Pseudouridine synthases upregulate 5′-tRF-Lys to inhibit YPEL3 and drive malignant progression in nasopharyngeal carcinoma

Abstract

Transfer RNA-derived fragments (tRFs) are a recently discovered class of short noncoding RNAs widely distributed in various tissues and cell types. They are involved in the regulation of gene expression and play important roles in both physiological and pathological processes, garnering growing attention. However, the functions and underlying mechanisms of most tRFs in tumorigenesis and progression remain largely unclear. Through small RNA sequencing of nasopharyngeal carcinoma (NPC) and adjacent tissues, we found that among the top 30 highly expressed tRFs in NPC tissues, 13 were derived from lysine tRNAs, forming the 5′-tRF-Lys cluster. This cluster was found to promote NPC cell proliferation, invasion, and migration. Mechanistically, 5′-tRF-Lys binds to the 3′-untranslated region (3′-UTR) of YPEL3 messenger RNA (mRNA), suppressing its expression and thereby activating the Hippo/YAP signaling pathway to drive tumor progression. The elevated expression of pseudouridine synthases PUS1 and PUS7 in NPC tissues catalyzes pseudouridine modification of tRNA-Lys, facilitating its cleavage into 5′-tRF-Lys and accounting for its upregulation. Notably, the PUS1-targeting small-molecule inhibitor mogroside IV-e effectively reversed malignant phenotypes in both in vitro and in vivo NPC models. This study uncovers a novel mechanism in which pseudouridine synthases PUS1 and PUS7 drive the biogenesis of the tRF-Lys cluster, promoting NPC malignancy by suppressing YPEL3 and activating the Hippo/YAP signaling pathway. These findings highlight the therapeutic potential of targeting pseudouridine synthases to reduce tRF-Lys production as a novel strategy for NPC treatment.

Supplementary Information

The online version contains supplementary material available at 10.1186/s11658-026-00861-8.

Background

Nasopharyngeal carcinoma (NPC) is a malignant tumor of the head and neck that arises from the nasopharyngeal mucosa and exhibits a strikingly uneven global distribution. It is particularly prevalent in Southeast Asia, with Southern China bearing the highest incidence. In these endemic regions, more than 95% of NPC cases are classified as nonkeratinizing undifferentiated carcinoma, a subtype characterized by aggressive local invasion and a high tendency for distant metastasis [1, 2]. The anatomical location of NPC—deep within the nasopharynx—makes early detection challenging through routine clinical examination. Moreover, its early symptoms are typically subtle and nonspecific, often leading to misdiagnosis or delayed diagnosis. As a result, a significant proportion of patients present with metastatic disease at the time of initial diagnosis [3, 4]. Invasion and metastasis are the primary contributors to treatment failure and mortality in NPC. Therefore, elucidating the molecular mechanisms underlying NPC pathogenesis—particularly those driving proliferation, invasion, and metastasis—may offer valuable insights for identifying therapeutic targets and developing more effective, personalized treatment strategies.

The progression of NPC is driven by dysregulated expression of numerous oncogenes and tumor suppressor genes, alongside disruptions in complex intracellular signaling networks. Emerging evidence highlights the critical involvement of noncoding RNAs (ncRNAs) in regulating gene expression and modulating signal transduction pathways, thereby contributing to the pathogenesis of a wide range of human diseases, including NPC. Among these, transfer RNAs (tRNAs)—a conserved class of ncRNAs typically 76–90 nucleotides in length—play a canonical role in protein synthesis by delivering specific amino acids to the ribosome. These amino acids are covalently linked to the 3′-end of tRNAs by their corresponding aminoacyl-tRNA synthetases.

Recent studies have revealed that tRNAs can be selectively cleaved by specific enzymes to produce various tRNA-derived fragments (tRFs). These tRFs have been shown to regulate gene expression through diverse mechanisms, including gene silencing, modulation of translation efficiency, involvement in ribosome biogenesis, and epigenetic regulation [5]. Accumulating evidence links tRFs to multiple pathological conditions, such as neurodegenerative diseases, metabolic disorders, viral infections, and cancer [6–8]. In the context of cancer, tRFs have been implicated in key cellular processes, including tumor cell proliferation, metastasis, and drug resistance, largely by influencing gene expression and translational control [9–11]. However, the biological functions and mechanistic underpinnings of many tRFs in tumor initiation and progression remain poorly understood. Further systematic investigation is needed to elucidate the dynamic regulatory networks involving tRFs and their downstream targets in cancer pathogenesis.

In this study, we identified differentially expressed tRFs from our previously generated small RNA transcriptomic data of NPC and found that the 5′-tRF-Lys cluster was significantly upregulated in NPC tissues. Functional assays in vitro and in vivo demonstrated that this cluster promotes NPC cell proliferation, invasion, and migration by facilitating the degradation of the tumor suppressor YPEL3 messenger RNA (mRNA), thereby activating the Hippo/YAP signaling pathway. The aberrant elevation of 5′-tRF-Lys was linked to increased pseudouridine (Ψ) modification on Lys-tRNA, catalyzed by the pseudouridine synthases PUS1 and PUS7, both of which were found to be upregulated in NPC. Importantly, the small-molecule compound mogroside IV-e, which targets PUS1, effectively suppressed NPC progression in experimental models. These findings uncover a novel regulatory axis in NPC pathogenesis and suggest potential therapeutic targets for clinical intervention.

Methods

NPC clinical samples

Fresh biopsy specimens, including 36 NPC tissues and 9 chronic nasopharyngitis tissues (Supplementary Table 1), were collected for total RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 5′-tRF-Lys expression. All NPC samples were pathologically confirmed and obtained from Hunan Cancer Hospital. The study was approved by the Institutional Review Board of Hunan Cancer Hospital, and written informed consent was obtained from all participants.

Cell culture

The NPC cell lines HK1 and 5-8F were maintained at the Cancer Research Institute of Central South University. Cells were cultured in RPMI-1640 medium (Life Technologies, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, MA, USA) and incubated under standard conditions at 37 °C in a humidified atmosphere containing 5% CO₂.

Plasmids, small interfering RNAs (siRNA), and transfection

The YPEL3-Flag overexpression plasmid was obtained from Sangon Biotech (Shanghai, China) and verified by Sanger sequencing. Small interfering RNAs (siRNAs) targeting YPEL3, PUS1, and PUS7 were purchased from Tsingke (Beijing, China), and their sequences are provided in Supplementary Table 3. Plasmid transfections were performed using Neofect™ DNA Transfection Reagent (Neofect Biotech Co., Ltd., Beijing, China), while siRNA transfections were carried out with HiPerFect^® Transfection Reagent (Qiagen, Hilden, Germany).

