tsRNAs: Novel small molecules from cell function and regulatory mechanism to therapeutic targets

Abstract tsRNAs are small fragments of RNAs with specific lengths that are generated by particular ribonucleases, such as dicer and angiogenin (ANG), clipping on the rings of transfer RNAs (tRNAs) in specific cells and tissues under specific conditions. Depending on where the splicing site is, tsRNAs can be segmented into two main types, tRNA‐derived stress‐induced RNAs (tiRNAs) and tRNA‐derived fragments (tRFs). Many studies have shown that tsRNAs are functional molecules, not the random degradative products of tRNAs. Notably, due to their regulatory mechanism in regulating mRNA stability, transcription, ribosomal RNA (rRNA) synthesis and RNA reverse transcription, tsRNAs are significantly involved in the cell function, such as cell proliferation, migration, cycle and apoptosis, as well as the occurrence and development of a variety of diseases. In addition, tsRNAs may represent a new generation of clinical biomarkers or therapeutic targets because of their stable structures, high conservation and widely distribution, particularly in the peripheral tissues, bodily fluids and exosomes. In this review, we describe the generation, function and mechanism of tsRNAs and illustrate the current research progress of tsRNAs in various diseases, highlight their potentials as biomarkers and therapeutic targets in clinical application. Although our understanding of tsRNAs is still in infancy, the application prospects shown in this field deserve further exploration.

chain keeps extending. 3 However, protein-coding genes and related transcripts in different species account for only about 2% of the entire human genome. 4 The vast majority of the remaining sequences do not encode proteins, suggesting that non-coding RNAs (ncRNAs) may dominate the eukaryotic transcriptome. 5 ncRNAs include long ncRNAs (lncRNAs) and small non-coding RNAs, divided by the length of 200 nucleotides (nts). There are several types of regulatory small non-coding RNAs, including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs). [6][7][8][9][10][11][12] These ncRNAs have been reported to be involved in a number of biological activities, such as cell proliferation and migration, the cell cycle, apoptosis and autophagy. [13][14][15] Of note, they are closely related to various diseases, such as cancer, cardiovascular diseases, nervous system diseases and inflammation. 13,[16][17][18][19][20][21][22] In recent years, with the rapid advancement of sequencing technology, a cluster of new small ncRNAs, namely, tsRNAs, has been discovered. 23 tsRNAs are not randomly degraded fragments of tRNAs but are rather a class of abundant and novel RNAs with precise sequence structures as well as specific biological functions. 24 These new small RNAs, as members of the ncRNA family, are not yet fully understood.
Based on the present understanding, tsRNAs undergo special cleavage and are then modified at the 5′-and 3′-ends to give them the property of stability. 25 tsRNAs were reported to be implicated in several mechanisms, such as regulating mRNA stability, translation, rRNA synthesis and RNA reverse transcription. Accordingly, tsRNAs participate in regulating biological functions through these mechanisms, including cell proliferation, migration, cycle and apoptosis. Similarly, many studies have shown that tsRNAs have tissue specificity and temporal specificity and are expressed abnormally in numerous diseases, such as cancer, neurological diseases, metabolic diseases and viral infectious diseases. 26,27 More recently, the essential roles of tsRNAs in functions and mechanisms of various diseases were investigated.
In this review, we generalize the biological features, biogenesis, biology function and regulatory mechanisms of tsRNAs and illustrate the critical involvement of tsRNAs in the progression and regulation of cancer and other diseases. Finally, we discuss the potential of tsRNAs for new diagnostic biomarkers and clinical therapeutic strategies, despite the limitations of current research.

