tsRNA, cell death and disease: Connecting the dots

Abstract

Cell death is essential for maintaining physiological homeostasis and regulating pathological processes. tRNA-derived small RNAs (tsRNAs), an emerging non-coding small RNA, play key roles in a various form of cell death. The relationship between tsRNAs and diseases has attracted growing attention. However, many challenges remain, particularly in understanding how tsRNAs regulate cell death and their newly discovered roles in these pathways. This paper reviews the generation and classification of tsRNAs and their roles in different diseases through cell death processes, such as ferroptosis, apoptosis, necroptosis, autophagy and pyroptosis. We discuss in detail the dysregulation of tsRNA expression in neurological disorders, metabolic diseases and cancer. We highlight the potential of tsRNAs as biomarkers and therapeutic targets in the context of cell death pathways.

Abbreviation

tsRNAs

tRNA-derived small RNAs

tiRNAs

tRNA-derived RNAs

tRFs

tRNA-derived fragments

pre-tRNA

Precursor tRNA

AGO

Argonaute

ROS

Reactive oxygen species

1. Introduction

Cell death is involved in both physiological and pathological processes [1]. Cell death helps eliminate damaged cells to maintain homeostasis but can also result from pathological responses to harmful stimuli [2,3]. The Cell Death Nomenclature Committee has developed guidelines to categorize cell death into two types: accidental cell death (ACD) and regulated cell death (RCD), based on morphology, biochemistry and function [4]. Cells may die from ACD or RCD, where ACD is a biologically unregulated process, and RCD involves organized signaling pathways and precisely targeted effector processes [5].

Transfer RNA (tRNA) is a non-coding RNA (76–93 nt) essential for mRNA translation [6]. Each tRNA contains an anticodon sequence that recognizes a specific codon triplet sequence on mRNA and transfers the corresponding amino acid to the growing polypeptide chain. Under stress or specific enzymatic activity, tRNA can be fragmented to yield several small RNAs known as tRNA-derived small RNAs (tsRNAs). tsRNA is a significant and emerging class of small non-coding RNA (sncRNA) [7] which demonstrates great potential in the regulation of gene expression, involvement in disease genesis and function of diagnostic biomarkers as well as therapeutic targets [8]. As research advances, the complexity of tsRNA bioregulatory networks may provide new strategies for disease diagnosis and treatment.

tsRNAs are closely linked to cell death and play a role in regulating the cell death process through various mechanisms. In this paper, we summarize the functions of tsRNAs in different diseases, examining their classification, characterization and role in cell death pathways.

2. Cell death types

Cell death may occur in various forms in response to different stresses, especially oxidative stress [9]. Dysregulation of cell death, whether singular or mixed, can lead to disorders like cancer, neurodegenerative diseases, autoimmune diseases and infections [[10], [11], [12], [13]]. Over the past decades, several novel RCDs have been linked to various human pathologies. For instance, necroptosis, apoptosis and pyroptosis are caused by activation of programmed cell death pathways [14], while ferroptosis and cuproptosis result from inactivation or destruction of basic cell survival mechanisms [15] (Table 1).

Table 1.

Biochemical characterization, morphological features and regulatory pathways of different cell death.

Type	Morphological features	Regulatory pathway	References
Ferroptosis	Cell membrane: breaks and vesicles are seen Nucleus: no obvious morphological changes, no chromatin condensation and nuclear rupture. Mitochondria: mitochondria will show atrophy and become smaller, mitochondrial membrane density increased, mitochondrial cristae decreased or even disappeared, outer membrane rupture.Under the electron microscope, the mitochondria as a whole will be atrophied, and the mitochondrial membrane potential will be obviously reduced or disappeared.	Positive: TFRC, ACSL4, LPCAT3 Negative: GPX4, FSP1, NRF2	[16,17]
Apoptosis	Cell size: the cell volume will be significantly reduced Cell membrane: the cell membrane lipid bilayer loses its two-sided asymmetry and phosphatidylserine is exposed on the cell surface, which leads to phagocytosis.On the surface of the cell membrane, many tiny vesicle-like structures called apoptotic vesicles will be formed. Nucleus: The chromatin of the nucleus will appear to be significantly condensed and take on a condensed appearance.At the same time, the nucleus will also undergo nuclear consolidation, and the chromatin within the nucleus will become denser.Another distinctive feature of apoptosis is the fragmentation of the nucleus, splitting into multiple small nuclear fragments (nucleosomes)	Endogenous: BAX, BAK, BIM, BID, PUMA, BCL-2, BCL-XL Exogenous: TNFR1/2, Fas, DR4, DR5, RIPK1	[18]
Necrotic apoptosis	Cell membrane: necrotic apoptotic cells experience rupture of the cell membrane, resulting in leakage of cell contents outside the cell Organelles: organelles such as mitochondria and endoplasmic reticulum become swollen Cytoplasm and nucleus: necrotic apoptotic cells undergo disintegration of the cytoplasm and nucleus, but unlike apoptosis, the nucleus does not form apoptotic vesicles	Positive: TNFR1, DR, RIPK1, RIPK3 and MLKL Negative: AURK4, ESCRT-III	[19]
Phagocytosis	Formation of double-membrane autophagic lysosomes, (autophagosome) with a double or multilayer membrane structure. Observed under TEM as crescent-shaped or cup-shaped with a tendency to encapsulate cytoplasmic components, including macroautophagy, microautophagy and chaperone-mediated autophagy	Positive: AMKP, ULK1, ULK2, FIP200, ATG13, ATG101, VPS34, PI3P Negative: mTOR	[20]
Pyroptosis	Cell size: under the light microscope, pyroptosis cells show swollen and enlarged cells, and there are many bubble-like protrusions. Cell membrane: under the electron microscope, it can be clearly seen that before the rupture of the plasma membrane of the cell, the pyroptosis cells form a large number of vesicles, i. e. pyroptosis vesicles. After that, pores will be formed on the cell membrane, the cell membrane ruptures, and the content flows out. Nucleus: nuclear condensation and DNA breaks. Mitochondria: intact and swollen, reduced matrix density and cristae collapse.	Positive: CASP1, CASP3, CASP4, CASP5 (mouse caspase-11), CASP6, CASP8, CASP11, Gasdermin D, NLRP3 Negative:ESCRT-III、GPX4	[21]