RNA extraction and qRT-PCR

Total RNA was extracted from cells and tissues using TRIzol Reagent (Life Technologies, NY, USA), followed by isopropanol precipitation. First-strand complementary DNA (cDNA) synthesis was performed using the HiScript cDNA Synthesis Kit (Vazyme, Nanjing, China). Quantitative real-time PCR (qRT-PCR) was carried out with 2× SYBR Green Master Mix (Bimake, TX, USA) on a CFX96™ Real-Time PCR System (Bio-Rad, CA, USA) to assess target RNA expression levels. Primer sequences used for qRT-PCR are listed in Supplementary Table 2.

Wound healing assay

Cells were seeded in 6-well plates and cultured until they reached appropriate confluence. Following transfection, cells were incubated for an additional 48 h. A sterile 10 μL pipette tip was then used to create uniform linear scratches across the cell monolayer. Microscopic images were captured at fixed positions at 0 h and 24 h post-scratch, and wound widths were measured to assess cell migration.

Transwell invasion assay

Transwell inserts (Millipore, MA, USA) were coated with a layer of diluted Matrigel (BD, Shanghai, China) applied to the bottom of the upper chamber. After the Matrigel solidified, cells were seeded into the upper chamber in serum-free medium, while Roswell Park Memorial Institute Medium (RPMI)−1640 supplemented with 20% fetal bovine serum was added to the lower chamber as a chemoattractant. Following incubation for 48–60 h, cells that had invaded through the membrane were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and imaged using an inverted phase-contrast microscope. Invaded cells were then quantified.

CCK8 cell proliferation assay

Cells were seeded at a density of 1000 cells per well in 96-well plates. Following cell attachment, 10 μL of CCK-8 reagent (Abiowell, Changsha, China) was added to each well daily from day 0 to day 5. Plates were incubated at 37 °C for 2 h in the dark, after which absorbance was measured at 490 nm using a microplate reader (Molecular Devices, CA, USA).

Actinomycin D treatment

Actinomycin D (Sigma, St. Louis, MO, USA) was added to the culture medium of NPC cells at a final concentration of 2 μg/mL. Cells were harvested at 0, 1, 2, 4, and 8 h post-treatment for RNA extraction and subsequent qRT-PCR analysis.

Animal experiments

All experimental animals were obtained from the Experimental Animal Center of Central South University. Female BALB/c nude mice aged 4 weeks were housed under specific pathogen-free (SPF) conditions. All animal procedures were approved by the Institutional Animal Care and Use Committee of Central South University. Female BALB/c nude mice were randomly assigned to four groups (n = 6 per group). Each mouse was subcutaneously injected with 2 × 10⁶ HK1 cells transfected with one of the following: mimic control (NC-m), tRF mimics (mi-tRF), inhibitor control (NC-i), or tRF inhibitors (in-tRF).

In a separate experiment, an additional cohort of 12 female BALB/c nude mice was randomly divided into two groups (n = 6 each). Each mouse received a subcutaneous injection of 2 × 10⁶ HK1 cells. One group was treated with intraperitoneal injections of mogroside IV-e (200 μmol/mL) every 3 days, while the control group received equal volumes of saline. Tumor growth was monitored every 3 days by measuring tumor volume.

At 28 days post-inoculation, all mice were euthanized by cervical dislocation. Subcutaneous tumors were excised, weighed, and subjected to statistical analysis. Tumors were then processed by graded dehydration, paraffin-embedded, and sectioned for histological preservation. For the footpad tumor model, hind footpads were photographed every 7 days. At 28 days post-inoculation, mice were euthanized by cervical dislocation, and primary footpad tumors along with ipsilateral popliteal lymph nodes were harvested. Lymph node volumes and weights were measured, followed by paraffin embedding and sectioning for preservation.

In situ hybridization (ISH)

Paraffin sections were dewaxed and rehydrated, followed by quenching of endogenous peroxidase activity with 3% hydrogen peroxide. Tissue digestion was performed using freshly prepared pepsin solution (3% citric acid containing concentrated pepsin), and the reaction was stopped with 0.1 mol/L glycine. Sections were then refixed in 4% paraformaldehyde and incubated with prewarmed prehybridization solution at 37 °C for 30 min. Hybridization was conducted using digoxigenin-labeled 5′-tRF-Lys probes at 37 °C for 12–16 h. After hybridization, sections were washed with saline-sodium citrate (SSC) buffer and sequentially incubated with biotinylated mouse anti-digoxigenin antibody, streptavidin–biotin complex (sABC), and biotinylated peroxidase. Color development was achieved by incubation with 3,3'-diaminobenzidine (DAB), followed by hematoxylin counterstaining. Finally, sections were mounted with neutral resin and imaged under a microscope.

Western blotting

Cellular proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (Roche Applied Science, Baden-Württemberg, Germany). Protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on 8–12% gels at 80–120 V for 1.5–2 h, then transferred onto 0.2 μm polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). Membranes were blocked with 5% nonfat milk at room temperature for 1–2 h and incubated overnight at 4 °C with primary antibodies. After washing with phosphate-buffered saline + Tween 20 (PBST) or Tris-buffered saline + Tween 20 (TBST), membranes were incubated with species-specific secondary antibodies for 2 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Millipore, MA, USA). Detailed antibody information is listed in Supplementary Table 3.

Northern blotting

RNA samples were electrophoresed at 200 V on a 15% TBE-urea gel for 1.5 h. The gel was stained with SybrGold (1:10,000 dilution) and imaged using the Champ Gel 7000 chemiluminescence system (SINSAGE, Beijing, China). Nucleic acids were then transferred onto a Hybond-N+ membrane and ultraviolet (UV)-crosslinked. After prehybridization, membranes were hybridized with 50 pmol/mL biotinylated 5′-tRF-Lys probe at 40 °C for 12–16 h. Following washes with SSC buffer, membranes were incubated with streptavidin–horseradish peroxidase (HRP) conjugate at room temperature for 15 min. After washing with wash buffer, signals were detected using the ECL detection system (Millipore, MA, USA).

