Ehrlichia modulation of RNA splicing to influence KIRREL1-regulated Hippo signaling - ABSTRACT Ehrlichia chaffeensis is a Gram-negative, obligately intracellular bacterium and the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging, life-threatening, tick-borne zoonosis. E. chaffeensis. preferentially infects mononuclear phagocytes and survives intracellularly by subverting innate immune defenses mediated in part by tandem repeat protein (TRP) effectors. Within the last decade, our laboratory has identified a multitude of molecular ehrlichial TRP-host interactions, many that are new to science, illuminating the breath and complexity of pathogen-host interaction dynamics that occur during infection. Notably, we have investigated TRP effector interactions with host cell posttranslational modification (PTM) machinery, transcription factors and epigenetic modifiers and immune and developmental signaling pathways. We have shown that TRP120 directly interacts with multiple eukaryotic proteins involved in transcription and post transcriptional regulation, including hnRNPA2B1, an RNA splicing factor. Understanding the molecular strategies E. chaffeensis has evolved to manipulate RNA splicing factors such as hnRNPA2B1 to modulate genes associated with regulation of cellular signaling and other responses linked to innate immunity is an unexplored gap in our knowledge. The long-term goal of this project is to understand post transcriptional regulatory mechanisms exploited by E. chaffeensis to modulate macrophage innate defenses. The objective of this proposal is to investigate RNA splicing factor hnRNPA2B1 as a substrate of TRP120 HECT E3 Ub ligase activity and determine the effects of pre-mRNA AS of KRILLEL1 on the Hippo/YAP pathway activity during infection. We hypothesize that E. chaffeensis TRP120 ubiquitinates nuclear hnRNPA2B1 for degradation to modulate KIRREL1 pre-mRNA AS to maintain YAP activation and to promote infection. This research will promote the rational development of innovative and mechanistically targeted therapeutics to combat intracellular pathogens that manipulate RNA splicing for infection. Our hypothesis will be examined with the following specific aims: Aim 1 will investigate hnRNPA2B1 as a substrate of E. chaffeensis TRP120 HECT E3 Ub ligase. Aim 2 will investigate functional role of hnRNPA2B1 in regulating mRNA stability by AS during infection; and Aim 3 will investigate pre-mRNA AS of KIRREL1 as a mechanism to regulate Hippo/YAP signaling. The significance of this research is also based on defining mechanistic strategies, whereby intracellular pathogens with small genomes and a limited number of effector proteins target host RNA regulatory proteins to manipulate host innate defenses for infection through regulation of evolutionarily conserved signaling pathways. In this investigation, we will begin to define the molecular and cellular mechanisms E. chaffeensis has evolved to modulate cell signaling and host innate defenses regulated by RNA splicing factors.
