Regulation of Pumilio RNA Binding Proteins by Long Noncoding RNA NORAD

The mammalian genome is extensively transcribed and encodes thousands of long noncoding RNAs (lncRNAs). Defining the mechanism of action of lncRNAs has been a critical challenge and priority for understanding their biological functions. An important example of this is the lncRNA NORAD, which is a highly conserved lncRNA that is required for maintaining genome stability in mammals. Work in this thesis begins by examining the physiologic function and molecular mechanism of NORAD in human cells. We clearly demonstrate that NORAD localizes predominantly to the cytoplasm where it functions by binding to and inhibiting PUMILIO (PUM1 and PUM2) RNA binding proteins (RBPs). Thorough dissection of the functional domains of NORAD establish the essentiality of PUM binding to NORAD for its function in maintaining genome stability. We further examine the mechanism by which NORAD is able to efficiently sequester and regulate PUM proteins. Through a multidisciplinary approach involving biochemical, microscopy, and mutational analysis experiments, we uncover how NORAD sequesters a super-stoichiometric amount of PUM proteins into novel liquid-like membraneless organelles through a multivalency-induced phase separation mechanism. Moreover, we provide a molecular understanding of the properties of NORAD that promote PUM phase separation, revealing new principles of lncRNA function and demonstrating the physiologic importance of RNA-driven phase separation as a regulatory mechanism in mammalian biology. Our findings, together with the widespread repetitive architecture of lncRNAs, suggest that the phase separation principles we establish may be broadly utilized for lncRNA-mediated regulation.