Decoupling expression and editing preferences of ADAR1 p150 and p110 isoforms

Abstract

Significance ADAR1, an A-to-I RNA editing enzyme, is an essential gene and also an attractive target for cancer therapy. Mutations in ADAR1 cause devastating autoinflammatory diseases, and inhibition of ADAR1 remarkably reduces tumor growth. ADAR1 binds and edits RNA through two isoforms: p150 (150 kDa) and p110 (110 kDa), but the function and RNA substrates of each isoform are incompletely understood. Here we report that the p150 mRNA is capable of coexpressing p150 and p110 isoforms. This finding raises the importance of investigating the biological significance behind coupled expression of p150 and p110. To this end, we developed a genetic strategy to decouple p150 and p110 expression that allows for determination of isoform-selective RNA editing events. Human adenosine deaminase acting on RNA 1 (ADAR1) catalyzes adenosine-to-inosine deamination reactions on double-stranded RNA molecules to regulate cellular responses to endogenous and exogenous RNA. Defective ADAR1 editing leads to disorders such as Aicardi-Goutières syndrome, an autoinflammatory disease that manifests in the brain and skin, and dyschromatosis symmetrica hereditaria, a skin pigmentation disorder. Two ADAR1 protein isoforms, p150 (150 kDa) and p110 (110 kDa), are expressed and can edit RNA, but the contribution of each isoform to the editing landscape remains unclear, largely because of the challenges in expressing p150 without p110. In this study, we demonstrate that p110 is coexpressed with p150 from the canonical p150-encoding mRNA due to leaky ribosome scanning downstream of the p150 start codon. The presence of a strong Kozak consensus context surrounding the p110 start codon suggests the p150 mRNA is optimized to leak p110 alongside expression of p150. To reduce leaky scanning and translation initiation at the p110 start codon, we introduced synonymous mutations in the coding region between the p150 and p110 start codons. Cells expressing p150 constructs with these mutations produced significantly reduced levels of p110. Editing analysis of total RNA from ADAR1 knockout cells reconstituted separately with modified p150 and p110 revealed that more than half of the A-to-I edit sites are selectively edited by p150, and the other half are edited by either p150 or p110. This method of isoform-selective editing analysis, making use of the modified p150, has the potential to be adapted for other cellular contexts.