RNA binding proteins and glycoRNAs form domains on the cell surface for cell penetrating peptide entry

Abstract

The composition and organization of the cell surface determine how cells interact with their environment. Traditionally, glycosylated transmembrane proteins were thought to be the major constituents of the external surface of the plasma membrane. Here, we provide evidence that a group of RNA binding proteins (RBPs) are present on the surface of living cells. These cell surface RBPs (csRBPs) precisely organize into well-defined nanoclusters that are enriched for multiple RBPs, glycoRNAs, and their clustering can be disrupted by extracellular RNase addition. These glycoRNA-csRBP clusters further serve as sites of cell surface interaction for the cell penetrating peptide TAT. Removal of RNA from the cell surface, or loss of RNA binding activity by TAT, causes defects in TAT cell internalization. Together, we provide evidence of an expanded view of the cell surface by positioning glycoRNA-csRBP clusters as a regulator of communication between cells and the extracellular environment.

Cell surface ribonucleoproteins cluster with heparan sulfate to regulate growth factor signaling

“…Siglec-4, Siglec-7, Siglec-9, and Siglec-11 strongly bound both cell types above the level shown by the control IgG-Fc (Figure 1A, S1A). A pooled RNase treatment 19 (RNase A, a single stranded RNase and RNase III, a double stranded RNase) resulted in a significant reduction of Siglec-11 binding ability, as measured by total dots per cell (Figure 1A, 1B) and intensity per cell (Figure 1A, S1B), on both cell types. Live cell RNase treatment had no impact on the binding of Siglec-4, Siglec-7, or Siglec-9 (Figure 1A, 1B, S1B).…”

“…Given the observed punctate nature of Siglec-11 binding and its RNA-dependency, we predicted that RNA should colocalize with Siglec-11 on the cell surface. We recently described a new domain on the cell surface, glycoRNA-csRBP clusters 19 , where cell surface RNA binding proteins (csRBPs) and glycoRNAs colocalize to facilitate the functional entry of molecules such as cell penetrating peptides. To address the possible association of Siglec-11 ligands near or within these glycoRNA-csRBP clusters, we used an anti-dsRNA antibody (9D5) that detects cell surface RNA 19 .…”

“…We recently described a new domain on the cell surface, glycoRNA-csRBP clusters 19 , where cell surface RNA binding proteins (csRBPs) and glycoRNAs colocalize to facilitate the functional entry of molecules such as cell penetrating peptides. To address the possible association of Siglec-11 ligands near or within these glycoRNA-csRBP clusters, we used an anti-dsRNA antibody (9D5) that detects cell surface RNA 19 . Costaining of U2OS and MOLM-13 cells with 9D5 and Siglec-11 showed that 42% and 61% of 9D5 puncta overlapped with Siglec-11 on MOLM-13 and U2OS cells, respectively.…”

“…On the cell surface, these sialoglycoRNAs are in physical proximity to specific RNA binding proteins on the plasma membrane of cells (csRBPs) 19 . The cell penetrating peptide (CPPs) TAT localizes to and enters cells in a manner partially dependent on RNA at sites where csRBPs cluster 19 . CPPs are classically reported to leverage HSPGs for cell surface association and entry 20 , highlighting the possibility that glycoRNA-csRBP clusters and HSPGs have some relationship.…”

“…We recently described a new negatively charged glycopolymer on the cell surface called sialoglycoRNA 17 , which present sialylated and fucosylated N-glycans on small RNAs, covalently attached to one another via the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) 18 . On the cell surface, these sialoglycoRNAs are in physical proximity to specific RNA binding proteins on the plasma membrane of cells (csRBPs) 19 . The cell penetrating peptide (CPPs) TAT localizes to and enters cells in a manner partially dependent on RNA at sites where csRBPs cluster 19 .…”