3. Nomenclature, biogenesis, modification effects and functions of tsRNAs

tsRNAs are mainly derived from mature tRNAs or tRNA precursors (pre-tRNAs) through cleavage by enzymes such as Angiogenin(ANG), Dicer, RNase Z and RNase P. The generation of tsRNAs is a conserved and regulated cellular process [[22], [23], [24]]. tsRNAs is classified based on their parental tRNAs and biogenesis source regions, including tRNA-derived fragments(tRFs) and tRNA-derived stress-induced RNA named tRNA halves(more commonly referred to as tiRNAs) [8]. The expression and modification of tsRNAs are tissue- and cell-specific [25]. They are also involved in a range of physiological and pathological processes, including stress response, protein translation regulation, ribosome biosynthesis, tumorigenesis, cell proliferation, cell death and transgenerational transmission of epigenetic information [24,26].

3.1. Nomenclature of tsRNAs

To standardize research and classification, tsRNA should follow certain naming rules. Overall, tsRNA naming usually involves the following aspects based on the tsRNA classification and source location: Source of the tRNA type (e.g., tRNA-Ala, tRNA-Gly), location of cutting (e.g., 5′ end, 3′ end, near the anticodon ring), type of fragment (e.g., tRF, tiRNA, tRNA halves), possible length or specific location number and the anticodon information or modification information (e.g., m⁵C-tRF 5-Thr-TGT means that the 5′-end tRF contains a 5-methylcytosine modification) [27].

Besides, there is also a standardized nomenclature system based on the molecular origin.This naming system is called the tDR (tRNA-derived RNA) naming system. First, tsRNA names should follow specific prefixes and suffixes. For example, prefix “tDR” is used for “small RNA derived from transfer RNA”. For mitochondrial or plastid-derived tsRNA, either “mtDR” or “ptDR” is used respectively. Next, the name includes the start and end positions of the tDR relative to its source tRNA like “tDR-4:33”, based on the Sprinzl numbering system. If present, the 5′ leader and 3′ trailer positions are labeled with “L” or “T” respectively. The source tRNA name is then added from the GtRNAdb, such as Lys-CTT-003. If multiple tsRNAs are derived from the same source tRNA, an “M” and the number of matching transcripts are included like “M7”. Lastly, any nucleotide variations from the reference source tRNA are annotated like “U10A” [28].

3.2. The biogenesis of tsRNAs

tsRNAs can be classified based on the cleavage site of the tRNA. tRNA-derived fragments (tRFs) are categorized into i-tRF, tRF-5, tRF-1, tRF-2, and tRF-3′ (Fig. 1) [24]. tiRNAs are differentiated into 5′-tiRNAs or 3′-tiRNAs based on whether they contain 5′ or 3′ sequences [26].

Fig. 1.

tsRNAs species and biogenesis. A. tRNA folds into a cloverleaf structure. B. tsRNAs can be classified into different types depending on the cleavage site in mature tRNAs. Additionally, the cleavage of pre-tRNAs can also produce tsRNAs.

As shown in Fig. 1, tRF-5 (14–30 nt), generated by Dicer cutting the D-loop, anticodon and D-loop stem, is divided into three isoforms based on length: tRF-5a (14–16 nt), tRF-5b (22–24 nt) and tRF-5c (28–30 nt) [29,30]. tRF-3 (18–22 nt), cleaved by Angiogenin, Dicer or exonucleases, includes the 3′ CCA sequence and is further divided into tRF-3a (18 nt) and tRF-3b (22 nt). tRF-2, produced by the anticodon loop under certain conditions like hypoxic [31], excludes the 5′ end and 3′ end regions but contains the anticodon loop and part of the anticodon stem. The precursor tRNA(16-48 nt) is the initial product of transcription of the tRNA gene and is cleaved by RNase Z to form tRF-1, which contains the 3′ tail of the polyU sequence. i-tRF, an internal tRF produced from mature tRNAs, includes D- and T-loop sequences but excludes the 5′ and 3′ end regions, and is less researched [32]. tsRNAs can also be classified into four types: Type I tsRNAs (including tRF-5 and tRF-3, from the D- or T-loop), tiRNAs, Type II tsRNAs (from tRF-1) and internal tsRNAs [33].

