tRNA Sequences Research Articles

MenT nucleotidyltransferase toxins extend tRNA acceptor stems and can be inhibited by asymmetrical antitoxin binding.

Mycobacterium tuberculosis, the bacterium responsible for human tuberculosis, has a genome encoding a remarkably high number of toxin-antitoxin systems of largely unknown function. We have recently shown that the M. tuberculosis genome encodes four of a widespread, MenAT family of nucleotidyltransferase toxin-antitoxin systems. In this study we characterize MenAT1, using tRNA sequencing to demonstrate MenT1 tRNA modification activity. MenT1 activity is blocked by MenA1, a short protein antitoxin unrelated to the MenA3 kinase. X-ray crystallographic analysis shows blockage of the conserved MenT fold by asymmetric binding of MenA1 across two MenT1 protomers, forming a heterotrimeric toxin-antitoxin complex. Finally, we also demonstrate tRNA modification by toxin MenT4, indicating conserved activity across the MenT family. Our study highlights variation in tRNA target preferences by MenT toxins, selective use of nucleotide substrates, and diverse modes of MenA antitoxin activity.

Direct sequencing of total Saccharomyces cerevisiae tRNAs by LC-MS/MS.

Among RNAs, transfer RNAs (tRNAs) contain the widest variety of abundant posttranscriptional chemical modifications. These modifications are crucial for tRNAs to participate in protein synthesis, promoting proper tRNA structure and aminoacylation, facilitating anticodon:codon recognition, and ensuring the reading frame maintenance of the ribosome. While tRNA modifications were long thought to be stoichiometric, it is becoming increasingly apparent that these modifications can change dynamically in response to the cellular environment. The ability to broadly characterize the fluctuating tRNA modification landscape will be essential for establishing the molecular level contributions of individual sites of tRNA modification. The locations of modifications within individual tRNA sequences can be mapped using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this approach, a single tRNA species is purified, treated with ribonucleases, and the resulting single-stranded RNA products are subject to LC-MS/MS analysis. The application of LC-MS/MS to study tRNAs is limited by the necessity of analyzing one tRNA at a time, because the digestion of total tRNA mixtures by commercially available ribonucleases produces many short digestion products unable to be uniquely mapped back to a single site within a tRNA. We overcame these limitations by taking advantage of the highly structured nature of tRNAs to prevent the full digestion by single-stranded RNA-specific ribonucleases. Folding total tRNA prior to digestion allowed us to sequence Saccharomyces cerevisiae tRNAs with up to 97% sequence coverage for individual tRNA species by LC-MS/MS. This method presents a robust avenue for directly detecting the distribution of modifications in total tRNAs.

Novel ELAC2 Mutations in Individuals Presenting with Variably Severe Neurological Disease in the Presence or Absence of Cardiomyopathy

Transcription of mitochondrial DNA generates long polycistronic precursors whose nucleolytic cleavage yields the individual mtDNA-encoded transcripts. In most cases, this cleavage occurs at the 5'- and 3'-ends of tRNA sequences by the concerted action of RNAseP and RNaseZ/ELAC2 endonucleases, respectively. Variants in the ELAC2 gene have been predominantly linked to severe to mild cardiomyopathy that, in its milder forms, is accompanied by variably severe neurological presentations. Here, we report five patients from three unrelated families. Four of the patients presented mild to moderate cardiomyopathy and one died at 1 year of age, one patient had no evidence of cardiomyopathy. The patients had variable neurological presentations that included intellectual disability, ataxia, refractory epilepsy, neuropathy and deafness. All patients carried previously unreported missense and nonsense variants. Enzymatic analyses showed multiple OXPHOS deficiencies in biopsies from two patients, whereas immunoblot analyses revealed a decreased abundance of ELAC2 in fibroblasts from three patients. Northern blot analysis revealed an accumulation of unprocessed mt-tRNAVal-precursor consistent with the role of ELAC2 in transcript processing. Our study expands the genetic spectrum of ELAC2-linked disease and suggests that cardiomyopathy is not an invariably present clinical hallmark of this pathology.

