Transfer RNA (tRNA) fragments (tRFs) are a group of small non-coding RNAs with biological functions. The involvement of tRNAs in cancer has also been recognized, but most studies focused on nuclear tRFs, very few on mitochondrial tRFs.

We analyzed the TCGA microRNAseq data to identify differentially expressed mitochondrial tRFs (mt-tRFs) in breast tumors and evaluated their associations with the disease outcome. Cox proportional hazards regression was used to determine the associations between mt-tRFs and patient survival while adjusting for clinicopathological variables. Quantitative RT-PCR was developed to measure a specific tRF expression in a validation study.

Our analysis of 1,060 tumor samples from TCGA revealed that mt-tRF-Tyr-GTA-001 (tRF-21-X3OJI8EWB or t00018104) expression, a tRF from mitochondrial tRNA with tyrosine anticodon GTA (mt-tRNA-Tyr-GTA), was significantly lower in breast tumors than the adjacent tissues (p< 0.0001). Patients with low expression had significantly higher risk of death (HR = 1.69, p = 0.0018) regardless of their age at diagnosis, disease stage, tumor grade, and hormone receptor status. This survival association was replicated in an independent study where mt-tRF-Tyr-GTA-001 expression was measured with qRT-PCR. Further analysis suggested that the mt-tRF expression was correlated with ribonuclease ANG and RNase 4 known to cleave tRNAs and upregulated under hypoxia. IPA interrogation of the mt-tRF-Tyr-GTA-001 expression signature indicated the inhibitory effects of mt-tRF-Tyr-GTA-001 on malignant transformation, tumor growth, and cell invasion. In silico analysis showed that the binding targets of mt-tRF-Tyr-GTA-001 included several oncogenic transcription factors (E2Fs, CCNE1, FOXM1). We also found the mt-tRF correlated with the abundances of M0 macrophages and resting mast cells, two of the immune cells known for innate immunity.

In summary, our study suggests that mt-tRF-Tyr-GTA-001, a mitochondrial tRF, may suppress breast cancer progression through its involvement in regulation of cell phenotype and tumor immunity.

Orofacial clefts are the most common form of congenital craniofacial malformation worldwide. The etiology of these birth defects is multifactorial, involving genetic and environmental factors. However, in most cases, the underlying causes remain unexplained, precluding a molecular understanding of disease mechanisms. Here, we integrated genome-wide association data, targeted resequencing of case and control cohorts, tissue- and cell-type-specific epigenomic profiling, and genome architecture analyses to molecularly dissect a genomic locus associated with an increased risk of non-syndromic orofacial cleft. We found that common and rare risk variants associated with orofacial cleft intersect with an enhancer (e2p24.2) that is active in human embryonic craniofacial tissue. We mapped e2p24.2 long-range interactions to a topologically associated domain harboring MYCN, DDX1, and CYRIA. We found that MYCN and DDX1, but not CYRIA, are required during craniofacial development in chicken embryos. We investigated the role of DDX1, a key component of the tRNA splicing complex, in cranial neural crest cells (cNCCs). The loss of DDX1 in cNCCs resulted in the accumulation of unspliced tRNA fragments, depletion of mature intron-containing tRNAs, and ribosome stalling at codons decoded by these tRNAs. This was accompanied by defects in both global protein synthesis and cNCC migration. We further showed that the induction of tRNA fragments is sufficient to disrupt craniofacial development. Together, these results uncovered a molecular mechanism in which impaired tRNA splicing affects cNCCs and craniofacial development and positioned MYCN, DDX1, and tRNA processing defects as risk factors in the pathogenesis of orofacial clefts.

Small non-coding RNAs can be categorized into two main classes: structural RNAs and regulatory RNAs. Structural RNAs, which are abundant and ubiquitously expressed, have essential roles in the maturation of pre-mRNAs, modification of rRNAs and the translation of coding transcripts. By contrast, regulatory RNAs are often expressed in a developmental-specific, tissue-specific or cell-type-specific manner and exert precise control over gene expression. Reductions in cost and improvements in the accuracy of high-throughput RNA sequencing have led to the identification of many new small RNA species. In this Review, we provide a broad discussion of the genomic origins, biogenesis and functions of structural small RNAs, including tRNAs, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), vault RNAs (vtRNAs) and Y RNAs as well as their derived RNA fragments, and of regulatory small RNAs, such as microRNAs (miRNAs), endogenous small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs), in animals.

MicroRNAs (miRNAs) are important regulators of gene expression whose dysregulation is widely linked to tumourigenesis, tumour progression and Epithelial-Mesenchymal Transition (EMT), a developmental process that promotes metastasis when inappropriately activated. However, controversy has emerged regarding how many functional miRNAs are encoded in the genome, and to what extent non-regulatory products of RNA degradation have been mis-identified as miRNAs. Central to miRNA function is their capacity to associate with an Argonaute (AGO) protein and form an RNA-Induced Silencing Complex (RISC), which mediates target mRNA suppression. We report that numerous "miRNAs" previously reported in EMT and cancer contexts, are not incorporated into RISC and are not capable of endogenously silencing target genes, despite the fact that hundreds of publications in the cancer field describe their roles. Apparent function can be driven through the expression of artificial miRNA mimics which is not necessarily reflective of any endogenous gene regulatory function. We present biochemical and bioinformatic criteria that can be used to distinguish functional miRNAs from mistakenly annotated RNA fragments.

Tumor metastasis is a significant contributor to increased cancer mortality. Transfer RNA-derived small RNAs (tsRNAs), a class of endogenous non-coding RNA molecules, play crucial functional roles in various physiological processes, including the regulation of transcription and reverse transcription, the modulation of translation processes, the modification of epigenetic inheritance, the regulation of the cell cycle, etc. Dysregulated tsRNAs are closely related to the occurrence and progression of human malignancies. Accumulating evidence indicates that the abnormal expression of tsRNAs is associated with tumor metastasis through a variety of mechanisms. Hence, we summarize the fundamental structure and biological functions of tsRNAs, with a focus on how tsRNAs influence the tumor metastasis process through downstream targets or the regulation of interactions between upstream and downstream molecules, thereby providing a novel perspective for targeted therapy for tumor metastasis.