3.3. Effects of tRNA modification on tsRNA

tRNA modifications not only influence its function but also prevent its cleavage by nucleases. Thus, the absence of tRNA modifications may induce the production of tsRNA [34].Certain RNA modifications protect tRNAs from cleavage, thereby affecting tsRNA production. For example, in human cells, the m⁵C modification plays a protective role in specific tRNA species, preventing them from being cleaved into tsRNAs [35].Conversely, certain modifications promote tRNA cleavage, which in turn affects tsRNA production. For example, in E. coli, the wobble U of tRNA-Lys is modified to 5-methylaminomethyl-2-thiouridine (mnm5s2U), a modification that makes tRNA-Lys a specific target of PrrC nuclease for cleavage upon phage infection [36].

Some RNA modifications can enhance the stability of tsRNAs, allowing them to function for a longer period of time in cells [37]. For example, pseudouridine (Ψ) modification in TOG (terminal oligoguanine motif)-containing tsRNAs stabilizes their structure and thus enhances their function in repressing translation.Modifications can also regulate tsRNA activity [38]. Besides, in mammalian cells, RNase L cleaves tRNA-His to generate tsRNAs, and the choice of cleavage site is partly dependent on the modification on tRNA-His. Specifically, G34 is modified to Q34, and this modification provides specificity for RNase L so that its cleavage occurs at a specific site, thereby affecting tsRNA activity [39].

RNA modifications can alter the binding ability of tsRNAs to proteins, thereby affecting their function. For example, TOG-containing tsRNA interacts with Y-box binding protein 1 (YB-1) through its TOG motif, and this interaction is essential for tsRNA-induced formation of stress granules (SGs) [40]. Certain modifications may alter the binding properties of tsRNAs to other RNAs. For example, tsRNAs can inhibit translation of mRNAs by complementary pairing with specific regions of the mRNA, and RNA modifications may affect the stability and specificity of this complementary pairing, thereby modulating the regulatory effects of tsRNAs on mRNAs [41].

3.4. The functions of tsRNAs

tsRNAs play diverse roles in biological processes (Fig. 2), including mRNA silencing, cell–cell communication, gene expression regulation, post-transcriptional modifications, cell proliferation and cell death [33,42]. For example, tsRNAs can inhibit mRNA translation in a sequence-specific manner by binding to AGO proteins, exerting miRNA-like regulatory effects [22]. Additionally, tsRNAs can also regulate protein translation initiation by binding to eIF4G/eIF4E or YBX1 [43,44], which in turn affects overall protein synthesis [45].

Fig. 2.

tsRNAs' functions. tsRNA has multiple functions in cell biology, including influencing cell proliferation and death, participating in cell communication, regulating gene expression like RNA silencing, translational regulation and reverse transcription.

Studies on the role of tsRNA in cell death, including apoptosis, are limited. However, research shows that tsRNAs can inhibit translation and apoptosis by interacting with cytochrome C (Cyt C), preventing apoptosome formation and caspase-9 activation [46]. Similarly, tiRNA can bind to Cyt C released from mitochondria, forming a Cyt C-ribonucleoprotein (RNP) complex that inhibits apoptosis [47] (Fig. 3).

Fig. 3.

tsRNAs and cell death pathways. tsRNAs can regulate various signaling pathways (such as NF-κB, p53, NLRP3 inflammasome, autophagy-related proteins and RIPK1) to affect ferroptosis, apoptosis, pyroptosis, necroptosis and autophagy, which cover different types of cell death mechanisms.

tsRNAs hold great potential as biomarkers for diagnosing diseases due to their high content and stability in a wide range of body fluids [[48], [49], [50]]. To date, tsRNAs have been discovered in numerous body fluids, such as the blood, serum, urine and saliva. Their expression patterns are altered in different diseases, such as cancers(especially lung, liver, colon and pancreatic cancers), neurodegenerative disorders and cardiovascular diseases [51]. While most research has focused on tsRNAs as tumor biomarkers for diagnosis, staging, and prognosis, they also have potential for therapeutic development and in understanding acquired metabolic diseases, neurological conditions and stress-related injuries, though research in these areas is still limited.

4. tsRNAs in disorders

4.1. tsRNAs and metabolic diseases

Obesity, diabetes, related cardiometabolic disorders and non-alcoholic fatty liver disease (NAFLD) are increasingly common in modern society. The etiology of these conditions is multifactorial, influenced by the interaction between genetic factors and environmental elements like diet and physical activity [52]. In metabolic diseases, tsRNAs are implicated not only in developmental processes but also play a vital role in regulating organ damage, partly by influencing cell death (Table 2).

Table 2.

List of diseases associated tsRNAs.