| THE IDENTIFI C ATI ON OF TS RNA S
The origin of tsRNAs dates back to the late 1970s, when they were originally discovered in tumour cells. 28,29 However, they were merely identified as random degradation products from tRNA sources and had not attracted intense attention in the field of scientific research.
Owing to the development of science and technology, tsRNAs were sequentially discovered in the human, 30 protists, 31 bacteria 32 and virus. 33 Some tsRNAs are evolutionarily conservative, despite their differential evolution between species. 34 In addition, they are not only widely expressed, but also have abundant sources. Because a single amino acid has many different tRNA acceptors, and an anticodon corresponds to many different tRNA acceptors, various types of tsRNAs can come from diverse tRNA sources. Moreover, accumulating evidence suggests that tsRNAs have special methylation modification and terminal modification, including cyclic phosphorylation modification, 5-OH modification and aminoacyl modification, 35 which modification occurs may be depended on the type of tsRNAs. In addition, the production of tsRNAs could be affected and activated by phosphate starvation, 36 hypoxia, 37 oxidative damage 38 and other malignant conditions. However, the level of tsRNAs rarely changes under normal conditions. 39 tsRNAs are considered to be functional molecules and not as by-products of non-functional degradation. 39 Thus, tsRNAs are not the tRNA random degradation products, 23 but a novel cluster of small molecular RNAs splits from tRNAs, with inherent conservation, high expression and good stability.

| THE B I OG ENE S IS OF TS RNA S: TS RNA S ARE USUALLY D IVIDED INTO T WO C ATEG ORIE S WITH S PECIAL S PLICING PAT TE R N S
The earliest of tRNA cleavage was observed in Escherichia coli (E coli) as a response to bacteriophage infection. 40 To better understand the biological production process of tsRNAs, it is necessary to first understand the structural characteristics of tRNAs. tRNA molecules are produced in the form of precursors and then go through a series of maturation events, including RNase P trimming the 5′ beginning end, RNase Z taking out the 3′ trailer, removing the introns and adding the 3′-terminal CCA sequence catalysed by CCA-adding enzymes. 41 Mature tRNAs have cloverleaf secondary structures with lengths of approximately 70-90 nts, and each tRNA includes the amino acid acceptor arm, the D-loop, the anti-codon loop, the variable loop and the TψC loop (also called the T-loop). 42 Furthermore, tRNAs have tertiary structures that look like an 'L', also called an L-shaped structure. tsRNAs usually can be divided into the following two subtypes depending on the cleavage sites in tRNAs and lengths: tiRNAs and tRFs. 20 tiRNAs are 28-36 nts long, generated by particular ribonucleases cleaving the anti-codon loops of mature tRNAs, 20,43 while tRFs, which are 15-32 nts in length, are produced by clipping the ends of precursor or mature tRNAs (Table 1). Accordingly, the two categories of tsRNAs are produced under different conditions and in different ways (Figure 1).

| The biogenesis of tiRNAs
The biogenesis processes of tiRNAs usually occur when cells experience stress conditions. 29 ANG is a member of the ribonuclease A superfamily, whose major function is promoting rRNA transcription and cell growth under growth conditions in the mammalian cell nucleus; however, ANG is mobilized to the cytosol to promote tRNA cleavage under stress conditions. [44][45][46][47] As a member of Ribonuclease T2 family, RNA, Ro60-associated Y1 (RNY1) can meditate the biogenesis processes of tiRNAs in yeast. 43 The ribonucleases above can cleave in the anti-codon loop of mature tRNA, forming two subtypes, called the 5′-tiRNAs and 3′-tiRNAs. 43 5′-tiRNAs are found from the 5′-ends to the cleavage sites, while 3′-tiRNAs are found from the cleavage sites to the 3′-ends. 47 These fragments are usually 28-36 nts long.

