PROJECT SUMMARY To ensure a robust immune response to pathogens without risking immunopathology, the kinetics and amplitude of inflammatory gene expression in macrophages need to be exquisitely well-controlled. To mount balanced inflammatory responses, macrophages need to be able to coordinate the transcription and processing of thousands of genes that are synthesized de novo following pathogen sensing. Yet, we have an incomplete understanding of the molecular mechanisms employed by macrophages to properly regulate the timing and amplitude of inflammatory gene expression. Key to organization and compartmentalization of the nucleus are biomolecular condensates. Condensates, which include structures like the nucleolus, Cajal body, and nuclear speckle, are known to concentrate functionally related nucleic acids and proteins. We have become interested in one such biomolecular condensate called the nuclear paraspeckle, which forms on a long lncRNA called Neat1. Studies of model cell types (e.g. HeLa and HEK293T) have shown that paraspeckles can aggregate in response to a variety of cellular stresses (e.g. osmotic stress, hypoxia, sodium arsenite), but how paraspeckle dynamics change in response to more physiological stresses—and how they may help regulate the cell’s response to stress—is not well-understood. My project aims to define the role of the paraspeckle in macrophage activation. Our lab recently discovered that macrophage paraspeckles are dynamically regulated over an early time-course of macrophage activation via lipopolysaccharide (LPS). We also found that in response to LPS treatment, Neat1 KO macrophages fail to properly express a large cohort of proinflammatory cytokines, chemokines, and antimicrobial mediators and consequently, cannot control replication of Salmonella enterica or vesicular stomatitis virus (VSV). I hypothesize that macrophage paraspeckles regulate innate immune gene expression by dynamically sequestering and/or releasing nuclear proteins/RN