Immunoprecipitation (IP)

Protein-specific antibodies were incubated with IP beads using a DH-II DNA rotator (Scienentz, Ningbo, China) at room temperature for 2 h to prepare antibody-conjugated beads. Meanwhile, nasopharyngeal carcinoma cells were lysed on ice with Glo Lysis Buffer (GLB +) supplemented with protease inhibitors. The lysates were centrifuged at low temperature to collect protein extracts. These extracts were incubated with the antibody-conjugated beads at 4 °C for 12–16 h. Following incubation, the antibody-bead complexes were washed with prechilled GLB +. Protein loading buffer was then added, and samples were boiled for 5–10 min to denature proteins, yielding co-immunoprecipitated samples. Target proteins were subsequently detected by western blotting. Detailed antibody information is provided in Supplementary Table 3.

Immunofluorescence (IF)

NPC cells were cultured on glass coverslips in cell culture plates for 12–24 h. Once optimal confluency was reached, cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.25% Triton X-100, and blocked with 5% bovine serum albumin (BSA). Cells were then incubated with primary antibodies at 4 °C overnight (12–16 h), followed by incubation with secondary antibodies at 37 °C for 1 h. Nuclei were counterstained with DAPI at room temperature in the dark. Coverslips were mounted using anti-fade mounting medium, and images were captured using a confocal microscope (Leica, HE, Germany). Antibody details are provided in Supplementary Table 3.

Hematoxylin and eosin staining (H&E)

Paraffin-embedded tissue sections were baked at 65 °C for 2 h, followed by deparaffinization in xylene and rehydration through a graded ethanol series. Nuclei were stained with hematoxylin solution (Biosharp, Anhui, China), and cytoplasm was counterstained with eosin solution (Biosharp, Anhui, China). Finally, sections were mounted with neutral balsam for preservation.

Immunohistochemistry (IHC)

Protein expression in tissues from animal models was assessed using the UltraSensitive™ SP (Mouse/Rabbit) immunohistochemistry (IHC) kit (MXB, Fujian, China). Following deparaffinization and rehydration of paraffin-embedded sections, antigen retrieval was performed using EDTA under high-temperature conditions. Sections were incubated with primary antibodies at 4 °C overnight (12–16 h). Staining was developed with DAB and counterstained with hematoxylin. Two pathologists independently scored all sections, and the final score for each tissue was calculated as the average of both evaluations. Detailed antibody information is provided in Supplementary Table 3.

Dual-luciferase reporter assay

Firefly and Renilla luciferase expression vectors were constructed and co-transfected into nasopharyngeal carcinoma cells. Luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (Vazyme, Nanjing, China).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.5.1. Comparisons between two groups were conducted using a two-tailed Student’s t-test. Data are presented as mean ± standard deviation (SD), and p < 0.05 was considered statistically significant. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.

Results

5′-tRF-Lys is significantly upregulated in NPC tissues

Re-analysis of our previously published small RNA sequencing dataset (GSE159746) [12] identified 60 most significantly differentially expressed tRFs in NPC, including 30 upregulated and 30 downregulated species. Notably, among the upregulated tRFs, 13 belonged to the 5′-tRF-Lys category (Fig. 1A), defined as fragments derived from the 5′-end of lysine tRNAs (Lys-tRNAs) (Fig. 1B). Sequence alignment revealed high homology among these 13 isoforms, with only minor nucleotide variations (Fig. 1C). To validate their expression in NPC cells, we designed specific primers targeting two representative isoforms: the longest and most abundant isoform, tRF-1:31-Lys-CTT-1-M2 (hereafter referred to as tRF-31), and the shortest isoform, tRF-1:28-Lys-TTT-3-M2 (tRF-28), reflecting anticodon diversity among source tRNAs. Quantitative RT-PCR analysis of 45 clinical NPC specimens (36 tumor tissues and 9 adjacent tissues) confirmed significant upregulation of the 5′-tRF-Lys cluster in tumor tissues compared with normal controls (Fig. 1D). Moreover, expression levels of tRF-31 and tRF-28 showed a strong positive correlation across NPC samples (Fig. 1E). Together, these results demonstrate that a group of 13 highly homologous 5′-tRF-Lys molecules are significantly upregulated in NPC and likely function cooperatively, leading us to designate them as the 5′-tRF-Lys cluster.

Fig. 1.

5′-tRF-Lys is significantly overexpressed in NPC. A Heat map analysis of small RNA high-throughput sequencing data from NPC tissues and adjacent non-tumor tissues (GSE159746). B Schematic diagram showing the biogenesis of 5′-tRF-Lys. C Multiple sequence alignment of 13 significantly upexpressed 5′-tRF-Lys in NPC tissues. D qRT-PCR analysis of tRF-1:28-Lys-TTT-3-M2 and tRF-1:31-Lys-CTT-1-M2 expression in 36 NPC tissues and 9 adjacent non-tumor tissues. Statistical significance was assessed using Student’s t-test (two-tailed). ^**p < 0.01. E Correlation analysis of qRT-PCR Cq values of tRF-1:28-Lys-TTT-3-M2 and tRF-1:31-Lys-CTT-1-M2 across 36 NPC tissues. Statistical analysis was performed using chi-squared test, p < 0.0001

5′-tRF-Lys promotes proliferation, invasion, and metastasis of NPC cells

To investigate the functional role of 5′-tRF-Lys cluster in NPC progression, we synthesized mimics (designated as mi-tRF-28 and mi-tRF-31) and designed sequence-specific inhibitors (in-tRF-28 and in-tRF-31) targeting tRF-28 and tRF-31, respectively (Supplementary Fig. S1). Functional assays in NPC cells revealed that overexpression of either tRF-28 or tRF-31 significantly enhanced cell proliferation, migration, and invasion, whereas knockdown of both tRFs markedly suppressed these malignant phenotypes. These findings were consistently validated by three independent experimental approaches: CCK-8 proliferation assays, scratch wound healing assays, and Transwell invasion assays (Fig. 2A, B; Supplementary Fig. S1B–D).

Fig. 2.

