RNA N-glycosylation enables immune evasion and homeostatic efferocytosis

Glycosylation is central to the localization and function of biomolecules¹. We recently discovered that small RNAs undergo N-glycosylation² at the modified RNA base 3-(3-amino-3-carboxypropyl) uridine (acp³U)³. However, the functional significance of N-glycosylation of RNAs is unknown. Here we show that the N-glycans on glycoRNAs prevent innate immune sensing of endogenous small RNAs. We found that de-N-glycosylation of cell-culture-derived and circulating human and mouse glycoRNA elicited potent inflammatory responses including the production of type I interferons in a Toll-like receptor 3- and Toll-like receptor 7-dependent manner. Furthermore, we show that N-glycans on cell surface RNAs prevent apoptotic cells from triggering endosomal RNA sensors in efferocytes, thus facilitating the non-inflammatory clearance of dead cells. Mechanistically, N-glycans conceal the hypermodified uracil base acp³U, which we identified as immunostimulatory when exposed in RNA. Consistent with this, genetic deletion of an enzyme (DTWD2) that synthesizes acp³U abrogated innate immune activation by de-N-glycosylated small RNAs and apoptotic cells. Furthermore, synthetic acp³U-containing RNAs are sufficient to trigger innate immune responses. Thus, our study has uncovered a natural mechanism by which N-glycans block RNAs from inducing acp³U-dependent innate immune activation, demonstrating how glycoRNAs exist on the cell surface and in the endosomal network without inducing autoinflammatory responses.