Defining the role of lncRNA Neat1 and the nuclear paraspeckle in regulating macrophage innate immune gene expresison

About This Grant

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/RNA binding proteins, thus impacting their concentration/bioavailability for participation in innate immune gene expression. Here, I will investigate how different stimuli impact paraspeckle dynamics in macrophages, how paraspeckle composition changes in response to LPS, and how differential sequestration of nuclear proteins in paraspeckles controls innate immune gene expression. In Aim 1, I will define the dynamics of macrophage paraspeckles in response to various cellular stresses, carrying out a high-throughput screen using macrophage cell lines that express GFP-tagged paraspeckles. In Aim 2, I will apply a cutting-edge biotin proximity labeling approach called O- MAP to compositionally define the paraspeckle over a time course of LPS and follow up on how novel paraspeckle-associated proteins contribute to macrophage antimicrobial responses. Together, these experiments will provide important insights into how cellular stresses are transmitted to the nucleus to control paraspeckle dynamics and how reorganization of proteins in and out of paraspeckles controls innate immune gene expression in macrophages.