On August 6, 2025, a collaborative team led by Vijay A. Rathinam of the UConn Health School of Medicine and Ryan A. Flynn of Harvard University published a study titled "RNA N-glycosylation enables immune evasion and homeostatic efferocytosis" in Nature. This study not only confirmed that RNA can be N-glycosylated (glycoRNA), but also revealed that these "sugar-coated RNAs" play a crucial role in immune regulation and maintaining body health. They act like an "invisibility cloak," cleverly helping the body avoid autoimmune attacks and ensuring the silent elimination of dying cells.

Research Background

How Does the Immune System Distinguish Friend from Foe?

Our immune system is constantly on guard against invading pathogens, such as viruses. Viruses often carry nucleic acids (DNA or RNA). Immune cells use specialized detectors—pattern recognition receptors (PRRs, such as TLR3, TLR7, TLR8, and RIG-I)—to recognize these foreign nucleic acids, sounding the alarm and triggering inflammation and interferon responses to eliminate the enemy. But the problem is that our own cells are also full of RNA. How does the immune system avoid mistaking these friendly cells for enemies and launching attacks, leading to autoimmune diseases? Cells have evolved various mechanisms, such as spatially isolating RNA sensors from their own RNA, expressing RNases to degrade free RNA, and chemically modifying their own RNA.

A Newly Discovered "Sugar Coating": N-Glycosylation on RNA

Glycosylation (adding glycan chains to molecules) has long been considered a modification exclusive to proteins and lipids, crucial for their localization and function within cells. However, a groundbreaking study in 2021 discovered that certain small RNAs (such as tRNA, gamma-RNA, snRNA, and snoRNA) can also be modified with complex N-glycan chains containing sialic acid, forming so-called "glycoRNAs." These glycoRNAs are transported to the cell surface.

Critically, scientists have identified the direct site of N-glycosylation on RNA: a rare modified base called 3-(3-amino-3-carboxypropyl)uridine (acp3U), typically located in the D-loop of tRNA. However, a core mystery remains: what exactly are these glycan chains on RNA used for?

Scientific Question

The core goal of this paper is to decipher the functional mystery of RNA N-glycosylation.

The researchers proposed a key hypothesis: these N-Glycans covering the RNA surface may serve as an identification marker that helps RNA hide from the immune system. Considering that RNA exists on the cell surface and in the bloodstream, when cells undergo apoptosis and are engulfed by macrophages (efferocytosis), the glycoRNAs on their surfaces readily enter the macrophage's endosomal compartments, which contain RNA sensors such as TLR3/TLR7. How can this crucial "garbage removal" process be prevented from triggering inflammation?

Experimental Strategy

Glycostripping Assay (PNGase F)

• GlycoRNA-rich small RNAs are isolated from cultured cells (e.g., HeLa cells) or mouse or human serum.
• The N-glycans on these RNAs are removed (deglycosylated) using an enzyme (PNGase F). These treated RNAs are then transfected into or directly added to macrophages or dendritic cells.
• Results: Deglycosylated RNA (but not untreated glycoRNA or non-glycoRNA) strongly induced the production of type I interferon (IFNβ) and other inflammatory cytokines (TNF, IL-6). This response was dependent on the RNA itself and the TLR3/TLR7 signaling pathway.

Efferocytosis Model

• Induce apoptosis in cells (e.g., HeLa cells).
• Treat these apoptotic cells with PNGase F to remove the glycanchains on their cell surface glycoRNA.
• Engulf these treated apoptotic cells by macrophages.
• Results: Macrophages engulfing deglycosylated apoptotic cells produced a robust IFNβ and inflammatory cytokine response, whereas macrophages engulfing normal or RNase-treated (RNA-destroying) apoptotic cells did not. Injection of deglycosylated apoptotic cells in an in vivomouse model also triggered an intraperitoneal interferon response. This suggests that the glycan chains on cell surface glycoRNA are key to the silent clearance of apoptotic cells.

