3'UTR RNA editing driven by ADAR1 modulates MDM2 expression in breast cancer cells

Abstract

Epitranscriptomic changes in the transcripts of cancer related genes could modulate protein levels. RNA editing, particularly A-to-I(G) editing catalyzed by ADAR1, has been implicated in cancer progression. RNA editing events in the 3’ untranslated region (3’UTR) can regulate mRNA stability, localization, and translation, underscoring the importance of exploring their impact in cancer. Here, we performed an in silico analysis to detect breast cancer enriched RNA editing sites using the TCGA breast cancer RNA-seq dataset. Notably, the majority of differential editing events mapped to 3’ untranslated regions (3’UTRs). We confirmed A-to-I(G) editing in the 3’UTRs of MDM2 (Mouse Double Minute 2 homolog), GINS1 (GINS Complex Subunit 1), and F11R (Junctional Adhesion Molecule A) in breast cancer cells. RNA immunoprecipitation with ADAR1 antibody confirmed the interaction between ADAR1 and MDM2, GINS1, and F11R 3’UTRs. ADAR1 knockdown revealed decreased editing levels, establishing ADAR1 as the editing enzyme. A reporter assay for MDM2, an oncogene overexpressed mostly in luminal breast cancers, demonstrated that RNA editing enhances protein expression, in agreement with reduced MDM2 protein levels in ADAR1 knockdown cells. Further exploration into the mechanisms of 3’UTR editing events revealed an interaction between ADAR1 and CSTF2, a core component of the polyadenylation machinery, as identified through biotin-based proximity labeling mass spectroscopy, and co-immunoprecipitation experiments. Furthermore, CSTF2 knockdown reduced both ADAR1 and MDM2 protein levels. Our findings highlight implications for MDM2 regulation by ADAR1-dependent 3’UTR RNA editing and present an interplay between RNA editing on 3’UTRs and the mRNA polyadenylation machinery. These results improve our understanding of ADAR1’s role in cancer-associated 3’ UTR RNA editing and its potential as a therapeutic target.