ADAR (adenosine deaminase acting on RNA) converts adenosine residues to inosine (A-to-I RNA editing) in double-stranded RNA. At the very beginning of the previous 27 years of this grant, we identified ADAR1, the first member of the ADAR gene family. This in turn led to the identification of ADAR2 and ADAR3. Since then we have made major contributions to development of the A-to-I RNA editing field, in particular by focusing on understanding the biological functions of ADAR1. ADAR1 seems to have multiple functions, some editing-dependent and the others editing-independent, in protein-recoding of select genes, editing of retrotransposon derived repeat elements, suppression of innate immunity, regulation of RNA interference, and stress response. Even so, it is not yet clear whether these already described ADAR1 functions are the reasons why the ADAR1 gene has been retained over the course of evolution of the vertebrate genome. Nascent RNA usually dissociates from its template DNA strand after transcription, but occasionally the newly transcribed RNA forms a stable RNA:DNA hybrid, one consequence of which is leaving the sense DNA in a single-stranded form. This structure is called an R-loop, and causes abortive transcription and instability of the genome, resulting in DNA damage, mutations, and replication stress. R-loop accumulation leads to human diseases such as Aicardi-Goutires syndrome (AGS), a severe autoimmune disease caused by inflammatory responses to nucleic acids. Interestingly, in a subgroup of AGS patients (AGS6), the disease is a result of mutations in ADAR1. Experimentally, we have obtained preliminary results suggesting that ADAR1 knockdown results in significant accumulation of R-loops and mitotic catastrophe. During the next grant support period, we will explore the R-loop regulatory function of ADAR1 and its relevance to the mechanisms that maintain the stability of the human genome. We will first investigate the R- loop dissociation mechanism in vitro using recombinant proteins and a reconstituted R-loop structure made with synthetic RNA/DNA oligonucleotides. We will examine how the efficiency of R-loop dissociation is affected by A-to-I RNA editing mediated by ADAR1. We will determine globally the precise locations of R-loops specifically regulated by ADAR1 by DRIP-seq of both the RNA and the DNA strands of isolated R-loops. We will visualize particular chromosome regions, such as centromeres and telomeres, where persistence of R- loops may be specifically regulated by ADAR1 using fluorescent proteins fused to region specific markers such as CENPA and TRF1. Finally, we will test our hypothesis that accumulated R-loops are the causative nucleic acids for aberrant IFN production and inflammatory responses detected in ADAR1 null mouse embryos and AGS6 patients. R-loops isolated from ADAR1 null mouse embryos and HeLa cells carrying the ADAR1 mutations of AGS6 will be investigated by DRIP-seq. Together, these experiments will reveal the evolutionarily most important in vivo function of ADAR1, namely maintenance of genome stability.

Public Health Relevance

R-loop accumulation causes human diseases such as Aicardi-Goutires syndrome (AGS), frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), ataxia-ocular apraxia type 2 (AOA2), and fragile X syndrome (FXS). The molecular mechanisms of R-loop formation and resolution are largely unknown, and definitive cures for the associated diseases are not available at this time. Our recent findings indicate that ADAR1 plays a major role in resolution of R-loops. Therefore, our proposal to expand our previous studies and define the exact function of ADAR1 in the control of R-loop persistence is highly relevant to the diseases caused by accumulation of R-loops, AGS, FTD, ALS AOA2, and FXS.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics B Study Section (MGB)
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Krasnewich, Donna M
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Wistar Institute
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Nishikura, Kazuko (2017) Oesophageal cancer: RNA editing of SLC22A3 mRNAs: causative relevance to familial ESCC? Nat Rev Gastroenterol Hepatol 14:569-570
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Watanabe, Yoshihisa; Yoshimoto, Kanji; Tatebe, Harutsugu et al. (2014) Enhancement of alcohol drinking in mice depends on alterations in RNA editing of serotonin 2C receptors. Int J Neuropsychopharmacol 17:739-51
Ota, Hiromitsu; Sakurai, Masayuki; Gupta, Ravi et al. (2013) ADAR1 forms a complex with Dicer to promote microRNA processing and RNA-induced gene silencing. Cell 153:575-89
Nishikura, Kazuko; Sakurai, Masayuki; Ariyoshi, Kantaro et al. (2013) Antagonistic and stimulative roles of ADAR1 in RNA silencing. RNA Biol 10:1240-7

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