Gastric cancer is one of the malignant tumors with the highest morbidity and mortality rates worldwide. Yet, there is a lack of diagnostic markers with high sensitivity in the clinic. tRNA-derived small RNAs are a novel type of non-coding small RNAs, which are abundant in tumor cells and body fluids. In this study, we explored the potential of 3'-tRFArg as a tumor marker for the diagnosis of GC. Differential expression of 3'-tRFArg was screened by high-throughput sequencing, and Quantitative real-time PCR confirmed its low expression in GC serum with good stability. Differential expression of serum 3'-tRFArg could distinguish between GC patients, gastritis patients, and healthy donors and was significantly correlated with clinical pathological features such as tumor differentiation, lymph node metastasis, and TNM staging. The receiver operating characteristic curve showed that 3'-tRFArg had a higher diagnostic value compared with conventional biomarkers, especially in the diagnosis of early gastric cancer. In conclusion, our results suggest that 3'-tRFArg can serve as a highly sensitive biomarker with a certain value for monitoring tumor development and prognosis.

Transfer-RNA-derived fragments (tRFs) are a relatively recently discovered class of non-coding RNAs derived from both precursor and mature transfer RNAs (tRNAs). Research on these molecules has been expanding rapidly, revealing their diverse roles in cellular processes, both in normal physiology and in disease states, often via post-transcriptional regulation of target genes. Altered tRFs abundances have been implicated in various conditions, where they may act as either drivers of disease progression or as protective agents. For instance, specific tRFs are associated with increased risk for cancer metastasis, while others may suppress tumor cell proliferation. Despite the growing recognition of tRFs as functional RNAs rather than sequencing noise, this field of study faces numerous challenges. Inconsistent naming conventions and variability in experimental approaches hinder the comparison of findings across studies, limiting our understanding of the common roles and mechanisms of tRFs. This review provides a comprehensive analysis of current literature on the various roles of tRFs in different diseases, particularly focusing on four broad areas: cancer, neurological, cardiovascular, and musculoskeletal disorders. We analyze studies that link specific tRFs to various aspects of human diseases and provide a convenient classification of these studies regarding the depth of the provided evidence. Further, we note gaps in current investigations and consider strategies to address methodological inconsistencies, including validation experiments and unified nomenclature. By consolidating research in this manner, we aim to facilitate comparisons across diverse studies, enhancing our ability to identify functional commonalities and furthering our understanding of the mechanisms by which tRFs act.

In Arabidopsis, effector-triggered immunity (ETI) against avirulent Pseudomonas syringae pv. tomato (Pst) correlates with the rapid, Dicer-Like 1 (DCL1)-dependent nuclear accumulation of a 31-nt 5'-tRNA fragment derived from Asp-tRNA (tRF31Asp2). Several tRFs, including tRF31Asp2, are induced at early stages of infection and associate with AGO2 in the nucleus. Infiltrating Arabidopsis leaves with synthetic tRF31Asp2 induces over 500 defense-associated genes, conferring immunity against virulent and avirulent Pst as well as aphids, while tRF31Asp2 depletion compromises resistance to avirulent Pst. The biological activity of tRF31Asp2 requires its 5' sequence and predicted stem-loop structure, and its loading into AGO2 or related clade members may contribute to activating defense responses. Chromatin affinity precipitation-sequencing revealed that tRF31Asp2 binds specific sequences in defense genes and the Gypsy superfamily of LTR retrotransposons, particularly at their primer binding sites (PBS). tRF31Asp2 binding appears to modulate transcriptional reprogramming, inducing neighboring tRF-responsive defense genes while suppressing active retrotransposons. Since Gypsy retrotransposon proliferation is primed by tRNA binding at PBS, tRF31Asp2 may exploit a similar mechanism to coordinate defense responses. Together, these findings reveal a role for DCL1 and tRF31Asp2 in regulating plant immunity and transcriptional dynamics at defense-associated loci and retrotransposons.

Glioblastoma multiforme (GBM) is the most common and aggressive primary central nervous system tumor. The formation of vasculogenic mimicry (VM) in GBM is closely related to poor patient prognosis. Therefore, it is urgently necessary to explore the mechanisms that promote VM formation in GBM and identify therapeutic targets. CGGA data analysis revealed that TRMT10A expression is significantly downregulated in WHO grade IV primary glioma samples compared to grade II samples, consistent with the protein expression levels. Additionally, GBM patients with low TRMT10A expression have poorer prognoses. In human glioma cells, TRMT10A expression is significantly lower than in human astrocytes. Knockdown of TRMT10A reduces m1G9 modification of tRNA-ArgCCT, upregulates tRF-22 expression, and promotes glioma cell proliferation, migration, invasion, and tube formation. Overexpression of tRF-22 in glioma cells significantly downregulates MXD1 expression. tRF-22 negatively regulates MXD1 expression by binding to its 3'UTR, reducing MXD1's transcriptional inhibition of HIF1A, thereby promoting glioma cell proliferation, migration, invasion, and tube formation. Overexpression of TRMT10A combined with tRF-22 inhibition significantly reduces the number of VM channels and inhibits tumor growth in xenograft models in nude mice. This study elucidates the mechanism by which TRMT10A affects VM formation in glioma and provides a novel therapeutic target for GBM.

tRNA-derived fragments (tRFs) play critical roles in cellular process, and we have previously reported that tRFs are involved in ischemia reperfusion injury induced acute kidney injury (IRI-AKI). However, the precise involvement of tRFs in IRI-AKI remains obscure. This study aims to elucidate the impact of tRF-Val-TAC-004 (tRF-Val) on IRI-AKI and uncover the underlying mechanisms. Our observations reveal a significant downregulation of tRF-Val in IRI-AKI mice and its overexpression mitigated renal dysfunction, morphological damage, and apoptosis in IRI-AKI mice, while its inhibition exacerbated these effects. Similar outcomes were replicated in CoCl2-treated BUMPT cells upon transfection with tRF-Val mimic or inhibitor. Mechanistically, dual-luciferase reporter assay and AGO-RIP qPCR analyses demonstrated that tRF-Val suppresses Apaf1 expression by targeting the 3'-UTR of Apaf1 mRNA. Furthermore, the protective efficacy of tRF-Val was notably weakened by Apaf1-overexpressing plasmids. In summary, these novel findings unveil the protective role of tRF-Val against IRI-AKI through inhibition of Apaf1-mediated apoptosis.

