biomarkers for tumor diagnosis and prognosis and as therapeutic targets. tRFs regulate the malignant progression of tumors via various mechanisms, primarily including modulation of noncoding RNA biogenesis, global chromatin organization, gene expression regulation, modulation of protein translation, regulation of epigenetic modification, and alternative splicing regulation. In conclusion, tRF-mediated regulatory pathways could present new avenues for tumor treatment, and tRFs could serve as promising therapeutic targets for cancer therapy., (© 2024 UICC.)

RTP801/REDD1 is a stress-responsive protein overexpressed in neurodegenerative diseases such as Alzheimer's disease (AD) that contributes to cognitive deficits and neuroinflammation. Here, we found that RTP801 interacts with HSPC117, DDX1 and CGI-99, three members of the tRNA ligase complex (tRNA-LC), which ligates the excised exons of intron-containing tRNAs and the mRNA exons of the transcription factor XBP1 during the unfolded protein response (UPR). We also found that RTP801 modulates the mRNA ligase activity of the complex in vitro since RTP801 knockdown promoted XBP1 splicing and the expression of its transcriptional target, SEC24D. Conversely, RTP801 overexpression inhibited the splicing of XBP1. Similarly, in human AD postmortem hippocampal samples, where RTP801 is upregulated, we found that XBP1 splicing was dramatically decreased. In the 5xFAD mouse model of AD, silencing RTP801 expression in hippocampal neurons promoted Xbp1 splicing and prevented the accumulation of intron-containing pre-tRNAs. Finally, the tRNA-enriched fraction obtained from 5xFAD mice promoted abnormal dendritic arborization in cultured hippocampal neurons, and RTP801 silencing in the source neurons prevented this phenotype. Altogether, these results show that elevated RTP801 impairs RNA processing in vitro and in vivo in the context of AD and suggest that RTP801 inhibition could be a promising therapeutic approach., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Due to its high modification content tRNAs are notoriously hard to quantify by reverse transcription and RNAseq. Bypassing numerous biases resulting from concatenation of enzymatic treatments, we here report a hybrid approach that harnesses the advantages of hybridization-based and deep sequencing-based approaches. The method renders obsolete any RNAseq related workarounds and correction factors that affect accuracy, sensitivity, and turnaround time. Rather than by reverse transcription, quantitative information on the isoacceptor composition of a tRNA pool is transferred to a cDNA mixture in a single step procedure, thereby omitting all enzymatic conversations except for the subsequent barcoding PCR. As a result, a detailed tRNA composition matrix can be obtained from femtomolar amounts of total tRNA. The method is fast, low in cost, and its bioinformatic data workup surprisingly simple. These properties make the approach amenable to high-throughput investigations including clinical samples, as we have demonstrated by application to a collection of variegated biological questions, each answered with novel findings. These include tRNA pool quantification of polysome-bound tRNA, of tRNA modification knockout strains under stress conditions, and of Alzheimer patients' brain tissues., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Adenosine-to-inosine (A-to-I) RNA editing plays an important role in the post-transcriptional regulation of eukaryotic cell physiology. However, our understanding of the occurrence, function and regulation of A-to-I editing in bacteria remains limited. Bacterial mRNA editing is catalysed by the deaminase TadA, which was originally described to modify a single tRNA in Escherichia coli. Intriguingly, several bacterial species appear to perform A-to-I editing on more than one tRNA. Here, we provide evidence that in the human pathogen Streptococcus pyogenes, tRNA editing has expanded to an additional tRNA substrate. Using RNA sequencing, we identified more than 27 editing sites in the transcriptome of S. pyogenes SF370 and demonstrate that the adaptation of S. pyogenes TadA to a second tRNA substrate has also diversified the sequence context and recoding scope of mRNA editing. Based on the observation that editing is dynamically regulated in response to several infection-relevant stimuli, such as oxidative stress, we further investigated the underlying determinants of editing dynamics and identified mRNA stability as a key modulator of A-to-I editing. Overall, our findings reveal the presence and diversification of A-to-I editing in S. pyogenes and provide novel insights into the plasticity of the editome and its regulation in bacteria., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Anti-phage defenses must rapidly sense and respond to diverse viruses. A recent pair of papers in Nature reveal via structural and functional assays how the PARIS defense system, a recently discovered toxin-antitoxin system, senses phage-associated molecular patterns (PhAMPs), thereby activating an endonuclease toxin that cleaves tRNA to block phage replication., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aminoacyl-tRNA synthetases hold the key to the genetic code and assign nucleic acid-based codons to amino acids, the building blocks of proteins. In their ability to recognize identity elements on transfer RNAs (tRNAs), some as simple as a single base pair, they ensure that the same proteins are formed each time information embedded in DNA is transcribed into messenger RNA (mRNA) and translated into proteins (Figure 1A). Thus, aminoacyl-tRNA synthetase active sites are conserved; however, since their evolutionary origin, their functions have been co-opted, expanded on and played novel roles during evolution. Below, we provide an overview of the many functions of aminoacyl-tRNA synthetases - from their role in translation, one of the most fundamental processes of all life, to newly discovered, diverse functions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Understanding the molecular circuitries that govern early embryogenesis is important, yet our knowledge of these in human preimplantation development remains limited. Small non-coding RNAs (sncRNAs) can regulate gene expression and thus impact blastocyst formation, however, the expression of specific biotypes and their dynamics during preimplantation development remains unknown. Here we identify the abundance of and kinetics of piRNA, rRNA, snoRNA, tRNA, and miRNA from embryonic day (E)3-7 and isolate specific miRNAs and snoRNAs of particular importance in blastocyst formation and pluripotency. These sncRNAs correspond to specific genomic hotspots: an enrichment of the chromosome 19 miRNA cluster (C19MC) in the trophectoderm (TE), and the chromosome 14 miRNA cluster (C14MC) and MEG8-related snoRNAs in the inner cell mass (ICM), which may serve as 'master regulators' of potency and lineage. Additionally, we observe a developmental transition with 21 isomiRs and in tRNA fragment (tRF) codon usage and identify two novel miRNAs. Our analysis provides a comprehensive measure of sncRNA biotypes and their corresponding dynamics throughout human preimplantation development, providing an extensive resource. Better understanding the sncRNA regulatory programmes in human embryogenesis will inform strategies to improve embryo development and outcomes of assisted reproductive technologies. We anticipate broad usage of our data as a resource for studies aimed at understanding embryogenesis, optimising stem cell-based models, assisted reproductive technology, and stem cell biology., (© 2024. The Author(s).)

