Revealing protein-protein interactions and RNA-protein interactions at genome-scale in two weeks Abstract Our ability to interpret the human genome is limited by our knowledge of the interaction networks of the products of the genome sequence, including RNAs and proteins. If we had a complete reference map of all DNA-DNA, protein-DNA, RNA-DNA, RNA-RNA, RNA-protein, and protein-protein interactions, we would have a completely new approach to reading the book of the human genome. Recent technology breakthroughs, including those led by NIH Common Fund programs, enabled genome-wide mapping of DNA-DNA, protein-DNA, RNA-DNA, and RNA-RNA interactions en masse. However, genome-scale mapping of RNA-protein interactions (RPI) and protein-protein interactions (PPI) remain laborious and resource-intensive. In this project, we propose two extremely high-throughput genomic-based technologies for mapping human RPI and PPI networks at genome scale. We will develop bioinformatics tools to analyze the data with statistical rigor. Specifically, we propose an “all-vs-all” approach to map PPI and RPI networks. These genomics technologies and their coupled genomic informatics tools will generate reference maps of human PPI and RPI networks. In the long term, such maps will greatly facilitate the interpretation of the functions of the human genome. In Aim 1, we will develop an extremely high-throughput technology called “PPI-seq” to map the PPI network at genome-scale. PPI-seq is expected to be capable of generating a reference map of the human PPI network within a single lab in 2 weeks' time, with an expected yield of ~50,000 high-confidence pairwise PPIs. In Aim 2, we will develop an extremely high- throughput technology called “RPI-seq” to map the RPI network at genome-scale. RPI-seq is expected to be capable of generating a reference map of the human RPI network within single lab in 2 weeks' time. RPI-seq will simultaneous reveal RNA binding proteins (RBP) and the RNAs bound by each RBP.