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