tRNA-derived small RNAs in human cancers: roles, mechanisms, and clinical application.

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are a new type of non-coding RNAs (ncRNAs) produced by the specific cleavage of precursor or mature tRNAs. tsRNAs are involved in various basic biological processes such as epigenetic, transcriptional, post-transcriptional, and translation regulation, thereby affecting the occurrence and development of various human diseases, including cancers. Recent studies have shown that tsRNAs play an important role in tumorigenesis by regulating biological behaviors such as malignant proliferation, invasion and metastasis, angiogenesis, immune response, tumor resistance, and tumor metabolism reprogramming. These may be new potential targets for tumor treatment. Furthermore, tsRNAs can exist abundantly and stably in various bodily fluids (e.g., blood, serum, and urine) in the form of free or encapsulated extracellular vesicles, thereby affecting intercellular communication in the tumor microenvironment (TME). Meanwhile, their abnormal expression is closely related to the clinicopathological features of tumor patients, such as tumor staging, lymph node metastasis, and poor prognosis of tumor patients; thus, tsRNAs can be served as a novel type of liquid biopsy biomarker. This review summarizes the discovery, production, and expression of tsRNAs and analyzes their molecular mechanisms in tumor development and potential applications in tumor therapy, which may provide new strategies for early diagnosis and targeted therapy of tumors.

Structural basis of translation inhibition by a valine tRNA-derived fragment.

Translational regulation by non-coding RNAs is a mechanism commonly used by cells to fine-tune gene expression. A fragment derived from an archaeal valine tRNA (Val-tRF) has been previously identified to bind the small subunit of the ribosome and inhibit translation in Haloferax volcanii Here, we present three cryo-electron microscopy structures of Val-tRF bound to the small subunit of Sulfolobus acidocaldarius ribosomes at resolutions between 4.02 and 4.53 Å. Within these complexes, Val-tRF was observed to bind to conserved RNA-interacting sites, including the ribosomal decoding center. The binding of Val-tRF destabilizes helices h24, h44, and h45 and the anti-Shine-Dalgarno sequence of 16S rRNA. The binding position of this molecule partially overlaps with the translation initiation factor aIF1A and occludes the mRNA P-site codon. Moreover, we found that the binding of Val-tRF is associated with steric hindrance of the H69 base of 23S rRNA in the large ribosome subunit, thereby preventing 70S assembly. Our data exemplify how tRNA-derived fragments bind to ribosomes and provide new insights into the mechanisms underlying translation inhibition by Val-tRFs.

Translation velocity determines the efficacy of engineered suppressor tRNAs on pathogenic nonsense mutations.

Nonsense mutations - the underlying cause of approximately 11% of all genetic diseases - prematurely terminate protein synthesis by mutating a sense codon to a premature stop or termination codon (PTC). An emerging therapeutic strategy to suppress nonsense defects is to engineer sense-codon decoding tRNAs to readthrough and restore translation at PTCs. However, the readthrough efficiency of the engineered suppressor tRNAs (sup-tRNAs) largely varies in a tissue- and sequence context-dependent manner and has not yet yielded optimal clinical efficacy for many nonsense mutations. Here, we systematically analyze the suppression efficacy at various pathogenic nonsense mutations. We discover that the translation velocity of the sequence upstream of PTCs modulates the sup-tRNA readthrough efficacy. The PTCs most refractory to suppression are embedded in a sequence context translated with an abrupt reversal of the translation speed leading to ribosomal collisions. Moreover, modeling translation velocity using Ribo-seq data can accurately predict the suppression efficacy at PTCs. These results reveal previously unknown molecular signatures contributing to genotype-phenotype relationships and treatment-response heterogeneity, and provide the framework for the development of personalized tRNA-based gene therapies.

Genetic Code Expansion in Pseudomonas putida KT2440.

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

Host-like RNA Elements Regulate Virus Translation.

