Shell protein composition specified by the lncRNA NEAT1 domains dictates the formation of paraspeckles as distinct membraneless organelles.

Many membraneless organelles (MLOs) formed through phase separation play crucial roles in various cellular processes. Although these MLOs co-exist in cells, how they maintain their independence without coalescence or engulfment remains largely unknown. Here, we investigated the molecular mechanism by which paraspeckles with core-shell architecture scaffolded by NEAT1_2 long noncoding RNAs exist as distinct MLOs. We identified NEAT1 deletion mutants that assemble paraspeckles that are incorporated into nuclear speckles. Several paraspeckle proteins, including SFPQ, HNRNPF and BRG1, prevent this incorporation and thus contribute to the segregation of paraspeckles from nuclear speckles. Shell localization of these proteins in the paraspeckles, which is determined by NEAT1_2 long noncoding RNA domains, is required for this segregation process. Conversely, U2-related spliceosomal proteins are involved in internalizing the paraspeckles into nuclear speckles. This study shows that the paraspeckle shell composition dictates the independence of MLOs in the nucleus, providing insights into the importance of the shell in defining features and functions of MLOs.

Paraspeckles are constructed as block copolymer micelles.

Paraspeckles are constructed by NEAT1_2 architectural long noncoding RNAs. Their characteristic cylindrical shapes, with highly ordered internal organization, distinguish them from typical liquid-liquid phase-separated condensates. We experimentally and theoretically investigated how the shape and organization of paraspeckles are determined. We identified the NEAT1_2 RNA domains responsible for shell localization of the NEAT1_2 ends, which determine the characteristic internal organization. Using the soft matter physics, we then applied a theoretical framework to understand the principles that determine NEAT1_2 organization as well as shape, number, and size of paraspeckles. By treating paraspeckles as amphipathic block copolymer micelles, we could explain and predict the experimentally observed behaviors of paraspeckles upon NEAT1_2 domain deletions or transcriptional modulation. Thus, we propose that paraspeckles are block copolymer micelles assembled through a type of microphase separation, micellization. This work provides an experiment-based theoretical framework for the concept that ribonucleoprotein complexes (RNPs) can act as block copolymers to form RNA-scaffolding biomolecular condensates with optimal sizes and structures in cells.