NORAD-induced Pumilio phase separation is required for genome stability

Liquid-liquid phase separation is a major mechanism of subcellular compartmentalization(1,2). Although the segregation of RNA into phase-separated condensates broadly affects RNA metabolism(3,4), whether and how specific RNAs use phase separation to regulate interacting factors such as RNA-binding proteins (RBPs), and the phenotypic consequences of such regulatory interactions, are poorly understood. Here we show that RNA-driven phase separation is a key mechanism through which a long noncoding RNA (lncRNA) controls the activity of RBPs and maintains genomic stability in mammalian cells. The lncRNA NORAD prevents aberrant mitosis by inhibiting Pumilio (PUM) proteins(5-8). We show that NORAD can out-compete thousands of other PUM-binding transcripts to inhibit PUM by nucleating the formation of phase-separated PUM condensates, termed NP bodies. Dual mechanisms of PUM recruitment, involving multivalent PUM-NORAD and PUM-PUM interactions, enable NORAD to competitively sequester a super-stoichiometric amount of PUM in NP bodies. Disruption of NORAD-driven PUM phase separation leads to PUM hyperactivity and genome instability that is rescued by synthetic RNAs that induce the formation of PUM condensates. These results reveal a mechanism by which RNA-driven phase separation can regulate RBP activity and identify an essential role for this process in genome maintenance. The repetitive sequence architecture of NORAD and other lncRNAs(9-11) suggests that phase separation may be a widely used mechanism of lncRNA-mediated regulation.

Automated summary

Liquid-liquid phase separation is a key mechanism through which long noncoding RNA controls RNA-binding proteins and maintains genomic stability in mammalian cells. The study shows that RNA-driven phase separation competitively sequesters RNA-binding proteins by nucleating the formation of phase-separated condensates, influencing RBP activity and genome maintenance. The repetitive sequence architecture of lncRNAs suggests that phase separation may be a widely used mechanism of lncRNA-mediated regulation.