The Many Virtues of tRNA-derived Stress-induced RNAs (tiRNAs): Discovering Novel Mechanisms of Stress Response and Effect on Human Health*

In mammalian cells, mature tRNAs are cleaved by stress-activated ribonuclease angiogenin to generate 5′- and 3′-tRNA halves: a novel class of small non-coding RNAs of 30–40 nucleotides in length. The biogenesis and biological functions of tRNA halves are emerging areas of research. This review will discuss the most recent findings on: (i) the mechanism and regulation of their biogenesis, (ii) their mechanism of action (we will specifically discuss their role in the protein synthesis inhibition and the intrinsic pathway of apoptosis), and (iii) their effects on the human physiology and disease conditions.

In mammalian cells, mature tRNAs are cleaved by stress-activated ribonuclease angiogenin to generate 5-and 3-tRNA halves: a novel class of small non-coding RNAs of 30 -40 nucleotides in length. The biogenesis and biological functions of tRNA halves are emerging areas of research. This review will discuss the most recent findings on: (i) the mechanism and regulation of their biogenesis, (ii) their mechanism of action (we will specifically discuss their role in the protein synthesis inhibition and the intrinsic pathway of apoptosis), and (iii) their effects on the human physiology and disease conditions.
Recent breakthroughs in high-throughput sequencing have led to a more comprehensive view of the cellular transcriptome. We are now aware of numerous additional non-protein-coding RNA (ncRNA) 3 candidates in all three domains of life, thus indicating a hidden layer of transcriptome complexity (1,2). A more recent development in our understanding of the complexity of cellular RNomes arose with the exciting discovery that ncRNA transcripts with well described functions, such as the tRNAs, can serve as precursors for downstream cleavage events, generating yet another class of functional RNA fragments. Two major classes of tRNA fragments have been identified in human cells. The 17-26-nucleotide long tRNA-derived RNA fragments (tRFs) are products of precise processing at the 5Ј-or 3Ј-end of mature or precursor tRNAs. The tRF-5 and tRF-3 are derived from terminal ends of mature tRNAs, whereas tRF-1 are 3Ј-trailer sequences of pre-tRNAs (3). The other important class of tRNA fragments found in mammalian cells and tissues is the tRNA-derived stress-induced RNAs (tiRNAs). tiRNAs were first reported in human fetus hepatic tissue (4) and human osteosarcoma cells (U2OS), respectively (5). tRFs and tiRNAs are the newest members of the cellular ncRNA repertoire that are found in several organisms and play prominent roles in various cellular functions (6). These tRNA fragments can be generated in cells under physiological conditions and also produced as part of the cellular stress response (4 -9). In mammalian cells, tiRNAs are produced by the cleavage of mature tRNAs at positions close to the anticodon, giving rise to the 30 -40-nucleotide-long 5Ј-and 3Ј-tRNA halves, a term used interchangeably with 5Ј-and 3Ј-tiRNAs throughout this review. The enzyme responsible for this endonucleolytic cleavage is angiogenin (ANG) (4,5). ANG is a member of the pancreatic RNase superfamily distinguished by its potent in vivo angiogenic activity as well as its prominent role in cancer development and neurodegeneration (10,11). Here we will review the ANG-induced tRNA halves (tiRNAs), their role in mammalian stress response mechanisms, and other cellular functions. Additionally, we will discuss their potential implications in the pathobiology of human diseases with a special focus on neurodegenerative disorders.

ANG-induced tRNA Cleavage in Mammalian Cells
Endonucleolytic cleavage of mammalian tRNAs by ANG was reported by two research groups in 2009 (4, 5). Yamasaki et al. (5) reported that ANG cleaves tRNAs non-specifically during arsenite treatment, heat shock, and UV irradiation. Fu et. al. (4) reported that ANG-induced tRNA cleavage also occurs during nutrition deficiency, hypoxia, and hypothermia. However, it is not a general stress response; tRNA cleavage was not observed in ␥-irradiated (4), etoposide-treated, or caffeine-treated human cells (5). Several groups have studied the mechanism of ANG-induced tRNA cleavage (12). The general consensus is that during some stress conditions, ANG-induced tRNA cleavage is either (a) activated by the translocation of ANG into the cytoplasm from the nucleus, thus bringing it into the proximity of its substrates or (b) activated by disassociation of ANG from its cytoplasmic inhibitor ribonuclease/angiogenin inhibitor 1 (RNH1), thus activating its RNase activity (6). However, there is still a gap in our knowledge about these pathways. For example, the molecular players involved in the translocation of ANG from the nucleus into the cytoplasm during the stress response, as well as the mechanisms of inactivation of ANG in the cytoplasm, still need to be identified. A recent study reported that the knockdown of RNH1 abolished stress-induced relocalization of ANG in HeLa cells (13), indicating a regulatory function of RNH1 on the subcellular distribution of ANG. The mechanism of ANG dissociation from its inhibitor during stress also needs to be characterized. Our own study showed that the RNH1 protein is degraded during hyperosmotic stress, which can lead to activation of ANG and subsequent tRNA cleavage (14). As an additional mechanistic insight, our study also showed that higher levels of tRNA cleavage correlate with higher translation rates (e.g. translation inhibitors down-regu-late both translation and tRNA cleavage), suggesting that ANG can better access the tRNA when protein synthesis is active (14).