Type	Disease	Expression	Function	Pathway	References
tRF-47 (tRF-47-58ZZJQJYSWRYVMMV5BO)	NASH	Up	Treatment target	Autophagy, Pyroptosis	[53]
tRF3-IleAAT	DKD	Down	Treatment target	Ferroptosis	[54]
tRF-5014a	DCM	Up	Diagnostic biomarker and treatment target	Autophagy	[55]
tRF3-Thr-AGT	AP	Down	Treatment target	Pyroptosis	[56]
tsRNA-3029b	Depression	Up	Treatment target	Ferroptosis	[57]
5′ Tyr-tRF	NDs	Down	Treatment target	Apoptosis	[58]
tRF-315 (tRF-Lys-CTT)	PCa	Up	Diagnostic biomarker and treatment target	Apoptosis	[59]
tRF-Glu-TTC-027	GC	Down	Treatment target	Apoptosis	[60]
tRF-Val (tRF-60:76-Val-CAC-2)	GC	Up	Treatment target	Apoptosis	[61]
tRF-23-Q99P9P9NDD	GC	Up	Diagnostic biomarker and Treatment target	Ferroptosis	[62]
tiRNA-Lys-CTT-003	AKI	Down	Treatment target	Ferroptosis	[63]
tRF-1:30-Gly-GCC-2	Follicular atresia	Up	Treatment target	Ferroptosis, Apoptosis	[64]
tRF-Gly-GCC	RILI	Up	Treatment target	Apoptosis	[65]
tRF-SeC-TCA-001 tiRNA-Gly-CCC-003 tRF-Gly-GCC-002 tRF-Tyr-GTA-007	AF	Up	Treatment target		[66]
3′-tiRNA-Leu-CAG	Aging	Up	Treatment target	Necroptosis	[67]

4.1.1. Non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease worldwide, and its prevalence continues to rise annually [68]. NASH is based on varying degrees of inflammation, hepatic steatosis, hepatocellular injury and fibrosis, and can progress to cirrhosis or even hepatocellular carcinoma [69]. Research on the role of tsRNAs in hepatic disorders is still in its early stages, requiring further investigation to understand their involvement in disease progression.

In NASH, Juanjuan Zhu [53] found that reduced expression of tRF-47 diminishes the beneficial effects of tectorigenin (TEC, a component of blueberry extract). TEC inhibits toll-like receptor 4 (TLR4) expression and the MAPK/NF-κB pathway, promoting autophagy and protecting against liver failure by significantly increasing the autophagy marker LC3B in NASH mice [70]. TEC upregulates tRF-47, which is a specific tsRNA that activates autophagy and inhibits pyroptosis. Pyroptosis mainly works as an inflammation-driven cell death, depending on the inflammasome complex (NLRP3, ASC and pre-caspase-1) [71]. In NASH mice, upregulation of NLRP3 and GSDME (markers of pyroptosis) activates inflammatory vesicles and TLR4, but TEC significantly inhibits pyroptosis and reduces their activation. Decreased tRF-47 expression impairs TEC's effects on NASH by inhibiting autophagy, activating pyroptosis and increasing the release of inflammatory factors like IL-6, TNF-α, and IL-1β [72]. Thus, tsRNAs may influence NASH progression by regulating the balance of autophagy and pyroptosis. Moreover, tRF-47 may reduce lipid accumulation in the liver by affecting lipid metabolism-related pathways, such as the PI3K-Akt signaling pathway. These findings suggest that tRF-47 could be a potential therapeutic target for NASH, with TEC as a possible treatment.

4.1.2. Complications of diabetes

Diabetic kidney disease (DKD) is a common complication of diabetes mellitus, closely linked to renal fibrosis, where the synthesis of extracellular matrix (ECM) components in glomerular cells plays a critical role in disease progression [73]. Yun-Yang Qiao [54] reported that overexpression of tRF-3 significantly reduces the key components of ECM synthesis and helps alleviate renal fibrosis in DKD. In their study, Db/db mice exhibit significant renal insufficiency, with significantly higher expression of GPX4 and SLC7A11 while lower expression of ACSL4 in renal tissue, indicating increased ferroptosis and ECM synthesis. Overexpression of tRF-3 reversed these effects.Former research revealed that tRF3-IleAAT inhibits ferroptosis by targeting the downstream gene ZNF281. tRF3-IleAAT is fully complementary to the 3′ UTR of ZNF281, a relationship confirmed by dual luciferase reporter assays [74]. Overexpression of tRF3-IleAAT reduced the key ECM components protein like levels of FN (fibronectin) and Col I (collagen I). This reduction in ECM synthesis contributes to alleviation of renal fibrosis in DKD [75]. Therefore, tRF3-IleAAT has emerged as a promising target for DKD as it directly targets ZNF281, downregulates its expression, inhibits ferroptosis and reduces ECM synthesis in DKD models.

