BPS2026 – Insoluble proteins compete for RNA binding to assemble multilayered condensates

Biomolecular condensates are assemblies of proteins and RNAs that mediate essential cellular processes. The biological functions of many condensates depend on their layered internal architectures, but the biophysical principles controlling internal layering are largely unknown. We sought to uncover these principles by studying nuclear paraspeckles, which regulate post-transcriptional gene expression. Paraspeckles are composed of several RNA-binding proteins arranged in distinct core and shell layers, with the proteins FUS and NONO in the core and TDP-43 in the shell. This layering is orchestrated by a long noncoding RNA scaffold called NEAT1. A previous model suggested that FUS and NONO directly bind to specific segments of NEAT1 in the core to assemble paraspeckle layers, but direct biochemical evidence for this model is missing. Using cell-free reconstitution, we discovered that FUS and NONO prefer to bind NEAT1 segments that correspond to the shell rather than the core. This preference is due to an enrichment of RNA binding motifs for these proteins within NEAT1 shell segments. However, the shell protein TDP-43 also binds to abundant motifs in NEAT1 shell segments, leading to a competition between core and shell proteins for NEAT1 binding. Notably, TDP-43 is also insoluble with FUS and NONO, causing the proteins to spontaneously separate from each other into distinct layers. Using physics-based modeling, we found that this combination of competitive RNA binding and mutual protein insolubility redirects FUS and NONO from their preferred binding sites in the shell to suboptimal sites in the core. This redistribution recapitulates the natural layered organization observed in cells. Our study reveals that NEAT1 encodes a binding logic that guides the spatial arrangement of insoluble proteins. We propose that other types of RNA scaffolds may operate similarly to build layered condensates with varied cellular functions.