Single-stranded circular RNAs (circRNA) are enriched in cancers, yet the regulation and biological function of most circRNA remains unclear. Our long-term goal is to understand the role of both human papillomavirus (HPV)-derived and endogenous circRNAs in both infectious and neoplastic diseases. The specific goal of this proposal is to generate a more comprehensive understanding of the function and regulation of circRNA that are present in head and neck squamous cell carcinoma (HNSCC). Our central hypothesis is that many circRNA are coding RNA that differ in regulation and function from their corresponding full-length RNAs in ways that promote the development of HNSCC. In preliminary studies, we have developed innovative circRNA-Seq and Polysome RNA-Seq protocols and found that many circRNA with coding potential are enriched in HNSCC tumors compared to adjacent non-tumor mucosa. In addition, our discovery of HPV16 circular E7 RNA (circE7), which is translated to the E7 oncoprotein, offers a novel tool to understand the mechanistic and physiological regulation of circRNA formation and function. With these innovative tools and extensive preliminary evidence supporting feasibility, we propose to expand our understanding of circRNAs in HNSCC in three related aims. First, a comprehensive transcriptomic profile from up to 50 HPV+ and 50 HPV- HNSCC tumors and adjacent tissue will be generated, including RNA-Seq approaches that enrich for circRNAs. Moreover, circRNA identified in this transcriptomic profiling and circE7 will be assessed for their potential as prognostic biomarkers in a validation cohort of archived HNSCC. Second, using the HPV16 circE7 locus, we will determine the cis and trans elements that regulate circRNA formation in HNSCC and determine how they are physiologically regulated. Third, we will combine HNSCC circRNA-Seq datasets with recently completed polysome RNA-Seq to identify endogenous circRNA that are likely to be translated. These prioritized candidates will be validated and characterized through rigorously controlled overexpression and knockdown experiments including the use of circRNA-derived specific peptide antibodies. The investigative team includes experts in circRNA metabolism, HPV biology, pathology, statistical genetics, head and neck cancer, and epidemiology, thus ensuring we have the skills and resources to execute the proposal. Completion of the proposal will provide both a global and detailed understanding of circRNA and their regulation in HNSCC.