5′-tRF-Lys promotes proliferation, invasion, and metastasis of NPC. A Transwell invasion assay assessing the invasive capacity of NPC cells following overexpression or knockdown of tRF-28 or tRF-31. Statistical analysis was performed using a two-tailed Student’s t-test. B Wound healing assay evaluating the migratory ability of NPC cells after overexpression or knockdown of tRF-28 or tRF-31. Statistical analysis was performed using a two-tailed Student’s t-test. C Representative images of subcutaneous tumors dissected from nude mice at the end of the experiment (n = 6 per group). D Tumor growth curves and tumor weight comparisons of subcutaneous xenografts in each group. Statistical analysis was performed using a two-tailed Student’s t-test. E Representative gross images of in situ tumors and ipsilateral popliteal lymph nodes following dissection of the injection-side popliteal fossa after euthanasia. F Images of dissected popliteal lymph nodes from nude mice at the experimental endpoint (n = 6 per group). G Quantification of lymph node volume and weight in each group. Statistical analysis was performed using a two-tailed Student’s t-test. H Representative histological images of subcutaneous xenograft tumors showing H&E staining, in situ hybridization for tRF-31, and Ki-67 immunohistochemical staining. I Representative images of H&E and pan-cytokeratin (CK-PAN) staining in popliteal lymph node sections from the “footpad-lymph node” metastasis model, highlighting metastatic tumor cells or tissues. Data are presented as mean ± SD. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001

To assess the effect of 5′-tRF-Lys cluster on the proliferative capacity of nasopharyngeal carcinoma (NPC) cells in vivo, we established a subcutaneous xenograft model using female BALB/c nude mice (4 weeks old). tRF-31, the most abundantly expressed isoform in NPC, was selected as the representative target. Tumor volumes were measured at regular intervals, and growth curves were generated for each group. The results showed that tumors in the tRF-31 overexpression group exhibited significantly accelerated growth, while those in the knockdown group grew markedly slower (Fig. 2C, D). Consistently, tRF-31 overexpression significantly increased both tumor volume and weight, whereas its knockdown produced the opposite effect. Histological analyses, including hematoxylin and eosin (H&E) staining, in situ hybridization for tRF-31, and Ki67 immunohistochemistry, revealed a substantial increase in Ki67 expression in tumors from the overexpression group and a notable decrease in the knockdown group (Fig. 2H). Collectively, these findings demonstrate that tRF-31 promotes the proliferation of NPC cells in vivo in a nude mouse xenograft model.

To evaluate the role of 5′-tRF-Lys cluster in promoting lymph node metastasis of NPC, we established a “footpad-lymph node” metastasis model in nude mice, using tRF-31 as a representative isoform owing to its high abundance and the clinical relevance of lymphatic spread in patients with NPC [13]. NPC cells from different experimental groups were injected into the left hind footpads of female BALB/c nude mice, and injection sites were monitored regularly (Supplementary Fig. S2A). After 4 weeks (Supplementary Fig. S2B), dissection of the ipsilateral popliteal lymph nodes revealed significantly greater lymph node enlargement in the tRF-31 overexpression group, consistent with enhanced metastatic activity. In contrast, lymph nodes in the tRF-31 knockdown group were significantly smaller (Fig. 2E–G). Histopathological examination of lymph nodes using H&E staining and CK-PAN immunohistochemistry (IHC) confirmed the presence of metastatic tumor foci in the tRF-31 overexpression group, with a significantly larger metastatic area compared with controls. Conversely, lymph nodes from the tRF-31 knockdown group exhibited no discernible metastatic foci and contained only scattered tumor cells, which were significantly fewer than in the control group (Fig. 2I). Together, these findings demonstrate that tRF-31 enhances lymph node metastasis of NPC cells in vivo.

5′-tRF-Lys promotes proliferation, invasion, and migration of NPC by downregulating YPEL3 expression

Previous studies have shown that tRNA-derived fragments (tRFs) can function similarly to microRNAs within cells. By associating with Argonaute (AGO) proteins, tRFs facilitate the formation of the RNA-induced silencing complex (RISC), which binds to the 3′-untranslated region (3′-UTR) of target mRNAs, decreasing their stability and thereby regulating gene expression at the post-transcriptional level [14]. To explore whether 5′-tRF-Lys exhibits microRNA-like functions, we used our tRF target prediction tool, tRF Prospect, to identify potential mRNA targets. We then analyzed the expression profiles of these predicted targets using the Gene Expression Omnibus (GEO) datasets GSE12452 and GSE53819. This analysis yielded 11 candidate genes that were downregulated in NPC and potentially regulated downstream of 5′-tRF-Lys (Fig. S3A, B). Using RNAhybrid, we predicted the binding free energy between these candidates and 5′-tRF-Lys. Among them, Yippee-like 3 (YPEL3), dual oxidase 1 (DUOX1), and Period circadian regulator 2 (PER2) exhibited the lowest minimum free energy (MFE) values for binding with 5′-tRF-Lys (Supplementary Fig. S3C). These three genes were thus selected for further in-depth investigation.

To confirm whether YPEL3, DUOX1, and PER2 are target genes of 5′-tRF-Lys, we overexpressed or knocked down tRF-28 or tRF-31 in NPC cells and examined the mRNA expression levels of YPEL3, DUOX1, and PER2. Results demonstrated that overexpression of either tRF-28 or tRF-31 reduced YPEL3 mRNA expression, whereas knockdown of tRF-28 or tRF-31 increased YPEL3 mRNA levels (Fig. 3A). In contrast, no significant effects were observed on DUOX1 or PER2 expression (Supplementary Fig. S3D). Western blot analysis further confirmed that both tRF-28 and tRF-31 inhibited YPEL3 protein expression (Fig. 3B). In clinical NPC tissue samples, qRT-PCR revealed significantly lower YPEL3 expression in tumor tissues compared with adjacent normal tissues, with YPEL3 levels showing a strong inverse correlation with tRF-28 and tRF-31 expression (Fig. 3C, D). Furthermore, in situ hybridization (ISH) and IHC analyses of subcutaneous xenografts in nude mice demonstrated a mutually exclusive expression pattern between tRF-28/tRF-31 and YPEL3 (Supplementary Fig. S4A, B). All together, these results indicate that the 5′-tRF-Lys cluster negatively regulates YPEL3 expression in NPC cells.

Fig. 3.

5′-tRF-Lys binds to the 3′-UTR of YPEL3 mRNA to promote its degradation. A qRT-PCR analysis of YPEL3 mRNA expression following overexpression or knockdown of tRF-28 or tRF-31 in NPC cells. Statistical analysis was performed using a two-tailed Student’s t-test. B Western blot analysis of YPEL3 protein expression after overexpression or knockdown of tRF-28 or tRF-31. C qRT-PCR detection of YPEL3 expression in 36 NPC tissues and 9 adjacent non-tumor tissues. Statistical analysis was performed using a two-tailed Student’s t-test. D Correlation analysis between YPEL3 and tRF-28 or tRF-31 qRT-PCR Cq values in 36 NPC samples. Statistical analysis was performed using the chi-squared test (p < 0.0001). E Dual-luciferase reporter assay measuring luciferase activity after overexpression or knockdown of tRF-28 or tRF-31. Statistical analysis was performed using a two-tailed Student’s t-test. F qRT-PCR analysis of YPEL3 mRNA stability following actinomycin D treatment (0–480 min) in cells overexpressing or silenced for tRF-28 or tRF-31. Statistical analysis was performed using a two-tailed Student’s t-test. Data are presented as mean ± SD. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.