Despite the numerous sequencing methods available, the diversity in RNA size and chemical modification makes it difficult to capture all RNAs in a cell. We developed a method that combines quasi-random priming with template switching to construct sequencing libraries from RNA molecules of any length and with any type of 3' modifications, allowing for the sequencing of virtually all RNA species. Our ligation-independent detection of all types of RNA (LIDAR) is a simple, effective tool to identify and quantify all classes of coding and non-coding RNAs. With LIDAR, we comprehensively characterized the transcriptomes of mouse embryonic stem cells, neural progenitor cells, mouse tissues, and sperm. LIDAR detected a much larger variety of tRNA-derived RNAs (tDRs) compared with traditional ligation-dependent sequencing methods and uncovered tDRs with blocked 3' ends that had previously escaped detection. Therefore, LIDAR can capture all RNAs in a sample and uncover RNA species with potential regulatory functions., Competing Interests: Declaration of interests J.E.W. serves as a consultant for Laronde., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Staphylococcus aureus is a major contributor to bacterial-associated mortality, owing to its exceptional adaptability across diverse environments. Iron is vital to most organisms but can be toxic in excess. To manage its intracellular iron, S. aureus , like many pathogens, employs intricate systems. We have recently identified IsrR as a key regulatory RNA induced during iron starvation. Its role is to reduce the synthesis of non-essential iron-containing proteins under iron-depleted conditions. In this study, we unveil IsrR's regulatory action on MiaB, an enzyme responsible for methylthio group addition to specific sites on transfer RNAs (tRNAs). We use predictive tools and reporter fusion assays to demonstrate IsrR's binding to the Shine-Dalgarno sequence of miaB RNA, thereby impeding its translation. The effectiveness of IsrR hinges on the integrity of a specific C-rich region. As MiaB is non-essential and has iron-sulfur clusters, IsrR induction spares iron by downregulating miaB . This may improve S. aureus fitness and aid in navigating the host's nutritional immune defenses.IMPORTANCEIn many biotopes, including those found within an infected host, bacteria confront the challenge of iron deficiency. They employ various strategies to adapt to this scarcity of nutrients, one of which involves regulating iron-containing proteins through the action of small regulatory RNAs. Our study shows how IsrR, a small RNA from S. aureus , prevents the production of MiaB, a tRNA-modifying enzyme containing iron-sulfur clusters. With this illustration, we propose a new substrate for an iron-sparing small RNA, which, when downregulated, should reduce the need for iron and save it to essential functions., Competing Interests: The authors declare no conflict of interest.