Recent progress in noncoding RNA research has highlighted transfer RNA-derived small RNAs (tsRNAs) as key regulators of gene expression, linking them to numerous cellular functions. tsRNAs, which are produced by ribonucleases such as angiogenin and Dicer, are classified based on their size and cleavage positions. They play diverse regulatory roles at the transcriptional, post-transcriptional, and translational levels. Furthermore, tRNAs undergo various modifications that influence their biogenesis, stability, functionality, biochemical characteristics, and protein-binding affinity. tsRNAs, with their aberrant expression patterns and modifications, act as both oncogenes and tumor suppressors. This review explores the biogenetic pathways of tsRNAs and their complex roles in gene regulation. We then focus on the importance of RNA modifications in tsRNAs, evaluating their impact on the biogenesis and biological functions on tsRNAs. Furthermore, we summarize recent data indicating that tsRNAs exhibit varied expression profiles across different cancer types, highlighting their potential as innovative biomarkers and therapeutic targets. This discussion integrates both existing and new knowledge about tsRNAs, emphasizing their importance in cancer biology and clinical advancement.

Recurrent spontaneous abortion (RSA) affects approximately 1-5% of childbearing women and poses a significant threat to global reproductive health. Transfer RNA-derived small RNAs (tsRNAs) are a novel class of noncoding RNAs implicated in various human diseases. However, the role and mechanism of tsRNAs in regulating trophoblast function during RSA development remain unknown.

High-throughput sequencing was performed to analyze the differential tsRNAs in the villous tissues of patients with RSA and controls. CCK-8, transwell assay, and flow cytometry were performed to detect the effects of tRF-5028c on proliferation, migration, invasion, and apoptosis of human extravillous trophoblast cell line HTR-8/SVneo. The target genes of tRF-5028c were predicted via bioinformatic analysis and verified by dual luciferase reporter gene assay. Moreover, pregnant mice were injected with tRF-5028c mimics to confirm the findings in vivo.

A total of 1907 tsRNAs were detected, of which 298 were differentially expressed in the villous tissues. tRF-5028c was significantly upregulated in the RSA group compared with control. Functionally, tRF-5028c overexpression inhibited HTR-8/SVneo cell proliferation, migration, and invasion and promoted apoptosis, whereas tRF-5028c knockdown showed opposite effects. Mechanically, tRF-5028c suppressed CRKL expression by directly binding to its 3'-untranslated region, thus inactivating the downstream C3G/Rap1 signaling pathway. Finally, tRF-5028c mimics injection increased embryo absorption rate in mice.

tRF-5028c upregulation impaired trophoblast function to facilitate RSA development by directly targeting CRKL-mediated Rap1 pathway. The findings provide the first evidence of tsRNA dysregulation in RSA pathogenesis and lay a foundation for potential targeted therapies.

Transfer RNA-derived small RNAs (tsRNAs) refer to a newly established family of non-coding RNAs that regulate a diverse set of biological processes. However, the function of tsRNAs in skin photoaging remains unclear. This research aims to investigate the potential correlation between tsRNAs and skin photoaging. Human dermal fibroblasts (HDFs) were irradiated with UVA at 10 J/cm2 once a day lasting for 14 days, resulting in the establishment of a photoaging model induced by UVA. To identify the expression profiles and functions of tsRNAs, tsRNA sequencing and bioinformatics analysis were conducted. qPCR was employed to validate the results of differentially expressed (DE) tsRNAs. A total of 34 tsRNAs exhibited significant differential expression between the UVA and control groups (n = 3), with nine upregulated and 25 downregulated (log2 fold change >1.5, p-value <0.05). Six tsRNAs were selected at random and validated by qRT-PCR. The enrichment analysis of DE tsRNAs target genes indicated that the dysregulated tsRNAs appeared to be connected with cell cycle, DNA replication and the AGE-RAGE signaling pathway. The expression of tsRNAs was found to be aberrant in UVA-HDF. These findings provide insights into the UVA-induced damage and potential target genes for skin photoaging.

tsRNA is a class of non-coding RNAs derived from mature or precursor tRNAs. In recent years, more and more studies have explored the correlation between tsRNAs and tumours. tsRNAs can affect the biological behaviours of tumour cells such as proliferation, apoptosis and metastasis by regulating gene expression, protein translation or post-transcriptional regulation. In this paper, we systematically review the production, biological function and research progress of tsRNA in tumour and discuss its prospects as biomarkers and therapeutic targets.

tRNA-derived small RNA (tsRNA) is a recently discovered small non-coding RNA (ncRNA) molecule that widely exists in prokaryotic and eukaryotic transcriptomes and is produced by specific cleavage of mature tRNA or precursor tRNA. In recent years, with the development of high-throughput sequencing technology, tsRNA has been found to have a variety of biological functions, including gene expression regulation, stress signal activation, etc. In addition, it has been found that these molecules are abnormally expressed in various diseases and participate in various pathological processes, which play an important role. At present, more and more studies have shown that the expression level of tsRNA changes significantly during the development of neurogenetic diseases. This review provides an overview of the classification and biological functions of tsRNAs, with a particular emphasis on their roles in neurogenetic disorders and their potential as diagnostic biomarkers and therapeutic targets. Despite the nascent stage of tsRNA research, their relevance to the diagnosis and treatment of neurogenetic diseases warrants further investigation.