To elucidate the molecular mechanism by which the 5′-tRF-Lys cluster regulates YPEL3 expression, we aligned the predicted binding sequence between tRF-31 and YPEL3 from RNAhybrid with the sequence of the 5′-tRF-Lys cluster. This analysis revealed that the tRF-31–YPEL3 binding site lies within the conserved region of the 5′-tRF-Lys cluster (Supplementary Fig. S3E). Accordingly, we constructed wild-type and mutant dual-luciferase reporter plasmids containing the predicted YPEL3 binding sequence (Supplementary Fig. S3F). Dual-luciferase reporter assays showed that transfection with tRF-28 or tRF-31 mimics significantly decreased luciferase activity in cells carrying the wild-type YPEL3 reporter, whereas transfection with their respective inhibitors increased luciferase activity. No significant changes were observed with the mutant reporter plasmids (Fig. 3E). Furthermore, actinomycin D assays following overexpression or knockdown of tRF-28 or tRF-31 in nasopharyngeal carcinoma cells demonstrated that both tRFs reduced YPEL3 mRNA stability (Fig. 3F). These results indicate that tRF-28 and tRF-31 suppress YPEL3 expression by binding to its mRNA and promoting its degradation.

To determine whether the 5′-tRF-Lys cluster promotes proliferation, invasion, and migration of NPC cells by suppressing YPEL3, we overexpressed YPEL3 in cells transfected with tRF-28 or tRF-31 mimics. Conversely, we knocked down YPEL3 in cells transfected with tRF-31 or tRF-28 inhibitors (Supplementary Fig. S5A). The results showed that upregulation of tRF-28 or tRF-31 enhanced NPC cell proliferation, invasion, and migration, while YPEL3 overexpression effectively reversed these effects. Similarly, YPEL3 knockdown counteracted the suppressive effects of tRF-28 or tRF-31 inhibition on these malignant phenotypes (Fig. 4A–C; Supplementary Fig. S5B–D). These findings demonstrate that the 5′-tRF-Lys cluster promotes NPC cell proliferation, invasion, and migration through downregulation of YPEL3.

Fig. 4.

5′-tRF-Lys promotes proliferation, invasion, and migration of NPC cells through downregulating YPEL3. A CCK-8 assay assessing the proliferation of NPC cells following co-overexpression or co-knockdown of YPEL3 and tRF-31. Statistical analysis was performed using a two-tailed Student’s t-test. B Transwell invasion assay evaluating NPC cell invasiveness after co-overexpression or co-knockdown of YPEL3 and tRF-31. Statistical analysis was performed using a two-tailed Student’s t-test. C Wound healing assay measuring NPC cell migration under conditions of co-overexpression or co-knockdown of YPEL3 and tRF-31. Statistical analysis was performed using a two-tailed Student’s t-test. Data are presented as mean ± SD. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001

5′-tRF-Lys activates the Hippo/YAP signaling pathway by suppressing YPEL3

Previous studies have shown that Yippee-like protein 3 (YPEL3) is downregulated in various cancers and is closely associated with the Hippo/YAP signaling pathway [15, 16]. However, the precise regulatory relationship between YPEL3 and the Hippo/YAP pathway remains poorly defined. To explore this, we employed AlphaFold-based protein–protein docking to predict potential interactions between YPEL3 and key components of the Hippo/YAP signaling cascade (Supplementary Fig. S6). The analysis revealed that YPEL3 has the highest predicted binding confidence with MOB1 (Supplementary Fig. S6, Fig. 5A), suggesting a strong likelihood of direct functional interaction. We validated the predicted interaction between YPEL3 and MOB1 through co-immunoprecipitation assays. Mutation of the predicted binding sites on YPEL3 abolished this interaction, demonstrating that their association is mediated by direct protein–protein binding (Fig. 5B). Additionally, immunofluorescence analysis confirmed the subcellular co-localization of YPEL3 and MOB1 in NPC cells (Fig. 5B, C).

Fig. 5.

5′-tRF-Lys activates the Hippo/YAP signaling pathway through suppressing YPEL3. A AlphaFold-based protein docking analysis predicting the interaction interface between YPEL3 and MOB1. B Co-immunoprecipitation assays demonstrating a physical interaction between YPEL3 and MOB1 in NPC cells, which was abolished when the predicted MOB1-binding sites in YPEL3 were mutated. Cells were transfected with a Flag-tagged YPEL3 wild-type (WT) or mutant (MUT) overexpression vector, immunoprecipitated using anti-Flag antibodies, and analyzed by western blotting with anti-Flag and anti-MOB1 antibodies. C Immunofluorescence staining showing the expression and subcellular localization of YPEL3 and MOB1 in NPC cells. D Western blot analysis of MOB1 and YAP phosphorylation levels following co-overexpression or co-knockdown of YPEL3 and tRF-31. E Nuclear-cytoplasmic fractionation assay assessing YAP nuclear translocation after co-overexpression or co-knockdown of YPEL3 and tRF-31/tRF-28. F Immunofluorescence staining to visualize YAP nuclear translocation in NPC cells following co-overexpression or co-knockdown of YPEL3 and tRF-31

To determine whether YPEL3 regulates MOB1 phosphorylation and activation of the Hippo/YAP signaling pathway, we overexpressed or silenced YPEL3 in nasopharyngeal carcinoma cells. Western blot analysis revealed that YPEL3 overexpression enhanced phosphorylation of MOB1 as well as YAP at Ser127 and Ser397, whereas YPEL3 knockdown led to reduced phosphorylation at these sites (Supplementary Fig. S7B). Immunofluorescence staining and nucleocytoplasmic fractionation further showed that YPEL3 overexpression decreased YAP nuclear translocation, while YPEL3 knockdown promoted its nuclear accumulation (Supplementary Fig. S7C, D). These findings suggest that YPEL3 inhibits YAP activity by binding to MOB1 and enhancing its phosphorylation, thereby activating the Hippo/YAP signaling pathway.

To further determine whether the 5′-tRF-Lys cluster activates the Hippo/YAP signaling pathway through suppression of YPEL3, we manipulated tRF-31 expression in nasopharyngeal carcinoma cells while concurrently overexpressing or silencing YPEL3. Western blotting, nucleocytoplasmic fractionation, and immunofluorescence staining were performed to assess phosphorylation of MOB1 and YAP (at Ser127 and Ser397) and YAP nuclear translocation (Fig. 5D–F; Supplementary Fig. S8A–C). The results showed that tRF-31 overexpression inhibited phosphorylation of MOB1 and YAP and promoted YAP nuclear translocation, whereas tRF-31 knockdown had the opposite effect. Importantly, co-expression of YPEL3 partially reversed the effects of tRF-31 on Hippo/YAP signaling. These findings support the conclusion that tRF-31 activates the Hippo/YAP pathway, at least in part, by suppressing YPEL3.

