Live-cell imaging of circular and long noncoding RNAs associated with FUS pathological aggregates by Pepper fluorescent RNA

Lately, important advancements in visualizing RNAs in fixed and live cells have been achieved. Although mRNA imaging techniques are well-established, the development of effective methods for studying noncoding RNAs (ncRNAs) in living cells is still challenging but necessary, as they show a variety of functions and intracellular localizations, including participation in highly dynamic processes like phase transition, which is still poorly studied in vivo. Addressing this issue, we tagged two exemplary ncRNAs with the fluorescent RNA (fRNA) Pepper. Specifically, we showed that circ-HDGFRP3 interacts with p-bodies and is recruited in pathological FUS aggregates in a dynamic fashion, and we super-resolved its distribution in such condensates via structured illumination microscopy. Moreover, we tracked the long noncoding RNA (lncRNA) nHOTAIRM1, a motor neuron–specific constituent of stress granules, monitoring its behavior throughout the oxidative-stress response in physiological and pathological conditions. Overall, as fRNA development progresses, our work demonstrates an effective use of Pepper for monitoring complex processes, such as phase transition, in living cells through the visualization of circular RNAs (circRNAs) and lncRNAs with super-resolution power.