Another important aspect of ANG-induced tRNA cleavage that needs to be further explored is the mechanism of enzymatic cleavage of tRNA by ANG in vivo. ANG targets singlestranded ribonucleic acid sequences with ϳ20-fold higher preference for CA over UA and 3-fold higher for CA over CG (15). It is generally believed that ANG cleaves the tRNAs around the anticodon during stress (4,5). In their recent study, Czech et al. (16) proposed an additional site of tRNA cleavage by ANG. They reported that during oxidative stress, the 3Ј-CCA end of tRNAs is cleaved by ANG before a second cleavage in proximity to the anticodon. They hypothesized that the cleavage of the 3Ј-end during stress is an immediate response to stress and is rapidly reversed by 3Ј-CCA-adding enzymes, whereas the cleavage at the anticodon is likely not reversible. However, the evidence indicating the cleavage of the 3Ј-CCA termini followed by the repair by 3Ј-CCA-adding enzymes was based on in vitro analyses. Additional in vivo evidence supporting the tandem activity of ANG and 3Ј-CCA-adding enzymes during stress response will provide solid confirmation to this two-step mechanism of tRNA cleavage.
The cleavage of tRNAs at the vicinity of the anticodon during stress is also regulated by the presence of specific tRNA modifications in the anticodon. DNA methyltransferase 2 (Dnmt2) and NOP2/Sun RNA methyltransferase 2 (NSun2) modify many tRNAs to generate the 5-methylcytidine (m 5 C) modification in flies and mammals, respectively. Knockdown of these modification enzymes has been shown to promote stress-induced cleavage of tRNAs (17,18). Thus modification of specific nucleosides can provide resistance to ANG-induced cleavage in tRNAs; this protective action against cleavage might play a regulatory role in the tiRNA-mediated stress responses.

Role of tiRNAs in the Cellular Stress Response and Other Pathways
The cellular response to environmental stress involves regulation of phosphorylation of the translation initiation factor 2 (eIF2). eIF2 delivers the initiator tRNA (Met-tRNA iMet ) for translation initiation of mRNAs. Phosphorylation of the ␣ subunit on Ser 51 sequesters eIF2 in an inactive complex, thus decreasing translation initiation (19). Concomitantly, phosphorylated eIF2␣ facilitates the preferential translation of select transcripts, including activating transcription factor 4 (ATF4), a transcriptional activator of stress response genes (20,21). Phosphorylation of eIF2␣ is considered the initiator of transcriptional and translational reprogramming during stress, known as the integrated stress response. Several studies, including our own, have shown that ANG-induced cleavage of tRNAs during stress conditions occurs independently of eIF2␣ phosphorylation, thus indicating that tiRNAs might participate in mechanisms of the stress response that do not involve the integrated stress response (5,14).
The functions of tiRNAs in the regulation of protein synthesis in vivo during stress conditions have not been extensively studied. Although stress induces tiRNA accumulation, the concentration of full-length tRNAs does not change (5,14). It is therefore more likely that tiRNAs play a role in other stressinduced cellular processes rather than regulating global rates of protein synthesis via changes in the abundance of full-length tRNAs. The Anderson lab has shown that transfection of 5Ј-tiRNAs, but not 3Ј-tiRNAs, modestly (ϳ20%) inhibits protein synthesis and promotes stress granule formation in a phospho-eIF2␣-independent manner (5,22). Further research in their lab showed that synthetic 5Ј-tiRNA Ala and tiRNA Cys inhibit translation in rabbit reticulocyte lysate by displacing the eukaryotic initiation factor eIF4G/A from mRNAs. The same study also reported that the 5Ј-terminal oligoguanine sequence is essential for the translational repression ability of these tiRNAs (23). The translation inhibitory effect of tRNA fragments has been also reported in other organisms; the 26-nucleotide 5Ј-tRFs derived from tRNA Val have been shown to inhibit translation in the archaeon Haloferax volcanii (24). Similar to tiRNAs, Val-tRF is produced in a stress-dependent manner. Its translation inhibitory action is reported to be due to its binding with the small ribosomal subunit both in vitro and in vivo. The existence of tRNA fragments capable of inhibiting translation across domains of life suggests that this might be an ancient mechanism of translation regulation in various species.