To assess whether 5′-tRF-Lys promotes NPC cell proliferation, invasion, and migration via the Hippo/YAP pathway, we overexpressed tRF-31 while silencing YAP, or knocked down tRF-31 while overexpressing YAP in nasopharyngeal carcinoma cells (Supplementary Fig. S9A). CCK-8 proliferation assays, Transwell invasion assays, and wound healing assays showed that YAP knockdown partially reversed the pro-tumorigenic effects of tRF-31, whereas YAP overexpression counteracted the suppressive effects of tRF-31 knockdown (Supplementary Figs. S9B–D and S10A–C).

Collectively, the above findings demonstrate that the 5′-tRF-Lys cluster activates the Hippo/YAP signaling pathway by suppressing YPEL3, thereby enhancing the proliferation, invasion, and migration of NPC cells.

PUS1 and PUS7 drive the biogenesis of the 5′-tRF-Lys cluster

RNases are key enzymes involved in tRNA cleavage, with angiogenin (ANG) and DICER1 being the primary RNases known to cleave tRNAs near the anticodon loop [14, 17, 18]. Analysis of the GEO dataset (GSE12452) and evaluation of ANG and DICER1 expression in NPC clinical samples showed no significant changes in their expression levels in NPC tissues (Supplementary Fig. S11A, B).

tRNAs harbor diverse chemical modifications that not only affect their structural stability and codon-decoding capacity but also influence tRNA fragmentation [5, 19, 20]. Predictive analysis of potential modifications on Lys-tRNA using the MODONOMICS database identified m¹G, m²G, pseudouridine (Ψ), m⁵C, and m¹A as possible modifications (Fig. 6A). Although m¹G and m²G modifications have not been associated with tRF biogenesis, we analyzed the expression of enzymes responsible for Ψ, m⁵C, and m¹A modifications. Analysis of the GEO dataset GSE12452 revealed significant upregulation of the Ψ-modifying enzymes PUS1 and PUS7 in nasopharyngeal carcinoma (Supplementary Fig. S11C). While the m⁵C-modifying enzyme NSUN2 and the m¹A-modifying enzyme TRMT61B were also upregulated in NPC, existing evidence indicates that m⁵C and m¹A modifications enhance tRNA stability by protecting against enzymatic cleavage and degradation, thereby inhibiting tRF biogenesis. Consequently, these modifications are unlikely to contribute to 5′-tRF-Lys generation [21–24]. Another m¹A-modifying enzyme, TRMT61A, showed no significant expression changes (Supplementary Fig. S11D–F) [25]. Given the reversible nature of m⁵C and m¹A modifications—regulated by demethylases—as opposed to the irreversible Ψ modification, we further examined the expression of eraser enzymes. Neither the m⁵C demethylase FTO nor the m¹A demethylases ALKBH1 and ALKBH3 exhibited significant changes in expression (Supplementary Fig. S11G) [25–29]. Collectively, these findings exclude m⁵C and m¹A modifications as drivers of elevated 5′-tRF-Lys expression.

Fig. 6.

PUS1/7 promotes the biogenesis of 5′-tRF-Lys. A Schematic diagram showing predicted pseudouridine (Ψ) modification sites on Lys-tRNA based on the MODOMICS database. B qRT-PCR analysis of PUS1 and PUS7 expression in 36 nasopharyngeal carcinoma (NPC) tissues and 9 adjacent non-tumor tissues. Statistical analysis was performed using a two-tailed Student’s t-test. C qRT-PCR detection of tRF-Lys expression following knockdown of PUS1 and/or PUS7 in NPC cells. Statistical analysis was performed using a two-tailed Student’s t-test. D Northern blot analysis of tRF-Lys expression after knockdown of PUS1 and/or PUS7 in NPC cells. E qRT-PCR analysis of tRF-Lys expression following treatment of NPC cells with mogroside IV-e (M-I), compared with DMSO control. Statistical analysis was performed using a two-tailed Student’s t-test. F Northern blot analysis of tRF-Lys expression after mogroside IV-e (M-I) treatment compared with DMSO control. G–H CCK-8 assay for cell proliferation (G) and Transwell assay for cell invasion (H) following mogroside IV-e treatment. Statistical analysis was performed using a two-tailed Student’s t-test. I Representative images of subcutaneous tumors dissected from nude mice at the end of the experiment (n = 6 per group). J Tumor growth curves and tumor weight measurements from each group. Statistical analysis was performed using a two-tailed Student’s t-test. K Representative gross images of in situ tumors and ipsilateral popliteal lymph nodes after dissection of the injection-side popliteal fossa. L Images of dissected popliteal lymph nodes from each group at the end of the experiment (n = 6 per group). M Quantification of volume and weight of popliteal lymph nodes in each group. Statistical analysis was performed using a two-tailed Student’s t-test. N Representative histological images of subcutaneous xenografts stained with H&E, in situ hybridization for tRF-Lys, and Ki-67 immunohistochemistry.O Representative H&E and CK-PAN staining of popliteal lymph node sections from the nude mouse footpad-lymph node metastasis model, indicating the presence of metastatic tumor cells. Data are presented as mean ± SD. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001

Emerging evidence indicates that pseudouridine (Ψ) modification facilitates tRNA cleavage to generate tRFs, consistent with our findings [30]. Accordingly, we examined clinical nasopharyngeal carcinoma tissue samples and observed upregulated expression of PUS1 and PUS7 in NPC tissues. Notably, expression levels of PUS1 and PUS7 positively correlated with those of tRF-28 and tRF-31 (Fig. 6B; Supplementary Fig. S11H). Knockdown of either PUS1 or PUS7 in NPC cells reduced 5′-tRF-Lys expression, with combined knockdown producing a more pronounced decrease (Supplementary Fig. S11E; Fig. 6C, D). These results demonstrate that aberrant overexpression of PUS1 and PUS7 promotes 5′-tRF-Lys biogenesis, likely through increased pseudouridylation of Lys-tRNA.

