RNA structure controls assembly of multi-layered paraspeckles.

Abstract

Biomolecular condensates mediate key cellular functions from gene expression to signaling. Condensates assemble via multivalent interactions amongst proteins and nucleic acids and often behave as liquid-like droplets. An essential requirement for condensate function is the establishment of a distinct compositional identity. How cells maintain coexisting populations of different condensates, despite the propensity of liquids to fuse, is poorly understood. Multi-layered condensates, comprising layers of distinct biomolecular composition, are useful for understanding the establishment of molecular identity and the opposition to mixing. We set out to uncover molecular cues that encode multi-layered condensates by studying nuclear paraspeckles, composed of a core and shell. Paraspeckles are scaffolded by a long noncoding RNA called NEAT1 that bridges the core and shell and recruits different proteins to each layer. The molecular features of NEAT1 that encode for each layer are unknown. We found that isolated core or shell fragments of NEAT1 can assemble condensates with the paraspeckle core-associated protein FUS. When both RNAs are present, condensates do not fuse but instead form a core and shell reminiscent of paraspeckles. Quantitative imaging showed that condensates formed with shell RNA display a higher protein/RNA ratio compared to core RNA. Probing RNA structure uncovered that shell RNA was more well-structured compared to core RNA, suggesting that RNA structure contributes to differential protein recruitment to paraspeckle layers. Consistent with this idea, disrupting the native structures of each RNA via melting and rapid cooling drove dramatic changes in protein and RNA recruitment. Thus, the structural features along an RNA strand likely control the spatial arrangement of the RNA within paraspeckles and tune protein recruitment to each layer. More broadly, our findings suggest that RNA structure is a powerful control knob that determines condensate identity and function in diverse contexts.