Introduction

Glycosylated transmembrane proteins are the main components of the outer surface of the plasma membrane. These proteins play an important role in the interaction between cells and the external environment. On April 3, 2025, Ryan A. Flynn's team from Harvard University published an article entitled "RNA-binding proteins and glycoRNAs form domains on the cell surface for cell-penetrating peptide entry" in Cell. The study proposed a new view that RNA-binding proteins (RBPs) exist on the cell surface. These proteins participate in the interaction between cells and the outside world by forming specific nanocluster structures, and have an important influence on the internalization process of cell-penetrating peptide trans-activator of transcription (TAT).

Background

The cell surface is the core interface for the interaction between cells and the external environment. The traditional view is that its components are mainly dominated by Transmembrane Glycoproteins, lipids and Glycolipids. Past studies have focused on how these classic molecules mediate signal transduction, material transport and immune recognition. However, breakthrough discoveries in recent years have gradually revealed the complexity of cell surface biology:

• The "cross-border" phenomenon of RBPs: As early as the 1990s, RBPs such as nucleolin (NCL) were accidentally discovered to exist on the surface of certain cancer cells, but their universality and functional significance have long been in doubt.
• The discovery of glycoRNA: In 2021, it was first reported that RNA can be modified by N-glycan chains to form glycoRNA and localize on the cell surface, but there is a lack of understanding of its anchoring mechanism and function.

Fig. 1 Expansive presentation of a select group of RNA binding proteins on the surface of living cells. (Perr, et al., 2025)

Scientific Question

To explore whether there are specific domains formed by the co-localization of RNA-binding proteins (csRBPs) and glycoRNA on the surface of mammalian cells, and how these structures regulate the cell entry mechanism of cell-penetrating peptides (such as TAT).

Experimental Ideas

• Multi-omics analysis: Integrate RBP database and Surface Proteomics data to screen candidate csRBPs.
• Screening and verification: Combine mass spectrometry, flow cytometry and confocal microscopy to verify the presence of csRBPs on the cell surface and their glycosylation modification.
• Structural analysis: Use super-resolution imaging and proximity labeling techniques (such as HRP-biotin phenol labeling) to reveal the composition and spatial distribution of nanoclusters.
• Functional verification: Through RNAse treatment and TAT peptide mutant experiments, the necessity of RNA dependence in cell penetration is demonstrated.
• Mechanism exploration: Combine protein interaction analysis and co-localization studies to propose a model for the formation of functional complexes between csRBPs and glycoRNA.

Innovation

• New discovery: Revealed the existence of domains formed by RBPs and glycoRNA on the Cell Surface, expanding the traditional understanding of the composition of the cell surface.
• New mechanism: The discovery of the csRBP-glycoRNA domain as a functional binding site for TAT provides a new RNA-dependent mechanism for the internalization of cationic cell-penetrating peptides.
• Technology integration: Combining surface proteomics, super-resolution imaging and functional experiments to analyze the molecular composition of the cell surface in multiple dimensions.

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Reference

1. Perr, J., et al. (2025). RNA-binding proteins and glycoRNAs form domains on the cell surface for cell-penetrating peptide entry. Cell, 188(7), 1878-1895. DOI: 1016/j.cell.2025.01.040.