7 research outputs found

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

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    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

    Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles

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    Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule assembly. In this paper, we report many PLDs in proteins associated with paraspeckles, subnuclear bodies that form around long noncoding RNA. We mapped the interactome network of paraspeckle proteins, finding enrichment of PLDs. We show that one protein, RBM14, connects key paraspeckle subcomplexes via interactions mediated by its PLD. We further show that the RBM14 PLD, as well as the PLD of another essential paraspeckle protein, FUS, is required to rescue paraspeckle formation in cells in which their endogenous counterpart has been knocked down. Similar to FUS, the RBM14 PLD also forms hydrogels with amyloid-like properties. These results suggest a role for PLD-mediated liquid-phase transitions in paraspeckle formation, highlighting this nuclear body as an excellent model system for understanding the perturbation of such processes in neurodegeneration

    Smchd1 Targeting to the Inactive X Is Dependent on the Xist-HnrnpK-PRC1 Pathway

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    Summary: We and others have recently reported that the SMC protein Smchd1 is a regulator of chromosome conformation. Smchd1 is critical for the structure of the inactive X chromosome and at autosomal targets such as the Hox genes. However, it is unknown how Smchd1 is recruited to these sites. Here, we report that Smchd1 localizes to the inactive X via the Xist-HnrnpK-PRC1 (polycomb repressive complex 1) pathway. Contrary to previous reports, Smchd1 does not bind Xist or other RNA molecules with any specificity. Rather, the localization of Smchd1 to the inactive X is H2AK119ub dependent. Following perturbation of this interaction, Smchd1 is destabilized, which has consequences for gene silencing genome-wide. Our work adds Smchd1 to the PRC1 silencing pathway for X chromosome inactivation. : Jansz et al. report that the chromatin protein Smchd1 depends on polycomb repressive complex 1-mediated ubiquitylation of histone H2A for its recruitment to the inactive X chromosome and for its protein stability. These data have implications for Smchd1 targeting genome-wide. Keywords: Smchd1, X inactivation, Xist, PRC1, Hnrnpk, Ring1
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