Functional Domains of NEAT1 Architectural lncRNA Induce Paraspeckle Assembly through Phase Separation

Abstract

A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction; however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation.

Membraneless nuclear organelles and the search for phases within phases

“…Both the electron microscopy and SRM data show that NEAT1 folds in half, with the 5′ and 3′ ends positioned in the outer shell, and the middle of the RNA in the inner core, which correlates with the self‐interaction data summarized above (Lin et al, ; Souquere et al, ; West et al, ). This correlates well with recent research that indicates the NEAT1 lncRNA middle domain is essential for NONO and SFPQ recruitment, and phase separation (Yamazaki et al, ). These data have also recently been aligned with NEAT1's role in pri‐microRNA processing, known as the bird's nest model, which situates the multi‐subunit microRNA biogenesis factory known as Microprocessor at the exterior of the paraspeckle (Jiang et al, ; Krol, ).…”

“…Protein complex coacervation is assessed in live cells by microscopic methods (i.e., immunofluorescence, live‐cell imaging), where the formation of large spherical foci and their ability to fuse or split is recorded (Sleeman et al, ). Aliphatic compounds that interfere with weak intermolecular interactions, such as 1,6‐hexanediol, are also used to confirm that a self‐organizing cellular droplet organelle is in a liquid‐like state (Krocschwalk & Alberti, ; Lin et al, ; Lu et al, ; Molliex et al, ; Wheeler, Matheny, Jain, Abrisch, & Parker, ; Yamazaki et al, ). However, many studies opt to investigate protein LLPS in vitro to tightly control experimental conditions.…”

RNAs, Phase Separation, and Membrane‐Less Organelles: Are Post‐Transcriptional Modifications Modulating Organelle Dynamics?

“…Indeed, particular protein domains that bind nucleic acids recognized structure over sequence identity . For example, particular conserved as well as repetitive sequences in the lncRNA NEAT1 were shown to be essential for protein binding resulting in nuclear paraspeckle formation . Moreover, structural changes in specific mRNAs can influence BioMC identity.…”

Biomolecular condensates in neurodegeneration and cancer

“…Accordingly, RNA promotes or represses the phase separation of RNA‐binding proteins (depending on its concentration) and modulates the material properties of their condensates . RNA splicing, secondary structure and modifications have been shown to modulate cellular condensate assembly and composition . Similarly, various protein modifications have been shown to modulate condensate assembly (reviewed in Reference ), composition (reviewed in Reference ) and material properties .…”