Illuminating Mitochondrial RNA G‐Quadruplexes as Structural Brakes on RNA Granule Assembly and OXPHOS

ABSTRACT G‐quadruplexes (G4s) have been extensively investigated in cells, with established methods available for studying nuclear DNA G4s, cytoplasmic RNA G4s, and even mitochondrial DNA G4s. However, mitochondrial RNA (mtRNA) G4s have remained largely unexplored in cells due to the lack of suitable tools, leaving their biological functions poorly understood. Here, through rational molecular design, we developed MitoQUMA , a fluorescent probe that allows the visualization of mtRNA G4 dynamics in live cells. Using this probe, we observed that, unlike cytoplasmic RNA G4s, which generally promote phase separation to form RNA granules, excessive formation of mtRNA G4s correlates with reduced assembly of mitochondrial RNA granules (MRGs). A MitoQUMA ‐based chemical genetic screen revealed that the Wnt/β‐catenin pathway regulates this mitochondrial event by modulating GRSF1 expression, thereby affecting mtRNA G4 abundance and processing. When RNA processing is compromised, mtRNA maturation is impaired, and MRG becomes unstable and undergoes disassembly, ultimately disrupting mitochondrial gene expression and energy metabolism. Collectively, our study introduces a tool for real‐time monitoring of mtRNA G4s in cells and identifies the Wnt/β‐catenin–GRSF1–mtRNA G4 axis as a previously unrecognized pathway coordinating MRG assembly and energy metabolism, providing new insights into phase separation within mitochondria.