One of the most common gastrointestinal tumors is gastric cancer (GC), which has a high lethality and a poor prognosis. Recently, it was discovered that mature tRNAs, which are expressed differently in a variety of malignancies, give rise to a novel class of tRNA-derived small RNAs (tsRNAs). In this study, we investigated the role of short RNAs produced from tRNA in GC and possible therapeutic uses. edgeR was used to screen the differentially expressed tsRNAs from the TCGA database and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was used to verify the levels of tsRNAs in GC samples. tRF-24-6VR8K09LE9 downregulated in GC was confirmed by detecting serum samples from 114 patients with gastric cancer, 40 patients with gastritis, and 100 healthy controls. The chi-square test displayed that tRF-24-6VR8K09LE9 was highly related to differentiation grade (P = 0.029), T-stage (P = 0.036), lymph node status (P = 0.036), TNM staging (P < 0.0001), and neurological/vascular invasion (P = 0.033). The receiver operating characteristic (ROC) curve indicated that tRF-24-6VR8K09LE9 is more effective than the current diagnostic markers for GC. Furthermore, mechanistic studies verified that tRF-24-6VR8K09LE9 affected the malignant progression of GC through the PI3K/AKT signaling pathway. In conclusion, tRF-24-6VR8K09LE9 can be served as a molecular marker for early GC auxiliary diagnosis. Over-expression of tRF-24-6VR8K09LE9 inhibits the malignant progression of GC, which may provide a new strategy for the adjuvant treatment of GC.

Transfer RNA is a class of non-coding RNA that plays a role in amino acid translocation during protein synthesis. After specific modification, the cleaved fragment is called tRNA-derived small RNA. The advancement of bioinformatics technology has led to an increase in the visibility of small RNA derived from tRNA, and their functions in biological processes are being revealed. These include gene silencing, transcription and translation, epigenetics, and cell death. These properties have led to the implication of tsRNAs in various diseases. Although the current research mainly focuses on the role of tRNA-derived small RNA in cancer, there is mounting evidence that they are also strongly associated with cardiovascular disease, including cardiac hypertrophy, atrial fibrillation, heart failure, and myocarditis. Therefore, the regulatory role of tRNA-derived small RNA in cardiovascular disease will become an emerging therapeutic strategy. This review succinctly summarizes the characteristics, classification, and regulatory effect of tsRNA. By exploring the mechanism of tsRNA, it will provide a new tool for the diagnosis and prognosis of cardiovascular disease.

Background: Liquid biopsy has gained significant attention as a non-invasive method for cancer detection and monitoring. IsomiRs and tRNA-derived fragments (tRFs) are small non-coding RNAs that arise from non-canonical microRNA (miRNAs) processing and the cleavage of tRNAs, respectively. These small non-coding RNAs have emerged as pro-mising cancer biomarkers, and their distinct expression patterns highlight the need for further exploration of their roles in cancer research. Methods: In this study, we investigated the differential expression profiles of miRNAs, isomiRs, and tRFs in plasma extracellular vesicles (EVs) from colorectal and prostate cancer patients compared to healthy controls. Subsequently, a combinatorial analysis using the CombiROC package was performed to identify a panel of biomarkers with optimal diagnostic accuracy. Results: Our results demonstrate that a combination of miRNAs, isomiRs, and tRFs can effectively di- stinguish cancer patients from healthy controls, achieving accuracy and an area under the curve (AUC) of approximately 80%. Conclusions: These findings highlight the potential of a combinatorial approach to small RNA analysis in liquid biopsies for improved cancer diagnosis and management.

In recent years, there has been a mounting occurrence of lung cancer, which stands as one of the most prevalent malignancies globally. This rise in incidence poses a significant hazard to human health, making lung cancer a matter of grave concern. It has been shown that tRNA-derived small non-coding RNA (tsRNA) is involved in the development of tumors, especially lung cancer, through mechanisms such as regulating mRNA stability, influencing protein translation, and acting as epigenetic regulators. Recent studies have shown that tsRNA is abnormally expressed in the plasma and tissues of lung cancer patients, and its expression level is closely related to the malignancy degree and postoperative recurrence of lung cancer. Therefore, for lung cancer patients, tsRNA represents a promising non-invasive biomarker, exhibiting significant potential for facilitating early diagnosis and prognostic evaluation, and for achieving precision treatment of lung cancer by regulating its expression. This article focuses on the biogenesis of tsRNA and its ability to promote lung cancer cell proliferation and invasion. In addition, the specific clinical significance of tsRNA in lung cancer was discussed. Finally, we discuss the need for further improvement of small RNA sequencing technology, and the future research directions and strategies of tsRNA in lung cancer and tumor diseases were summarized.

tRNA-derived RNAs (tDRs) are known for their diverse regulatory roles in many organisms. These small RNA transcripts have been identified mainly by high-throughput RNA sequencing, numbering hundreds to thousands of unique molecules in any given biological sample. As such, bioinformatic analysis is essential in understanding the features, complexity, and unexplored biological patterns of tDRs. This chapter describes use of tRAX: tRNA Analysis of eXpression, a specially designed comprehensive end-to-end software pipeline for tDR abundance estimation, differential expression comparison, and inference of RNA modifications from raw small RNA sequencing data. We also demonstrate tDRnamer, a web- and command-line-based companion tool that provides automated, standardized tDR naming and annotations based on source tRNAs and related tDRs.

tRNA-derived fragments (tRFs), generated from the cleavage of mature or precursor tRNAs are a category of regulatory noncoding RNAs with diverse functions in physiological or pathophysiological conditions. Here we describe a framework for the over-expression of tRFs from their parental tRNAs in mammalian cells. The process involves bioinformatics analysis to identify specific tRNAs that produce the tRF, PCR amplification of corresponding tRNA genes, and insertion into expression vectors. Transfection is carried out in HEK293T cells and detection of tRFs is achieved through northern blotting and dual luciferase reporter assays. In the latter, a complementary sequence to the tRF of interest is inserted into the luciferase reporter. By observing the reduction in luciferase activity, we can validate the expression of tRFs. This method enables precise study of tRF functions and their roles in cellular processes.