| The biogenesis of tRFs
The biogenesis of tRFs occurs under similar conditions as that of tiR-NAs, but the fragments are smaller with lengths of 15-32 nts. 48,49 tRFs have precise cleavage sites at the tRNA terminus, mainly determined by ribonucleases. According to the type of ribonuclease, tRFs contain type I tRFs and type II tRFs. From these, type I tRFs require Dicer to participate in the generation process, while instead, the 5′end of type II tsRNA is produced by the cleavage of RNaseZ, and RNA polymerase III is required to terminate transcription at the 3′end. 30 But the specific cleavage mechanisms are still under investigation. 26,30 Apart from above classification, tRFs can also be generally divided into three major types based on cleavage sites on the precursor or mature tRNA transcripts:tRF-5,tRF-3 and tRF-1.
The first type is tRF-5, which is 14-30 nts long and is produced by cutting the D-loop or the site between the D-loop and anti-codon loop of the mature tRNA transcript. This type is further divided into three subtypes of tRF-5 with different specific lengths; for example, tRF-5a has a length of 14-16 nts, tRF-5b is 22-24 nts, and tRF-5c is 28-30 nts. 48,49 tRF-3 has two subclasses: tRF-3a and tRF-3b, produced by ANG as well as other ribonucleases of the Ribonuclease A superfamily cutting the T-loop of mature tRNAs. They are ~18 or ~22 nts in length, which are smaller than tRF-5. 48,50,51 tRF-1 is generated from 3′-trailer fragment during the pre-tRNA maturation process, and the 5′-ends are cleaved by RNase Z or ELAC2, and the 3′-ends are transcriptional termination signals of RNA pol III (UUUUU, UUCUU, GUCUU or AUCUU). Therefore, the length of tRF-1 is very different. 23,52,53 Moreover, there are other types of tRFs, for example, tRF-2 and i-tRF. 54,55 However, the exact mechanism is not entirely clear. The specific subcellular localization of tRFs is also important for their biogenesis; for example, tRF-5 usually exists in the nucleus, while tRF-3 and tRF-1 are usually in the cytoplasm. 48,49 In addition, these fragments have 5′ phosphates and 3′ hydroxyls similar to the structure and size of miRNA, so that they have received attention recently.

| The relationship between tRNA modifications and tsRNA biogenesis
It is known to be more than 170 kinds of RNA modifications so far, of them, 93 are existed in tRNAs with different frequency and distribution depending on organisms or tRNA species. 56 Recently, tRNA modification abnormalities were found to not only influence the stability and function of tRNAs, but also affect the biogenesis of tsR-NAs. Recent study showed that NOP2/Sun RNA methyltransferase 2 (NSun2) and DNA methyltransferase-2 (Dnmt2) are both methyltransferases which can methylate C5 at cytosine residues (called m5C) to sustain tRNA stability and functions. 57 By disturbing these two methyltransferases, NSun2 and Dnmt2, cytosine-C5 tRNA methylation was deficient in the mice, which resulted in reduced tRNA stability and decrease in protein synthesis. 24,25 Except for the influence of tRNA, reduction in stability due to deficiency of m5C mediated by Dnmt2 could also result in a significant increase in tiRNAs derived from the cleavage tRNAs of ANG. 58,59 Deletion of Dnmt2 would induce the deletion of m5C at C38 on tRNA, which finally promote the shearing of tRNA, leading to the increase and accumulation of tsRNAs. 57 On the contrary, upregulation of m5C modifications decreased the levels of tiRNAs by suppressing the cleavage anti-codon loop of tRNAs by ANG. 60 Moreover, Alkbh1 and ALKBH3 were reported as 1-methyl adenosine (m1A) demethylases of tRNA. m1A demethylated tRNA is more sensitive to angiogenin (ANG) cleavage, which can reduce the stability of tRNA and increase tRNA cleavage, and finally produce tsRNAs in the anti-codon region of tRNA. 61,62 There is another study found that the lack of the pseudouridine synthase 7 (PUS7) can also interfere with different types of tRFs, and the pseudouridylation (Ψ) regulates the levels of multiple tRFs in stress-induced situations or specific cell types. 63 In addition, studies have shown that certain modifications may promote the activity of RNAases. For example, in order to prevent the population from being infected by bacteriophages, bacteria will specifically cut their own tRNAs in a suicidal manner. 64 Similarly, in order to avoid the proliferation of a non-self yeast species, the yeast Kluyveromyces lactis uses endonuclease to cut the anti-codons of various Saccharomyces cerevisiae tRNAs. 65 The above two cases indicate that the modification of tRNA anti-codon at the wobble uridine (U34) position, namely 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), is necessary for the enzyme protein tRNase to cleave tRNA. This modification promotes the activity of tRNase, but the defect of mcm5s2U at position U34 inhibits the activity of tRNase and the cleavage of tRNA. 66,67 The production of tRNAs is closely related to the activity of RNases. Although these studies do not clearly explain the molecular mechanism of the interaction between RNases and tRNA modifications, they do prove that tRNA modifications can affect the cleavage of tRNAs, which lead to the production of tRNAs. Collectively, the above studies show that abnormal tRNA modifications can cause self-splicing, which supports the view that specific tRNA modifications can affect tRNA cleavage; on the other hand, it suggests that the production of tsRNAs is a coordinated process when the body is stimulated. Then, this feature also provides evidence for tsRNAs as diagnostic markers; that is, when the body is attacked by pathogenic factors, the body may cooperate and undergo programmed tRNA cleavage to produce tsRNAs as markers for disease diagnosis.

