loading . . . Inositol hexakisphosphate Functions as a Cofactor and Modulator of ADAR1 Activity Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by adenosine deaminases acting on RNA (ADARs), is a key post-transcriptional modification that regulates RNA splicing, stability, and translation. Dysregulation of ADAR activity caused by mutations in ADAR1 leads to Aicardi-Goutieres syndrome (AGS), an autoimmune disorder characterized by aberrant activation of Melanoma differentiation-associated protein 5 (MDA5) by self RNA and excessive type I interferon production. Despite its biological and clinical relevance, the regulation of ADAR1 activity remains incompletely understood. Here, we show that ADAR1 protein levels and RNA editing activity in mammalian cells critically depend on the cofactor inositol hexakisphosphate (IP6). Using Inositol-pentakisphosphate 2-kinase (IPPK)-knockout cells, next-generation sequencing (NGS), and a cell-permeable IP6 prodrug (Pro-IP6), we demonstrate that IP6 depletion drastically reduces global RNA editing, while supplementation with Pro-IP6 restores and even enhances editing levels. In vitro ADAR1 translation and RNA editing assays revealed that IP6 contributes to the folding and full catalytic activity of ADAR1, and that inositol pentakisphosphate (1,3,4,5,6-IP5) can partially substitute IP6 as a cofactor. Molecular dynamics simulations and biochemical analyses identified the C6-phosphate of IP6 as a critical determinant of ADAR1 catalytic efficiency, functioning within a hydrogen-bonding network that indirectly governs Zn2+-ion positioning through interactions with key residues, including K1039 and N907. Notably, the AGS-associated N907S mutation impairs RNA editing, by altering IP6 coordination and introducing a more dynamic situation in the hydrogen-bonding network that linked IP6 and Zn2+-ion. Together, these findings identifies IP6 as an essential cofactor and regulator of ADAR1 activity and highlights cofactor availability and interaction networks as potential strategies for therapeutically modulating RNA editing in disease. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, Project-ID 369799452 TRR237 - A02, Project-ID 548714673, CIBSS EXC-2189 Project ID 390939984 Volkswagen Foundation, VW Momentum Grant 98604 Israel Science Foundation, 2637/23 Medical Research Council, MR/T028904/1 https://www.biorxiv.org/content/10.64898/2026.01.17.699700v1
