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An in-cell helicase reporter system for quantifying DDX3X and DDX3Y activities
Zhi Sheng Poh1, James Chia Wei Tan1, Brandon Han Siang Wong1,2
1Lee Kong Chian School of Medicine Nanyang Technological University Singapore Singapore.
Abstract:
Genome sequencing has identified numerous mutations in the DEAD-box RNA helicases, DDX3X and DDX3Y, associated with cancer and other diseases, but monitoring of their functional consequences remains a challenge. Conventional helicase assays are laborious, often technically difficult, and are performed in cell-free systems that do not address biologically relevant questions. Here, we developed an engineered DDX3 reporter cell system capable of interrogating helicase activities of DDX3X and DDX3Y and their mutational variants. For this, we deleted the endogenous DDX3X in human 293T cells using CRISPR/Cas9. DDX3Y is absent in 293T cells being a female-derived line. We transfected cells with firefly luciferase plasmids that provided bioluminescence signals, depending on helicase activities of exogenously expressed wild-type or mutant DDX3X or DDX3Y, and inserted Aequorea coerulescens Green Fluorescent Protein (AcGFP) as an internal control separated by an internal ribosome entry site (IRES). The developed reporter system can be applied to screen compound libraries targeting DDX3X or DDX3Y in living cells and study their functional roles in health and disease.
Insights
Researchers developed a novel reporter cell system to monitor the function of DEAD-box RNA helicases DDX3X and DDX3Y. This system allows for the study of mutations linked to diseases like cancer in living cells.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Mutations in DEAD-box RNA helicases DDX3X and DDX3Y are linked to various diseases, including cancer.
- Assessing the functional impact of these mutations is challenging due to limitations of conventional cell-free helicase assays.
Purpose of the Study:
- To develop a robust reporter cell system for interrogating the helicase activities of DDX3X and DDX3Y and their variants.
- To enable the study of DDX3X and DDX3Y functions and mutational consequences in a biologically relevant cellular context.
Main Methods:
- Engineered human 293T cells with endogenous DDX3X deleted using CRISPR/Cas9.
- Transfected cells with reporter plasmids encoding firefly luciferase, dependent on DDX3X/DDX3Y helicase activity, and Aequorea coerulescens Green Fluorescent Protein (AcGFP) as an internal control via IRES.
Main Results:
- Successfully established a DDX3 reporter cell system that measures helicase activity through bioluminescence.
- The system can differentiate between wild-type and mutant DDX3X and DDX3Y activities in living cells.
Conclusions:
- The developed reporter system provides a powerful tool for studying DDX3X and DDX3Y functional consequences in cellular models.
- This system facilitates screening of drug libraries targeting DDX3X/DDX3Y and aids in understanding their roles in health and disease.
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