NORAD-induced phase separation of PUMILIO proteins is required for genome maintenance

Long noncoding RNAs (lncRNAs) can regulate the activity of interacting RNA binding proteins (RBPs), but how lncRNAs efficiently compete against other transcripts to bind and regulate RBP activity is poorly understood. This problem is exemplified by NORAD, a lncRNA that maintains genomic stability by inhibiting PUMILIO (PUM) proteins. Here we show that NORAD is able to out-compete other PUM-binding transcripts 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 sequester a super-stoichiometric amount of PUM in these condensates. Accordingly, synthetic RNAs that induce PUM phase separation rescue genome instability in NORAD-deficient cells. These results illuminate mechanisms of RBP regulation by RNA-driven phase separation and uncover an essential role for this process in genome maintenance. Total RNA was isolated from biological triplicates of each cell line using the miRNeasy Mini RNA isolation kit (QIAGEN). Library preparation and next generation sequencing were performed by DNA Link using NEB Next Small RNA Library Prep for Illumina (New England BioLabs).