Pre-eclampsia (PE) is a gestational disorder that significantly endangers maternal and fetal health. Transfer ribonucleic acid (tRNA)-derived small RNAs (tsRNAs) are important in the progression and diagnosis of various diseases. However, their role in the development of PE is unclear. Consequently, we detected the expression profiles of tsRNAs in the plasma of patients with PE as well as those in the plasma of the healthy control group, and a multiplicity of experiments were conducted with the aim of clarifying their roles in the occurrence and development of PE and the feasibility of serving as predictive biomarkers for this disorder.

High-throughput sequencing of tsRNA in plasma from PE cases was performed to evaluate its potential as a diagnostic or therapeutic biomarker. The function of tsRNA in trophoblasts was explored using the HTR-8/SVneo cell line. Plasma from pregnant women with suspected PE was analyzed to assess the potential of tsRNA to act as a predictive marker of PE.

High-throughput sequencing of tsRNA was performed on plasma from pregnant women with PE and from healthy pregnant controls. Analysis revealed a significant reduction in the level of tRNA-derived stress-inducing RNA (tiRNA)-Gln-CTG in the plasma (p < 0.001) and placenta (p < 0.001) of pregnant women with PE, suggesting its potential involvement in the development of this condition. tiRNA-Gln-CTG was identified in the cytoplasm and nucleus of HTR-8/SVneo cells. In vitro experiments revealed that tiRNA-Gln-CTG influences the proliferation, cycling, migration, and invasion of HTR-8/SVneo cells, possibly by targeting the 3'UTR region of thrombospondin-2 messenger ribonucleic acid (mRNA) for degradation. Extracellular vesicle (EV) carriers may mediate the level of tiRNA-Gln-CTG in the circulation. Y-box binding protein-1 (YBX1) may be involved in loading tiRNA-Gln-CTG into EVs. The sensitivity of low tiRNA-Gln-CTG levels for predicting the onset of PE in suspected cases was 91.7% within 1 week of delivery, 85.7% within 4 weeks of delivery, and 89.3% before delivery, with corresponding specificities of 84.5%, 79.2%, and 73.4%, respectively.

tiRNA-Gln-CTG significantly influences trophoblast function and is associated with the development of PE. It can serve as an effective biomarker for predicting PE progression within one week of delivery in women with suspected PE.

tRNA-derived fragments (tRFs) are novel short noncoding RNAs that play pivotal roles in cell proliferation and survival. However, knowledge of the biological roles of tRFs in anterior cruciate ligament (ACL) cells is limited. Here, we intended to investigate the function of tRF-3031B in ACL cell. We used the tRF and tiRNA array to analyze tRF and tiRNA expression profiles in osteoarthritis (OA) ACL cells and normal ACL cells, and qRT-PCR and fluorescence in situ hybridization (FISH) were used to determine tRF-3031B expression. The results showed that tRF-3031B was expressed at low levels in OA ACL and Interleukin-1β (IL-1β) treated ACL cells. We found that RELA was the target of tRF-3031B. When ACL cells were transfected with tRF-3031B mimics, RELA expression was suppressed, whereas transfection with tRF-3031B inhibitors had the opposite effect. The rescue and dual-luciferase reporter assays showed that tRF-3031B silenced the RELA expression by binding to its untranslated region (3'-UTR). Hence, this study showed the novel function of tRF-3031B in regulating ACL cell proliferation and survival by targeting RELA, and these findings may offer a new direction for the study of ACL degeneration and pathophysiological of OA.

tRNA molecules are among the most fundamental and evolutionarily conserved RNA types, primarily facilitating the translation of genetic information from mRNA into proteins. Beyond their canonical role as adaptor molecules during protein synthesis, tRNAs have evolved to perform additional functions. One such non-canonical role for tRNAs is through the generation of tRNA-derived fragments via specific cleavage processes. These tRNA-derived small RNAs (tsRNAs) are present across all three domains of life, including in protozoan parasites. They are formed through the cleavage of the parent tRNA molecules at different sites, resulting in either tRNA halves or smaller fragments. The precise mechanisms underlying the synthesis of various tRNA-derived fragments, including the specific RNases involved, as well as their distinct functions and roles in parasite physiology, are not yet fully understood and remain an active area of ongoing research. However, their role in modulating gene expression, particularly during stress responses, is becoming increasingly evident. In this context, we discuss recent findings on the roles of tRNA-derived small RNA in various protozoan parasites. Furthermore, we investigate how these tsRNAs either modulate gene expression within the parasite itself or are packaged into extracellular vesicles to alter host gene expression, thereby promoting parasite survival and adaptation.

The authors have withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2024/592122. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The correct preprint can be found at doi: https://doi.org/10.1101/2024.05.01.592122 .

Recently, members of a unique species of non-coding RNA, known as transfer RNA-derived small RNAs (tsRNAs) have been reported to serve multiple molecular functions, including in cells that mediate immunity. Because of their low molecular weights, tsRNAs were previously difficult to detect and were thus overlooked, until now. In this review, we delve into the biogenesis of tsRNAs and their diverse biological functions, ranging from transcriptional regulation to modulation of mRNA translation. We highlight the current evidence demonstrating their involvement in the immune response, as well as how tsRNAs modulate immunity to influence tumor growth and spread, autoimmune disease pathology and infection by pathogens. We surmise that tsRNAs are likely informative as diagnostic markers of cellular homeostasis and disease, and that therapeutic targeting of tsRNAs could be beneficial for a range of human diseases. Improved knowledge on the functions for tsRNAs in the mammalian immune system will enable us to leverage tsRNAs for their effective clinical use as treatments for human health challenges.

tRNA-derived RNAs (tDRs), resulting from enzyme-mediated hydrolysis of tRNAs, have been implicated as active small RNAs in various molecular processes. While the molecular modes of action for these small RNAs remain unclear, attempts to decipher the mechanistic details of tDR functionality have mostly used synthetic tDR sequences. Since parental tRNAs are extensively post-transcriptionally modified, tDR functionality is likely affected by chemical modifications. To help approach the biological function of endogenously modified tDRs, this contribution details a protocol that allows purifying specific tDRs carrying post-transcriptional modifications from both in vivo and in vitro sources. Purified tDRs can be used for various downstream applications including differential affinity capture of tDR-binding proteins, the details of which are also described in this contribution.