Viruses are obligate, intracellular parasites that co-opt host cell machineries for propagation. Critical among these machineries are those that translate RNA into protein and their mechanisms of control. Most regulatory mechanisms effectuate their activity by targeting sequence or structural features at the RNA termini, i.e., at the 5' or 3' ends, including the untranslated regions (UTRs). Translation of most eukaryotic mRNAs is initiated by 5' cap-dependent scanning. In contrast, many viruses initiate translation at internal RNA regions at internal ribosome entry sites (IRESs). Eukaryotic mRNAs often contain upstream open reading frames (uORFs) that permit condition-dependent control of downstream major ORFs. To offset genome compression and increase coding capacity, some viruses take advantage of out-of-frame overlapping uORFs (oORFs). Lacking the essential machinery of protein synthesis, for example, ribosomes and other translation factors, all viruses utilize the host apparatus to generate virus protein. In addition, some viruses exhibit RNA elements that bind host regulatory factors that are not essential components of the translation machinery. SARS-CoV-2 is a paradigm example of a virus taking advantage of multiple features of eukaryotic host translation control: the virus mimics the established human GAIT regulatory element and co-opts four host aminoacyl tRNA synthetases to form a stimulatory binding complex. Utilizing discontinuous transcription, the elements are present and identical in all SARS-CoV-2 subgenomic RNAs (and the genomic RNA). Thus, the virus exhibits a post-transcriptional regulon that improves upon analogous eukaryotic regulons, in which a family of functionally related mRNA targets contain elements that are structurally similar but lacking sequence identity. This "thrifty" virus strategy can be exploited against the virus since targeting the element can suppress the expression of all subgenomic RNAs as well as the genomic RNA. Other 3' end viral elements include 3'-cap-independent translation elements (3'-CITEs) and 3'-tRNA-like structures. Elucidation of virus translation control elements, their binding proteins, and their mechanisms can lead to novel therapeutic approaches to reduce virus replication and pathogenicity.

RAPP-containing arrest peptides induce translational stalling by short circuiting the ribosomal peptidyltransferase activity.

Arrest peptides containing RAPP (ArgAlaProPro) motifs have been discovered in both Gram-positive and Gram-negative bacteria, where they are thought to regulate expression of important protein localization machinery components. Here we determine cryo-EM structures of ribosomes stalled on RAPP arrest motifs in both Bacillus subtilis and Escherichia coli. Together with molecular dynamics simulations, our structures reveal that the RAPP motifs allow full accommodation of the A-site tRNA, but prevent the subsequent peptide bond from forming. Our data support a model where the RAP in the P-site interacts and stabilizes a single hydrogen atom on the Pro-tRNA in the A-site, thereby preventing an optimal geometry for the nucleophilic attack required for peptide bond formation to occur. This mechanism to short circuit the ribosomal peptidyltransferase activity is likely to operate for the majority of other RAPP-like arrest peptides found across diverse bacterial phylogenies.

Bioorthogonal labeling and profiling of N6-isopentenyladenosine (i6A) modified RNA.

Chemical modifications in RNAs play crucial roles in diversifying their structures and regulating numerous biochemical processes. Since the 1990s, several hydrophobic prenyl-modifications have been discovered in various RNAs. Prenyl groups serve as precursors for terpenes and many other biological molecules. The processes of prenylation in different macromolecules have been extensively studied. We introduce here a novel chemical biology toolkit that not only labels i6A, a prenyl-modified RNA residue, by leveraging the unique reactivity of the prenyl group, but also provides a general strategy to incorporate fluorescence functionalities into RNAs for molecular tracking purposes. Our findings revealed that iodine-mediated cyclization reactions of the prenyl group occur rapidly, transforming i6A from a hydrogen-bond acceptor to a donor. Based on this reactivity, we developed an Iodine-Mediated Cyclization and Reverse Transcription (IMCRT) tRNA-seq method, which can profile all nine endogenous tRNAs containing i6A residues in Saccharomyces cerevisiae with single-base resolution. Furthermore, under stress conditions, we observed a decline in i6A levels in budding yeast, accompanied by significant decrease of mutation rate at A37 position. Thus, the IMCRT tRNA-seq method not only permits semi-quantification of i6A levels in tRNAs but also holds potential for transcriptome-wide detection and analysis of various RNA species containing i6A modifications.

Extended stop codon context predicts nonsense codon readthrough efficiency in human cells.

Protein synthesis terminates when a stop codon enters the ribosome's A-site. Although termination is efficient, stop codon readthrough can occur when a near-cognate tRNA outcompetes release factors during decoding. Seeking to understand readthrough regulation we used a machine learning approach to analyze readthrough efficiency data from published HEK293T ribosome profiling experiments and compared it to comparable yeast experiments. We obtained evidence for the conservation of identities of the stop codon, its context, and 3'-UTR length (when termination is compromised), but not the P-site codon, suggesting a P-site tRNA role in readthrough regulation. Models trained on data from cells treated with the readthrough-promoting drug, G418, accurately predicted readthrough of premature termination codons arising from CFTR nonsense alleles that cause cystic fibrosis. This predictive ability has the potential to aid development of nonsense suppression therapies by predicting a patient's likelihood of improvement in response to drugs given their nonsense mutation sequence context.

Beyond the Anticodon: tRNA Core Modifications and Their Impact on Structure, Translation and Stress Adaptation.

Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional chemical modifications. Approximately 100 different modifications have been identified in tRNAs, and each tRNA typically contains 5-15 modifications that are incorporated at specific sites along the tRNA sequence. These modifications may be classified into two groups according to their position in the three-dimensional tRNA structure, i.e., modifications in the tRNA core and modifications in the anticodon-loop (ACL) region. Since many modified nucleotides in the tRNA core are involved in the formation of tertiary interactions implicated in tRNA folding, these modifications are key to tRNA stability and resistance to RNA decay pathways. In comparison to the extensively studied ACL modifications, tRNA core modifications have generally received less attention, although they have been shown to play important roles beyond tRNA stability. Here, we review and place in perspective selected data on tRNA core modifications. We present their impact on tRNA structure and stability and report how these changes manifest themselves at the functional level in translation, fitness and stress adaptation.

In silico approach for the identification of tRNA-derived small non-coding RNAs in SARS-CoV infection.

tsRNAs (tRNA-derived small non-coding RNAs), including tRNA halves (tiRNAs) and tRNA fragments (tRFs), have been implicated in some viral infections, such as respiratory viral infections. However, their involvement in SARS-CoV infection is completely unknown. A comprehensive analysis was performed to determine tsRNA populations in a mouse model of SARS-CoV-infected samples containing the wild-type and attenuated viruses. Data from the Gene Expression Omnibus (GEO) dataset at NCBI (accession ID GSE90624 ) was used for this study. A count matrix was generated for the tRNAs. Differentially expressed tRNAs, followed by tsRNAs derived from each significant tRNAs at different conditions and time points between the two groups WT(SARS-CoV-MA15-WT) vs Mock and ΔE (SARS-CoV-MA15-ΔE) vs Mock were identified. Notably, significantly differentially expressed tRNAs at 2dpi but not at 4dpi. The tsRNAs originating from differentially expressed tRNAs across all the samples belonging to each condition (WT, ΔE, and Mock) were identified. Intriguingly, tRFs (tRNA-derived RNA fragments) exhibited higher levels compared to tiRNAs (tRNA-derived stress-induced RNAs) across all samples associated with WT SARS-CoV strain compared to ΔE and mock-infected samples. This discrepancy suggests a non-random formation of tsRNAs, hinting at a possible involvement of tsRNAs in SARS-CoV viral infection.

Structural and mechanistic insights into activation of the human RNA ligase RTCB by Archease.

RNA ligases of the RTCB-type play an essential role in tRNA splicing, the unfolded protein response and RNA repair. RTCB is the catalytic subunit of the pentameric human tRNA ligase complex. RNA ligation by the tRNA ligase complex requires GTP-dependent activation of RTCB. This active site guanylylation reaction relies on the activation factor Archease. The mechanistic interplay between both proteins has remained unknown. Here, we report a biochemical and structural analysis of the human RTCB-Archease complex in the pre- and post-activation state. Archease reaches into the active site of RTCB and promotes the formation of a covalent RTCB-GMP intermediate through coordination of GTP and metal ions. During the activation reaction, Archease prevents futile RNA substrate binding to RTCB. Moreover, monomer structures of Archease and RTCB reveal additional states within the RNA ligation mechanism. Taken together, we present structural snapshots along the reaction cycle of the human tRNA ligase.

tRNA epitranscriptome determines pathogenicity of the opportunistic pathogen Pseudomonas aeruginosa.

The success of bacterial pathogens depends on the coordinated expression of virulence determinants. Regulatory circuits that drive pathogenesis are complex, multilayered, and incompletely understood. Here, we reveal that alterations in tRNA modifications define pathogenic phenotypes in the opportunistic pathogen Pseudomonas aeruginosa. We demonstrate that the enzymatic activity of GidA leads to the introduction of a carboxymethylaminomethyl modification in selected tRNAs. Modifications at the wobble uridine base (cmnm5U34) of the anticodon drives translation of transcripts containing rare codons. Specifically, in P. aeruginosa the presence of GidA-dependent tRNA modifications modulates expression of genes encoding virulence regulators, leading to a cellular proteomic shift toward pathogenic and well-adapted physiological states. Our approach of profiling the consequences of chemical tRNA modifications is general in concept. It provides a paradigm of how environmentally driven tRNA modifications govern gene expression programs and regulate phenotypic outcomes responsible for bacterial adaption to challenging habitats prevailing in the host niche.

Sexual stage-specific A-to-I mRNA editing is mediated by tRNA-editing enzymes in fungi.

