Live-cell imaging of circular and long non-coding RNAs associated to FUS pathological aggregates by Pepper fluorescent RNA

Lately, important advancements in visualizing RNAs in fixed and live cells have been achieved. While mRNA imaging techniques are well-established, the development of effective methods for studying non-coding RNAs (ncRNAs) in living cells are still challenging but necessary, as they cover a variety of function and of intracellular localization, including highly dynamic processes like phase-transition, still poorly studied in vivo . Addressing this issue, we tagged two exemplary ncRNAs with the innovative fluorescent RNA (fRNA) Pepper. Specifically, we show circ-HDGFRP3 interaction with p-bodies, we recapitulate its recruitment in pathological FUS aggregates in a dynamic fashion and we super-resolve its distribution in such aggregates via Structured Illumination Microscopy. Moreover, we tracked the long non-coding RNA (lncRNA) nHOTAIRM1, a motor neurons-specific constituent of stress granules (SG), monitoring its behavior throughout the oxidative-stress response in physiological and pathological conditions. Overall, as fRNAs development advances, our work shows a successful use of Pepper for the monitoring of complex processes, as phase-transition, of paradigmatic molecules like circular RNAs (circRNAs) and lncRNAs with super-resolution potential in living mammalian cells.