tiRNAs also play an important role in the cellular intrinsic apoptotic pathway. Apoptosis occurs if the cellular damage incurred during stress exceeds the capacity of the repair mechanisms. In the intrinsic mechanism of apoptosis, mitochondria undergo biochemical and structural changes, leading to the release of various proteins from the intermembrane space, including cytochrome c (cyt c) (25,26). Our recent study showed that during hyperosmotic stress, ANG-induced tiRNAs bind cyt c and competitively inhibit the binding of cyt c to the apoptotic protease-activating factor 1 (APAF1) protein (27). The binding of cyt c and APAF1 is essential for the formation of the apoptosome and the downstream activation of cell death (28). RNA deep sequencing of the cyt c-bound tiRNAs showed that certain tiRNAs exhibit higher binding affinity than others, but no enrichment of specific isodecoders of tRNA halves was observed. Both 5Ј-tiRNAs and 3Ј-tiRNAs exhibited cyt c binding. Deeper understanding of these tiRNA-cyt c complexes will provide insights helpful for designing RNA-based therapeutics for diseases that involve deregulation of the apoptotic pathway such as various cancers and neurodegenerative diseases (29,30).
In addition to stress response pathways, tiRNAs are also speculated to participate in the cellular RNAi pathway by associating with the Argonaute (Ago) proteins and other proteins involved in the silencing pathway (7). Increased tRNA cleavage during heat shock in flies lacking the Dnmt2 modification enzyme has been shown to interfere with the siRNA pathway. tiRNAs bind to Dicer and saturate its binding pocket, thus reducing its ability to cleave dsRNAs (31). In an alternative mechanism, tRFs serve as substrates for the cellular RNAi machinery. Due to their smaller size and double-stranded structure, tRFs might be better suited to act as substrates for the RNAi machinery as compared with tiRNAs (7, 32, 33).

tiRNAs in Human Biology and Disease Etiology
Unbiased deep sequencing of human small RNAs has shown the presence of tRNA fragments of various sizes and structures (34). The nature of cleavage is regulated by the tRNA type and tissue expression (34). tRNA halves have been identified in various mammalian cells and tissue samples. Surveys of different somatic tissues have revealed that 5Ј-tRNA halves are concentrated within blood cells and hematopoietic tissues of mice (35). The level and composition of the tRNA halves change with age and calorie intake, and the tRNA halves circulate as a part of a large nucleoprotein complex. Another study reported that the composition of circulating small RNAs in patients with stable and advanced heart failure consists of tRNA fragments in addition to microRNAs (36). These findings suggest that analogous to circulating microRNAs (37,38), tRNA halves may act as signaling molecules that participate in cell-to-cell communication. The presence of tRNA halves in hematopoietic tissues and the bloodstream in normal (unstressed) conditions also point toward a possible role in immune signaling (reviewed in Ref. 39).
Although tRNA halves are constitutively generated in human cells and tissues, their accumulation increases by ANG-induced cleavage during stress (4,5,14). This suggests that tiRNAs might play an important role in stress-related disease and injury. Using a 1-methyladenosine (m 1 A) antibody, Mishima et al. (40) studied the circulating tRNA and tiRNA composition in various animal models of tissue damage (e.g. toxic injury, irradiation, and ischemic reperfusion). m 1 A modification of tRNA is highly conserved in most organisms and has been found to occur in almost all tRNAs (41)(42)(43). The study showed that the production of tiRNAs correlates with the degree of damage. The authors also showed that during oxidative stress, a change in the tRNA conformation promotes ANG-induced tiRNA production. Additionally, renal ischemia/reperfusion injury and cisplatin-mediated nephrotoxicity (both of which induce tissue damage via oxidative stress) generate tiRNAs in damaged kidneys. Thus tiRNAs can be used as biomarkers to detect stress-induced tissue damage in humans.
