PROJECT SUMMARY Cell fate transitions prompt widespread changes in gene expression programs that require multiple levels of regulation to specify and ultimately restrict cell identity. Post-transcriptional mechanisms are fundamental regulators of gene expression and have recently emerged as critical to establishing and maintaining cell identity. However, the role of post-transcriptional mechanisms in development and tissue homeostasis remains poorly understood. The long-term goal of my research program is to define the role of post-transcriptional regulation in mammalian cell fate decisions in order to develop new therapeutic strategies for regenerative medicine and cancer. The objective of this proposal is to characterize the function of post-transcriptional mechanisms in embryogenesis and adult tissue homeostasis. Our central hypothesis is that the sequestration of RNAs in cytoplasmic condensates has crucial roles during developmental transitions and is important to maintain cellular identity and tissue homeostasis. Although cytoplasmic RNA condensates are thought to play important roles in various RNA-related processes, their molecular function and role in mammalian cell fate transitions are poorly understood. In the first Project, we will elucidate how RNA sequestration instructs embryonic cell fate transitions. We will track early lineage decisions following perturbation of RNA sequestration in early embryos. Moreover, we will define the first landscape of RNA condensates in embryonic stem cells as well as endoderm, ectoderm, and mesoderm progenitors. We then will assess how transcript isoforms and RNA methylation influence compartment-specific regulation of RNAs in pluripotent stem cells. In the second Project, we will determine the role of post-transcriptional mechanisms during tissue homeostasis. Specifically, we will explore the cellular, molecular, and functional role of RNA sequestration in hematopoiesis. Further, we will utilize several innovative tools and cutting-edge technologies, including profiling of the RNA content of cytoplasmic condensates and translatome mapping in hematopoietic stem cells, to dissect the mechanisms by which RNA sequestration controls hematopoietic cell identity. Collectively, this work will reveal how RNA processing drives key aspects of lineage-specific mammalian cell fate trajectories. Uncovering and understanding novel regulatory mechanisms that control cell fate decisions is a crucial step to develop approaches for regenerative medicine and cancer. We, therefore, anticipate that the proposed work will have important implications for both basic science and translational medicine.