m ⁵ C‐Modified tRF3b‐Cys ^GCA ‐23 Suppresses Bladder Cancer Malignancy by Repressing H3K18 Lactylation via Stabilizing RBM4

ABSTRACT The epigenetic modification of transfer RNA (tRNA) and tRNA‐derived small RNAs (tsRNAs) is associated with the initiation and development of cancer. However, the biological role of m ⁵ C‐modified tsRNAs, especially in bladder cancer (BC), and their regulatory mechanisms remain unclear. Here, we identify a novel m ⁵ C‐modified tsRNA, m ⁵ C‐tRF3b‐Cys ^GCA ‐23 (mtRC), whose expression is significantly downregulated in both tumor tissues and urine samples of BC patients and is strongly negatively correlated with the malignant progression of bladder cancer. In vitro and in vivo functional experiments reveal that mtRC, but not its unmodified counterpart (tRC), exhibits a tumor‐suppressive role. Furthermore, NOP2/Sun RNA methyltransferase 6 (NSUN6) regulates mtRC abundance and suppresses cell proliferation. Mechanistically, mtRC directly binds the oncosuppressor protein RNA‐binding motif 4 (RBM4) and improves its stability by preventing RBM4 ubiquitination, thereby upregulating RBM4 protein levels. RBM4 reduces the levels of glycolytic genes and decreases glycolysis, thereby inhibiting histone H3 lysine 18 lactylation (H3K18la). This reduction in H3K18 lactylation attenuates the transcriptional activation of the downstream oncogenes IL1RAP and VASH2, thereby ultimately suppressing tumor malignancy in BC. Together, our results not only underscore the critical role of mtRC in BC but also unravel a novel and coherent regulatory signaling axis—mtRC/RBM4/H3K18la/IL1RAP&VASH2—that orchestrates BC malignancy, suggesting mtRC may serve as a candidate therapeutic target for BC treatment.