Diabetic cardiomyopathy (DCM) is a severe complication of diabetes mellitus, characterized by cardiomyocyte dysfunction, which can lead to arrhythmias and sudden death. Yongting Zhao [76] discovered that tRF-5014a, the most significantly upregulated tsRNA in injured cardiomyocytes, negatively regulates the expression of ATG5 (a key protein in autophagy). Reduced ATG5 expression impairs autophagy function [77]. Inhibition of tRF-5014a restore autophagy, increases cell viability and reduces cell death as well as decreases the release of pro-inflammatory cytokines (e.g. , IL-1b and IL-18) under high glucose (HG) conditions [78].

These results indicate that tRF-5014a is a crucial factor in cardiomyocyte damage induced by high glucose, and blocking it may counteract autophagy suppression, improve cell survival and reduce inflammation.

4.1.3. Acute pancreatitis

Acute pancreatitis (AP) is a pancreas-related disease characterized by severe inflammatory, with 20 % of cases progressing to severe AP (SAP), which has a high mortality rate [79]. NLRP3-mediated pyroptosis cell death and inflammation play a critical role in the pathogenesis of AP, and targeting this biological process is a potential therapeutic strategy for SAP [80]. Studies show that NLRP3 activation exacerbates AP by promoting pyroptosis [81]. Related studies have shown that tsRNA can inhibit AP progression through the cell death pathway. Boshi Sun's study [56] reported that upregulation of tRF3-Thr-AGT was able to inhibit NLRP3-mediated cellular pyroptosis and inflammatory responses by targeting the 3′UTR of ZBP1(a protein closely associated with inflammation and cellular pyroptosis) [82]. This leads to the degradation of ZBP1 and inhibits AP progression. Overexpression of tRF3-Thr-AGT in AR42J cells reversed STC-induced damage, improving cell viability, reducing pyroptosis and suppressing inflammation [83]. These findings demonstrate that tRF3-Thr-AGT regulates pyroptosis and inflammation through ZBP1 stability, establishing a direct connection between tsRNA and pyroptosis. Given NLRP3 inflammasome plays a regulatory role in a diversity of inflammatory diseases [84]. tsRNAs could potentially serve as a therapeutic target for conditions, including rheumatoid arthritis, inflammatory bowel disease and atherosclerosis.

Previous research on non-coding RNAs in metabolism has focused on their roles in processes such as browning promotion, glucose tolerance improvement and insulin regulation, suggesting a growing interest in the role of tsRNAs in metabolism [85]. However, our understanding of tsRNAs in metabolic diseases, aging, reproduction and stress-related cell death pathways remains limited. Additionally, some areas that could potentially be influenced by tsRNAs have yet to be identified. More research is needed to fully explore the role and mechanisms of tsRNAs in disease and physiological processes.

4.2. tsRNAs in nervous system diseases

The nervous system is highly complex and consists of different regulatory pathways that govern neurobiological functions. In recent decades, the role of tsRNAs in the development of the central nervous system (CNS) and related diseases has gained significant attention. tsRNA is distributed specifically in nervous system-related tissues, with levels in the hippocampus of primates being nearly six times higher than in other brain regions and organs. The most prevalent types are 5′ tiRNA-Gly-GCC and 5′ tiRNA-Glu-CTC [86]. 5′-tRNA halves have similar expression levels in all tissues, while 3′-tRNA halves are highly aggregated in the amygdala/hippocampus complex, and tRF accumulates in brain tissues but not in the others [87]. Besides, tsRNAs are implicated in various neurological disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS), stroke and other neurological disorders [88,89]. tsRNAs contribute to neuronal cell death through multiple pathways (Table 2), with a reduction in tRNA ligation leading to increased tRNA fragment formation. These fragments inhibit protein translation and regulate cell death in both yeast and mammalian cells [90,91].

4.2.1. Depression

Depression is a prevalent mental health disorder characterized by persistent low mood, lack of pleasure in previously enjoyable activities, including both somatic (e.g., weight changes, sleep disturbances) and cognitive symptoms (e.g., inattention) [92]. Enze Li [57] reported that ferrostatin-1 (Fer-1), a ferroptosis inhibitor, reduced chronic unpredictable mild stress (CUMS)-induced depression-like behaviors and promoted neuronal growth in mice. CUMS leads to disturbed tsRNA expression profiles in mouse hippocampal tissues, whereas Fer-1 downregulates aberrant tsRNA expression. Specifically, tsRNA-3029b was identified as an effective target. Inhibiting tsRNA-3029b lowered reactive oxygen species (ROS) levels and increased the expression of SLC7A11 and GPX4 [93], thereby preventing ferroptosis in neuronal cells, promoting neuronal regeneration and reducing neuronal damage [89]. Silencing tsRNA-3029b may offer a new approach for treating depression by protecting neurons from ferroptosis.

4.2.2. Neurodegenerative diseases

Neurodegeneration, associated with aging, leads to the progressive deterioration of CNS myelin and neuronal dysfunction. Aberrant tRNA metabolism and mutations in tRNA-processing enzymes have been linked to neurodegenerative diseases (NDs) [94,95].

