Demethylase ALKBH4 Inhibits Cell Proliferation via Induction of 5'-tsRNAGlu in Non-Small Cell Lung Cancer

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are an emerging class of small non-coding RNAs that play critical roles in tumor progression; however, the functions and underlying mechanisms of tsRNAs in lung cancer development remain poorly understood. Thus, this study aimed to elucidate the molecular mechanism through which alkB homolog 4, lysine demethylase (ALKBH4) influences non-small cell lung cancer (NSCLC) progression through 5'-tsRNA^Glu, with a particular focus on the role of tsRNA in translational regulation and malignant phenotype formation.

Cell proliferation and cell cycle progression were assessed using Cell Counting Kit-8 (CCK-8), colony formation, and flow cytometry assays. Global translation efficiency was evaluated using nascent protein synthesis assays and polysome profiling. Northern blotting was used to confirm 5'-tsRNA^Glu expression post-transfection, and downstream targets of 5'-tsRNA^Glu were screened using a dual-luciferase reporter system. RNA pull-down and RNA immunoprecipitation (RIP) assays were employed to identify proteins that interacted with 5'-tsRNA^Glu. Data were analyzed using Student's t-test or one-way analysis of variance (ANOVA).

The demethylase ALKBH4 promoted the biogenesis of 5'-tsRNA^Glu in NSCLC cells. Overexpression of ALKBH4 or 5'-tsRNA^Glu inhibited cell proliferation, induced cell cycle arrest, and reduced global translational efficiency, whereas knockdown of 5'-tsRNA^Glu attenuated the tumor-suppressive effects of ALKBH4. Mechanistically, 5'-tsRNA^Glu did not function in a miRNA-like manner but instead interacted with ribosomal protein L17 (RPL17). Furthermore, 5'-tsRNA^Glu was predominantly localized in the cytoplasm, and 5'-tsRNA^Glu overexpression significantly increased the associated distribution in the 60S ribosomal subunit while diminishing the associated presence in the 80S monosome. Predictive modeling indicated that 5'-tsRNA^Glu binds to the region of RPL17 spanning residues 137-142, the RPL17-rRNA interaction interface, suggesting that binding of 5'-tsRNA^Glu to RPL17 interferes with RPL17-rRNA interaction, thereby disrupting the 80S ribosome assembly and global translational efficiency.

ALKBH4 suppresses malignant phenotypes in NSCLC cells via 5'-tsRNA^Glu, likely through an interaction between 5'-tsRNA^Glu and RPL17.