To investigate whether pseudouridine synthases influence the malignant progression of NPC, we knocked down PUS1 or PUS7 in NPC cells. CCK-8 proliferation assays, Transwell invasion assays, and wound healing assays demonstrated that knockdown of either PUS1 or PUS7 significantly suppressed NPC cell proliferation, invasion, and migration (Supplementary Fig. S12A–C). Treatment of NPC cells with mogroside IV-e, a small-molecule inhibitor of PUS1 [31], resulted in markedly reduced expression of tRF-31 and tRF-28, accompanied by inhibited proliferation, invasion, and migration (Fig. 6E–H; Supplementary Fig. S12D). In BALB/c nude mouse models bearing NPC subcutaneous xenografts and footpad-lymphatic metastasis tumors, mogroside IV-e administration (Supplementary Fig. S13A, B) significantly slowed tumor growth and reduced lymphatic metastasis compared with saline-treated controls, without causing significant weight loss (Fig. 6I–O). These findings suggest that mogroside IV-e is a promising therapeutic agent for clinical NPC treatment with minimal toxicity.

Discussion

In this study, we identified significant overexpression of the 5′-tRF-Lys cluster in NPC, which promotes malignant progression by targeting YPEL3 and thereby activating the Hippo/YAP signaling pathway. The elevated levels of 5′-tRF-Lys are driven by upregulation of the pseudouridine synthases PUS1 and PUS7 in NPC. Treatment with mogroside IV-e [31], a specific inhibitor of PUS1, effectively suppresses NPC malignant progression. This research is the first to elucidate the mechanism behind the aberrant overexpression of the 5′-tRF-Lys cluster in NPC and its oncogenic role, providing novel insights into the molecular basis and potential therapeutic strategies for NPC.

tRFs (tRNA-derived fragments) are a recently recognized class of small noncoding RNAs that have long been overlooked. However, growing evidence suggests that their distinct expression patterns and regulatory roles in tumors position them as potential “dark matter” within cells, contributing significantly to tumorigenesis and cancer progression. tRFs can interact directly with proteins, be packaged into exosomes for extracellular transport to facilitate intercellular communication, or more commonly, regulate gene expression by targeting mRNAs in a microRNA (miRNA)-like manner [10, 32, 33]. For instance, in gastric cancer, tRF-3019a inhibits FBXO47, while tRF-Glu-TTC-027 promotes tumor progression by suppressing the MAPK signaling pathway. In gallbladder cancer, tRF-His-GTG-012 suppresses cell proliferation by silencing TPRG1L mRNA [10, 34, 35]. Despite these insights, the roles and mechanisms of tRFs in NPC remain unexplored.

In this study, we conducted high-throughput small RNA sequencing to characterize the differential expression profile of tRFs in NPC for the first time. Among the top 30 most significantly upregulated tRFs, we identified 13 5′-tRF-Lys fragments derived from Lys-tRNA and confirmed their high expression in clinical samples. Multiple sequence alignment revealed that these 13 5′-tRF-Lys fragments share a high degree of sequence similarity. This striking pattern prompted an intriguing question: Why does NPC display such a unique expression profile dominated by highly upregulated and conserved 5′-tRF-Lys fragments? Could these fragments exert collective biological effects through their conserved sequences?

This phenomenon is closely tied to the origin of tRFs, which are derived from tRNAs. tRNA was the first noncoding RNA discovered and, although it does not encode proteins, it plays a central role in translation through codon–anticodon complementary pairing [35, 36]. The human nuclear genome encodes over 500 tRNA genes, with an additional 22 encoded by the mitochondrial genome—far exceeding the number of distinct tRNA types defined by their anticodons and the amino acids they carry [37, 38]. Notably, human tRNAs with the same anticodon are often encoded by multiple genomic loci. These tRNA genes share high sequence similarity but are not identical, resulting in a diverse pool of tRNA transcripts. The biological rationale for this redundancy remains unclear. One hypothesis is that conserved sequences maintain the structural integrity necessary for tRNA function, while sequence diversity supports tissue- or cell type-specific expression, enabling tRNAs to fulfill additional regulatory roles—including serving as precursors for tRFs generated by precise enzymatic cleavage under specific conditions [39, 40]. This dual nature—where tRNAs produce a diverse array of transcripts and individual tRFs can arise from multiple tRNA genes—gives rise to a large repertoire of tRFs that are similar in sequence yet differ subtly in origin and function. As a result, tRF nomenclature typically reflects sequence length, the source tRNA’s amino acid and anticodon, and may indicate derivation from multiple tRNA genes [41]. While most studies have focused on the function of individual tRFs, there is growing evidence for coordinated or “group effects” among tRFs. For example, a group of 22-nucleotide, m1A-modified 3′-tRFs has been shown to collectively regulate bladder cancer progression via the unfolded protein response. Similarly, 5′-tRFs derived from tRNA-Ala, tRNA-Cys, and tRNA-Val containing a 5′-terminal oligoguanine (TOG) motif can inhibit translation initiation by displacing the eIF4F complex [30, 42]. These findings suggest that sequence conservation among tRFs may endow them with shared functional properties. However, this concept of group effects has not yet been explored in the context of nasopharyngeal carcinoma (NPC). In this study, we identified a cluster of highly conserved 5′-tRF-Lys fragments in NPC, derived from multiple Lys-tRNA transcripts. Through bioinformatic analysis and experimental validation, we demonstrate that these 13 5′-tRF-Lys molecules specifically target the 3′-untranslated region (3′-UTR) of YPEL3 mRNA, collectively promoting its degradation. This, in turn, facilitates the malignant progression of NPC. Their shared targeting mechanism and oncogenic function led us to define them as the 5′-tRF-Lys cluster, a term that underscores the functional cooperativity among these tRF molecules.

YPEL3 is a highly conserved gene across eukaryotes and is known to play roles in cell cycle regulation, proliferation, and differentiation. It has been reported to be downregulated in various cancers [16, 43–46]. Since its discovery in 2004, YPEL3 has been identified as a potential tumor suppressor, possibly acting through modulation of the Hippo/YAP signaling pathway [15], although its precise mechanism of action remains incompletely understood. In this study, we found that YPEL3 interacts with MOB1—a core component in the activation of the Hippo/YAP signaling cascade—enhancing its phosphorylation and thereby suppressing activation of the Hippo/YAP pathway. Aberrant activation of this pathway results in uncontrolled cell growth and impaired differentiation, contributing to tumor cell proliferation, invasion, and metastasis [47–49]. Previous research has demonstrated that noncoding RNAs, such as long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs), can modulate the Hippo/YAP signaling pathway [48, 50, 51]. Here, we provide the first experimental evidence that tRFs—an emerging class of small noncoding RNAs—also participate in the regulation of this pathway. Specifically, we show that blocking the generation of 5′-tRF-Lys can inhibit upstream activation of the Hippo/YAP pathway, thereby mitigating the malignant behaviors of NPC cells.

