Mitochondria-specific targeting of noncanonical ntmRNA-coordinated mitophagy receptor BNIP3 homodimerization disrupts mitochondrial metabolism and suppresses hepatocellular carcinoma growth and .

Hypoxia-driven metabolic reprogramming is a hallmark of hepatocellular carcinoma (HCC) and depends critically on mitochondrial signaling. We sought to identify RNA-based molecular factors that orchestrate the hypoxia-mitochondria crosstalk and regulate metabolic adaptation in HCC cells. An integrated mtRNA-seq and mitochondria-specific LwaCas13a-BN-MLS RNA targeting approach was employed to profile RNA molecules aberrantly enriched in HCC mitochondria. Mitophagy was assessed via mt-Keima assay, immunofluorescence, transmission electron microscopy, and Western blotting of key autophagic markers. RNA-protein interactions were examined using RNA immunoprecipitation (RIP), electrophoretic mobility shift assays (EMSA), and computational structural modeling. and tumorigenicity was evaluated using colony formation, transwell invasion, wound healing, and subcutaneous xenograft models in nude mice. Nuclear-encoded mRNA was aberrantly translocated to mitochondria, where it functions as a non-translating mRNA (ntmRNA) essential for mitophagy. Mitochondria-specific targeting disrupted mitochondrial homeostasis by accumulating damaged mitochondria, lowering ATP, increasing ROS, reducing membrane potential, diminishing spare respiratory capacity, and impairing hypoxia-induced mitophagy. Mechanistically, ntmRNA promoted mitophagy through the HIF-1α/BNIP3/NIX axis by recruiting BNIP3 to mitochondria and coordinating its homodimerization via a 3'-UTR MRE. A synthetic MRE oligonucleotide rescued BNIP3 dimerization after depletion. Finally, we demonstrated that loss suppressed malignant phenotypes and reduced xenograft tumor growth . This study reveals a noncanonical role for mRNA as an epigenetic regulator of mitophagy in HCC, thus expanding the functional repertoire of mRNA molecules beyond protein coding. Targeting this noncanonical ntmRNA-BNIP3 homodimerization mechanism may suggest new therapeutic strategies for treating HCC.