On distinguishing between canonical tRNA genes and tRNA gene fragments in prokaryotes

ABSTRACT Automated genome annotation is essential for extracting biological information from sequence data. The identification and annotation of tRNA genes is frequently performed by the software package tRNAscan-SE, the output of which is listed for selected genomes in the Genomic tRNA database (GtRNAdb). Here, we highlight a pervasive error in prokaryotic tRNA gene sets on GtRNAdb: the mis-categorization of partial, non-canonical tRNA genes as standard, canonical tRNA genes. Firstly, we demonstrate the issue using the tRNA gene sets of 20 organisms from the archaeal taxon Thermococcaceae. According to GtRNAdb, these organisms collectively deviate from the expected set of tRNA genes in 15 instances, including the listing of eleven putative canonical tRNA genes. However, after detailed manual annotation, only one of these eleven remains; the others are either partial, non-canonical tRNA genes resulting from the integration of genetic elements or CRISPR-Cas activity (seven instances), or attributable to ambiguities in input sequences (three instances). Secondly, we show that similar examples of the mis-categorization of predicted tRNA sequences occur throughout the prokaryotic sections of GtRNAdb. While both canonical and non-canonical prokaryotic tRNA gene sequences identified by tRNAscan-SE are biologically interesting, the challenge of reliably distinguishing between them remains. We recommend employing a combination of (i) screening input sequences for the genetic elements typically associated with non-canonical tRNA genes, and ambiguities, (ii) activating the tRNAscan-SE automated pseudogene detection function, and (iii) scrutinizing predicted tRNA genes with low isotype scores. These measures greatly reduce manual annotation efforts, and lead to improved prokaryotic tRNA gene set predictions.

IDHU-Ensem: Identification of dihydrouridine sites through ensemble learning models.

Dihydrouridine (D) is one of the most significant uridine modifications that have a prominent occurrence in eukaryotes. The folding and conformational flexibility of transfer RNA (tRNA) can be attained through this modification. The modification also triggers lung cancer in humans. The identification of D sites was carried out through conventional laboratory methods; however, those were costly and time-consuming. The readiness of RNA sequences helps in the identification of D sites through computationally intelligent models. However, the most challenging part is turning these biological sequences into distinct vectors. The current research proposed novel feature extraction mechanisms and the identification of D sites in tRNA sequences using ensemble models. The ensemble models were then subjected to evaluation using k-fold cross-validation and independent testing. The results revealed that the stacking ensemble model outperformed all the ensemble models by revealing 0.98 accuracy, 0.98 specificity, 0.97 sensitivity, and 0.92 Matthews Correlation Coefficient. The proposed model, iDHU-Ensem, was also compared with pre-existing predictors using an independent test. The accuracy scores have shown that the proposed model in this research study performed better than the available predictors. The current research contributed towards the enhancement of D site identification capabilities through computationally intelligent methods. A web-based server, iDHU-Ensem, was also made available for the researchers at https://taseersuleman-idhu-ensem-idhu-ensem.streamlit.app/.

Single-read tRNA-seq analysis reveals coordination of tRNA modification and aminoacylation and fragmentation.

Transfer RNA (tRNA) utilizes multiple properties of abundance, modification, and aminoacylation in translational regulation. These properties were typically studied one-by-one; however, recent advance in high throughput tRNA sequencing enables their simultaneous assessment in the same sequencing data. How these properties are coordinated at the transcriptome level is an open question. Here, we develop a single-read tRNA analysis pipeline that takes advantage of the pseudo single-molecule nature of tRNA sequencing in NGS libraries. tRNAs are short enough that a single NGS read can represent one tRNA molecule, and can simultaneously report on the status of multiple modifications, aminoacylation, and fragmentation of each molecule. We find correlations among modification-modification, modification-aminoacylationand modification-fragmentation. We identify interdependencies among one of the most common tRNA modifications, m1A58, as coordinators of tissue-specific gene expression. Our method, SingLe-read Analysis of Crosstalks (SLAC), reveals tRNAome-wide networks of modifications, aminoacylation, and fragmentation. We observe changes of these networks under different stresses, and assign a function for tRNA modification in translational regulation and fragment biogenesis. SLAC leverages the richness of the tRNA-seq data and provides new insights on the coordination of tRNA properties.

