An RNA-centric view
of the cell surface

A major current avenue of research has focused on exploring the hypothesis that RNAs can localize to the luminal spaces of organelles, such as the ER or Golgi. Taking a chemical biology approach, and leveraging the fact that proteins trafficked through these luminal spaces are glycosylated, I searched for evidence of RNA glycosylation. This effort resulted in the surprising identification of sialylated glycans conjugated to RNAs in mammalian cells. These glycoRNA conjugates are present in all cell types and tissues tested across human, mouse, and hamster, and show differential accumulation in primary compared to cancer cell lines.

Sequencing revealed a distinct set of small RNAs including Y-RNAs, snRNAs, tRNAs, and snoRNAs as modified putatively at guanosine. Chemical, genetic, and enzymatic approaches defined the glycan as an N-linked structure produced by the oligosaccharyltransferase (OST) complex in the ER lumen. To enable more facile examination of glycoRNA biology we developed a sensitive and scalable protocol — leveraging periodate oxidation and aldehyde ligation (rPAL) and SWATH-MS — identifying the modified RNA base acp3U as a site of N-glycan attachment.

Collectively, these findings suggest the existence of a previously unknown interface of RNA biology and glycobiology, and an expanded role for glycosylation beyond canonical lipid and protein scaffolds.

Traditionally, glycosylated transmembrane proteins were thought to be the major constituents of the external surface of the plasma membrane. We have begun to challenge this view, providing evidence that a group of RNA binding proteins (RBPs) are present on the surface of living cells. These cell surface RBPs (csRBPs) are detectable across a variety of mammalian cells and can be isolated by numerous chemical strategies to selectively label the cell surface. csRBPs are themselves enriched for glycosylation, suggesting a mechanistic path for their cell surface presentation.

The csRBPs are organized into well-defined nanoclusters enriched for multiple RBPs, glycoRNAs, and can be disrupted by extracellular RNase addition. These glycoRNA–csRBP clusters serve as sites of interaction for the cell penetrating peptide TAT. By leveraging a genome-wide knockout screen, we discovered that heparan sulfate proteoglycans (HSPGs) are a major organizing force for these clusters.

We have established that (1) signal transduction by HS-dependent growth factors, such as VEGF-A165, is regulated by cell surface RNAs, (2) VEGF-A165 selectively interacts with glycoRNAs in vitro, and (3) key amino acids in VEGF-A165 control glycoRNA binding in vivo. Our findings uncover a new molecular mechanism controlling signal transduction of specific growth factors via the regulated assembly of glycoRNAs, csRBPs, and heparan sulfate clusters.