Paraspeckles are stress-induced subnuclear bodies that form around a long noncoding RNA (lncRNA) NEAT1. Once formed, paraspeckles affect gene expression, at least in part, by the sub-nuclear sequestration of specific transcription factors. But what are the molecular mechanisms driving proteins into paraspeckles?

NEAT1 ‘seeds’ paraspeckle formation by acting as a scaffold for specific proteins to bind and then use protein-protein interactions to build up a micron-scale RNP particle. To reveal the network of protein-protein interactions in paraspeckles we carried out a combinatorial yeast-two-hybrid screen of the forty known paraspeckle proteins. Surprisingly, only a few important hub proteins seem to link the paraspeckle interactome together. Accordingly, the main hub proteins, HNRNPK, FUS and RBM14, are also essential for paraspeckle formation: knocking them down results in loss of paraspeckles. Interestingly, the hub proteins are also enriched in low complexity prion-like domains.

We have focused our downstream analysis on one key hub protein, RBM14 (RNA Binding Motif Protein 14; also known as CoAA/PSP2/SIP). Crucially, RBM14 is the only protein linking the essential DBHS proteins (NONO and SFPQ) to the rest of the network. We found that the RBM14 prion-like domain is responsible for mediating the interaction with DBHS proteins and is also required for targeting RBM14 to paraspeckles. As with another hub protein, FUS, the prion-like domain of RBM14 forms a hydrogel when expressed in vitro. Mutations in critical tyrosines in the FUS and RBM14 prion-like domains disrupt both hydrogel formation and paraspeckle targeting by these proteins. Finally, we show that paraspeckle formation is dependent on an intact RBM14 prion-like domain. Together these data indicate that specific prion-like domains in RNA binding proteins are intimately involved in the formation of paraspeckles.