Differentially expressed tRNA-derived fragments in bovine fetuses with assisted reproduction induced congenital overgrowth syndrome.

Background: As couples struggle with infertility and livestock producers wish to rapidly improve genetic merit in their herd, assisted reproductive technologies (ART) have become increasingly popular in human medicine as well as the livestock industry. Utilizing ART can cause an increased risk of congenital overgrowth syndromes, such as Large Offspring Syndrome (LOS) in ruminants. A dysregulation of transcripts has been observed in bovine fetuses with LOS, which is suggested to be a cause of the phenotype. Our recent study identified variations in tRNA expression in LOS individuals, leading us to hypothesize that variations in tRNA expression can influence the availability of their processed regulatory products, tRNA-derived fragments (tRFs). Due to their resemblance in size to microRNAs, studies suggest that tRFs target mRNA transcripts and regulate gene expression. Thus, we have sequenced small RNA isolated from skeletal muscle and liver of day 105 bovine fetuses to elucidate the mechanisms contributing to LOS. Moreover, we have utilized our previously generated tRNA sequencing data to analyze the contribution of tRNA availability to tRF abundance. Results: 22,289 and 7,737 unique tRFs were predicted in the liver and muscle tissue respectively. The greatest number of reads originated from 5' tRFs in muscle and 5' halves in liver. In addition, mitochondrial (MT) and nuclear derived tRF expression was tissue-specific with most MT-tRFs and nuclear tRFs derived from LysUUU and iMetCAU in muscle, and AsnGUU and GlyGCC in liver. Despite variation in tRF abundance within treatment groups, we identified differentially expressed (DE) tRFs across Control-AI, ART-Normal, and ART-LOS groups with the most DE tRFs between ART-Normal and ART-LOS groups. Many DE tRFs target transcripts enriched in pathways related to growth and development in the muscle and tumor development in the liver. Finally, we found positive correlation coefficients between tRNA availability and tRF expression in muscle (R = 0.47) and liver (0.6). Conclusion: Our results highlight the dysregulation of tRF expression and its regulatory roles in LOS. These tRFs were found to target both imprinted and non-imprinted genes in muscle as well as genes linked to tumor development in the liver. Furthermore, we found that tRNA transcription is a highly modulated event that plays a part in the biogenesis of tRFs. This study is the first to investigate the relationship between tRNA and tRF expression in combination with ART-induced LOS.

Introducing multiple-gene mutations in Pleurotus ostreatus using a polycistronic tRNA and CRISPR guide RNA strategy.

The white-rot fungus Pleurotus ostreatus is an agaricomycete that is frequently used in molecular genetics studies as many useful tools are applicable to the fungus. In particular, efficient gene targeting using homologous recombination and CRISPR/Cas9 enables the introduction of a mutation in the gene of interest for functional analysis. Multiple genes encoding various lignocellulose-degrading enzymes are predicted to be present in the genome; therefore, analyses of multiple-gene mutants are required to elucidate the mechanisms underlying lignocellulose degradation by P. ostreatus. Conventional tools for generating multiple-gene mutations in P. ostreatus are laborious and time-consuming. Therefore, more efficient and practical methods are needed. In this study, we introduced CRISPR/Cas9-assisted multiple-gene mutations using a polycistronic tRNA and CRISPR guide RNA approach. The frequency (triple-gene mutation in fcy1, vp2, and 62347) was only 3.3% when a tetracistronic tRNA-sgRNA containing four different sgRNAs targeting fcy1, vp2, vp3, or 62347 was expressed. It increased to 20% (triple-gene mutation in vp1, vp2, and vp3) after a tricistronic tRNA-sgRNA was expressed with replaced/modulated promoter and tRNA sequences. This study demonstrated, for the first time, the applicability of a strategy to induce multiple-gene mutations in P. ostreatus in a transformation experiment.

