Cellular RNA ‘coat’ attracts immune receptors

In recent years, researchers have been finding ever more evidence that RNA plays a role not only inside but also outside the cell in a variety of processes. RNA has now been detected on the surface of various mammalian cells. Whether this truly points to a functional role remained disputed. Some researchers argued that the RNA could originate from dead cells and subsequently attach to living cells via non-specific interactions.

A team led by biochemists Jack Li of Utrecht University and Bhagyashree S. Joshi, who was affiliated with Delft University of Technology at the time of the research and is now at the Whitehead Institute (USA), has now demonstrated that RNA molecules do indeed perform a function at the surface of various cells. Amongst other things, the extracellular RNA helps immune receptors bind more effectively to their target.

The findings stem from a close collaboration between Utrecht University, Delft University of Technology, Leiden University Medical Centre, and Radboud University, among others. Together, the researchers show that the RNA chains form part of a three-component complex. In this complex, long sugar molecules — heparan sulphates — attach to the cell surface, whilst RNA-binding proteins provide the connection between the RNA and the sugar chain. Li: ‘We have demonstrated that RNA molecules are present at cell surfaces through specific interactions, and we provide a molecular model for the complexes they form.’

Sensor

For their study, the researchers developed a sensor that binds to the surface RNA. They drew inspiration from Toll-like receptor 7 (TLR7), a protein of the immune system that binds to, amongst other things, viral or bacterial RNA in order to trigger an immune response. ‘We created a recombinant, fluorescently tagged sensor from this, added it to living mammalian cells, and picked up a signal,’ says Li. ‘As soon as we then added an enzyme that degrades RNA, the signal disappeared. That showed us the sensor was working.’

Using the sensor and genetic screening, the researchers searched for molecules involved in the presence of surface RNA. To this end, they worked with different cell populations, switching off only one protein in each population. Using their TLR7 sensor, they then determined which populations showed a loss of signal. They discovered this occurred in the absence of enzymes involved in the production of heparan sulphates. Li: ‘When we observed under the microscope that these sugars co-localised with surface RNA, we concluded — following multiple validation experiments — that they are required for the presence of RNA at the cell surface.’

Checkpoint

Since RNA molecules and heparan sulphates are both negatively charged, the researchers suspected that a protein must be binding them together. Using proximity labelling and proteomics, they therefore identified all proteins located near the surface RNA. ‘To do this, we coupled the TLR7 sensor to ascorbate peroxidase, an enzyme that catalyses a chemical reaction to attach fluorescent tags to surrounding proteins,’ says Li. ‘We discovered that many of the proteins around the sensor were RNA-binding proteins. In further experiments, we found that these are indeed required to link the sugars and RNA together.’

Amongst the many neighbouring proteins, the researchers also identified the immune checkpoint protein poliovirus receptor (PVR), or CD155 — a ‘brake’ on the immune system. Li: ‘We wondered whether the surface RNA regulates PVR’s interactions with other receptors.’

They then tested various recombinant receptors known to bind to PVR. In doing so, they found that KIR2DL5, a receptor that inhibits natural killer cells, bound far more effectively to PVR when RNA was present. The results thereby suggest that RNA influences how immune cells recognise other cells.

Broadly accessible

Whilst Li began this research as a postdoc in Leiden and Delft, he is now developing this line of research further with his own research group in Utrecht. ‘We are working hard to optimise the sensor protocol and make it broadly accessible. In doing so, we are “democratising” this field.’

With his group, he aims to determine how surface RNA regulates receptor binding. They will also delve deeper into the RNA complexes. ‘We want to know what types of RNA these consist of, and to study further which RNA-binding proteins bind to which RNA sequences.’

Z. Li, B.S. Joshi, et al., Cell-surface RNA forms ternary complex with RNA-binding proteins and heparan sulfate to recruit immune receptors, Molecular Cell (2025), doi:10.1016/j.molcel.2025.11.020