The role of RNA in the nanoscale organization of alpha-synuclein phase separation

The accumulation of alpha-synuclein (aS) protein aggregates in cells is a typical hallmark of several neurodegenerative diseases. Recent studies have shown that the aberrant transition of monomeric aS into solid-like aggregates can occur via an intermediate liquid-like state, where the protein partitions between dense and dilute phases. Although aS is not typically recognized as an RNA-binding protein, it can bind RNA under aggregation conditions. However, the impact of RNA on aS dynamics within dilute and dense phases remains unclear. Here, we employ a combination of fluorescence spectroscopy techniques to explore aS dynamics in both phases in the presence of RNA. Our sensitive analysis of the dilute phase revealed the formation of precursor nanoclusters involved in initiating phase separation. We also uncovered heterogeneity within the dense phase, discovering that aS molecules exist in two distinct mobility states: freely diffusing molecules or confined within micro-domains. Additionally, RNA was found to induce morphological changes in the dense phase. Our quantitative analysis, based on fluorescence lifetime measurements, showed that RNA alters the liquid-like properties of the aS dense phase, promoting a liquid-to-solid transition. These findings indicate the active role of RNA in modulating the material properties of aS and influencing its phase transitions.