The Evolutionary Dynamics of the Mitochondrial tRNA in the Cichlid Fish Family.

Simple SummaryCichlids are a unique example of fish diversity and species richness which have been explained by sympatric speciation at different freshwater sources in Africa. The mitochondria contribute to cell vitality by providing energy. It contains a circular genome with an established translation system that is spatially independent of the cytosolic counterpart. The current study aimed to investigate the evolutionary dynamics of the mitochondrial tRNA and its role in cichlids’ diversity. The available cichlid mitogenomes in the public database were filtered, in addition to newly sequenced accessions from a specific cichlid group known as the haplotilapiine lineage that is widely distributed in the Egyptian sector of the Nile River. Based on the comparative analysis of mitogenomic data, we identified 22 tRNA genes, in which a single gene was D-armless, while the cloverleaf secondary structure subdivided into stem-loop formations was predicted and used to define the levels of genetic divergence for the remained tRNAs. Peculiarly, in cichlids, the formation known as “T-arm” showed the lowest polymorphism levels among other structures in contrast to other organisms (e.g., scorpions). Comparing the whole family to the specific haplotilapiine lineage showed that the tryptophan tRNA was the most conserved tRNA, with signatures of possible purifying selection.The mitochondrial transfer RNA genes (tRNAs) attract more attention due to their highly dynamic and rapidly evolving nature. The current study aimed to detect and evaluate the dynamics, characteristic patterns, and variations of mitochondrial tRNAs. The study was conducted in two main parts: first, the published mitogenomic sequences of cichlids mt tRNAs have been filtered. Second, the filtered mitochondrial tRNA and additional new mitogenomes representing the most prevalent Egyptian tilapiine were compared and analyzed. Our results revealed that all 22 tRNAs of cichlids folded into a classical cloverleaf secondary structure with four domains, except for trnSGCU, missing the D domain in all cichlids. When consensus tRNAs were compared, most of the mutations were observed in the trnP at nucleotide levels (substitutions and indels), in contrast to trnLUAA. From a structural perspective, the anticodon loop and T-loop formations were the most conserved structures among all parts of the tRNA in contrast to the A-stem and D-loop formations. The trnW was the lowest polymorphic unneutral tRNA among all cichlids (both the family and the haplotilapiine lineage), in contrast with the neutral trnD that was extremely polymorphic among and within the haplotilapiine lineage species compared to other cichlids species. From a phylogenetic perspective, the trnC was extremely hypervariable and neutral tRNA in both haplotilapiine lineage and cichlids but was unable to report correct phylogenetic signal for the cichlids. In contrast to trnI and trnY, less variable neutral tRNAs that were able to cluster the haplotilapiine lineage and cichlids species as previously reported. By observing the DNA polymorphism in the coding DNA sequences (CDS), the highest affected amino acid by non-synonymous mutations was isoleucine and was equally mutated to valine and vice versa; no correlation between mutations in CDS and tRNAs was statistically found. The current study provides an insight into the mitochondrial tRNA evolution and its effect on the cichlid diversity and speciation model at the maternal level.

