RNA-PROTEIN CONDENSATION PATTERNS THE CYTOSOLIC LANDSCAPE OF A SYNCYTIUM

To function properly and survive in differing environmental conditions, every cell must organize their cytoplasm in one form or another. This requirement is increasingly necessary in large, multinucleated cells. Traditionally, this has been thought to be mainly driven by membrane-bound compartments (i.e. organelles) that help eukaryotic life organize into distinct biochemical spaces. More recently, it’s become apparent that the continuous cytosol is also organized into distinct compartments, through entirely separate means. In the multinucleated Drosophila embryo, approximately 70% of mRNA transcripts were determined to be heterogeneously localized across the cell, and mRNA spatial organization in the multinucleate fungus Ashbya gossypii has implicated phase separating RNA binding proteins (RBPs), where transcript heterogeneity is critical for autonomous nuclear division and polarized growth in these syncytial cells. The ability of RNAs to condense into droplets is in many instances contributing to previously appreciated mRNA localization phenomena. Phase separation enables mRNAs to selectively and efficiently co-localize and be co-regulated allowing control of gene expression in time and space. The work presented here demonstrates that mRNA sequence not only drives the localized condensation of RNA-protein droplets in A. gossypii, but also, governs the identity of these specialized RNA granules. Work in this thesis provides evidence that the RNA binding protein, Whi3, exhibits differential phase-separation behavior depending on which RNA target it binds and that this differential behavior is specified by features within the mRNA sequence. In addition, this work investigates the possibility of an auto feedback mechanism by which Whi3 phase separates with its own mRNA to drive differential crowding within the cytosol to promote droplet condensation in crowded cytosolic regions, thus creating individual territories of cytosol within a common syncytial cytoplasm. These data suggest mechanisms by which cells can employ asymmetric RNA localization, specifically the localization of RNAs via phase-separating RNA binding proteins, to generate functionally distinct domains to achieve efficient, and timely cytosolic organization.Doctor of Philosoph