RNA Editing by ADAR Adenosine Deaminases in the Cell Models of CAG Repeat Expansion Diseases: Significant Effect of Differentiation from Stem Cells into Brain Organoids in the Absence of Substantial Influence of CAG Repeats on the Level of Editing

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Viacheslav V. Kudriavskii^1,2,4,a*, Anton O. Goncharov^1,2, Artem V. Eremeev², Evgenii S. Ruchko², Vladimir A. Veselovsky², Ksenia M. Klimina², Alexandra N. Bogomazova², Maria A. Lagarkova², Sergei A. Moshkovskii^2,3,5,b*, Anna A. Kliuchnikova^1,2,3

¹Pirogov Russian National Research Medical University, 117997 Moscow, Russia

²Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435 Moscow, Russia

³Institute of Biomedical Chemistry, 119121 Moscow, Russia

⁴Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology, 121205 Moscow, Russia

⁵Max Planck Institute for Interdisciplinary Research, Göttingen, 37077, Germany

^* To whom correspondence should be addressed.

Received: April 1, 2024; Revised: June 24, 2024; Accepted: July 11, 2024

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Expansion of CAG repeats in certain genes is a known cause of several neurodegenerative diseases, but exact mechanism behind this is not yet fully understood. It is believed that the double-stranded RNA regions formed by CAG repeats could be harmful to the cell. This study aimed to test the hypothesis that these RNA regions might potentially interfere with ADAR RNA editing enzymes, leading to the reduced A-to-I editing of RNA and activation of the interferon response. We studied induced pluripotent stem cells (iPSCs) derived from the patients with Huntington’s disease or ataxia type 17, as well as midbrain organoids developed from these cells. A targeted panel for next-generation sequencing was used to assess editing in the specific RNA regions. Differentiation of iPSCs into brain organoids led to increase in the ADAR2 gene expression and decrease in the expression of protein inhibitors of RNA editing. As a result, there was increase in the editing of specific ADAR2 substrates, which allowed identification of differential substrates of ADAR isoforms. However, comparison of the pathology and control groups did not show differences in the editing levels among the iPSCs. Additionally, brain organoids with 42-46 CAG repeats did not exhibit global changes. On the other hand, brain organoids with the highest number of CAG repeats in the huntingtin gene (76) showed significant decrease in the level of RNA editing of specific transcripts, potentially involving ADAR1. Notably, editing of the long non-coding RNA PWAR5 was nearly absent in this sample. It could be stated in conclusion that in most cultures with repeat expansion, the hypothesized effect on RNA editing was not confirmed.

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KEY WORDS: RNA editing, ADAR, trinucleotide repeat diseases, Huntington’s disease, ataxia type 17, induced pluripotent stem cells, midbrain organoids

DOI: 10.1134/S0006297924080078

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