Innate immune roles of glycoRNA

PROJECT SUMMARY/ABSTRACT GlycoRNAs are newly discovered biopolymers that are comprised of small non-coding RNAs and a sialic- acid containing N-glycan. These RNAs decorate the mammalian cell surface and exhibit double-stranded regions that are capable of detection by anti-dsRNA antibodies. Currently, there are no known functions linked to

glycoRNAs. However, diverse pathogens employ glycosidases to breakdown host cell surface glycans to enable efficient intracellular invasion. Further, intracellular infection results in dramatic transcriptional changes and increased deployment of glycosylated molecules on the cell surface to aid in host defense. To this point, we

hypothesize that the abundance and diversity of glycoRNA is altered upon intracellular infection. Our preliminary data show that infection of HeLa or THP-1 cells via Salmonella Typhimurium (STm) or Herpes Simplex Virus-1 (HSV-1) results in a dramatic increase in cell surface dsRNA, suggesting a change in glycoRNA

abundance. Building on this data, we aim to investigate this phenomenon both in vitro and in vivo using a sepsis model. Here, we will use click-chemistry to isolate glycoRNA from splenocytes of C57BL/6J and Toll-like receptor 4 knockout mice injected with lipopolysaccharide or STm. This will allow us to examine how pathogens influence

the expression of glycoRNA at the physiological level. Aim 1 will explore and characterize these changes through flow cytometry and RNA sequencing by employing click-chemistry isolation of glycoRNA. In immunology, the “guard hypothesis” postulates that host-derived sentinel molecules can monitor

(guard) for pathogen invasion by being targeted by virulence factors. Damage or perturbations to these guarded molecules then activate potent immune responses. We hypothesis that glycoRNAs may function in part as guard molecules. We found that treating total small RNA fractions from HeLa cells with the N-glycan-specific

glycosidase PNGase F resulted in greater innate immune activation than untreated (glycosylated) RNA. This effect was abrogated by treatment of this RNA fraction with RNase, suggesting that glycoRNAs are endogenous RNAs guarded by their associated glycan which prevents recognition as immunostimulatory self-RNA at steady

state. To further characterize this mechanism, we will define the RNA sensors that detect glycoRNA in the absence of N-glycans and downstream signaling pathways responsible for initiating an innate immune response. Lastly, we will identify the secondary structures of glycoRNA that confer cellular sensitivity. Thus, Aim 2 will

investigate the mechanism by which N-glycans shield endogenous RNA from triggering an innate immune response, and our hypothesis that N-glycans serve as a determinant of self- or non-self nucleic acid. Our proposed experiments will enhance our knowledge of these new cell surface molecules and expand

our understanding of host-pathogen interactions. This fellowship and funding will enable me to explore a newly discovered field while taking full advantage of the mentorship and training opportunities offered by UConn Health and the Jackson Laboratory, directly helping to advance my career as an independent researcher.