Decoupling expression and editing preferences of ADAR1 p150 and p110 isoforms

Abstract

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.

ADAR1 interaction with Z-RNA promotes editing of endogenous double-stranded RNA and prevents MDA5-dependent immune activation

“…In this study, we performed a transcriptome-wide analysis of thousands of A-to-I sites in human and mouse cells that express Za domain mutant ADAR1 and found that the overall editing efficiency was reduced by loss of ADAR1/Z-RNA interaction. While the reduction in ADAR1 activity in mouse cells applied to whole transcripts, this was particularly evident for 3 0 UTRs in human transcripts, which is in agreement with a recent study (Sun et al, 2021). As introns are spliced out before nuclear export, SINE-derived dsRNA structures in 3 0 UTRs are most likely responsible for the activation of the cytosolic dsRNA sensor MDA5.…”

The ADAR1 editome reveals drivers of editing-specificity for ADAR1-isoforms

“…We compared the outcome of our RIP-Seq results that were performed in HEK293 cells with the currently published ADAR1-isoform-specific editome that was also generated in HEK293 cells (Sun et al, 2021). To do so, we employed CrossMap (v0.5.4) (Zhao et al, 2014) to convert the set of identified editing sites to the GRCh38 assembly.…”

An I for an A: Dynamic Regulation of Adenosine Deamination-Mediated RNA Editing

“…The NES-bearing cytoplasmic ADAR1-p150 isoform is expressed from an interferon-regulated promoter [ 54 ]. Inflammation-mediated interferon signaling thus leads to the cytoplasmic accumulation of this isoform and therefore will boost editing of cytoplasmically localized RNAs [ 55 , 56 ]. In contrast, the nucleo-cytoplasmic shuttling of ADAR1 p110 is controlled by its RNA-bound state [ 45 ].…”