Cell-Surface RNA Forms Ternary Complex with RNA-Binding Proteins and Heparan Sulfate to Recruit Immune Receptors

Recent discoveries have shown the presence of RNA molecules on the cell surface, defying the traditional view that RNA only functions intracellularly. However, it is not well understood how cell-surface RNA (csRNA) is stably present on the plasma membrane and what functions it performs. We answer the pressing questions in the emerging field by taking integrated omic-wide approaches and multiple orthogonal validatory methods. Firstly, we exploited the RNA-sensing ability of TLR7 as a specific recombinant probe to detect csRNA. Coupling it with a genome-wide CRISPR-Cas9-knockout screening, we identified heparan sulfate (HS) as a crucial factor for RNA presentation on cells. Using the TLR7 probe, cell surface proximity labelling revealed that these HS-associated csRNAs (hepRNAs) are in vicinity with a plethora of RNA-binding proteins. The compelling observation led us to a molecular model where HS, RNA and RBP form ternary complexes at cell surface. A photochemical RNA-protein crosslinking technology termed SCOOPS were then established to validate the termolecular model in a TLR7-orthogonal manner. Moreover, enabled by SCOOPS, we unveiled identities of hepRNA using next-generation sequencing, and identified traits in RNA primary structures that facilitate HS association. We further show that hepRNA binds to killer cell immunoglobulin-like receptor 2DL5 (KIR2DL5), recruiting the protein to cell surface and potentially enhancing receptor-ligand interactions. Our findings provide a foundation for exploring how cell-surface ribonucleoproteins contribute to immune modulation.