Regulation of the Inflammasome by Gasdermin D mRNA Chimerism - ABSTRACT Appreciation for the complexity of RNA processing is essential for understanding regulatory mechanisms of innate immunity. We uncovered novel splice variants that occur through the fusion of two distinct RNA transcripts (chimeric mRNA) in murine macrophages, including a fusion transcript containing Gsdmd and Tmem106a (Gsdmd:Tmem106a), two genes situated on separate chromosomes. Following inflammasome assembly, triggered by microbial signals and cellular damage, gasdermin D (GSDMD) is cleaved into its active form by inflammatory caspases. GSDMD N-terminal (GSDMD-NT) fragments oligomerize and create pores in the plasma membrane allowing for the release of IL-1β. This often triggers inflammatory cell death but occasionally allows for longer lasting states of pro-inflammatory cytokine release. GSDMD is essential for host defense against a variety of bacterial pathogens but excessive inflammasome activation and GSDMD pore formation is linked to autoinflammatory diseases, sepsis, and cytokine release syndrome. Regulatory mechanisms of pore formation, acting downstream of GSDMD cleavage, are a topic of intensive investigation. To functionally assess the role of Gsdmd:Tmem106a downstream of inflammasome activation, we selectively knocked-down this chimeric mRNA using RNA interference and discovered that it is required for optimal IL-1β release. Based on our preliminary results, this proposal will test the hypothesis that Gsdmd:Tmem106a facilitates GSDMD pore formation and contributes to physiological inflammation. To robustly investigate the function of this chimeric mRNA and protein, we generated two mouse models: endogenous GSDMD:TMEM106A protein tag mice (GTMyc) and GSDMD:TMEM106A-deficient mice (GTstop). Aim 1 of this proposal will mechanistically interrogate Gsdmd:Tmem106a function by first characterizing chimeric protein localization and interacting partners during inflammasome activation using GTMyc mice (Aim 1.1) and secondly, by assessing GSDMD-NT oligomerization and pore formation in GTstop bone marrow derived macrophages, as well as by using in vitro systems of GSDMD- NT pore formation that function independently of the upstream inflammasome (Aim 1.2). As Gsdmd:Tmem106a encodes a novel protein with a truncated GSDMD-NT, we hypothesize that it promotes native GSDMD pore formation through direct interaction. Aim 2 of this proposal will evaluate the in vivo expression landscape of Gsdmd:Tmem106a following systemic LPS exposure (Aim 2.1), as well as use GTstop mice to assess its physiological role in LPS sepsis induced inflammation and death (Aim 2.2). We hypothesize that Gsdmd:Tmem106a will be robustly expressed following LPS treatment, and that this chimeric mRNA plays a role in exacerbating inflammation and LPS sepsis lethality, as native GSDMD has been reported to. Taken together, this work will uncover a novel mechanism of GSDMD pore formation, and demonstrate that chimeric mRNA are present and functional during inflammation, highlighting an entirely new class of mRNA for further exploration in the immune system.
