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 directly measure RNA structure in DDX3X-dependent mRNAs, we used SHAPE-MaP to determine the secondary structures present in DDX3X-sensitive 5' UTRs and then used HART to investigate how sequence alterations influence 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 translational machinery and 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 in regulating translation through its interaction with the translational machinery during ribosome scanning and establish the HART reporter as a robust, lentivirally encoded, colorimetric measurement of DDX3X-dependent translation in cells.
Subject(s)
5' Untranslated Regions , DEAD-box RNA Helicases , Genes, Reporter , Protein Biosynthesis , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , Humans , Nucleic Acid Conformation , RNA, Messenger/genetics , RNA, Messenger/metabolism , HEK293 Cells , Protein BindingABSTRACT
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.
ABSTRACT
Translation initiation is the first step in protein synthesis, during which the small subunit of the ribosome scans the 5' untranslated region (5'UTR) of an mRNA to identify a start codon and commence translation elongation. By unwinding and modulating secondary structures and other RNA features present in the 5'UTR, RNA helicases can regulate ribosome scanning and start codon selection. This chapter presents an approach to measure the effect of RNA helicases on mRNA translation initiation. 5'UTR luciferase reporters are transcribed in vitro and employed in either of two assays. The in vitro assay translates the reporters in a cell-free whole-cell lysate system, which allows for greater biochemical manipulation and tighter control over confounding effects. In the alternative cell-based approach, the reporters are transfected and translated in living cells, which provides a more physiological setup. Either method can be used to investigate how the perturbation of a helicase, such as changes in protein levels or mutations, affects translation initiation at the 5'UTR level. The chapter also discusses alternative approaches, troubleshooting, and further applications of these methods. These assays will provide insights on the role of helicases and other translational factors as regulators of the proteome both in physiological and diseased settings.