Ancestral archaea expanded the genetic code with pyrrolysine

The pyrrolysyl-tRNA synthetase (PylRS) facilitates the cotranslational installation of the 22nd amino acid pyrrolysine. Owing to its tolerance for diverse amino acid substrates, and its orthogonality in multiple organisms, PylRS has emerged as a major route to install noncanonical amino acids into proteins in living cells. Recently, a novel class of PylRS enzymes was identified in a subset of methanogenic archaea. Enzymes within this class (ΔPylSn) lack the N-terminal tRNA-binding domain that is widely conserved amongst PylRS enzymes, yet remain active and orthogonal in bacteria and eukaryotes. In this study, we use biochemical and invivo UAG-readthrough assays to characterize the aminoacylation efficiency and substrate spectrum of a ΔPylSn class PylRS from the archaeon Candidatus Methanomethylophilus alvus. We show that, compared with the full-length enzyme from Methanosarcina mazei, the Ca. M. alvus PylRS displays reduced aminoacylation efficiency but an expanded amino acid substrate spectrum. To gain insight into the evolution of ΔPylSn enzymes, we performed molecular phylogeny using 156 PylRS and 105 pyrrolysine tRNA (tRNAPyl) sequences from diverse archaea and bacteria. This analysis suggests that the PylRS•tRNAPyl pair diverged before the evolution of the three domains of life, placing an early limit on the evolution of the Pyl-decoding trait. Furthermore, our results document the coevolutionary history of PylRS and tRNAPyl and reveal the emergence of tRNAPyl sequences with unique A73 and U73 discriminator bases. The orthogonality of these tRNAPyl species with the more common G73-containing tRNAPyl will enable future efforts to engineer PylRS systems for further genetic code expansion.

Viruses Ubiquity and Diversity in Atacama Desert Endolithic Communities.

Viruses are key players in the environment, and recent metagenomic studies have revealed their diversity and genetic complexity. Despite progress in understanding the ecology of viruses in extreme environments, viruses’ dynamics and functional roles in dryland ecosystems, which cover about 45% of the Earth’s land surfaces, remain largely unexplored. This study characterizes virus sequences in the metagenomes of endolithic (within rock) microbial communities ubiquitously found in hyper-arid deserts. Taxonomic classification and network construction revealed the presence of novel and diverse viruses in communities inhabiting calcite, gypsum, and ignimbrite rocks. Viral genome maps show a high level of protein diversity within and across endolithic communities and the presence of virus-encoded auxiliary metabolic genes. Phage-host relationships were predicted by matching tRNA, CRISPR spacer, and protein sequences in the viral and microbial metagenomes. Primary producers and heterotrophic bacteria were found to be putative hosts to some viruses. Intriguingly, viral diversity was not correlated with microbial diversity across rock substrates.

DNA methylation differences during development distinguish sympatric morphs of Arctic charr (Salvelinus alpinus).

Changes in DNA methylation in specific coding or non-coding regions can influence development and potentially divergence in traits within species and groups. While the impact of epigenetic variation on developmental pathways associated with evolutionary divergence is the focus of intense investigation, few studies have looked at recently diverged systems. Phenotypic diversity between closely related populations of Arctic charr (Salvelinus alpinus), which diverged within the last 10,000 years, offers an interesting ecological model to address such effects. Using bisulphite sequencing, we studied general DNA methylation patterns during development in the four sympatric morphs of Arctic charr from Lake Thingvallavatn. The data revealed strong differences between developmental timepoints and between morphs (mainly along the benthic-limnetic axis), both at single CpG sites and in 1000bp-regions. Genes located close to differentially methylated CpG sites were involved in nucleosome assembly, regulation of osteoclast differentiation, and cell-matrix adhesion. Differentially methylated regions were enriched in tRNA and rRNA sequences, and half of them were located close to transcription start sites. The expression of 14 genes showing methylation differences over time or between morphs was further investigated by qPCR and nine of these were found to be differentially expressed between morphs. Four genes (ARHGEF37-like, H3-like, MPP3 and MEGF9) showed a correlation between methylation and expression. Lastly, histone gene clusters displayed interesting methylation differences between timepoints and morphs, as well as intragenic methylation variation. The results presented here provide a motivation for further studies on the contribution of epigenetic traits, such as DNA methylation, to phenotypic diversity and developmental mechanisms.

Identification of Novel tRNA-Leu-CAA-Derived tsRNAs for the Diagnosis and Prognosis of Diffuse Gliomas.