Recent studies suggest that tsRNAs influence ND progression by regulating cell death through tRNAs-protein interactions. BEX (brain-expressed X-linked proteins) has become an emerging research target on this issue.The BEX family consists of five proteins—BEX1, BEX2, BEX3, BEX4 and BEX5—expressed in various tissues and involved in signaling pathways related to NDs and cancer [96,97]. Mariana J. do Amaral [98]'s studies found that tRFs co-purified with hBEX3(an X-linked protein 3 expressed in the human brain). tRFs induce a structural transformation of hBEX3 from a disordered to a compact, globular fold. In its compact form, hBEX3 becomes the p75NTR-associated cell death executor, which can self-aggregate to form oligomers rich in intrinsic disorder [99] and interact with the p75NTR receptor. Meanwhile, it binds to hamartin, a protein expressed by the TSC1 genes that regulates Neurotrophins (NGF)-induced apoptosis and differentiation. Their interactions impair the function of the nervous system and causes the onset and progression of neurological disorders, psychiatric disorders and cancers [100]. These findings highlight new avenues for studying tsRNAs in the pathophysiology of neurodegenerative diseases.

tsRNAs are involved in the normal functioning of motoneurons and their response to oxidative stress, with certain tsRNAs modulating the susceptibility of motoneurons to cell death induced by oxidative stress. Schaffer AE [101] found that the accumulation of low levels of mature tRNAs increased the sensitivity of motor neurons to oxidative stress. These tRNAs were derived from the RNA kinase CLP1, which is crucial for tRNA, mRNA and siRNA maturation as well as tRNA splicing [102]. CLP1 deficiency leads to stress-induced cell death and genetic diseases like PCH10 (pontocerebellar hypoplasia type 10) [103]. In some cases, CLP1 mutations lead to the production of tRF, which increases neuronal susceptibility to p53-mediated oxidative damage, resulting in reduced familial NDs [104]. Additionally, the Tyr tRNA 5′-Phospho-3′-exon fragment protects CLP1R104H mutant cells from stress-induced cell death. Transfection with a 5′ tRNA fragment from tyrosine precursor tRNA (5′ Tyr-tRF) similarly protects CLP mutant cells from oxidative stress-induced death [105]. Impaired tRNA maturation due to CLP1 dysfunction is associated with neurodegenerative changes in the cerebellum and brainstem. Mutations in CLP1 affect tsRNA maturation, disrupting the cellular stress response and cell death, which may contribute to the onset and progression of neurodegenerative diseases [106].

4.2.3. Neurodevelopmental disorders

In addition to their role in nervous system diseases, tsRNAs also influence neuronal development. Masanori Inoue [58] reported that 5′ Tyr-tRF caused p53-dependent neuronal cell death more significantly than other types of tRNA fragments. Under different stresses, p53 triggers various cellular processes, including metabolism, apoptosis/non-apoptotic cell death and migration/invasion [107]. During neural development, PKM2 (pyruvate kinase M2) provides energy and metabolic intermediates for neuronal cell growth, differentiation and functional maintenance by regulating the glycolytic pathway. 5′ Tyr-tRF binds directly to PKM2, causing apoptosis in human SH-SY5Y cells, particularly before they differentiate into neurons. Embryonic developmental abnormalities and neurological defects caused by 5′ Tyr-tRF-induced cell death could be prevented by knocking down p53 and injecting PKM2 mRNA [108]. In addition to their role in nervous system diseases, tsRNAs also influence neuronal development.

tsRNAs are crucial components in the central nervous system (CNS), where they are implicated in a range of neurological disorders through cell death. In depression, tsRNAs contribute to ferroptosis, while in neurodegenerative diseases (NDs), they primarily regulate apoptosis. tsRNAs also show potential as biomarkers in disorders like Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and epilepsy [[109], [110], [111]]. For example, AS-tDR-013428 is a potential biomarker in ALS [[110], [112]]. However, more research is needed to understand how tsRNAs influence disease processes through cell death pathways.

4.3. tsRNAs and cancers

Numerous studies have demonstrated that tsRNA levels are dysregulated in various cancers, such as lung, liver, colon and pancreatic cancers, indicating that tsRNAs are significantly involved in cancer development [113]. Some tsRNAs may serve as potential tumor biomarkers, assisting in diagnosis, staging and prognostic monitoring (Table 2).

4.3.1. Colorectal cancer

Colorectal cancer (CRC), a common malignancy of the colon or rectum, is the second leading cause of cancer-related deaths worldwide, with higher incidence rates in developed countries. Tao [114] found that 5′ tiRNA-His-GTG expression is upregulated in CRC tissues, and inhibiting this tsRNA induces the progress of apoptosis. They discovered that 5′tiRNA-His-GTG is mediated by AGO1 and AGO3, and it targets LATS22 via the HIF1α/ANG axis, thereby inactivating the Hippo signaling pathway [115]. This leads to reduced phosphorylation and increased total protein levels of the Yes-Associated Protein (YAP), promoting cell division and inhibiting cell death, thus contributing to CRC progression [116]. These findings suggest potential therapeutic targets for CRC. Three tRFs (tRF-3022b, tRF-3030b and tRF-5008b) were found to be expressed at higher levels in CRC tissues than in neighboring normal tissues and tended to be elevated in plasma exosomes of CRC patients in Sicheng Lu's research [117]. These tRFs may be upregulated in cancer cells and then secreted via exosomes. Among them, tRF-3022b regulates M2-type macrophage polarization by binding to galectin 1 (LGALS1) and macrophage migration inhibitory factor (MIF) in CRC cells [118]. This suggests that tRF may influence the development of colon cancer through immunogenic cell death (ICD), which may be a novel discovery on the tRF-mediated cell death pathway.

