ABOVE: Cell surface glycoRNAs help neutrophils reach their destinations. © iStock, ALIOUI Mohammed Elamine

RNA is thought to be a stay-at-home kind of molecule, often comfortably confined within the cell’s interior. So, when RNA molecules were detected on the surface of several cell types, researchers wondered what purpose they might serve. A recent study has revealed one of their functions: mobilizing immune cells to inflamed tissue. 1

Published last month in Cell, a Yale University team led by geneticist Jun Lu  and pharmacologist Dianqing Wu described how cell surface RNA helps neutrophils latch onto endothelial cells and infiltrate tissue. Removing the molecules prevents the immune cells from reaching inflammatory sites in mice, highlighting their role in the immune system’s response to potential threats.

After remaining a mystery for so long, it is encouraging that researchers are paying attention to the functions of cell-surface RNAs, stated Carolyn Bertozzi, a biochemist at Stanford University, who was not involved in the new study. “I’m really excited that this group started thinking of [them] as a potential mediator of neutrophils.”

Proof of cell-surface RNAs first came to light in 2020, when they were detected on immune cells in human blood.2 The following year, Bertozzi’s group found the molecules littering the surface of cancer cells and stem cells, where they are welded to a sugar chain.3 Like glycoproteins and glycolipids, this new category of molecule, christened glycoRNAs, appeared to bind to immune receptors, pointing to potential immunoregulatory functions.

When Lu came across these papers, he responded with a healthy dose of skepticism. After all, he reasoned that any exposed RNA should be torn apart by RNases, RNA-degrading enzymes that roam the blood plasma.

“I wondered how it was even possible. At the time we had a postdoc joining the lab and we said maybe you want to spend two months proving them wrong, then move onto something else,” said Lu with a smile.

His team began by using a chemical marker called biotin to label any sugar chains present on the neutrophil surface, tagging glycoproteins, glycolipids, and—potentially—glycoRNAs. Without rupturing the cell membrane, they purified RNA from labeled cells and then applied RNase at concentrations far higher than typically found in the human body. If the enzyme diminished the biotin signal, some sugars on the cells must be bound to extracellular RNA. To Lu’s surprise, the signal vanished, confirming the presence of glycoRNAs on the cell surface. 

If glycoRNAs share similar functions to glycoproteins and glycolipids, they may help immune cells reach inflammatory sites. To test this, the researchers dyed some neutrophils red and degraded their extracellular glycoRNAs using RNase. They labeled other neutrophils green but left their surface RNAs intact. After injecting the dyed cells into a mouse that had abdominal inflammation, Lu’s team found that the cells lacking glycoRNAs were less likely to reach the stomach.

To infiltrate tissue, neutrophils must first latch onto exterior cells and then traverse through several cell layers. Lu wondered whether glycoRNAs contributed to both stages of this process, so his team placed neutrophils on one side of a cultured endothelial layer and a chemoattractant on the other. Immune cells lacking glycoRNAs showed less adhesion and reduced migration through the cell layer in vitro. Without the endothelial barrier, the cells migrated normally, suggesting that glycoRNAs do not affect cell mobility. 

To uncover how neutrophil glycoRNAs mediate attachment to endothelial cells, the researchers split the molecule into its sugar and RNA components. Saturating the same cell culture system with glycans blocked the immune cells from migrating through the endothelial layer, while RNA saturation had no effect. The findings suggested that—in a similar manner to other glycoconjugates—the glycan portion latches onto endothelial cells, while the RNA tethers the sugar to the membrane.

Serving as a scaffold, the actual RNA sequence—at least in this context—appears less important, the researchers explained. “There may be a hidden life for a sequence-based function too,” Lu said. “This is just the tip of the iceberg.” 

The researchers wondered whether glycoRNAs originate intracellularly or from captured fragments released extracellularly by dying cells. To explore this, they again labeled groups of neutrophils red or green. They chemically labeled the glycoRNAs on only the green cells. After culturing the cells together for three days, the team detected the chemical tags on only green cells, suggesting that the RNA is produced in-house rather than transferred from the external environment.

Sequencing the glycoRNA molecules turned up hits for ribosomal RNA, transfer RNA, and small nucleolar RNAs, suggesting that they may be repurposed fragments of noncoding nucleic acids. Yet the rules that determine which bits of RNA end up on the membrane, and how they are protected from degradation, are unclear. 

These are questions that the Yale team hopes to address in future work. The researchers also plan to investigate whether glycoRNA expression is altered in various disease states. But owing to a lack of real-time detection methods, this will be experimentally challenging. “Methodologies need to be improved before we can address many of those questions,” Wu said.