A to I RNA editing by ADAR1 is influenced by inositol hexaphosphate and prevents RIG-I activation

• Applicant: Dr. Francisco Venegas Solis
• Subject Area: Immunology ; Biochemistry
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 548714673

Adenosine deaminase acting on RNA 1 (ADAR1) catalyzes the conversion of adenosine to inosine in both endogenous and exogenous double-stranded RNA (dsRNA). This enzyme plays a crucial role in preventing the activation of the receptor MDA5 by endogenous dsRNA, thereby mitigating the overexpression of type I interferon (IFN) observed in mouse models with ADAR1 deficiency. Of note, ADAR1 has been implicated in various conditions including cancer, autoimmune diseases, and exhibits antiviral properties in specific contexts. Despite its significance, the regulation and modulation of ADAR1 and its potential implications in various functions and diseases remain understudied. In 2005, the crystal structure of human ADAR2, an enzyme belonging to the same family as ADAR1, revealed the presence of inositol hexaphosphate (IP6) in the catalytic domain (Macbeth et al., 2005). This study highlighted the involvement of IP6 in the correct folding of hADAR2 and its modulating role in editing activity. However, the role of IP6 in ADAR1, the major RNA editing enzyme in humans, and in mammalian cells, remains unexplored. Here, we aim to investigate the role of IP6 in RNA editing by ADAR1 in mammalian cells using WT and KO Inositol-pentakisphosphate 2-kinase (IPPK) HeLa cells, where IPPK is the enzyme responsible for IP6 production. Additionally, we plan to assess potential correlations between editing levels, ADAR1/2 expression, and IP6 levels using primary human immune cells and cell lines. Furthermore, we will explore the effects of IP6 and other factors such as inositol phosphates, pH, and temperature on in vitro editing reactions by ADAR1/2. Finally, HEK293-RIG-I luciferase reporter cells, PBMCs, and edited dsRNA by in vitro translated ADAR1 will be utilized to study the impact of edited RNA on RIG-I activation. The project has the potential to enhance our understanding of the regulatory influence of IP6 on the editing function of ADAR1/2, as well as shed new light on the role of editing in either activating or inhibiting RIG-I. These insights may be valuable for the new therapeutic strategies using endogenous ADAR activity, while also deepening our comprehension of ADARs' functional dynamics.

• DFG Programme: WBP Position