PROJECT SUMMARY There is increasing appreciation for the role of RNA binding proteins (RBPs) in shaping the transcriptome of specialized cell types and in helping cells respond to stress. Macrophages are innate immune cells that mount rapid and robust gene expression programs following detection of pathogen- and damage-associated molecular patterns (PAMPs and DAMPs). We know that this response is subject to post-transcriptional regulation at the levels of alternative splicing, alternative polyadenylation, RNA editing, etc. We also know that many of the RBPs that control post-transcriptional regulation of gene expression in macrophages are differentially phosphorylated downstream of PAMP and DAMP sensing. We hypothesize that post-translational modification of RBPs functionalizes them to coordinate the macrophage innate immune response. Tight regulation is essential for a functional immune response: a weak response may be insufficient to fight infection whereas a strong response risks cytokine storms, autoimmunity, and chronic inflammation. The role of RBPs in post-transcriptional regulation of innate immune gene expression remains an understudied feature of this important aspect of human health and disease. In the next five years, my lab will work to implicate new RBPs in the macrophage innate immune response and to define new paradigms through which macrophages reorganize their nuclei to activate innate gene expression. We have identified a cohort of RBPs in the SR/hnRNP families of splicing regulatory factors that are differentially phosphorylated in macrophages in response to PAMP/DAMP sensing. In the next funding period, we will work to uncover the molecular mechanisms through which each of these factors (hnRNP C, hnRNP F, RALY, and U2SURP) influence macrophage activation. We will study two complexes with the capacity to regulate many macrophage RBPs at once: the biomolecular condensate/suborganelle known as the nuclear paraspeckle and the nuclear RNA exosome. Our preliminary data demonstrate that the paraspeckle is dynamically up- and down-regulated over the course of early macrophage activation, can sequester specific RBPs in response to PAMP sensing, and is required for proper amplification of key inflammatory transcripts including Il6, Cxcl1, and Cxcl9. We found that the RNA exosome can control paraspeckle dynamics and is required to maintain normal homeostatic levels of antiviral transcripts in macrophages. Pursuing these lines of investigation will provide fundamental insights into how specific RBPs and ribonucleoprotein complexes control the ability of macrophages to sense and response to pathogens. By furthering our understanding of cellular stress responses and identifying novel nodes that control of innate immune gene expression outcomes, our work may contribute to efforts to develop therapeutics to help patients battling immune disorders and infection.