F I G U R E 1
The biogenesis of tsRNAs. tsRNAs can be generally classified into the following two categories according to the cleavage sites in tRNAs and lengths: tiRNAs and tRFs. tiRNAs are classed by their source sequences from the 5′-or 3′-end of tRNA cleaved by ANG. tRFs are classified into three major types depending on the size and location of the source: tRF-5, tRF-3 and tRF-1. The current generation mechanisms of 2-tRF and i-tRF are still not completely clear

| THE REG UL ATORY MECHANIS MS OF TS RNA
It is known that tsRNAs are specific fragments derived from tRNAs cleaved by ribonucleases, but they are not the products of random degradation. Indeed, studies have successfully clarified that tsRNAs are small non-coding RNAs with regulatory functions, such as acting in mRNA binding (similar to miRNA) or as protein 'sponges,' regulating mRNA stability, inhibiting translation initiation and regulating ribosome biogenesis and RNA reverse transcription ( Figure 2).

| Regulating mRNA stability
tRFs are similar to miRNAs in structure and size, and, in particular, tRF-3s derived from tRNA Leu and tRNA Lys are the same as the 3′-end sequences of miR-1280 and miR-1274a/b, respectively. [68][69][70] Additionally, miRNAs are a class of small non-coding RNAs that regulate the combination of the miRNA-induced silencing complex (miRISC) with the 3′ untranslated region (3′UTR) of partially complementary sites in target genes to regulate mRNA stability. 71 Similarly, in a recent study, tRFs were found to have miRNA-like biological functions. For example, 3-tRF (also called CU1276) was a DICER1-dependent tRNA Gly-GCC -derived tRNA fragment expressed in mature B lymphocytes to modulate proliferation and the DNA damage response. 72 miRNA-like functions of tRFs in Drosophila have also been verified: tRFs suppressed mRNA translation preferentially by binding their 5′-and 3′-ends to conserved regions of 3′-UTRs in mRNAs, and most tsRNAs are conserved, imminent and plentiful in Drosophila according to an analysis of 495 common small RNA libraries. 73 Although most tRFs regulate the expression of target genes in a miRNA-like way, recent studies showed that tRF-3 played a role in inhibiting gene expression in a Dicer-independent manner. 25 In human HEK293 cells, tRFs were associated with Argonautes (Ago) 1, 3 and 4, but not with Ago 2, indicating that tRFs might play an important role in RNA silencing. 48 Additionally, a novel class of tRFs has been discovered recently, displacing the 3′-UTRs of YBX1 in breast cancer cells to suppress the stability of multiple oncogenic transcripts. 54 However, the mechanism by which endogenous tRFs regulate target mRNA is still unclear, and further studies are pending to elucidate the regulatory network of tRFs.

| tsRNAs regulate translation procedure
In addition to regulating mRNA stability by binding mRNA, tsRNAs also inhibit translation initiation by connecting with the translation initiation complex. 46

| tsRNAs participate in regulating rRNA synthesis
Ribosomes decode the genetic information carried on mRNAs into a polypeptides chain, while ribosome biogenesis, from the transcription of pre-rRNAs to the assembly of ribosomes, is regulated coordinately. 79 Recent research has started to show that tsRNAs participate in the process of rRNA synthesis by acting as a part of the pre-rRNA splicing complex (TXT) in protozoa (Tetrahymena). 80 TXT is a complex with many components that contains 3-tRF specifically binding with the Twi12 protein and exonuclease Xrn2. Upon binding, Twi12 stabilizes and localizes Xrn2 to stimulate the exonuclease, which cleaves the precursor rRNAs to regulate rRNA synthesis. 81 Another piece of evidence showed that tRNA Leu -derived tRF-3 enhances ribosomal protein translation by binding to RPS28 and RPS15 and inducing apoptosis in splitting cells in vitro as well as in vivo. 82

