Sensitive ADAR editing reporter in cancer cells enables high-throughput screening of small molecule libraries

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Adenosine-to-Inosine (A-to-I) RNA editing is crucial in the human transcriptome, and its dysregulation is linked to diseases like Aicardi-Goutières syndrome and various cancers.
• ADAR1 mutations cause severe disease, while its overexpression correlates with increased malignancy in breast, lung, and liver cancers. ADAR1 silencing in breast cancer cells promotes apoptosis, indicating a pro-cancer role.

Purpose of the Study:

• To develop a high-throughput screening assay for identifying chemical modifiers of ADAR1-mediated A-to-I RNA editing.
• To establish a sensitive and quantitative reporter system for assessing endogenous RNA editing activity in cancer cell lines.

Main Methods:

• Development of a bioluminescent reporter system utilizing Nanoluciferase, conditionally expressed upon successful reporter-transcript editing.
• Stable integration of the reporter system into cancer cell lines to monitor endogenous ADAR1 editing activity under various conditions (e.g., interferon induction, gene knockdown).
• Screening of a 33,000-compound small molecule library using the reporter system in a single-well setup with HeLa cells.

Main Results:

• The reporter system accurately reflects changes in endogenous ADAR1 and ADAR2 editing activity.
• The assay demonstrated a primary hit rate of 0.9% with 70% inhibition of editing when screening a large chemical library.
• Successful identification of potential modifiers of A-to-I editing activity.

Conclusions:

• The developed bioluminescent reporter system is a key tool for high-throughput identification of compounds that modulate A-to-I RNA editing.
• This assay facilitates the discovery of novel therapeutic agents targeting RNA editing in cancer progression.
• The system provides a rapid and accurate method for assessing endogenous editing activity, crucial for disease-related research.