PROJECT SUMMARY / ABSTRACT The composition and organization of the cell surface are critically important for cell-to-cell communication and for the ability of cells to interact with their surrounding environment. The cell surface is decorated with myriad glycosylated transmembrane proteins for which there is a molecular understanding of their function and cell surface presentation. Surprisingly, RNA and RNA-binding proteins (RBPs) are also glycosylated and components of the cell surface. However, is unknown what glycosylated RNAs and RBPs do at the cell surface. This unexplored area of biology represents a major knowledge gap in our understanding of both the RNA and cell surface fields. Thus, the immediate goal of this proposal is to close this knowledge gap and to create the conceptual and technical foundations that will enhance our understanding and exploration, respectively, of the biology of RNA at the surface of the cell. To achieve this, we propose to explore three fundamental, but outstanding, hypotheses. Importantly, these hypotheses are established on sound logic of the known biology of RNA and RBPs and supported by the published literature and preliminary data. Hypothesis 1 explores the molecular, biochemical, and biophysical activities of the cell surface localized RBPs DDX21 and NCL in the extracellular space. Hypothesis 2 takes an unbiased genetic approach to determine how cell surface RBPs are trafficked to the cell surface, given that they lack a classical membrane-targeting signal. Hypothesis 3 explores the intersection between cell surface RBPs and RNA in disease pathogenesis by focusing on the functional characterization of cell surface RBPs that possess cell-penetrating activities. The expected outcomes of this research are to reveal that the biochemical activities of RBPs are not restricted to the intracellular space of the cell, but also to the cell surface. The significance of the proposed research lies in expanding the field of RNA to the surface of cells and providing proof of concept of the importance of cell surface RBPs in human disease. While high-risk, the proposed research addresses an important problem and has the potential to be transformative. Hence its suitability for the Transformative Research Award.