Deciphering the RNA Landscapes on Mammalian Cell Surfaces.

Abstract

Cell surface RNAs, notably glycoRNAs, have been reported, yet the precise compositions of surface RNAs across different primary cell types remain unclear. Here, we introduce a comprehensive suite of methodologies for profiling, imaging, and quantifying specific surface RNAs. We present AMOUR, a method leveraging T7-based linear amplification, to accurately profile surface RNAs while preserving plasma membrane integrity. By integrating fluorescently labeled DNA probes with live primary cells, and employing imaging along with flow cytometry analysis, we can effectively image and quantify representative surface RNAs. Utilizing these techniques, we have identified diverse non-coding RNAs present on mammalian cell surfaces, expanding beyond the known glycoRNAs. We confirm the membrane anchorage and quantify the abundance of several representative surface RNA molecules in cultured HeLa cells and human umbilical cord blood mononuclear cells (hUCB-MNCs). Our imaging and flow cytometry analyses unequivocally confirm the membrane localization of Y family RNAs, spliceosomal snRNA U5, mitochondrial rRNA MTRNR2, mitochondrial tRNA MT-TA, VTRNA1-1, and the long non-coding RNA XIST. Our study not only introduces effective approaches for investigating surface RNAs but also provides a detailed portrayal of the surface RNA landscapes of hUCB-MNCs and murine blood cells, paving the way for future research in the field of surface RNAs.