Live-cell imaging of non-coding RNAs dynamics in ALS condensates

In the last few years, interest around non-coding RNAs (ncRNAs) has been growing as they have been found to be involved in several physiological and pathological processes. In fact, their expression is highly enriched in neuronal tissues and, thanks to their complex and modular secondary structure, they can work as scaffold for other RNAs and proteins for the assembly of ribonucleoparticles (RNPs). These supramolecular structures are known to participate in axonal trafficking, a process usually impaired in neurodegenerative diseases such as Amyotrophic Lateral Sclerosis and in particular in the presence of mutations of several RNA binding proteins, among which FUS. In this context, it is crucial to investigate ncRNA dynamics and kinetics in live cells, in order to unveil novel mechanisms for the understanding of neurodegeneration. For this purpose, we managed to engineer the motor neuron enriched circRNA circ-Hdgfrp3 and the lncRNA HOTAIRM1 with an array of Pepper, a novel fluorescent aptamer that shows enhanced stability and brightness if compared with previously described fluorescent RNAs, allowing robust RNA imaging with minimal target perturbation. Combining widefield and structured illumination microscopy, we were able to confirm in live mammalian cells that circ-Hdgfrp3 is loaded in G3BP1 and FUSmut RNPs, possibly determining the mechanism through which it is recruited in pathological aggregates in motor neurons. Moreover, we also observed its interaction with DCP1A-tagged processing bodies, raising promising insight about its function and metabolism. Notably, we also determined HOTAIRM1 constitutive participation in stress granules, while we observed its involvement in the dynamics of FUSmut aggregation. Moreover, we were able to follow its behavior throughout the induction of oxidative stress, an event that leads to the production of aggregates containing several RNA binding proteins, including FUSmut. Via live imaging assays, we determined that its recruitment in stress granules is not mediated neither by G3BP1 nor by FUS, consistent with the observation that it does not interfere with stress granules assembly and that it preferentially joins the outer layers of such structures. Overall, as fluorescent RNA technologies are rapidly spreading and are improved for live-imaging applications, our work provides a novel approach for the investigation of ncRNAs’ implication in neurodegenerative diseases with a super-resolution potential in live neuronal cells.