| Other potential regulatory mechanisms
Additionally, tsRNAs also participate in other potential functional mechanisms. For example, they could regulate cell proliferation and migration by inhibiting the expression of target genes. 85 They were proven to participate in RNA transcription and DNA-specific binding of proximal promoter of RNA polymerase II. Encouragingly, tsRNAs enveloped in exosomes were proved to be involved in communication signalling among cells. 86 Another study showed patrilineal metabolic diseases may be passed on to offspring through spermatozoa, and this epigenetic inheritance was regulated by tsRNAs. 87 Moreover, tsRNAs serve as important roles in immune response mediated by regulating T-cell activation. 88 As mentioned above, tsRNAs were reported to be implicated in several mechanisms, such as regulating mRNA stability, translation, rRNA synthesis and RNA reverse transcription. In the course of disease occurrence and development, the above mechanisms were more or less involved. Therefore, we have reasons to believe that tsRNAs may be used as a therapeutic method to inhibit the development and deterioration of diseases by regulating the expression of disease signalling pathways or their related genes.

| t sRNA S IN H UMAN D IS E A S E S
The discovery of tsRNAs is a recent breakthrough. The biological function and the regulatory mechanisms of different tsRNAs have been described above, along with these mechanisms corresponding to their roles in diseases. At present, a large amount of research shows that tsRNAs may play roles in the occurrence and development of various types of diseases, such as cancer, metabolic diseases, neurological diseases and viral infection diseases. Next, we will gradually explore the mechanisms of tsRNAs in diseases and potential therapeutic targets from the perspective of different diseases (Table 2).

| tsRNAs in cancer
Cancer is a group of diseases characterized by abnormal cell prolif- residues in tRNA instead of combining target sites of mRNA. 91 Collectively, tsRNAs can regulate cell proliferation by binding target genes, participating in pathways and performing other functions that are related to cell cycle, proliferation and migration, indicating tsRNAs have the potential to be detection biomarkers and (or) therapeutic targets; however, much more work needs to be performed to clarify the regulatory mechanisms and specific functional roles.

| tsRNAs in metabolic diseases
It has long been understood that an individual's environment is not only influenced by their own metabolism but also increased the probability of their offspring developing metabolic diseases. 92  study showed that in mice fed a low protein diet, tRFGly-GCC increased as spermatozoa matured in the epididymis, and genes associated with the endogenous retroelement MERVL were repressed not only in embryonic stem cells but also in embryos. Furthermore, tRFGly-GCC was cleaved in the epididymis and then transported to spermatozoa to carry out its function, inhibiting MERVL-regulated genes that affected placental size or function and then stimulating downstream effects on metabolism following altered placentation. 94 Another study suggested that the offspring of mice fed a high-fat diet (HFD) experienced the destruction of glucose tolerance and insulin secretion. Moreover, glucose tolerance was associated with sperm 5′tsRNAs. These tsRNAs contain numerous RNA modifications, such as 5-methylcytidine (m5C) and N2-methylguanosine (m2G), and the first offspring with metabolic disorders were created by influencing gene expression within metabolic pathways in the early embryo stage. 87 As previously reported, intergenerational inheritance becomes possible because spermatozoa may obtain environmental information mediated by tsRNAs. These studies provide theoretical support for the regulatory function of tsRNAs in metabolic diseases and provide a new idea that metabolic diseases can reduce the hereditary rate of offspring by regulating the level of tsRNAs.

| tsRNAs in neurological diseases
Neurological diseases are diverse and complex, and of these, neu- neurotrophic signalling pathways by targeting brain-derived neurotrophic factor. 103 Additionally, we have reason to believe that tsRNAs have a potential therapeutic effect on neurodegenerative diseases caused by NSun2 deficiency and mutation, but more studies are still needed to confirm how to regulate tsRNA levels and the precise mechanism of action. Besides, in order to better study tsRNAs, many animal models and types are needed to verify that they can be used as potential detection indicators and therapeutic targets.