Roughly 54,000 individuals are diagnosed with head and neck cancers in the United States yearly. Transfer RNA-derived fragments (tRF) are the products of enzymatic cleavage of precursor tRNAs, and have been proposed for use as biomarkers of head and neck cancer. In this study, we aim to further analyze the utility that tRFs might provide as biomarkers of head and neck cancer. tRF read counts were obtained for 453 tumor and 44 adjacent normal tissue samples and used to construct a gradient boosting diagnostic model. Although we identified 129 tRFs that were significantly dysregulated between these samples, the model achieved a sensitivity of only 69 % and a specificity of 59 %. tRFs are thought to induce the degradation of mRNA transcripts containing a complementary "seed" region. Despite the above performances, we chose to explore this concept of translational regulation by analyzing these tRFs for inverse correlation to the expression of select oncogenes and tumor suppressor genes implicated in head and neck cancer. Among others, CysGCA 5'-half and LysCTT 3'-tRF were upregulated in the tumor samples, and corresponded to decreased expression of PIK3R1, AKT1, and CPEB3. These transcripts were further found to contain numerous significantly complementary sites at which tRF-mediated mRNA degradation might occur. Although these tRFs did appear to correlate to many of the oncogenic metrics analyzed, we believe that additional research is needed before they might be used to improve the diagnosis, treatment, and survival of patients with this disease.

This article explores the important functions of transfer RNA and - transfer RNA derived small RNAs (tsRNAs) in cellular processes and disease pathogenesis, with a particular emphasis on their involvement in cerebrovascular disorders. It discusses the biogenesis and structure of tsRNAs, including types such as tRNA halves and tRNA-derived fragments, and their functional significance in gene regulation, stress response, and cell signaling pathways. The importance of tsRNAs in neurodegenerative diseases, cancer, and cardiovascular diseases has already been highlighted, while their role in cerebrovascular diseases is in early phase of exploration. This paper presents the latest advancements in the field of tsRNAs in cerebrovascular conditions, such as ischemic stroke, intracerebral hemorrhage, and moyamoya disease. Furthermore, revealing the aptitude of tsRNAs as biomarkers for the prediction of cerebrovascular diseases and as targets for therapeutic intervention. It provides insights into the role of tsRNAs in these conditions and proposes directions for future research.

Protein-RNA interactions are central to all RNA processing events, with pivotal roles in the regulation of gene expression and cellular functions. Dysregulation of these interactions has been increasingly linked to the pathogenesis of human diseases. High-throughput approaches to identify RNA-binding proteins and their binding sites on RNA - in particular, ultraviolet crosslinking followed by immunoprecipitation (CLIP) - have helped to map the RNA interactome, yielding transcriptome-wide protein-RNA atlases that have contributed to key mechanistic insights into gene expression and gene-regulatory networks. Here, we review these recent advances, explore the effects of cellular context on RNA binding, and discuss how these insights are shaping our understanding of cellular biology. We also review the potential therapeutic applications arising from new knowledge of protein-RNA interactions.

The prevalence of diabetic kidney disease (DKD) is increasing annually. Damage to and loss of podocytes occur early in DKD. tRNA-derived fragments (tRFs), originating from tRNA precursors or mature tRNAs, are associated with various illnesses. In this study, tRFs were identified, and their roles in podocyte injury induced by high-glucose (HG) treatment were explored. High-throughput sequencing of podocytes treated with HG was performed to identify differentially expressed tRFs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. The expression levels of nephrin, podocin, and desmin were measured in podocytes after overexpression of tRF-1:24-Glu-CTC-1-M2 (tRF-1:24) and concomitant HG treatment. A total of 647 tRFs were identified, and 89 differentially expressed tRFs (|log2FC| ≥ 0.585; p ≤ .05) were identified in the HG group, of which 53 tRFs were downregulated and 36 tRFs were upregulated. The 10 tRFs with the highest differential expression were detected by real-time quantitative polymerase chain reaction (RT-qPCR), and these results were consistent with the sequencing results. GO analysis revealed that the biological process, cellular component, and molecular function terms in which the tRFs were the most enriched were cellular processes, cellular anatomical entities, and binding. KEGG pathway analysis revealed that tRFs may be involved in signaling pathways related to growth hormones, phospholipase D, the regulation of stem cell pluripotency, and T-/B-cell receptors. Overexpression of tRF-1:24, one of the most differentially expressed tRFs, attenuated podocyte injury induced by HG. Thus, tRFs might be potential biomarkers for podocyte injury in DKD.

