Quantification of ongoing APOBEC3A activity in tumor cells by monitoring RNA editing at hotspots

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.

Similarity between mutation spectra in hypermutated genomes of rubella virus and in SARS-CoV-2 genomes accumulated during the COVID-19 pandemic

“…Unlike A to G changes, in-depth analysis of C to U substitutions revealed that they were predominantly present in the RNA plus-strands of both viruses and demonstrated a preference for loops versus stems in the RNA secondary structure (Fig 3 and S5 Table). This phenomenon is similar to the preference of ssDNA and mRNA editing in loops by the APOBEC3A cytidine deaminase [35, 38]. Agnostic analysis of enrichments in all 192 possible trinucleotide mutation motifs highlighted statistically significant excess of uCa to uUa motif in rubella, however these changes were not prevalent in SARS-CoV-2.…”

“…C to U mutations in RNA can be also caused by non-enzymatic deamination of cytidines similar to such deamination described in DNA [15, 34]. Recently it was revealed that APOBEC3A has a preference for deaminating cytosines in regions prone to forming loops in ssDNA secondary structure [35]. Therefore we annotated all positions in the SARS-CoV-2 and rubella genomes for either preference for loop or stem location in potential secondary structure formed by the RNA plus-strand ([29] S1B and S3B Tables and Methods).…”

Similarity between mutation spectra in hypermutated genomes of rubella virus and in SARS-CoV-2 genomes accumulated during the COVID-19 pandemic

“…is similar to the preference of ssDNA and mRNA editing in loops by the APOBEC3A cytidine deaminase [39,43], which so far is the only RNA-editing APOBEC explored for this feature. Agnostic analysis of enrichments in all 192 possible trinucleotide mutation motifs highlighted statistically significant excess of uCa to uUa motif in rubella, however these changes were not prevalent in SARS-CoV-2.…”

“…C to U mutations in RNA can be also caused by non-enzymatic deamination of cytidines similar to such deamination described in DNA [16,38]. Recently it was revealed that APOBEC3A has a preference for deaminating cytosines in regions prone to forming loops in ssDNA secondary structure [39]. Therefore we annotated all positions in the SARS-CoV-2 and rubella genomes for either preference for loop or stem location in potential secondary structure formed by the RNA plus-strand ([33] S1B and S3B Tables and Methods).…”

A mark of disease: how mRNA modifications shape genetic and acquired pathologies

“…Yet, no inactivating mutations have been identified in humans so far. Destefanis et al Beyond APOBEC1, other AID/APOBECs target RNA (Sharma et al 2015(Sharma et al , 2016Asaoka et al 2019;Jalili et al 2020). Among them, APOBEC3A is induced by hypoxia and interferon in monocytes and macrophages (Sharma et al 2015).…”