Gas-Liquid Phase Transitions at a Subcellular Level – Molecular Mechanisms By Single Molecule FRET

In germ cell development, ribonucleoprotein (RNP) complex termed p-body plays a critical role in mRNA storage, splicing, degradation and translation repression. Many proteins within p-body contain RNA binding domains and low complexity (LC) sequences of unknown function. We employed single molecule fluorescence to characterize LAF1 helicase of C. elegans as a model system to investigate p-body assembly process. LAF-1 is a DEAD box helicase which possess N-terminal RGG box and C-terminal poly-glutamine tract and a helicase core. Our results reveal that LAF-1 specifically binds single strand (ss) RNA and induces unexpected compaction and dynamics of the RNA strand. LAF-1 displays no unwinding of double stranded RNA, yet it promotes annealing of complementary ssRNA. Series of truncation mutants reveal that the N-terminal RGG box is responsible for providing the dynamic interaction and RNA annealing whereas other domains contribute to the compaction of RNA and oligmerization. Native gel analysis indicates that LAF-1 and the mutants form higher order structure, reflecting their inherent propensity to oligomerize. Together, we unravel the molecular mechanism how LAF-1 may contribute to gas-liquid transition-like behavior of p-granule.