Biomolecular condensates are cellular compartments formed through liquid-liquid phase separation of macromolecules, typically RNA-binding proteins and nucleic acids. The eukaryotic RNA-binding protein Pumilio1 (PUM1) is a post-transcriptional repressor of gene expression that exhibits efficient phase separation in the presence of NORAD, an abundant, highly conserved mammalian long noncoding RNA. To negatively regulate PUM activity, NORAD serves as a multivalent binding platform on which PUM proteins assemble, enabling additional PUM-PUM interactions via intrinsically disordered regions. This promotes sequestration of PUM proteins within cytoplasmic phase-separated condensates termed NORAD-Pumilio (NP) bodies. The mechanisms through which NORAD-induced Pumilio phase separation is dynamically regulated are still unknown. Post-translational modifications of RNA-binding proteins, such as phosphorylation, are important regulators of biomolecular condensate dynamics. We have made preliminary observations that NP bodies rapidly disperse during mitosis and reassemble at the start of G1, and I have recently identified by mass spectrometry putative PUM1 phosphorylation sites exclusively detected in mitotic cells. These observations lead us to hypothesize that PUM1 phosphorylation rapidly regulates NP body dynamics. In Aim 1, I will quantify our preliminary qualitative observation of NP body dispersal during mitosis by adapting a fluorescent cell cycle marker to accurately capture cell entry and exit from mitosis during live-cell imaging. Additionally, I have previously generated a quantitative image analysis pipeline that I will adapt to measure condensate size, number, and localization over the course of the cell cycle. Based on my mass spectrometry results, in Aim 2, I will simultaneously mutate nine phosphorylated residues to generate recombinant phosphomimetic and phospho- dead PUM1 variants and study their behavior in isolation through in vitro phase separation assays. To determine the key sites regulating NP body dynamics, I will generate additional PUM1 variants by systematically mutating fewer sites. Each of the phospho-mutants will be expressed in HCT116 cells, and through live-cell imaging using confocal microscopy, I will determine whether perturbation of the phosphorylation sites impacts NP body dynamics in unsynchronized cells and over the course of the cell cycle. Lastly, in Aim 3, I will identify the kinase responsible for PUM1 phosphorylation by knocking out candidate kinases in HCT116 cells using CRISPR/Cas9 and probing for measurable impacts on PUM1 phosphorylation and NP body dynamics during the cell cycle. Through the proposed work, I hope to uncover a paradigm for rapid regulation of biomolecular condensate dynamics that can be broadly applied to other physiologically relevant systems, including pathological protein aggregation in disease.