| tsRNAs in viral infection diseases
Viruses invade the body in a variety of ways and proliferate in susceptible host cells, eventually causing diseases in a manner or via a mechanism that is not yet clear. 51 Recently, tsRNAs were found to have a potential relationship with viral diseases. tsRNA abundance increases markedly in hepatitis B or hepatitis C and HBV-and HCVassociated cancers, and this growth trend is consistent with ANG levels, suggesting there are specific HBV-meditated mechanisms regulating tRNA biogenesis directly. 104 Additionally, human respiratory syncytial virus (RSV), which often causes children to develop bronchiolitis and pneumonia, leads to abundant production of tsR-NAs, especially tRF-5 Glu-CTC (Figure 4). As a target of tRF-5 Glu-CTC , the gene expression of apolipoprotein E receptor 2 (APOER2) would be inhibited by the 3′-portion of tRF-5 Glu-CTC recognizing and binding its 3′-UTR. This inhibition benefits RSV replication because APOER2 is a kind of anti-RSV protein. 51,104 A subsequent study also found that the quantity of tRF-5 increases with rickettsia infection. 105

| sRNAs are potential ideal biomarkers for diagnosis and prognosis
At present, many clinical diseases lack specific diagnostic indicators, and most of them rely on pathological biopsy for diagnosis, which not only takes time but also poses invasive harm to patients.
Especially for tumours, the lack of symptoms in the early stage causes the loss of the best time for treatment. When patients come to see a doctor due to symptoms, they are mostly in the late stage.
Since cancer has not yet been conquered by human beings, most late-stage cancers are treated with surgery, radiotherapy or chemical drugs. These long-term treatments impose economic burden on patients and bring great physical and psychological pain to patients.
Therefore, if diseases can be detected at an early stage through routine physical examination, it will be a great boon to patients. With Nucleotide modification, such as 5-methylcytidine (m5C) and N2methylguanosine (m2G), increases the stability of tsRNAs. The levels of tsRNAs are substantially reduced without methylation, which indicates unmethylated tsRNAs are degraded by ribonucleases. 25 This stability can also be sustained by binding a large-size serum protein complex. 27 The presence of such modifications greatly improves the stability of tsRNAs and lays an important foundation for their role as clinical detection, as well as differential diagnosis and prognosis. 27 In conclusion, all of these properties indicate that tsRNAs have the potential to become specific and accurate biomarkers for diagnosing cancer and other diseases in different systems.

| tsRNA sequencing analysis in patient samples
Based on the above analysis, since tsRNAs have the potential for diagnosis and prognosis, many studies have also been carried out in clinical samples to find tsRNA biomarkers that can be applied in clinical practice ( could be used not only as a diagnostic indicator for breast cancer staging but also as a tumour inhibitor to suppress the progression of breast cancer. In addition, 86 gastric cancer tissues and adjacent paired non-tumour tissues were collected, and the expression level of tiRNA5034-GluTTC-2 was observed to be significantly lower in gastric cancer tissues. 89 Then, paired plasma samples were collected from gastric cancer patients 1 day before and 7 days after surgery. The results showed that the levels of tiRNA5034-GluTTC-2 before and after surgery were significantly downregulated compared with those in the healthy group. More importantly, multivariate analysis of the study data showed the potential value of tiRNA5034-GluTTC-2 in the diagnosis of gastric cancer. Apart from these tsRNAs above, several tsRNAs were found dysregulated in CLL blood and EC tissue. 86,114 These clinical studies show that, whether in tissue samples or in plasma samples, tsRNAs are significantly differentially expressed in diseases and control groups. Taken together, these results indicate that tsRNAs exhibit significant advantages in disease detection and (or) therapeutic treatment.