Uncontrolled tissue damage is a common underlying cause of cancer (44,45). tRNA fragments were detected in the urine of cancer patients more than three decades ago and were proposed to be oncogenic molecules (46 -49). However, the role of these fragments in tumor growth and cancer progression still remains unclear. Genome-wide analysis of tRNA levels in breast cancer cells versus normal breast tissues revealed that both nuclear-encoded and mitochondrial tRNAs are significantly increased in transformed cells (50,51). However, it is not known whether increases in tRNA levels in transformed cells lead to increases in tRNA fragments. Because ANG level is upregulated in several kinds of cancer, it is possible that ANGinduced tRNA halves might be enriched in these scenarios (12,52). Two recent studies have revealed that endogenous tRNA fragments can play important roles in breast cancer and prostate cancer etiology. The first study identified a group of tRNA fragments that are up-regulated under hypoxia in non-transformed mammary epithelial and breast cancer cells (53). This study showed that these tRNA fragments competitively bind the RNA-binding protein Y box-binding protein 1 (YBX1 or YB-1). This binding causes disassociation of YB-1 from several of its substrate oncogenic transcripts, thus causing destabilization and down-regulation of these transcripts. Introduction of tRNA fragments into breast cancer cells led to decreased cancer growth under serum starvation, another evidence of their role as stress response molecules, whereas inhibition of these fragments by antisense locked nucleic acids increased the cancer phenotype. Additionally, these fragments also exhibited metastasis-suppressive action. The authors proposed that endogenous tRNA fragments that bind YB-1 are generated during oncogenic stress as a mechanism for tumor suppression. Interestingly, this is not the first study to report the interaction of tRNA fragments with YB-1. Ivanov et al. (23) have previously shown that translational repression exhibited by certain tiRNAs is YB-1-dependent. However, the types of tRNA fragments reported to associate with YB-1 are different in these two studies. tRNA fragments derived from tRNA Glu , tRNA Asp , tRNA Gly , and tRNA Tyr promote the destabilization of oncogenic transcripts via YB-1 binding, whereas tiRNA Ala and tiR-NA Cys are responsible for translation inhibition. These findings suggest that different classes of tRNA fragments can bind the same protein to modulate diverse signaling pathways in the cell.
A recent study revealed a novel tRNA fragment-mediated pathway in tumorigenesis of hormone-dependent cancers (54). Honda et al. (54) reported that estrogen receptor-positive breast cancer and androgen receptor-positive prostate cancer cell lines specifically and abundantly express a class of tRNAderived small RNAs, which they termed sex hormone-dependent tRNA-derived RNAs (SHOTRNAs). SHOTRNAs are produced in breast cancer cells by ANG-induced anticodon cleavage of amino-acylated mature tRNAs. The resulting 5Ј-tRNA halves contain a phosphate group at the 5Ј-end and a 2Ј, 3Ј-cyclic phosphate at the 3Ј-end, whereas the 3Ј-halves contain a 5Ј-hydroxyl group at the 5Ј-end and an amino acid at the 3Ј-end. Another important highlight of this study is the new method developed by the authors called cP-RNA-seq that exclusively amplifies and sequences RNAs containing a 3Ј-terminal cyclic phosphate. Development of newer and more efficient methods for detection of tRNA halves will facilitate early detection of these biomarkers.
ANG-induced tRNA halves have been also implicated in the cellular response to virus infection. Wang et al. (55) have shown that fragments corresponding to the 5Ј-half of mature tRNAs are abundantly produced in cells infected with human respiratory syncytial virus (RSV). RSV is paramyxovirus that causes respiratory tract infections in children (56) and increases the morbidity and mortality rate in immune-compromised patients and the elderly (57). The study showed that the induction of tRNA halves was virus-specific, only a subset of tRNAs were cleaved, and the cleavage was centered around the anticodon of the tRNAs. Wang et al. (55) also showed that a 5Ј-tRNA half derived from tRNA Glu-CTC represses target mRNAs in the cytoplasm and promotes RSV replication in cells. When ANG was suppressed by the use of siRNA, the induction of tRF5-GluCTC by RSV infection was significantly decreased (Ͼ50%), thus validating the ANG-mediated biogenesis of this tRNA fragment.