Transfer RNA‑derived small RNAs (tsRNAs) are novel non‑coding RNAs that are associated with the pathogenesis of various diseases. However, their association with lung adenocarcinoma (LUAD) has not been studied comprehensively. Therefore, the present study aimed to explore the diagnostic value of a tsRNA, hsa_tsr011468, in LUAD. The OncotRF database was used to screen tsRNAs and reverse transcription‑quantitative PCR (RT‑qPCR) was performed to detect the expression levels of hsa_tsr011468 in various samples. Subsequently, the diagnostic and prognostic values of hsa_tsr011468 for LUAD were determined via receiver operating characteristic (ROC) curve and survival curve analyses, and by assessing clinicopathological parameters. In addition, both nuclear and cytoplasmic RNA were extracted to assess the location of hsa_tsr011468. The OncotRF database identified high expression of hsa_tsr011468 in LUAD. In addition, the results of RT‑qPCR showed that the relative expression levels of hsa_tsr011468 in the serum and tissues of patients with LUAD were higher than those in normal controls. Furthermore, its expression was lower in postoperative serum samples than in preoperative serum samples from patients with LUAD. ROC and survival curves indicated that hsa_tsr011468 had good diagnostic and prognostic value. Furthermore, the clinicopathological analysis revealed that hsa_tsr011468 was associated with tumor size. In addition, hsa_tsr011468 was mainly localized in the cytoplasm of LUAD cells. The present study indicated that hsa_tsr011468 has good diagnostic value and, therefore, could be employed as a serum marker for LUAD.

Neurological disorders known as neurodegenerative diseases (NDDs) result in the slow loss of neurons in the central nervous system (CNS) or peripheral nervous system (PNS), as well as the collapse of neural networks in terms of structure and function. NDDs are expected to surpass cancer as the second biggest cause of mortality by 2040, according to World Health Organization (WHO) estimations. Neurons cannot effectively regenerate themselves because they are terminally differentiated. Accordingly, it is challenging to find medications that could stop or slow neurodegeneration.

The tsRNAs are a type of small non-coding RNAs derived from mature tRNAs or tRNA precursors. tsRNAs control gene expression and have a role in many physiological and pathological processes, including neurological illnesses. Antisense oligonucleotides are effective therapeutic agents for neurological diseases, and they may be the treatment of choice for neurodegenerative diseases in the future. Here, we review the biogenesis of tsRNA, its physiological and pathological functions in the central nervous system and neurological disorders, and its prospective use as a nucleic acid medication to treat NDDs, providing theoretical support and guidance for further exploration of tsRNAs in therapeutic intervention.

tsRNAs are emerging as important regulatory molecules in neurodegenerative diseases. Understanding the functions of tsRNAs in neurodegenerative diseases may provide new insights into disease mechanisms and lead to the development of novel treatment strategies.

The main function of transfer RNAs (tRNAs) is to carry amino acids into the ribosome and synthesize proteins under the guidance of messenger RNAs (mRNAs). In addition to this, it has been observed that tRNAs undergo precise cleavage at specific loci, giving rise to an extensive array of distinct small RNAs, termed tRNA-derived small RNAs (tsRNAs). Existing studies have shown that tsRNAs are widely present across various organisms and comprehensively regulate gene expression, aberrant expression of tsRNAs is inextricably linked to tumorigenesis and development, thus, a systematic understanding of tsRNAs is necessary. This review aims to comprehensively delineate the genesis and expression patterns of tsRNAs, elucidate their diverse functions and emphasize their prospective clinical application as biomarkers and targets for therapy. It is noteworthy that we innovatively address the roles played by tsRNAs in human cancers at the level of the hallmarks of tumorigenesis proposed by Hanahan in anticipation of a broad understanding of tsRNAs and to guide the treatment of tumors.

Chronic obstructive pulmonary disease (COPD) is characterized by lung remodeling induced by chronic inflammation, presenting challenges for effective treatment. Mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) have shown promise in mitigating inflammation and tissue repairing in various diseases, including COPD. However, the optimal therapeutic pathways for different stages of COPD remain unclear. Transfer RNA-derived small RNAs (tsRNAs) are emerging as key regulators of cellular processes. However, their role in COPD and MSC therapy remains poorly understood.

This study explored the optimal administration routes and efficacy of bone marrow mesenchymal stem cells (BMSCs) and their extracellular vesicles (BMSC-EVs) in treating inflammatory or emphysematous COPD stages in mouse models. Male C57BL/6 mice were exposed to cigarette smoke daily for 6 or 16 weeks, with intraperitoneal CSE injections every 10 days, to model different stages of COPD. Mice were then treated with tracheal or intravenous injections of BMSCs or BMSC-EVs. PKH26 fluorescent dye labeled BMSCs and BMSC-EVs for pulmonary distribution observation. Lung tissue inflammation, apoptosis, EMT, and collagen deposition were assessed using HE staining, TUNEL assay, immunohistochemistry, and Sirius Red staining. Gene and tsRNA expression in lung tissues were analyzed by high-throughput sequencing. Differentially expressed tsRNAs (DE-tsRNAs) were validated by RT-qPCR. Statistical analysis was performed using GraphPad Prism 9.0. Data are presented as mean ± standard deviation (SD).

In 16-week COPD mice characterized by emphysema, tracheal administration of BMSC-EVs showed more significant lung distribution and inhibition of emphysematous pathology. In 6-week COPD mice characterized by inflammation, intravenous injection of BMSCs led to significant pulmonary homing, significantly reduced lung inflammation, apoptosis, EMT, and collagen deposition (P < 0.05). High-throughput sequencing indicated BMSC treatment downregulated genes related to these processes while upregulating mitochondrial function genes. Co-expression networks of DE-tsRNAs and target genes suggested potential roles in COPD. RT-qPCR confirmed significant differential expression of two DE-tsRNAs during COPD progression and BMSC treatment (P < 0.05).

Our study provides insights into selecting MSC and MSC-EV administration routes for different COPD stages. High-throughput sequencing supports BMSCs' inhibitory effects on lung remodeling and identifies the first tsRNA expression profile in a COPD model, warranting further investigation.