Traditional ribonucleases, such as ANG and DICER1, can cleave tRNAs at specific recognition sites to generate tRFs [52, 53]. In addition to enzymatic cleavage, chemical modifications of tRNAs also regulate tRF biogenesis by affecting tRNA structural stability and altering the accessibility of cleavage sites [39, 40]. Among these modifications, pseudouridylation (Ψ) is the most prevalent. Catalyzed by pseudouridine synthases, Ψ affects tRNA function in multiple ways, including enhancing stability, facilitating aminoacylation, maintaining translational fidelity, and influencing the generation of tRFs [54–58]. However, the complex secondary and tertiary structures of tRNAs pose a technical challenge for precisely mapping modification sites and identifying their corresponding enzymes. As a result, the site-specific functions and mechanisms of Ψ modifications remain largely unresolved.

In this study, we predicted potential Ψ modification sites on Lys-tRNAs and experimentally demonstrated that the elevated production of 5′-tRF-Lys in nasopharyngeal carcinoma (NPC) is driven by increased expression of the pseudouridine synthases PUS1 and PUS7. Silencing PUS1 and PUS7 effectively reduced 5′-tRF-Lys levels and suppressed the malignant phenotypes of NPC cells, highlighting these enzymes as upstream regulatory targets. To explore potential therapeutic strategies, we identified mogroside IV-e as a specific inhibitor of PUS1 [31]. Mogroside IV-e is a natural saponin extracted from Siraitia grosvenorii (monk fruit) [59], and has demonstrated broad pharmacological activities—including anticancer, antibacterial, anti-inflammatory, antidiabetic, and antioxidant effects—with low toxicity in various models [60–62]. Notably, its application in NPC has not been previously reported. Upon treatment with mogroside IV-e, we observed a significant reduction in 5′-tRF-Lys expression, accompanied by markedly decreased proliferation, invasion, and migration of NPC cells in vitro. In vivo, intraperitoneal administration of mogroside IV-e significantly inhibited NPC growth and metastasis in mouse models, without evident toxic side effects. Collectively, these findings suggest that mogroside IV-e may serve as a promising therapeutic agent for NPC by targeting pseudouridine synthase-mediated tRF biogenesis.

In summary, tRFs constitute a novel class of noncoding RNAs with significant research value and strong potential as molecular biomarkers and therapeutic targets. This study is the first to elucidate the biogenesis and functional role of 5′-tRF-Lys in NPC. We further demonstrate that the small-molecule compound mogroside IV-e suppresses 5′-tRF-Lys expression by inhibiting pseudouridylation, thereby markedly attenuating NPC progression both in vitro and in vivo. These findings highlight mogroside IV-e as a promising candidate for NPC therapy. On the basis of our results, we propose a novel therapeutic approach: Targeting the biogenesis of tRF-Lys may represent an effective strategy to mitigate NPC malignancy and improve clinical outcomes.

Conclusions

This study uncovers a novel oncogenic axis in NPC, demonstrating for the first time that pseudouridine synthases PUS1 and PUS7 promote tumor progression by catalyzing pseudouridylation (Ψ) of tRNA-Lys, thereby facilitating the biogenesis of oncogenic 5′-tRF-Lys fragments. Mechanistically, the upregulated 5′-tRF-Lys cluster directly targets the 3′-UTR of YPEL3 mRNA, leading to its downregulation. Loss of YPEL3 disrupts the Hippo/YAP signaling pathway by reducing MOB1 phosphorylation and activating YAP, ultimately driving NPC cell proliferation, invasion, and metastasis. Importantly, we demonstrate the therapeutic potential of targeting this pathway: Mogroside IV-e, a specific PUS1 inhibitor, effectively blocks 5′-tRF-Lys production and reverses malignant phenotypes both in vitro and in vivo. These findings reveal a previously unrecognized Ψ modification-dependent mechanism underlying NPC pathogenesis and highlight PUS1/PUS7 inhibition as a promising therapeutic strategy. Our work provides a compelling rationale for the development of agents targeting pseudouridine synthase activity or 5′-tRF-Lys function to combat NPC progression.

Abbreviations

tRNA

Transfer RNA

tRF

Transfer RNA-derived fragment

NPC

Nasopharyngeal carcinoma

ncRNA

Noncoding RNA

Ψ

Pseudouridine

qRT-PCR

Quantitative real-time polymerase chain reaction

ISH

In situ hybridization

IHC

Immunohistochemistry

H&E

Hematoxylin and eosin staining

YPEL3

Yippee-like 3

MOB1

MOB kinase activator 1

LATS1/2

Large tumor suppressor kinase 1/2

MST1/2

Mammalian sterile 20-like kinase 1/2

YAP

Yes-associated protein

PUS1

Pseudouridine synthase 1

PUS7

Pseudouridine synthase 7

Author contributions

D.R., P.C., W.X., and Z.Z. designed the project; D.R. completed most of the experiments. M.Y., Y.M., Q.Y., L.S., S.Z., Z.G., and C.G. performed some of the experiments and collected tissue samples. D.R., M.Z., B.X., and M.T. analyzed the data and wrote the manuscript. D.R., M.T., G.L., P.C., W.X., and Z.Z. revised the manuscript. All authors read and approved the final manuscript.

Funding

This study was funded by the National Natural Science Foundation of China (82472789 and 82472711), the Natural Science Foundation of Hunan Province (2025JJ20099, 2024PT5102, 2024JJ3035, and 2023ZJ1120), the Graduate Student Scientific Research Innovation Project of Central South University (1053320242771), and the Changsha Science and Technology Project (kh2301025).

Availability of data and materials

Not applicable.

Declarations

Ethics approval and consent to participate

The research project has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Central South University. Animal experiments were strictly conducted in accordance with the 3R principles proposed by the International Council for Laboratory Animal Science. The ethics committee reviewed the purpose of the animal study, the number of animals used, and the measures implemented to minimize all foreseeable pain and distress throughout the experimental procedures. The maximum permitted tumor size was 2000 mm³ (approval no. CSU-2024-0328, 2024, Animal Welfare and Ethics Committee of Central South University). The use of human materials in this study was approved by the Medical Ethics Committee of Xiangya Hospital, Central South University (approval no. CSU-CRI-IRB-2021-06, 2021) and complied with the ethical principles outlined in the Declaration of Helsinki.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.