In our general understanding, RNA always functions in the nucleus or cytoplasm. Even if it appears on the cell membrane, it is considered to have only weak and unstable interactions with the cell membrane. However, in 2020, researchers found that RNA does exist on the cell membrane of human monocytes, and the corresponding Oligonucleotide Fragments antagonized the function of RNA, which would affect the adhesion between monocytes and endothelium.
In 2021, Carolyn R. Bertozzi's team found N-glycosylation of some microRNAs on the surface of cancer cell lines and embryonic stem cells, and named such RNAs glycoRNAs, whose Glycosylation occurs in association with several enzymes that cause protein glycosylation.
As an emerging research field, the specific mechanism of GlycoRNA production and its physiological effects at the in vivo level are still largely unknown. On January 22, 2024, the team of Jun Lu and Dianqing Wu from Yale University published a paper titled "Cell surface RNAs control neutrophil recruitment" in Cell. This work took neutrophils as the research object and found that glycoRNA on their surface affects their adhesion with endothelial cells, thereby affecting their chemotaxis to the site of inflammation. At the same time, the researchers also found that glycoRNA interacts with P-selectin (Selp) on endothelial cells, is produced in the cell and transported to the cell surface via Sidt.
Fig.1 Cell surface RNAs control neutrophil recruitment. (Zhang, et al., 2024)
The researchers first used the method reported in 2021 to verify whether glycoRNA2 exists on the surface of immortalized myeloid cells (HOXB8), neutrophils differentiated from the myeloid cells, and primary mouse neutrophils. Briefly, the researchers first labeled the glycosyl group with Ac4ManNAz, and after extracting total cellular RNA, they labeled the azide group of Ac4ManNAz using DBCO-PEG4-Biotin, which allowed them to determine the presence of glycoRNA by detecting the biotin signal. It was found that the biotin signal was present in all three cells and that the signal was sensitive to RNaseA. Further experiments revealed that treatment with RNaseA without disrupting the cell membrane also resulted in the disappearance of the biotin signal, and when it was stained, the signal was found to be mainly present on the Cell Surface, further verifying the cell membrane localization of glycoRNA.
So, does glycoRNA on the surface of neutrophils affect their biological functions in vivo? Normally, when inflammation occurs in vivo, neutrophils are recruited to the site of inflammation and undergo a series of processes such as rolling, adhesion, and invasion on the surface of vascular endothelial cells to reach the site of inflammation. After inducing peritonitis in a mouse model using thioglycolate (TG), researchers injected RNaseA-treated (red fluorescence) and non-RNaseA-treated (green fluorescence) neutrophils into the blood at a 1:1 ratio, and then examined the ratio of the two types of cells to the peritoneum by flow cytometry 2.5 hours later. It was found that the proportion of RNaseA-treated cells reaching the peritonitis site was significantly reduced.
When verified using Transwell assay, the researchers found that after RNaseA removed cell surface glycoRNA, the ability of neutrophils to migrate across the endothelial cells to the lower layers was significantly weakened under the stimulation of the chemotactic signal fMLP. And in in vivo experiments, by injecting a mixture of RNaseA-treated neutrophils and mock-treated neutrophils into the carotid arteries of mouse and detecting the fluorescence signals of the two types of cells in the blood vessels at the cremaster muscle, it was found that the proportion of neutrophils on endothelial cells that changed from free flow to rolling was significantly reduced after RNaseA treatment.
Integrins on the surface of neutrophils or selectins on the surface of endothelial cells usually play a key role in the adhesion of neutrophils to endothelial cells.Treatment with RNaseA did not significantly alter the levels of the integrins Cd11a and Cd11b on the surface of neutrophils, nor did it alter their interaction with the ligand ICAM-1. However, the binding of glycoRNA to endothelial cells was partially attenuated after blocking P-selectin with antibodies (no corresponding phenomenon was observed with E-selectin). In subsequent animal experiments, Knockout of Selp also led to a weakening of neutrophil rolling on vascular endothelial cells.
Past studies on glycoRNA have suggested that glycoRNA is released from dead cells into the extracellular environment and is then captured by cells on the cell surface. However, there may be another answer to this question,that is, the glycoRNA on the cell surface is synthesized by the cell itself. To elucidate this question, the researchers used Ac4ManNAz to label the glycosyl groups, while labeling the cells with green fluorescence and the glycosyl-unlabeled cells with red fluorescence, and co-cultured the two types of cells. It was found that biotin signals were only detected in the Ac4ManNAz-labeled, green-fluorescent cells, whereas the red-fluorescent cells did not capture RNA carrying the corresponding marker, suggesting that glycoRNAs are most likely produced by the cells themselves and transported to the cell surface. When the homologue of C. elegans RNA transporter protein Sid-1 in mammals was knocked out, the amount of glycoRNA on the cell surface was significantly reduced, and the migration ability of neutrophils was significantly weakened.
Fig.2 Experimental design to explore the source of glycoRNA. (Zhang, et al., 2024)
This article answers a number of important questions about glycoRNAs, including where they come from and what functions they are intended to perform, etc. RNA glycosylation is an important complement to traditional protein- and lipid-based Glycobiology. Given the wide variety of forms and types of RNAs, the biological functions of RNAs should go far beyond this, and more details are still needed to be added to future research.
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