Role of tiRNAs in Neurons: Implications for Neurodegenerative Disorders
The neuroprotective actions of ANG have been well established. In 2004, Greenway et al. (58) identified ANG as a susceptibility gene for amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by adult-onset loss of motor neurons (59,60). In a subsequent study, they showed that the ALS-associated ANG mutations affect functionally important residues essential for its ribonuclease activity (61). From this point onward, the role of ANG in neuron maintenance and survival was studied extensively. The protective action of ANG was observed under various stress treatments; for example, ANG protects motor neurons against excitotoxic injury in a PI 3-kinase/Akt kinase-dependent manner (62). ANG also protects neurons against hypoxic injury and endoplasmic reticulum stress-induced and trophic factor withdrawal-induced cell death, whereas ALS-associated ANG mutants exert no protective activity (62,63). Our own study showed that ANG protects primary neurons from hyperosmotic stress-induced cell death (27). A subset of ALS-associated ANG mutants have also been found in Parkinson disease (PD) patients (64). Most ALS/PD-associated ANG mutations involve RNase lossof-function mutations, in agreement with the neuroprotective function of ANG via its RNase activity (65). This collective evidence allows us to speculate that ANG-induced production of tiRNAs is essential for neuron survival during stress. Two potential mechanisms of protective actions exhibited by ANG might be due to the interaction of tiRNAs with cyt c, thus preventing apoptosis (27), or via translation regulation exhibited by tiRNAs during stress (23).
Recent work by Ivanov et al. (66) provides an interesting mechanistic explanation of how ANG-induced tRNA halves might contribute to neuron survival and maintenance. They found that 5Ј-tiRNA Ala and 5Ј-tRNA Cys , the tiRNAs that cooperate with YB-1 protein to displace the cap-binding complex eIF4F from capped mRNAs and thus inhibit translation initiation, are assembled into putative G-quadruplex structures. G-quadruplex structures do not exhibit the conformational differences between DNA and RNA double helices and are known to be able to efficiently enter cells (67). Ivanov et al. (66) showed that human motor neurons spontaneously uptake 5-tiDNA Ala (the DNA equivalent of 5Ј-tiRNA Ala ) and that this effectively rescues the motor neurons from stress-induced death. This study can pave the way for testing of tiRNA-based therapeutics in the treatment of neurodegenerative diseases.
The relationship between ANG-induced tiRNAs, cellular stress, and neurodevelopmental disorders has been also studied in the context of tRNA modifications. Human NSun2 methylates cytosine residues in the anticodon loop (position C34) and at the intersection of the variable loop and the T arm of tRNA (68 -70). Mutations in NSun2 cause microcephaly and other neurological abnormalities in mice and humans (71)(72)(73). A recent study reported that 5Ј-tiRNAs are enriched in NSun2 knock-out cells and these fragments are derived from nonmethylated tRNAs (18). The study showed that NSun2 Ϫ/Ϫ cells are more sensitive to various stress stimuli (UV radiation and oxidative stress) and that loss of NSun2 decreases neuron sur-vival and impairs brain development. The authors hypothesized that ANG-induced tRNA halves repress translation and trigger a stress response and cell death in cortical, hippocampal, and striatal neurons. They showed that injecting pregnant NSun2 ϩ/Ϫ females with the ANG inhibitor rescued some of these phenotypes in the NSun2 Ϫ/Ϫ embryo. The authors proposed ANG inhibitors as candidate drugs for patients exhibiting loss-of-function mutations in NSun2. This is a very attractive scenario; however further in vivo studies are needed to establish a direct link between tiRNAs and NSun2-associated neurodegenerative disorders.
In addition to ANG-induced tRNA halves, tRFs derived from intron-containing pre-tRNAs have been implicated in neurodegeneration (74 -76). Homozygous missense mutation (p.R140H) in the CLP1 gene in humans has been associated with severe motor sensory defects, cortical dysgenesis, and microcephaly. CLP1 is an RNA kinase involved in tRNA splicing. Patient-derived neurons displayed both depletion of mature tRNAs and accumulation of unspliced pre-tRNAs. Transfection of partially processed tRNA fragments into patient cells exacerbated oxidative stress-induced cell death (76). The biogenesis of these tRNA fragments is distinct from that of ANG-induced tiRNAs; however, collective findings in the field suggest that tRNA metabolism and development of neurodegenerative diseases are strongly related.

Perspectives and New Directions
tRNA fragments are the newest members of the cellular short ncRNA club (3,34,77,78). Derived from different parts of the tRNA molecule via different processing pathways, these RNAs come in all shapes and sizes (7)(8)(9)43) and have been observed in all domains of life (6, 79 -81). Although a natural concern might be that these fragments are products of random RNA degradation, several key observations substantiate that tRNA fragments (especially tiRNAs) represent biologically relevant entities. i) Their production is specifically induced during certain environmental (stress) conditions. ii) There is no clear correlation between their abundance and the codon usage and/or gene copy number of the corresponding full-length tRNAs. iii) Cleavage is centered at specific sites in the tRNA (around the anticodon for ANG-induced tiRNAs). iv) Most importantly, the pool of available mature tRNAs is not significantly altered by cleavage.