Targeting the Key Molecule: acp3U

• Gene Knockout(DTWD2 KO): DTWD2 is the key enzyme for acp3U synthesis. In cells with DTWD2 knockout, RNA lacks the acp3U modification. Even after removing the glycan chains (theoretically leaving nothing exposed), the RNA or apoptotic cells fail to effectively activate the immune response.
• Partial Glycostripping (Endo-F2/F3) Assay: Using Endo-F2/F3 enzymes, only the majority of the glycan chainsare removed, but an N-acetylglucosamine (GlcNAc) cap remains on acp3U. RNA or apoptotic cells treated in this manner also fail to activate the immune response, demonstrating that complete acp3U exposure is crucial.
• Synthetic RNA Verification: Short RNA fragments containing acp3U were artificially synthesized. Synthetic RNA containing acp3U (regardless of sequence context) effectively activated macrophages to produce inflammatory cytokines, while control RNA lacking acp3U had only a weaker effect. RNA sequencing analysis showed that the gene expression profile induced by acp3U-RNA was highly similar to that of deglycosylated natural small RNAs, and both strongly activated TLR and interferon signaling pathways.
• Chemical Modification Validation: Synthetic acp3U monomers or derivatives (e.g., blocking the carboxyl group with GlcNAc or amide groups) were used.
• Results: Free acp3U exhibited some immunostimulatory activity when conjugated to uridine, but modification of the carboxyl group completely abolished its activity. This explains why PNGase F was effective, while Endo-F2/F3 was ineffective.

Targeting Immune Receptors: TLR3 & TLR7

• Using Macrophages from Knockout Mice: When TLR3 or TLR7 or their key adaptor proteins (MyD88 for TLR7, TRIF for TLR3) were absent, the ability of deglycosylated small RNAs or apoptotic cells to induce IFNβ was significantly reduced or even abolished.
• Synthetic long RNA containing acp3U can also activate macrophages, similarly in a TLR3- and TLR7-dependent manner.
• Interestingly, the two receptors require synergistic action to generate a sufficiently strong signal to trigger the interferon response. This may be because the deglycosylated natural RNA pool or the designed synthetic RNA contains both single-stranded RNA (ssRNA) features recognized by TLR7 and double-stranded RNA (dsRNA) features recognized by TLR3.

Fig. 1 TLR3 and TLR7 sense de-N-glycosylated RNAs. (Graziano, et al. 2025)

Innovation

Functional Discovery

This study reveals the core biological functions of RNA N-glycosylation, including immune evasion and maintenance of immune homeostasis, particularly ensuring the silent clearance of apoptotic cells.

Key Molecular Mechanism

This study pinpoints acp3U as a key immunostimulatory site on RNA, and the role of N-glycans in "caging" acp3U, shielding it from TLR3/TLR7 recognition.

Connecting Physiology and Pathology

This study elucidates how glycoRNAs resolve the immunological paradox of apoptotic cell clearance, a crucial physiological process. Furthermore, the study discovered that glycoRNAs are present in circulating blood, and that their deglycosylated products are immunogenic, providing new insights into autoimmune diseases such as Systemic Lupus Erythematosus (SLE), one of the hallmarks of which is the abnormal activation of immune responses by autologous nucleic acids.

New Tools and Models

This study rigorously demonstrated its conclusions by combining enzymatic treatment (PNGase F, Endo-F2/F3), gene knockout (DTWD2, TLR3/7, MyD88, TRIF), and Chemical Synthesis (acp3U-containing RNA and monomers).

Conclusion

This study has deciphered the core functional mystery of RNA N-glycosylation, revealing a sophisticated immune regulatory mechanism:

• "Sugar-coating" Invisibility: Cells coat specific small RNAs with an "invisibility cloak" by adding N-glycans (glycoRNAs) to the acp3U base of these RNAs. This "sugar coating" physically obscures acp3U, preventing it from being recognized by the "sentinels" TLR3 and TLR7 within immune cell endosomes.
• Ensuring Silence and Cleanliness: This mechanism is crucial for the immune silencing and elimination of apoptotic cells (efferocytosis). Even when glycoRNAs on the surface of apoptotic cells are engulfed by macrophages into endosomes containing TLR3/TLR7, their glycan chainsprevent the exposure of acp3U, thereby preventing inflammation and maintaining tissue homeostasis.
• acp3U Acts As an Alarm Trigger: When the "sugar coating" is removed (e.g., in pathological conditions or experimental manipulations), the exposed acp3U becomes a potent endogenous immune alarm signal, triggering interferon and inflammatory responses by simultaneously activating TLR3 and TLR7.
• Connecting to Autoimmune Diseases: This mechanism reveals a new pathway by which self-RNA triggers immune responses. Circulating glycoRNA or its deglycosylated products (acp3U-exposed RNA or free acp3U) may play a key role in diseases such as SLE, where autologous nucleic acids activate the immune system. This provides new molecular targets and insights for understanding and treating these diseases.

In summary, this study elevates RNA N-glycosylation from a novel modification phenomenon to a critical, long-overlooked art of immune regulation, profoundly transforming our understanding of how the immune system distinguishes self from non-self and maintains homeostasis.

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Reference

1. Graziano, V. R., et al. (2025). RNA N-glycosylation enables immune evasion and homeostatic efferocytosis. Nature, 1-9. DOI: 1038/s41586-025-09310-6.