PurposetsRNA is a type of small non-coding RNA derived from tRNA. Diffuse gliomas are the most common brain tumors. This investigation focused on tsRNA identification and characterization within gliomas.MethodsThe sequences of human tRNA and tsRNAs were taken from GtRNAdb, tRFdb and tRFexplorer databases. Data processing and bioinformatic analysis were performed with R or Python software. The expression of tsRNAs in glioma tissues was analyzed by qRT-PCR assay.ResultsWith computational approaches, we identified hundreds of tsRNAs with available expression abundance in the glioma datasets, most of them derived from the 3′ end or 5′ end of mature tRNA. Among the tsRNAs derived from tRNA-Leu-CAA, ts-26, tRFdb-3012a, and tRFdb-3012b (tRFdb-3012a/b) were significantly decreased in diffuse gliomas. The clinical survivals of glioma patients with low tsRNA (ts-26, tRFdb-3012a, and tRFdb-3012b) expression were remarkably worse than that of those with high expression. Expression of tRFdb-3012a/b was correlated with IDH mutant status and MGMT promoter mutation in gliomas, and tRFdb-3012a/b and ts-23 tended to be highly expressed in patients with the IDH mutant. The enrichment analysis showed that some tRFdb-3012a/b-related genes were enriched in RNA splicing and processing, the spliceosome pathway and astrocyte molecular signatures. Moreover, the 3′ untranslated region of the RBM43 gene was predicted to contain putative binding sites of tRFdb-3012a/b, ts-26 may directly bind to the 3′ untranslated region of the HOXA13 gene, and the expressions of both RBM43 and HOXA13 were up-regulated in diffuse gliomas. High RBM43 and HOXA13 expressions were significantly associated with poor survival outcome of glioma patients.ConclusionThese results suggest that tRNA-Leu-CAA-derived tsRNAs (ts-26, tRFdb-3012a, and tRFdb-3012b) could be explored as diagnostic and prognostic biomarkers for diffuse gliomas, and tRFdb-3012a/b and ts-26 may play an important role in the progression of gliomas, through binding RBM43 and HOXA13, respectively.

PtRNAdb: a web resource of plant tRNA genes from a wide range of plant species.

The online version contains supplementary material available at 10.1007/s13205-022-03255-7.

Machine Learning Algorithms Highlight tRNA Information Content and Chargaff's Second Parity Rule Score as Important Features in Discriminating Probiotics from Non-Probiotics.

Simple SummaryProbiotics are a group of beneficial microorganisms that are symbionts of the human gut microbiome. The identification of new probiotics is therefore of paramount importance from both public health and commercial perspectives. In this study, we show for the first time that Artificial Intelligence algorithms can identify novel probiotics and also discriminate them from pathogenic organisms in the human gut. We were also able to determine the information content within tRNA sequences as the key genomic features capable of characterizing probiotics.Probiotic bacteria are microorganisms with beneficial effects on human health and are currently used in numerous food supplements. However, no selection process is able to effectively distinguish probiotics from non-probiotic organisms on the basis of their genomic characteristics. In the current study, four Machine Learning algorithms were employed to accurately identify probiotic bacteria based on their DNA characteristics. Although the prediction accuracies of all algorithms were excellent, the Neural Network returned the highest scores in all the evaluation metrics, managing to discriminate probiotics from non-probiotics with an accuracy greater than 90%. Interestingly, our analysis also highlighted the information content of the tRNA sequences as the most important feature in distinguishing the two groups of organisms probably because tRNAs have regulatory functions and might have allowed probiotics to evolve faster in the human gut environment. Through the methodology presented here, it was also possible to identify seven promising new probiotics that have a higher information content in their tRNA sequences compared to non-probiotics. In conclusion, we prove for the first time that Machine Learning methods can discriminate human probiotic from non-probiotic organisms underlining information within tRNA sequences as the most important genomic feature in distinguishing them.

TREPs-A New Class of Functional tRNA-Encoded Peptides.

