Abstract
DDX3X regulates the translation of a subset of human transcripts containing complex 5' untranslated regions (5' UTRs). In this study we developed the helicase activity reporter for translation (HART) which uses DDX3X-sensitive 5' UTRs to measure DDX3X mediated translational activity in cells. To dissect the structural underpinnings of DDX3X dependent translation, we first used SHAPE-MaP to determine the secondary structures present in DDX3X-sensitive 5' UTRs and then employed HART to investigate how their perturbation impacts DDX3X-sensitivity. Additionally, we identified residues 38-44 as potential mediators of DDX3X's interaction with the translational machinery. HART revealed that both DDX3X's association with the ribosome complex as well as its helicase activity are required for its function in promoting the translation of DDX3X-sensitive 5' UTRs. These findings suggest DDX3X plays a crucial role regulating translation through its interaction with the translational machinery during ribosome scanning, and establish the HART reporter as a robust, lentivirally encoded measurement of DDX3X-dependent translation in cells.