Revealing important functions of cell surface RNAs
Published: 2024-03-29 07:09:22

The vast majority of RNA in living organisms is located inside the cell and is a key molecule essential to the cell. However, new research has revealed that RNA is also present on the cell surface of mammalian cells, including RNA with glycosylated modifications, known as glycosylated RNA (glycoRNA). Why are RNAs present on the surface of mammalian cells, and how are such RNAs produced? Do they have important functions? These questions have become a focus of scientific interest.
 
On January 22, 2024, a team of researchers from Yale University published a paper in the journal Cell entitled "Cell Surface RNAs Control Neutrophil Recruitment".
 
The study reveals that most glycosylated RNAs exist in a stable form on the outer surface of neutrophils and that such cell surface RNAs are essential for neutrophil recruitment to inflammatory tissues in mice, as well as revealing an important mechanism that regulates the expression level of glycosylated RNAs.
 
Neutrophils are the predominant type of leukocyte in mammalian blood. As the host's first line of defense against invading pathogens, neutrophils are required to travel from peripherally circulating blood via vascular exudation to the adjacent site of infection or injury. This study reports that RNA is present on the outer surface of mouse neutrophils and that the elimination of cell surface RNA significantly reduces neutrophil recruitment to sites of inflammation in vivo and reduces neutrophil attachment to and migration from endothelial cells (EC). Neutrophil glycoRNAs are predominantly located on the extracellular surface and can be recognized by endothelial cell-expressed P-selectin, which is critical for neutrophil-endothelial cell interactions. This study also revealed that neutrophil surface glycoRNA is produced by the cells themselves and not by trapping free RNA molecules outside the cell. Knockout of the Sidt gene resulted in the loss of neutrophil glycoRNA and showed defective neutrophil recruitment at sites of inflammation, similar to the loss of cell surface RNA.
 
To test whether neutrophils contain glycoRNA, the team first labeled salivary acid-containing glycans using an azide-labeled salivary acid precursor (Ac4ManNAz) and detected an azide signal in highly purified RNA, suggesting that neutrophils contain glycoRNA. The team further verified that glycoRNA is predominantly distributed on the extracellular surface. They labeled cellular RNA using the nucleoside homologue 5'-bromouridine (BrU) and detected RNA signals directly on the cell surface of neutrophils with [BrU antibodies](https://mrna.creative-biolabs.com/therapeutic-antibody-coding-mrna.htm).
 
To investigate whether cell surface RNA can have a function in regulating neutrophils in vivo, the team used a variety of inflammation models in order to investigate the effect of cell surface RNA on neutrophil inflammatory responses at sites of inflammation. It was found that extracellular RNase A (exRNaseA) treatment of cells to eliminate cell surface RNA significantly reduced (~9-fold) neutrophil recruitment at sites of inflammation in vivo.
 
In vitro experiments showed that neutrophil surface RNA plays an important role in their attachment to endothelial cells and migration across endothelial cells. In contrast, pretreatment of the endothelial cell surface with purified neutrophil glycoRNA effectively neutralized the effects of neutrophil surface RNA, demonstrating that the RNA function of the neutrophil surface is mainly derived from glycoRNA. In vivo real-time imaging revealed that the elimination of cell surface RNA resulted in a shift of neutrophils from free-flowing to rolling on the endothelium of the vessel wall without altering their ratio from rolling to stable adhesion on vessel wall endothelial cells.
 
Further studies showed that the glycoRNA of neutrophils can be recognized by P-selectin on endothelial cells, thus facilitating neutrophil-endothelial cell interactions. These data suggest that cell surface RNAs play an important role in regulating neutrophil capture and migration on endothelial cells in the vessel wall.
 
Next, the team demonstrated that glycoRNA is produced and transported to the cell surface in a cell-autonomous manner. The Sidt genes (Sidt1 and Sidt2) are homologous to the Sid-1 RNA transporter protein in mammals. Knockdown of the Sidt genes results in loss of neutrophil glycoRNA, affects neutrophil-endothelial cell interactions, and at inflammatory sites presents a defect in neutrophil recruitment similar to that caused by loss of cell surface RNA. ExRNaseA treatment no longer regulates neutrophil attachment to endothelial cells and transendothelial cell migration after Sidt gene knockdown. This suggests that the Sidt gene is required for both glycoRNA expression and cell surface RNA function in neutrophils.
 
Taken together, this study reveals the biological importance of cell-surface glycoRNAs and highlights a novel area of RNA-mediated regulation of cellular function. The study of cell surface RNAs is still in its infancy. GlycoRNAs can be detected in a variety of mammalian cells. It can be expected that this area of research will rapidly progress in future studies.