Mechanisms and functions of cell surface glycoRNAs

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PROJECT SUMMARY/ ABSTRACT The cell surface is a platform for physical and regulatory control over cell biology, positioning it to be a key interface for diagnostic targeting and therapeutic intervention. While RNA is a central polymer in biology most thought and experimental effort devoted to RNA biology has been confined to intracellular spaces and excluded from participating in cell surface biology. On the cell surface, carbohydrate polymers (glycans) are of critical importance due to biophysical and signaling activities. Interestingly, despite both polymers playing central roles in biology, RNA and glycans have largely existed in entirely non-overlapping fields of study. However, my work has provided evidence of a hybrid molecule, an RNA-glycan conjugate (glycoRNA); this new class of biomolecule represents a direct link between RNA and glycobiology. Critically, glycoRNAs are localized to the external surface of living cells and can engage with immunomodulatory Siglec receptors. Thus, glycoRNAs are positioned on a surface of critical regulatory importance, with access to cell-cell interactions, pathogens, and signaling receptors on the cell surface. However, we currently lack facile tools to study this new cell surface molecule, we do not understand the molecular or atomic composition of glycoRNAs, and we have a poor understanding of how many species biosynthesize glycoRNAs. This MIRA proposal is focused on developing and implementing methods to uncover functional roles of RNA glycosylation and we will approach the complex biology of glycoRNAs in a systematic fashion. Initially we will develop novel chemical approaches to label glycans in the context of RNA. My proposed strategy of selective carboxylic acid labeling represents an innovative new approach to detecting glycoRNA, without the need for synthetic metabolic reporters. These tools will be easily implemented across cell types and species enabling others in the scientific community. We will apply these tools and other molecular assays to expand our understanding of the composition of the cell surface in the context of glycoRNA. Biochemical, biophysical, and imaging-based strategies will be used to define the molecular neighborhoods of glycoRNAs as well as the chemical nature of the RNA-glycan linkage; all together providing a more complete picture of the mammalian cell surface. Finally, we will develop the first evidence of glycoRNAs in non-mammalian organisms. First focusing on two major strains of yeast (S. cerevisiae and S. pombe) with robust culturing, functional, and genetic tools that will allow for rapid dissection of the biogenesis pathway for eventual engineering purposes. Expanding to other organisms including prokaryotes (pathogenic and not) as well as other multicellular eukaryotes like C. elegans will better define the scope of glycoRNA biosynthesis and more robustly equip us to generate synthetic glycoRNAs. More broadly, we intend to advance the general model of how cells interact with each other, pathogens, and exogenous molecules, as without cell surface glycoRNAs, they are likely incomplete. This proposal develops innovative methods to establish new conceptual and physical layers of regulation between a cell and its environment.