Project Summary/Abstract The overarching goal of my research is to define how the molecular grammar of RNA molecules regulates gene expression. To accomplish this goal, I have pursued extensive and interdisciplinary training in the molecular, cellular, and systems biology understanding of human RNA biology. I seek to build upon my documented track record in RNA biology and method development to address pressing questions in RNA biology. This proposal identifies two emphasis areas of importance for inquiry in the next five years. The first emphasis of this proposal builds on my decade of research into the RNA–binding protein DDX3 by identifying and exploring critical gaps in understanding. DDX3 is an essential ATP-dependent RNA–binding protein that couples ATP binding to local RNA duplex unwinding and ribonucleoprotein remodeling. Prior work from my group and others has implicated DDX3 in translational control for mRNA molecules containing a variety of mRNA elements, but the precise mechanism, important mRNA features, and genetic interactions remain incompletely understood. Here, we seek to define how DDX3 interacts with the ribosome to mediate translational control, to use a new assay we developed to define DDX3-dependent translation in an unbiased manner, to define how depletion of DDX3 versus missense variants differ in genetic interactions, and to establish the mechanism leading to changes in RNA levels following DDX3 depletion. The second emphasis of this proposal advances new developments in my group that enable single-molecule measurement of RNA- protein interactions in cells. We evolved a new deamination-based molecular recorder to capture RNA-protein interactions by modifying the sequence of RNA adjacent to an RNA-protein interaction. Through long-read sequencing we can then identify regions in RNA that were bound by a protein tagged with the deaminase. Using this approach, we find unexpected heterogeneity in RNA–binding protein sites on individual mRNA molecules. We propose to build upon these findings, both to understand the mechanistic and functional implications of this heterogeneity and to extend our approach to new RNA–binding proteins. Overall, the proposed research is aligned with my research goal by defining the mechanism of important RNA–binding proteins and by developing new approaches to measure single-molecule RNA biology. I expect the results of the proposed research to advance the understanding of RNA biology with implications for the fundamental understanding of RNA, human disease, and mRNA therapeutics.