Biological roles and mechanisms of cell surface RNAs and RNA-binding proteins in human papillomavirus endocytic trafficking

The subcellular localization of a biomolecule often reveals its functional significance. RNA has typically been confined to the cytoplasm and nucleus, where it is critically involved in numerous biochemical processes. Recently, glycosylated RNAs (glycoRNAs) have been discovered on the cell surface of various cell types, raising important questions about their functional relevance, mechanisms of action, and the trafficking pathways that maintain their dynamic equilibrium on the cell surface. The long-term goal of this proposal is to discover and define the functional mechanisms of cell surface RNAs in endocytosis and pathogen endocytic trafficking. Preliminary evidence suggests that cell surface RNAs are critically required in human papillomavirus (HPV) infection. This MIRA proposal is firstly focused on deciphering the functional roles of cell surface RNA in HPV endocytic trafficking. Here a combination of advanced imaging strategies, biochemical techniques, and genetic analysis will be used to dissect and determine the specific step in HPV cell entry that is regulated by cell surface RNAs. We are also exploring the mechanisms underlying this process by leveraging recent discoveries from my lab, including the identification of the cell surface RNA binding protein (RBP) DDX3X, a potential recognition site for cell surface RNA-DDX3X clustering on the HPV cell-penetrating peptide (CPP), and glycoRNA transcripts identified through RNA sequencing. Lastly, we are investigating the trafficking modes utilized by cell surface RNAs, which are critical for their cell surface presentation and functions. Collectively, this research addresses fundamental questions in cell membrane trafficking and provides valuable insights into the recently discovered cell surface RNAs. It may also introduce a new concept in which cell surface RNAs and RBPs cluster to form an endocytic receptor complex involved in pathogen cell entry. While many exciting questions lie beyond the scope of this MIRA proposal, the findings could stimulate broad interest, offer proof-of-concept frameworks, and develop techniques that enhance our understanding of the role of cell surface RNAs in endocytosis and pathogen entry mechanisms.