tRNA-derived fragment tRF-24 drives CELF1 phase separation to promote oncogenic splicing in esophageal squamous cell carcinoma.

RATIONALE: Esophageal squamous cell carcinoma (ESCC) is characterized by poor prognosis. tRNA-derived fragments (tRFs), a novel class of non-coding RNAs generated by tRNA cleavage, have emerged as key regulators of cancer development. However, the functional landscape of tRFs remains underexplored in ESCC. We here identified tRF-24-RPM8309M2S (tRF-24), a 5' tRF derived from mature tRNA(LeuAAG/TAG), which promotes the malignant progression of ESCC and offers a promising therapeutic target. METHODS: The public GSE207635 dataset from Gene Expression Omnibus (GEO) database was analyzed to identify tsRNAs involved in ESCC progression. The clinical significance of tRF-24 was investigated in samples from 96 ESCC patients. CUGBP Elav-like family member 1 (CELF1) was validated as a tRF-24 interactor through RNA pull-down assays. CCK-8 and transwell assays were applied to measure malignant cell phenotypes. mCherry-GFP-LC3 reporter assay was performed to examine the autophagy. Colocalization between LC3 and mitochondria was employed to detect mitophagy. Immunofluorescent and colony formation assay were conducted to assess the impact of DNA damage repair and cisplatin resistance in ESCC. Extracellular acidification rate (ECAR), lactate production and glucose consumption were performed to analyze changes in glycolysis. Fluorescence recovery after photobleaching (FRAP) was adopted to evaluate CELF1 phase separation. Additionally, RNA sequencing and alternative splicing analyses were conducted to determine global transcriptome alterations following tRF-24 or CELF1 overexpression. RESULTS: Our findings demonstrate that tRF-24 is significantly upregulated in tumor samples and is associated with poorer survival of ESCC patients. Depletion of tRF-24 suppresses malignant cell phenotypes in ESCC cells both in vitro and in vivo. Mechanistically, tRF-24 binds to the Ser28 residue of CELF1, inhibiting AKT1-mediated phosphorylation at this site, which facilitates CELF1 nuclear translocation and subsequent liquid-liquid phase separation (LLPS) formation. These CELF1-enriched nuclear condensates potently regulate the alternative splicing of BIN1 and BECN1 pre-mRNAs, generating pro-oncogenic BIN1-L and pro-autophagic/mitophagic BECN1-α isoforms that collectively enhance tumor malignancy by promoting tumor cell EMT, DNA damage repair, cisplatin resistance and glycolysis. Targeting tRF-24 with an antagomir significantly suppresses tumor progression in ESCC xenograft models, highlighting its therapeutic potential. CONCLUSIONS: Our findings establish tRF-24 as a promising therapeutic target in the comprehensive treatment of ESCC.