Transfer-RNA-derived fragments (tRFs) are a novel class of small non-coding RNAs that have been implicated in oncogenesis. tRFs may act as post-transcriptional regulators by recruiting AGO proteins and binding to highly complementary regions of mRNA at seed regions, resulting in the knockdown of the transcript. Therefore, tRFs may be critical to tumorigenesis and warrant investigation as potential biomarkers. Meanwhile, the incidence of papillary thyroid carcinoma (PTC) has increased in recent decades and current diagnostic technology stands to benefit from new detection methods. Although small non-coding RNAs have been studied for their role in oncogenesis, there is currently no standard for their use as PTC biomarkers, and tRFs are especially underexplored. Accordingly, we aim to identify dysregulated tRFs in PTC that may serve as biomarker candidates. We identified dysregulated tRFs and driver genes between PTC primary tumor samples (n = 511) and adjacent normal tissue samples (n = 59). Expression data were obtained from MINTbase v2.0 and The Cancer Genome Atlas. Dysregulated tRFs and genes were analyzed in tandem to find pairs with anticorrelated expression. Significantly anticorrelated tRF-gene pairs were then tested for potential binding affinity using RNA22-if a heteroduplex can form via complementary binding, this would support the hypothesized RNA silencing mechanism. Four tRFs were significantly dysregulated in PTC tissue ( p < 0.05), with only AsnGTT 3'-tRF being upregulated. Binding affinity analysis revealed that tRF-30-RY73W0K5KKOV (AsnGTT 3'-tRF) exhibits sufficient complementarity to potentially bind to and regulate transcripts of SLC26A4, SLC5A8, DIO2, and TPO, which were all found to be downregulated in PTC tissue. In the present study, we identified dysregulated tRFs in PTC and found that AsnGTT 3'-tRF is a potential post-transcriptional regulator and biomarker.

Human cytoplasmic tRNAs contain dihydrouridine modifications at positions 16 and 17 (D16/D17). The enzyme responsible for D16/D17 formation and its cellular roles remain elusive. Here, we identify DUS1L as the human tRNA D16/D17 writer. DUS1L knockout in the glioblastoma cell lines LNZ308 and U87 causes loss of D16/D17. D formation is reconstituted in vitro using recombinant DUS1L in the presence of NADPH or NADH. DUS1L knockout/overexpression in LNZ308 cells shows that DUS1L supports cell growth. Moreover, higher DUS1L expression in glioma patients is associated with poorer prognosis. Upon vector-mediated DUS1L overexpression in LNZ308 cells, 5' and 3' processing of precursor tRNA Tyr(GUA) is inhibited, resulting in a reduced mature tRNA Tyr(GUA) level, reduced translation of the tyrosine codons UAC and UAU, and reduced translational readthrough of the near-cognate stop codons UAA and UAG. Moreover, DUS1L overexpression increases the amounts of several D16/D17-containing tRNAs and total cellular translation. Our study identifies a human dihydrouridine writer, providing the foundation to study its roles in health and disease., (© 2024. The Author(s).)