| tsRNAs exist in microvesicles and exosomes
Exosomes are exocytic microvesicles (30-100 nm) released by healthy and pathological cells, which carry DNAs, RNAs and proteins to promote communication between different cells which are generally considered as promising therapeutics in future. 17,115 Studies had already found several ncRNAs, such as miRNAs and lncRNAs, are abundant in exosomes and interact with recipient cells to regulate cell function and disease progresses. [116][117][118] Likewise, recent studies showed tsRNAs are widely exist in microvesicles and exosomes, which could protect themselves from degradation and make them stable components of body fluids. 88 What's more, current studies had also confirmed that tsRNAs could transfer from transfected cells to recipient cells through exosomes, 119 and play roles by this special delivery system ( Figure 5). For example, Fusobacterium nucleatum, a kind of Gram-negative oral bacteria, could stimulate the release of tsRNA-000794 and tsRNA-020498 embedded in exosomes in human normal oral keratinocyte cells. In reverse, these two tsRNAs inhibited the growth of F nucleatum by disturbing protein synthesis. 120 Moreover, further study verified tRFs in EVs had the potential to regulate immune response by affecting T-cell activation. 88 Surprisingly, exosomal tsRNAs could be released by Schistosoma mansoni to regulate gene expression in eukaryotic cells. 121 Especially, the study found that the expression levels of four tsRNAs (tRNA-ValTAC-3, tRNA-GlyTCC-5, tRNA-ValAAC-5 and tRNA-GluCTC-5) were significantly increased in exosomes extracted from the blood of patients with liver cancer compared with the control group, 122 implying they had the potential of becoming diagnostic biomarkers.
However, there is no unified exosome separation method, and the low purity of exosomes is one of the unsolved problems. In addition,   of differentially expressed tRFs/tiRNAs. 86 The bioinformatic analysis of predicting downstream targets is not yet complete, which hinders the study of tsRNAs. Therefore, tsRNA research needs to overcome these limitations in order to achieve long-term development.

| CON CLUS I ON S AND PROS PEC TS
Although at present our research on tsRNAs is still in its infancy, the expression levels of tsRNAs in physiological and pathological conditions are obviously different, and we have reason to believe that tsRNAs have very good research prospects in cancers, metabolic diseases, neurodegenerative diseases, viral infections and additional diseases. First, tsRNA can be used as a biomarker for disease occurrence, development and prognosis. For example, a study on gastric cancer showed that tiRNA5034-GluTTC-2 may be a new biomarker because the expression level of tiRNA-5034-GluTTC-2 is negatively correlated with the size of the tumour. In addition, in the survival curve, the overall survival time of patients in the high expression group of tiRNA5034-GluTTC-2 was significantly higher than that of the low expression group, suggesting that tiRNA5034-GluTTC-2 is an independent predictor of gastric cancer prognosis. 89 Second, tsRNAs may be used as a means of clinical treatment target. It is reported that extracting tsRNAs from spermatozoa of HFD male mice and injecting it into the fertilized eggs of normal mice will cause metabolic disorders in the F1 generation offspring and changes in the expression of genes related to islet metabolism. Suppose that in order to block the occurrence of paternal inherited metabolic diseases, we may achieve this by promoting tsRNA degradation or inhibiting tsRNA production. 87 In addition, tsRNAs can be used in clinical therapy, not only as direct therapeutic targets but also to regulate disease progression through indirect regulation of signalling pathways. By regulating the expression of tsRNAs, blocking or delaying the occurrence and development of diseases can be a suitable clinical application strategy for tsRNAs. It is worth mentioning that tsRNAs are abundantly expressed, widely distributed in the body and modified to improve stability. These characteristics are strong evidence that tsRNAs have the potential to become non-invasive diagnostic or therapeutic methods. 89 With the development of science and technology, more new technologies will emerge and see constant improvement, which will be conducive to the identification of more tsRNAs that can be developed further into clinically available biomarkers and therapeutic targets, providing a new direction and perspective for the diagnosis and treatment of diseases.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflicting interests.

AUTH O R CO NTR I B UTI O N S
ZT, FX, LL and WZ collected articles and designed the manuscript.
ZT, YY and ZH wrote the manuscript. TG and LM prepared tables and figures. YT, ALHH, ZH, LP and WJ revised and approved the manuscript. All authors read the manuscript and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article, so no new data were created or analysed in this study.