Deciphering Phase-Separated Mitochondrial RNA Granules under Stress Conditions with the Mitoribosome-Targeting Small Molecule

Abstract

RNA granules are liquid–liquid-phase-separated condensates comprising RNA and proteins. Despite growing insights into their biological functions, studies have predominantly relied on biological methodologies lacking adequate chemical tools. Here, we introduce ICP-CHARGINGS, a concept for efficiently identifying chemical probes to characterize RNA granules of interest among nucleic acid-targeting agents. Focusing on mitochondrial RNA granules (MRGs), whose functions remain elusive, we developed a methodology within this framework and identified NATA, a new fluorescent molecule that, following mechanistic studies, was found to bind to the mitoribosome, enabling MRG labeling and recognition. Using NATA to reveal the potential buffering roles of MRGs, we demonstrated a close correlation between MRG maintenance and assembly and cellular survival and proliferation under cold shock and hypoxic stress. Overall, the introduction and implementation of the ICP-CHARGINGS strategy provide a specialized chemical tool for advancing our comprehension of MRG biology and establish a paradigm for elucidating RNA structures within RNA granules that can be targeted by small molecules, paving the way for developing tailored chemical probes for diverse RNA granules in future research.