We asked if transfer RNA (tRNA) ever got an opportunity of translating its own sequence during evolution, what would have been the function of such tRNA-encoded peptides (tREPs)? If not, could one artificially synthesize tREPs to study the corresponding functional outcomes? Here, we report a novel, first-in-the-class, chemically synthesized tREP-18 molecule originating from the Escherichia coli tRNA sequence showing potent antileishmanial property. As a first step, E. coli tRNAs were computationally translated into peptide sequence equivalents and a database of full-length hypothetical tREPs was created. The tREP sequences were sent into sequence, structure, and energy filters to narrow down potential peptides for experimental validation. Based on the functional predictions, tREPs were screened against antiparasitic targets, leading to the identification of tREP-18 as a potential antiparasitic peptide. The in vitro assay of chemically synthesized tREP-18 on the Ag83 strain of Leishmania donovani showed its potent antileishmanial property (IC50 value of 22.13 nM). The atomic force microscopy and scanning electron microscopy images indicated significant alteration in the cytoskeletal architecture of tREP-18-treated parasites. Also, tREP-18 seems to destabilize the mitochondrial membrane potential of parasites, disrupting their cellular integrity and leading to parasitic death. The cellular assays of the tREP-18 peptide on the BS12 strain, a clinical isolate of post-kala azar dermal leishmaniasis, demonstrated its significant efficacy at an IC50 value of 15 nM. The tREP-18 peptide showed a toxic effect on the amastigote stage of the parasite, showing macrophage pathogen clearance at a concentration of 22.5 nM. This study provides the proof of the concept of making a new class of functional peptides from tRNA sequences. It also opens a huge untapped tRNA-peptide space toward novel discoveries and applications. In the future, it would be interesting to perform tREP edits and redesign tREPs toward specific applications.

Bacterial tRNA landscape revisited

The updated Wobble Hypothesis reasonably explains why some 40 tRNA species are sufficient to decode the 61 amino acid codons of the Universal Genetic Code. However, we still have no clue why eubacteria lack tRNA isoacceptors with ANN anticodons, whereas eukaryotes universally lack eight GNN anticodons, only one of which is also absent in bacteria. Direct tRNA sequencing could resolve the patterns of nucleoside modification that had been driving the divergent evolution in prokaryotes and eukaryotes, but this task will require the development of AI-supported base-callers that can recognize modified nucleosides without any subsequent analytical verification. Our knowledge of the bacterial tRNA landscape is moreover broadened by the recent discovery of antisense tRNAs and tRNA-derived fragments that should be examined in their roles for gene expression, translation, bacterial physiology or metabolism.

Characterization of tRNA expression profiles in large offspring syndrome

BackgroundAssisted Reproductive Technologies (ART) use can increase the risk of congenital overgrowth syndromes, such as large offspring syndrome (LOS) in ruminants. Epigenetic variations are known to influence gene expression and differentially methylated regions (DMRs) were previously determined to be associated with LOS in cattle. We observed DMRs overlapping tRNA clusters which could affect tRNA abundance and be associated with tissue specificity or overgrowth. Variations in tRNA expression have been identified in several disease pathways suggesting an important role in the regulation of biological processes. Understanding the role of tRNA expression in cattle offers an opportunity to reveal mechanisms of regulation at the translational level. We analyzed tRNA expression in the skeletal muscle and liver tissues of day 105 artificial insemination-conceived, ART-conceived with a normal body weight, and ART-conceived bovine fetuses with a body weight above the 97th percentile compared to Control-AI.ResultsDespite the centrality of tRNAs to translation, in silico predictions have revealed dramatic differences in the number of tRNA genes between humans and cattle (597 vs 1,659). Consistent with reports in human, only a fraction of predicted tRNA genes are expressed. We detected the expression of 474 and 487 bovine tRNA genes in the muscle and liver with the remainder being unexpressed. 193 and 198 unique tRNA sequences were expressed in all treatment groups within muscle and liver respectively. In addition, an average of 193 tRNA sequences were expressed within the same treatment group in different tissues. Some tRNA isodecoders were differentially expressed between treatment groups. In the skeletal muscle and liver, we categorized 11 tRNA isoacceptors with undetected expression as well as an isodecoder that was unexpressed in the liver (SerGGA). Our results identified variation in the proportion of tRNA gene copies expressed between tissues and differences in the highest contributing tRNA anticodon within an amino acid family due to treatment and tissue type. Out of all amino acid families, roughly half of the most highly expressed tRNA isoacceptors correlated to their most frequent codon in the bovine genome.ConclusionAlthough the number of bovine tRNA genes is nearly triple of that of the tRNA genes in human, there is a shared occurrence of transcriptionally inactive tRNA genes in both species. We detected differential expression of tRNA genes as well as tissue- and treatment- specific tRNA transcripts with unique sequence variations that could modulate translation during protein homeostasis or cellular stress, and give rise to regulatory products targeting genes related to overgrowth in the skeletal muscle and/or tumor development in the liver of LOS individuals. While the absence of certain isodecoders may be relieved by wobble base pairing, missing tRNA species could increase the likelihood of mistranslation or mRNA degradation.

