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A Nonsequencing Approach for the Rapid Detection of RNA Editing
Published on: April 21, 2022
Quantification of ongoing APOBEC3A activity in tumor cells by monitoring RNA editing at hotspots
Pégah Jalili1, Danae Bowen1, Adam Langenbucher2
1Department of Biological Chemistry, Center for Epigenetics and Metabolism, Chao Family Comprehensive Cancer Center, University of California Irvine, Irvine, CA, USA.
Abstract:
APOBEC3A is a cytidine deaminase driving mutagenesis, DNA replication stress and DNA damage in cancer cells. While the APOBEC3A-induced vulnerability of cancers offers an opportunity for therapy, APOBEC3A protein and mRNA are difficult to quantify in tumors due to their low abundance. Here, we describe a quantitative and sensitive assay to measure the ongoing activity of APOBEC3A in tumors. Using hotspot RNA mutations identified from APOBEC3A-positive tumors and droplet digital PCR, we develop an assay to quantify the RNA-editing activity of APOBEC3A. This assay is superior to APOBEC3A protein- and mRNA-based assays in predicting the activity of APOBEC3A on DNA. Importantly, we demonstrate that the RNA mutation-based APOBEC3A assay is applicable to clinical samples from cancer patients. Our study presents a strategy to follow the dysregulation of APOBEC3A in tumors, providing opportunities to investigate the role of APOBEC3A in tumor evolution and to target the APOBEC3A-induced vulnerability in therapy.
Insights
Researchers developed a sensitive assay to measure APOBEC3A activity in tumors by detecting RNA mutations. This new method accurately reflects APOBEC3A
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- APOBEC3A (A3A) is a cytidine deaminase implicated in cancer mutagenesis, DNA replication stress, and DNA damage.
- The APOBEC3A-induced cancer vulnerability presents a therapeutic target.
- Quantifying APOBEC3A protein and mRNA in tumors is challenging due to low abundance.
Purpose of the Study:
- To develop a quantitative and sensitive assay for measuring ongoing APOBEC3A activity in tumors.
- To establish an assay superior to protein and mRNA quantification for predicting APOBEC3A's DNA activity.
- To validate the assay's applicability to clinical cancer patient samples.
Main Methods:
- Identification of hotspot RNA mutations specific to APOBEC3A-positive tumors.
- Development of a droplet digital PCR (ddPCR) assay to quantify APOBEC3A RNA-editing activity.
- Comparison of the RNA mutation-based assay with APOBEC3A protein and mRNA assays.
Main Results:
- The developed ddPCR assay accurately quantifies APOBEC3A RNA-editing activity.
- The RNA mutation-based assay demonstrates superior predictive value for APOBEC3A's DNA activity compared to protein/mRNA assays.
- The assay is successfully applied to clinical samples from cancer patients.
Conclusions:
- A novel, sensitive RNA mutation-based assay for APOBEC3A activity has been established.
- This assay overcomes limitations of protein and mRNA quantification in tumors.
- The assay provides a strategy to monitor APOBEC3A dysregulation, aiding cancer evolution studies and therapeutic targeting.
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