4.3.2. Prostate cancer

Prostate cancer is the most common urologic tumor among men, especially affects those over 60 years old [119]. Changwon Yang [59] found that tRF-315 expression was higher in prostate cancer cells (e.g., LNCaP and DU145) compared to normal cells. tRF-315 inhibited cisplatin-induced apoptosis and reduced mitochondrial damage in cancer cells. tRF-315 inhibitor led to changes in apoptotic pathway proteins, including increased GADD45A and BAX expression while reduced BCL-2 expression mediated by P53 in LNCaP and DU145 cells [120]. Overall, tRF-315 targets the tumor suppressor gene GADD45A to regulate the cell cycle, protecting prostate cancer cells from cisplatin-induced apoptosis.

4.3.3. Gastric cancer

Gastric cancer (GC) is a prevalent form of malignancy that arises from the epithelial cells of the gastric mucosa. It ranks as the third leading cause of cancer mortality worldwide and is particularly prevalent in East Asia [121]. Recent studies highlight the role of tRFs in GC, with some serving as potential treatment targets and others as biomarkers for early diagnosis and poor prognosis [60].

One study identified tRF-Glu-TTC-027, which downregulates key MAPK signaling proteins (ERK1/2, JNK, p38), thereby suppressing GC progression [60]. Huaiping Cu [61] found that tRF-Val was significantly upregulated in gastric cancer tissues and cell lines, correlating with tumor size and invasion depth. tRF-Val directly binds to the molecular chaperone EEF1A1, facilitating its entry into the nucleus and enhancing the interaction of EEF1A1 with MDM2 (E3 ubiquitin ligase for p53). As a result, tRF-Val can inhibit the p53 downstream molecular pathway, thereby reducing apoptosis and promoting gastric cancer progression. Reducing or blocking tRF-Val may restore p53's tumor-suppressor function, inhibiting cell proliferation and promoting apoptosis [122]. Additionally, tRF-23-Q99P9P9NDD was found to be expressed at a higher level in gastric cancer cells compared to normal cells [62]. tRF-23 binds with the 3′ untranslated region (UTR) site of ACADSB(A dehydrogenase short/branched chain) and promotes GC cell proliferation, migration and invasion. Downregulation of ACADSB led to increased lipid accumulation by inhibiting fatty acid catabolism and ferroptosis. Therefore, this study implicates that tRF-23-Q99P9P9NDD may affect GC lipid metabolism and ferroptosis by targeting ACADSB, thereby promoting GC progression [123].

These findings highlight the strong connection between tsRNAs and cell death in cancer, offering new insights into RNA-based therapeutic strategies, particularly for diseases involving apoptotic pathway dysregulation. tsRNAs are often dysregulated in cancer, and many studies have explored their potential as tumor biomarkers, focusing on how their levels are altered during disease progression.

4.4. tsRNAs and multiple organ injuries

Multiple studies have linked tsRNA to kidney injury. In particular, the relationship between tiRNAs (tRNA-derived small RNAs) and the study of kidney injury is emerging as a significant area within the realm of nephrology (Table 2).

Acute kidney injury (AKI) represents a clinical condition marked by a swift deterioration in kidney function stemming from diverse etiologies, potentially affecting individuals with no prior kidney disorders or exacerbating existing chronic kidney disease [124]. AKI has a high mortality rate and is one of the acute and critical conditions in kidney disease. Treatment of AKI is primarily based on its etiology, including fluid and electrolyte management, and sometimes dialysis is required [125]. Lidan [63] found that in a mouse model of cisplatin-induced AKI, tiRNA-Lys-CTT-003 (tiR-Lys) interacted with the RNA-binding protein GRSF1 to directly attenuate Eastin-induced ferroptosis and MDA accumulation in renal tubular epithelial cells [126]. This effect was accompanied by restoration of GPX4 expression. Additionally, overexpression of tiR-Lys and pretreatment with FG-4592 (HIF-1 inducer) could ameliorate the mitochondrial damage and lipid peroxidation in cisplatin-induced AKI mice [127]. These interventions also helped protect against renal function decline and morphological damage.

In addition, tsRNA also has effects on cell death in many organ damage diseases. Lin Deng [65], found that tRF-Gly-GCC may influence the PI3K/AKT and FOXO1 signaling pathways, promoting reactive oxygen species (ROS) production and increasing apoptosis during radiation-induced lung injury (RILI) through oxidative stress. Similarly, Feng Jiang [66], observed in patients with atrial fibrillation (AF) that tsRNAs, including tRF-SeC-TCA-001, tiRNA-Gly-CCC-003, tRF-Gly-GCC-002 and tRF-Tyr-GTA-007, significantly regulate myocardial cell adhesion and plasma membrane adhesion molecules, impacting various cellular processes [128]. These target genes may contribute to AF pathogenesis through mechanisms like glycosaminoglycan biosynthesis, AMP-activated protein kinase activity and insulin signaling pathways.

