Abstract

Subcellular compartments organize RNAs into phase-separated condensates, significantly influencing RNA metabolism. However, the study of how specific RNAs regulate interacting factors and their phenotypic outcomes is hindered by the lack of advanced imaging and regulation tools. To address this, we developed an orthogonal RNA aptamer system, Clivia-HT, which integrates a fluorescent imager with a ribonuclease-targeting chimera (RIBOTAC) degrader. This platform enables simultaneous RNA visualization and targeted degradation in living cells. Furthermore, we engineered light-activatable and light-inactivatable RIBOTACs to achieve temporal RNA degradation control. Applying this system, we demonstrated that Activating Transcription Factor 4 ( ATF4 ) mRNA localizes to stress granules and regulates their dynamics. We also detected the role of Non-Coding RNA Activated by DNA Damage ( NORAD ) in RNA–protein condensate assembly. Our approach establishes a versatile method for spatiotemporal RNA manipulation, providing a powerful tool for probing RNA function in dynamic cellular environments.