Eukaryotic tRNA sequences present conserved and amino acid-specific structural signatures.

Metazoan organisms have many tRNA genes responsible for decoding amino acids. The set of all tRNA genes can be grouped in sets of common amino acids and isoacceptor tRNAs that are aminoacylated by corresponding aminoacyl-tRNA synthetases. Analysis of tRNA alignments shows that, despite the high number of tRNA genes, specific tRNA sequence motifs are highly conserved across multicellular eukaryotes. The conservation often extends throughout the isoacceptors and isodecoders with, in some cases, two sets of conserved isodecoders. This study is focused on non-Watson–Crick base pairs in the helical stems, especially GoU pairs. Each of the four helical stems may contain one or more conserved GoU pairs. Some are amino acid specific and could represent identity elements for the cognate aminoacyl tRNA synthetases. Other GoU pairs are found in more than a single amino acid and could be critical for native folding of the tRNAs. Interestingly, some GoU pairs are anticodon-specific, and others are found in phylogenetically-specific clades. Although the distribution of conservation likely reflects a balance between accommodating isotype-specific functions as well as those shared by all tRNAs essential for ribosomal translation, such conservations may indicate the existence of specialized tRNAs for specific translation targets, cellular conditions, or alternative functions.

AAV-delivered suppressor tRNA overcomes a nonsense mutation in mice.

Gene therapy is a potentially curative medicine for many currently untreatable diseases, and recombinant adeno-associated virus (rAAV)is the most successful gene delivery vehicle for in vivo applications1-3. However, rAAV-based gene therapy suffers from several limitations, such as constrained DNA cargo size and toxicities caused by non-physiological expression of a transgene4-6. Here we show that rAAV delivery of a suppressor tRNA (rAAV.sup-tRNA) safely and efficiently rescued a genetic disease in a mouse model carrying a nonsense mutation, and effects lasted for more than 6 months after a single treatment. Mechanistically, this was achieved through a synergistic effect of premature stop codon readthrough and inhibition of nonsense-mediated mRNA decay. rAAV.sup-tRNA had a limited effect on global readthrough at normal stop codons and did not perturb endogenous tRNA homeostasis, as determined by ribosome profiling and tRNA sequencing, respectively. By optimizing the AAV capsid and the route of administration, therapeutic efficacy in various target tissues was achieved, including liver, heart, skeletal muscle and brain. This study demonstrates the feasibility of developing a toolbox of AAV-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) to rescue pathogenic nonsense mutations and restore gene function under endogenous regulation. As nonsense mutations account for 11% of pathogenic mutations, AAV-NoSTOP can benefit a large number of patients. AAV-NoSTOP obviates the need to deliver a full-length protein-coding gene that may exceed the rAAV packaging limit, elicit adverse immune responses or cause transgene-related toxicities. It therefore represents a valuable addition to gene therapeutics.