4.5. tsRNAs and aging

The metabolism and function of tRNAs are altered with age, and these changes are closely related to the aging process. For example, reduced levels of tRNA modification may promote the entry of tRNAs into the RTD pathway, thereby accelerating their degradation, causing insufficient supply of tRNAs and activation of the integrative stress response (ISR) and the ribosomal toxicity stress response (RSR), leading to senescence [129]. Hanqing He [67], found that 3′-tiRNA-Leu-CAG can damage hematopoietic stem cells(HSCs) through activation of the RIPK1-RIPK3-MLKL-mediated necrotic apoptotic cascade, a process that has been linked to senescence in HSCs. 3′-tiRNA-Leu-CAG upregulates the expression of RIPK1 and RIPK3, while decreasing the expression of the key proteins in the necroptosis pathway like caspase-8 and caspase-3, thereby contributing to the progression of cells toward necroptosis [130]. This suggests that tRNA fragments may be involved in regulating the senescence process by affecting necrotic apoptosis, which provides a new vision for delaying aging.

4.6. tsRNAs and reproduction

Research indicates that changes in tRF-related sequences could impact both reproductive health and the health of offspring [131]. Yuheng Pan [64], found that tRF-1:30-Gly-GCC-2 was involved in apoptosis and ferroptosis in Granulosa Cells (GCs) in ovarian dysfunction-related studies. tRF-1:30-Gly-GCC-2 inhibits granulosa cell proliferation and promotes ferroptosis by inhibiting mitogen-activated protein kinase 1 (MAPK1). These findings suggest that tRF-1:30-Gly-GCC-2 could be a novel target for improving atretic follicle development and granulosa cell dysfunction in ovarian disorders.

5. Discussion and future perspectives

Recent advancements in high-throughput technologies and the establishment of relevant databases have greatly facilitated the discovery and characterization of tsRNAs. These small RNAs act as “double-edged swords” in disease progression, either promoting or inhibiting cell death. As this review highlights, tsRNAs play key roles in various cell death pathways, including ferroptosis, apoptosis, necrosis, autophagy and pyroptosis. While they are involved in regulating cell death, tsRNAs also function as biomarkers for cellular stress and disease states. However, several challenges remain in the study of tsRNAs: (1) Their role in newly discovered cell death pathways, such as immunogenic cell death (ICD) and cuproptosis, is not yet fully understood and requires further exploration. (2) Research on different types of tsRNAs is uneven, with more focus on tRFs compared to tiRNAs, and even within tRFs, there is limited research on certain subtypes. For example, research on tRF-2 lags behind that of other tRF categories. (3) The extensive modifications of tsRNAs, inherited from their tRNA precursors, complicate their detection and expression analysis. (4) Most studies have concentrated on the biological functions of tsRNAs rather than their direct relationship with disease pathogenesis. Few have explored the regulatory mechanisms and networks involved. As an emerging subject within the field of non-coding RNA research, tsRNA holds great promise in understanding cell death and their potential implications in disease diagnostics and therapy. It is believed that the gradual increasing attention to tsRNA and the improvement of sequencing technology will contribute to the discovery of more tsRNA. By conducting thorough research into the functions and regulatory mechanisms of tsRNAs, their significance in biological and medical contexts, along with their potential in disease diagnostics and therapeutics, can be clarified.

FACTS

• 1.tsRNA is an emerging and significant class of small non-coding RNA that involve in a range of physiological and pathological processes, including cell death, stress response, cell proliferation and so on.
• 2.tsRNAs play key roles in various cell death pathways, including ferroptosis, apoptosis, necrosis, autophagy and pyroptosis, which influence the progress of different diseases.
• 3.tsRNAs exhibit significant potential as biomarkers for both the diagnosis and monitoring of different diseases such as neurological disorders, metabolic diseases and cancer through cell death pathways.

Open questions

• 1.How does tsRNA participate in the progress of different cell death pathways? What is the specific mechanism by which tsRNA affects the cell death pathway?
• 2.Although most studies have pointed to tsRNA as a possible criterion for disease diagnosis or prognosis, there has been less research on treatment. Could tsRNA be a target for treating disease through different pathways, such as cell death?
• 3.In addition to the type of death studied so far, does tsRNA affect disease progression through other cell death pathways, such as ICD and cuproptosis?

CRediT authorship contribution statement

Xiaoyu Ma: Writing – review & editing. Jiesi Xu: Writing – review & editing. Guoping Li: Writing – review & editing.

Declaration of generative AI and AI-assisted technologies in the writing and drawing process

Generative AI and AI-assisted technologies was only used in the writing process to improve the readability and language of the manuscript.

Funding

This work was supported by the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1–008) for Dr. Guoping Li, National High Level Hospital Clinical Research Funding (BJ-2024-219, BJ-2023-237) for Dr. Guoping Li and National Natural Science Foundation of China (82370877, 81970739) for Dr. Guoping Li.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.