Graziano Vincent R, Porat Jennifer, Ah Kioon Marie Dominique, Mejdrová Ivana, Matz Alyssa J, Lebedenko Charlotta G, Chai Peiyuan, Pluvinage John V, Ricci-Azevedo Rafael, Harrison Andrew G, Wright Skylar S, Wang Xinzheng, Strine Madison S, Wang Penghua, Wilson Michael R, Vanaja Sivapriya Kailasan, Zhou Beiyan, Barrat Franck J, Carell Thomas, Flynn Ryan A, Rathinam Vijay A
Department of Immunology, UConn Health School of Medicine, Farmington, CT, USA.
Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA.
Nature. 2025 Aug 6. doi: 10.1038/s41586-025-09310-6.
Glycosylation is central to the localization and function of biomolecules. We recently discovered that small RNAs undergo N-glycosylation at the modified RNA base 3-(3-amino-3-carboxypropyl) uridine (acpU). 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 acpU, which we identified as immunostimulatory when exposed in RNA. Consistent with this, genetic deletion of an enzyme (DTWD2) that synthesizes acpU abrogated innate immune activation by de-N-glycosylated small RNAs and apoptotic cells. Furthermore, synthetic acpU-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 acpU-dependent innate immune activation, demonstrating how glycoRNAs exist on the cell surface and in the endosomal network without inducing autoinflammatory responses.
糖基化对于生物分子的定位和功能至关重要。我们最近发现，小RNA在修饰的RNA碱基3-(3-氨基-3-羧丙基)尿苷（acpU）处发生N-糖基化。然而，RNA的N-糖基化的功能意义尚不清楚。在这里，我们表明糖基化RNA上的N-聚糖可防止对内源性小RNA的天然免疫感应。我们发现，对源自细胞培养物以及循环中的人和小鼠糖基化RNA进行去N-糖基化会引发强烈的炎症反应，包括以Toll样受体3和Toll样受体7依赖性方式产生I型干扰素。此外，我们表明细胞表面RNA上的N-聚糖可防止凋亡细胞触发吞噬细胞中的内体RNA传感器，从而促进死细胞的非炎性清除。从机制上讲，N-聚糖掩盖了高度修饰的尿嘧啶碱基acpU，我们发现当acpU在RNA中暴露时具有免疫刺激作用。与此一致的是，合成acpU的酶（DTWD2）的基因缺失消除了去N-糖基化的小RNA和凋亡细胞引起的天然免疫激活。此外，含合成acpU的RNA足以触发天然免疫反应。因此，我们的研究发现了一种天然机制，通过该机制N-聚糖可阻止RNA诱导acpU依赖性天然免疫激活，证明了糖基化RNA如何存在于细胞表面和内体网络中而不引发自身炎症反应。