Retrotransposons are mobile DNA elements that are more active with increasing age and exacerbate aging phenotypes in multiple species. We previously reported an unexpected extension of chronological lifespan in the yeast, Saccharomyces paradoxus , due to the presence of Ty1 retrotransposons when cells were aged under conditions of mild stress. In this study, we tested a subset of genes identified by RNA-seq to be differentially expressed in S. paradoxus strains with a high-copy number of Ty1 retrotransposons compared with a strain with no retrotransposons and additional candidate genes for their contribution to lifespan extension when cells were exposed to a moderate dose of hydroxyurea (HU). Deletion of ADE8 , NCS2 , or TRM9 prevented lifespan extension, while deletion of CDD1 , HAC1 , or IRE1 partially prevented lifespan extension. Genes overexpressed in high-copy Ty1 strains did not typically have Ty1 insertions in their promoter regions. We found that silencing genomic copies of Ty1 prevented lifespan extension, while expression of Ty1 from a high-copy plasmid extended lifespan in medium with HU or synthetic medium. These results indicate that cells adapt to expression of retrotransposons by changing gene expression in a manner that can better prepare them to remain healthy under mild stress.

Methylation of cytosine 32 in the anticodon loop of tRNAs to 3-methylcytosine (m 3 C) is crucial for cellular translation fidelity. Misregulation of the RNA methyltransferases setting this modification can cause aggressive cancers and metabolic disturbances. Here, we report the cryo-electron microscopy structure of the human m 3 C tRNA methyltransferase METTL6 in complex with seryl-tRNA synthetase (SerRS) and their common substrate tRNA Ser . Through the complex structure, we identify the tRNA-binding domain of METTL6. We show that SerRS acts as the tRNA Ser substrate selection factor for METTL6. We demonstrate that SerRS augments the methylation activity of METTL6 and that direct contacts between METTL6 and SerRS are necessary for efficient tRNA Ser methylation. Finally, on the basis of the structure of METTL6 in complex with SerRS and tRNA Ser , we postulate a universal tRNA-binding mode for m 3 C RNA methyltransferases, including METTL2 and METTL8, suggesting that these mammalian paralogs use similar ways to engage their respective tRNA substrates and cofactors., (© 2024. The Author(s).)

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 (s 2 U), 4-thiouridine (s 4 U), 2-thiocytidine (s 2 C), and 2-methylthioadenosine (ms 2 A), 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., 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. Published by Elsevier B.V.)

N 4 -acetylcytidine (ac 4 C), a conserved but recently rediscovered RNA modification on tRNAs, rRNAs and mRNAs, is catalyzed by N-acetyltransferase 10 (NAT10). Lysine acylation is a ubiquitous protein modification that controls protein functions. Our latest study demonstrates a NAT10-dependent ac 4 C modification, which occurs on the polyadenylated nuclear RNA (PAN) encoded by oncogenic DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV), can induce KSHV reactivation from latency and activate inflammasome. However, it remains unclear whether a novel lysine acylation occurs in NAT10 during KSHV reactivation and how this acylation of NAT10 regulates tRNAs ac 4 C modification. Here, we showed that NAT10 was lactylated by α-tubulin acetyltransferase 1 (ATAT1), as a writer at the critical domain, to exert RNA acetyltransferase function and thus increase the ac 4 C level of tRNA Ser-CGA-1-1 . Mutagenesis at the ac 4 C site in tRNA Ser-CGA-1-1 inhibited its ac 4 C modifications, translation efficiency of viral lytic genes, and virion production. Mechanistically, KSHV PAN orchestrated NAT10 and ATAT1 to enhance NAT10 lactylation, resulting in tRNA Ser-CGA-1-1 ac 4 C modification, eventually boosting KSHV reactivation. Our findings reveal a novel post-translational modification in NAT10, as well as expand the understanding about tRNA-related ac 4 C modification during KSHV replication, which may be exploited to design therapeutic strategies for KSHV-related diseases., (© 2024. The Author(s).)

Viruses compete with each other for limited cellular resources, and some deliver defence mechanisms that protect the host from competing genetic parasites 1 . The phage antirestriction induced system (PARIS) is a defence system, often encoded in viral genomes, that is composed of a 55 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB) 2 . However, the mechanism by which AriA and AriB function in phage defence is unknown. Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-electron microscopy to determine the structure of this complex, thereby explaining how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving host lysine transfer RNA. Phage T5 subverts PARIS immunity through expression of a lysine transfer RNA variant that is not cleaved by PARIS, thereby restoring viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids 3 ., (© 2024. The Author(s).)