A novel reporter for helicase activity in translation uncovers DDX3X interactions
- 1Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, California 94143, USA
- 2Graduate Division, University of California, San Francisco, San Francisco, California 94143, USA
- 3Faculty of Chemistry and Pharmacy, Julius-Maximilians-University of Würzburg, Würzburg 97070, Germany
- 4Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA
- 5Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California 94143, USA
- Corresponding author: stephen{at}floorlab.org
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.
Keywords
- Received September 13, 2023.
- Accepted April 12, 2024.
This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.