A-to-I RNA editing catalyzed by adenosine-deaminase-acting-on-RNA (ADARs) was assumed to be unique to metazoans because fungi and plants lack ADAR homologs. However, genome-wide messenger RNA (mRNA) editing was found to occur specifically during sexual reproduction in filamentous ascomycetes. Because systematic characterization of adenosine/cytosine deaminase genes has implicated the involvement of TAD2 and TAD3 orthologs in A-to-I editing, in this study, we used genetic and biochemical approaches to characterize the role of FgTAD2, an essential adenosine-deaminase-acting-on-tRNA (ADAT) gene, in mRNA editing in Fusarium graminearum. FgTAD2 had a sexual-stage-specific isoform and formed heterodimers with enzymatically inactive FgTAD3. Using a repeat-induced point (RIP) mutation approach, we identified 17 mutations in FgTAD2 that affected mRNA editing during sexual reproduction but had no effect on transfer RNA (tRNA) editing and vegetative growth. The functional importance of the H352Y and Q375*(nonsense) mutations in sexual reproduction and mRNA editing were confirmed by introducing specific point mutations into the endogenous FgTAD2 allele in the wild type. An in vitro assay was developed to show that FgTad2-His proteins purified from perithecia, but not from vegetative hyphae, had mRNA editing activities. Moreover, the H352Y mutation affected the enzymatic activity of FgTad2 to edit mRNA but had no effect on its ADAT activity. We also identified proteins co-purified with FgTad2-His by mass spectrometry analysis and found that two of them have the RNA recognition motif. Taken together, genetic and biochemical data from this study demonstrated that FgTad2, an ADAT, catalyzes A-to-I mRNA editing with the stage-specific isoform and cofactors during sexual reproduction in fungi.

Misacylation of tRNA with Ser-Pro Dipeptide for In Vitro Transcription-Translation.

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

The tsRNAs (tRFdb-3013a/b) serve as novel biomarkers for colon adenocarcinomas.

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

RNA therapeutics for infectious diseases.

Ribonucleic acids (RNAs), including the messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA), play important roles in living organisms and viruses. In recent years, the RNA-based technologies including the RNAs inhibiting other RNA activities, the RNAs targeting proteins, the RNAs reprograming genetic information, and the RNAs encoding therapeutical proteins, are useful methods to apply in prophylactic and therapeutic vaccines. In this review, we summarize and highlight the current application of the RNA therapeutics, especially on mRNA vaccines which have potential for prevention and treatment against human and animal infectious diseases.

The protein-only RNase Ps, endonucleases that cleave pre-tRNA: Biological relevance, molecular architectures, substrate recognition and specificity, and protein interactomes.

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

Microarray analysis of tRNA-derived small RNA (tsRNA) in LPS-challenged macrophages treated with metformin.

Metformin, a widely used anti-diabetic drug, has demonstrated its efficacy in addressing various inflammatory conditions. tRNA-derived small RNA (tsRNA), a novel type of small non-coding RNA, exhibits diverse regulatory functions and holds promise as both a diagnostic biomarker and a therapeutic target for various diseases. The purpose of this study is to investigate whether the abundance of tsRNAs changed in LPS versus LPS + metformin-treated cells, utilizing microarray technology. Firstly, we established an in vitro lipopolysaccharide (LPS)-induced inflammation model using RAW264.7 macrophages and assessed the protective effects of metformin against inflammatory damage. Subsequently, we extracted total RNA from both LPS-treated and metformin + LPS-treated cell samples for microarray analysis to identify differentially abundant tsRNAs (DA-tsRNAs). Furthermore, we conducted bioinformatics analysis to predict target genes for validated DA-tsRNAs and explore the biological functions and signaling pathways associated with DA-tsRNAs. Notably, metformin was found to inhibit the inflammatory response in RAW264.7 macrophages. The microarray results revealed a total of 247 DA-tsRNAs, with 58 upregulated and 189 downregulated tsRNAs in the Met + LPS group compared to the LPS group. The tsRNA-mRNA network was visualized, shedding light on potential interactions. The results of bioinformatics analysis suggested that these potential targets of specific tsRNAs were mainly related to inflammation and immunity. Our study provides compelling evidence that metformin exerts anti-inflammatory effects and modulates the abundance of tsRNAs in LPS-treated RAW264.7 macrophages. These findings establish a valuable foundation for using tsRNAs as potential biomarkers for metformin in the treatment of inflammatory conditions.

Identification of HDV-like theta ribozymes involved in tRNA-based recoding of gut bacteriophages.

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

Novel insights into transfer RNA-derived small RNA (tsRNA) in cardio-metabolic diseases.

Cardio-metabolic diseases, including a cluster of metabolic disorders and their secondary affections on cardiovascular physiology, are gradually brought to the forefront by researchers due to their high prevalence and mortality, as well as an unidentified pathogenesis. tRNA-derived small RNAs (tsRNAs), cleaved by several specific enzymes and once considered as some "metabolic junks" in the past, have been proved to possess numerous functions in human bodies. More interestingly, such a potential also seems to influence the progression of cardio-metabolic diseases to some extent. In this review, the biogenesis, classification and mechanisms of tsRNAs will be discussed based on some latest studies, and their relations with several cardio-metabolic diseases will be highlighted in sequence. Lastly, some future prospects, such as their clinical applications as biomarkers and therapeutic targets will also be mentioned, in order to provide researchers with a comprehensive understanding of the research status of tsRNAs as well as its association with cardio-metabolic diseases, thus presenting as a beacon to indicate directions for the next stage of study.