RNA N-glycosylation enables innate 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 TLR3- and TLR7-dependent manner. Further, we show that N-glycans of 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 of small RNAs and apoptotic cells. Additionally, 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-driven innate immune activation, demonstrating how glycoRNAs exist on the cell surface and in the endosomal network without inducing autoinflammatory responses. Overall design: THP1 monocytes were differentiated into macrophages with phorbol 12-myristate-13-acetate (PMA) for 24 hours. THP1-derived macrophages were differentiated via phorbol 12-myristate-13-acetate (PMA) for 24 hours then transfected with either of the following: 1. small RNA harvested from HeLa cells (UntxtRNA) 2. PNGase F-treated small RNA harvested from HeLa cells (PNGaseRNA) 3. Unmodified synthetic 18-mer tRNA fragment (Unmod-tRNA) 4. Acp3U-8C modified synthetic 18-mer tRNA fragment (Acp3U8C-tRNA) Following 6-hour incubation, RNA was harvested via Zymo Directzol RNA kit.