Transfer RNA (tRNA)-derived small RNAs (tDRs) are emerging as a novel class of regulatory molecules with significant implications in gene expression and cellular processes. These tDRs are generated through precise cleavage of precursor or mature tRNAs and can function in a sequence dependent manner or structure dependent manner. Recent studies have uncovered a unique subset of tDRs that can form tetramolecular assemblies, adding a new layer of complexity to their functional repertoire. Tetramolecular tDRs exhibit remarkable stability and functional diversity, influencing processes such as translation regulation, stress response, and cellular signaling. The assembly of these tDRs into tetramers is facilitated by guanine-rich sequence motifs which promote intermolecular interactions essential for their structure and biological activity. Understanding the formation, structural dynamics, and functional roles of tetramolecular tDRs offers new insights into tDR-mediated gene regulation and the potential development of RNA-based therapeutic strategies. This article aims to discuss a set of biochemical, biophysical, and reporter assay-based techniques that can be used to characterize G-quadruplex structures formed by tDRs.

In recent years, tRNA-derived small RNAs (tsRNAs) including tRNA-derived stress-induced RNAs (tiRNAs) and tRNA-derived fragments (tRFs), with specific structure and enriched in body fluids, have been found to have specific biological functions. In this paper, the biogenesis, classification, subcellular localization, and biological functions of tsRNAs were summarized. It has been proved that tsRNAs affected tumor cells in proliferation, apoptosis, migration and invasion, and played roles in regulating the occurrence and development of various tumors. In gastric cancer (GC), the imbalance of tsRNAs, such as tRF-33-P4R8YP9LON4VDP, tRF-17-WS7K092, tRF-23-Q99P9P9NDD and others, was closely related to the clinicopathological characteristics of GC patients. Some tsRNAs, such as tRF-23-Q99P9P9NDD, tRF-31-U5YKFN8DYDZDD, and tRF-27-FDXXE6XRK45 promoted the proliferation, migration and invasion of GC cells. Other tsRNAs, such as tRF-41-YDLBRY73W0K5KKOVD, tRF-18-79MP9PO4, and tRF-Glu-TTC-027 inhibited the proliferation, migration and invasion of GC cells. The tsRNAs played roles in the occurrence of GC were through several signaling pathways, such as phosphoinositide 3-kinase (PI3K)-AKT serine/threonine kinase (AKT), Wnt-β-Catenin, and mitogen-activated protein kinase (MAPK) pathways. These findings may provide new strategies for the diagnosis and treatment of GC.

Transfer RNA-derived small RNAs (tsRNAs) - categorized as tRNA-derived fragments (tRFs), tRNA-derived stress-induced RNAs (tiRNAs) and internal tRF (itRF) - are small non-coding RNAs that participate in various cellular processes such as translation inhibition and responses to cellular stress. We here identified tsRNA profiles within susceptible tissues in animal models of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Parkinson's disease (PD) to pinpoint disease-specific tsRNAs and those shared across neurodegenerative diseases. We performed small RNA sequencing in the SOD1G93A and TDP43A315T mouse models of ALS (spinal cord), the TauP301S model of FTD (hippocampus), and the parkin/POLG model of PD (substantia nigra). Bioinformatic analysis showed higher expression of 5' tiRNAs selectively in the two ALS models, lower expression of 3' tRFs in both the ALS and FTD mouse models, and lower expression of itRF Arg in the PD model. Experimental validation confirmed the expression of tsRNAs. Gene Ontology analysis of targets associated with validated 3' tRFs indicated functions in the regulation of synaptic and neuronal pathways. Our profiling of tsRNAs indicates disease-specific fingerprints in animal models of neurodegeneration, which require validation in human disease.

Salvianolic Acid B (SalB) has been proven to delay the progression of atherosclerosis. The therapeutic mechanisms of this compound are unclear. A novel class of short non-coding RNAs, pre-transfer RNA and mature transfer RNA (tsncRNAs) may regulate gene expression. TsncRNAs-sequencing revealed novel therapeutic targets for SalB. This is the first study focusing on tsncRNAs to treat atherosclerosis using SalB.

To explore the potential mechanism of SalB treating atherosclerosis through tsncRNAs.

Five groups of mice were created at random: control group (CON), atherosclerosis model group (MOD), SalB with high dose-treated group (SABH), SalB with low dose-treated group (SABL), and Simvastatin-treated group (ST). Aortic sinus plaque, body weight and inflammatory cytokines were evaluated. The Illumina NextSeq equipment was used to do expression profiling of tsncRNAs from serum. The targets of tsncRNAs were then predicted using tRNAscan and TargetScan. The KEGG pathway and GO analysis were utilized to forecast the bioinformatics analysis. Potential tsncRNAs and associated mRNAs were validated using quantitative real-time PCR.

tRF-Glu-CTC-014 and tRF-Gly-GCC-074 were markedly increased by SalB with high dose treatment and validated with quantitative real-time PCR. Two mRNAs SRF and Arrb related to tRF-Glu-CTC-014 changed consistently. GO analysis revealed that the altered target genes of the selected tsncRNAs were most enriched in protein binding and cellular process. Moreover, KEGG pathway analysis demonstrated that altered target genes of tsncRNAs were most enriched in MAPK signaling pathway.

SalB can promote the expression of tRF-Glu-CTC-014 to treat atherosclerosis.

Transfer RNA halves (tRHs) have various biological functions. However, the biogenesis of specific 5'-tRHs under certain conditions remains unknown. Here, we report that inositol-requiring enzyme 1α (IRE1α) cleaves the anticodon stem-loop region of tRNAGly(GCC) to produce 5'-tRHs (5'-tRH-GlyGCC) with highly selective target discrimination upon endoplasmic reticulum (ER) stress. Levels of 5'-tRH-GlyGCC positively affect cancer cell proliferation and modulate mRNA isoform biogenesis both in vitro and in vivo; these effects require co-expression of two nuclear ribonucleoproteins, HNRNPM and HNRNPH2, which we identify as binding proteins of 5'-tRH-GlyGCC. In addition, under ER stress in vivo, we observe simultaneous induction of IRE1α and 5'-tRH-GlyGCC expression in mouse organs and a distantly related organism, Cryptococcus neoformans. Thus, collectively, our findings indicate an evolutionarily conserved function for IRE1α-generated 5'-tRH-GlyGCC in cellular adaptation upon ER stress.