Our review is especially geared toward providing the readers with the exciting prospects of tiRNAs in human biology. There is burgeoning evidence that tiRNAs are enriched in multiple human cells and tissues (34). tRNA halves have been found in the serum as part of circulating macromolecular complexes (35), and the abundance of specific circulating tRNA halves changes in the serum of breast cancer patients (82). Although not well studied, there is also evidence of 5Ј-tRNA halves being enriched in the sperm of the mouse, rat, and human (83). The levels of these tRNA halves are relatively constant during the early stages of spermatogenesis, but substantially increase at late-or post-spermatogenesis and are found localized at the sperm head, which suggests delivery to the oocyte upon fertilization. Their level decreases upon fertilization, suggesting a physiological role in early embryogenesis. Additionally, 5Ј-tRNA halves have been found in exosomes within human semen (84) and in the urine of cancer patients (46 -49).
As more and more evidence of their existence surfaces, the list of the possible functions of these tiRNAs is also growing (85). One avenue that deserves further exploration is the functional interaction of the RNA-binding protein YB1 with tiR-NAs. A recent study showed that certain tRNA halves in association with YB-1 can play an important role in preventing breast cancer progression (53). YB-1 has an essential role in the acquisition of malignant characteristics by breast cancer cells, through epidermal growth factor receptor 2 (HER2)-Akt-dependent pathways (86,87). The underlying mechanism involves YB-1-mediated translational regulation of oncogenic transcripts (88,89). tRNA fragments associate with YB-1 to reduce its binding with oncogenic transcripts, thus inhibiting their translation (53). Deeper molecular understanding of tRF-YB-1 binding will allow the development of diagnostic and therapeutic tools based on tRNA fragments. Future studies will also determine whether tiRNAs can join the ranks of microRNAs in the regulation of mRNA translation in health and disease (90,91).
Looking into the future, the prospect of applying ANG-induced tRNA halves in the treatment of neurodegenerative diseases such as ALS is particularly exciting. ALS, often referred to as Lou Gehrig disease in the United States, is a paralyzing and ultimately fatal disease characterized by the progressive loss of motor neurons (92). ANG mutations that lead to loss of its RNase activity have been implicated in the pathophysiology of ALS (61,93). Loss of its RNase activity will reduce the production of ANG-induced tiRNAs. tiRNAs have been implicated in inhibition of protein synthesis (23), formation of stress granules (22), and inhibition of apoptosis via interaction with cyt c (27). The mechanisms for neurodegeneration in ALS are not completely understood; however, it has been reported that motor neuron degeneration in ALS structurally resembles apoptosis (94,95). The anti-apoptotic role of tiRNAs might thus play an important role in preventing neuronal death, and this aspect perhaps contributes to why loss-of-function mutations of ANG are causatively linked with ALS. As described above, the finding by Ivanov et al. (66) that 5Ј-tiDNA Ala (the DNA equivalent of 5Ј-tiRNA Ala ) via forming G-quadruplex structures can enter human motor neurons and protect them from stress-induced death introduces an exciting avenue for the use of tiRNA mimicry aptamers for the treatment of neurodegenerative diseases. Aptamers based on G-quadruplex structure have been used for the detection and treatment of severe pathologies including vascular, cancer, and viral diseases (96 -98). The observation that tiRNAs can assume the G-quadruplex structure opens up a plethora of new opportunities. For example, high throughput screens can be set up to examine whether specific tiRNAs can modulate the activity of enzyme superoxide dismutase 1 (SOD1). SOD1 mutations underlie almost 20% of familial ALS (92,99,100). Administration of ANG to SOD1 (G93A) mice, a standard laboratory model for ALS, significantly promotes both lifespan and motor function (62). Based on this observation and other reported findings, we believe that in-depth studies on tiRNAs and their regulatory roles in neurons could provide significant breakthroughs in ALS prevention and therapy. There is a lot more to learn about the generation and function of tRNA fragments in mammalian cells. This review highlighted the recent discoveries in this field (Fig. 1) and the potential use of tRNA fragments as biomarkers in development of diseases that involve stress-induced tissue damage.