ADAR (adenosine deaminase acting on RNA) converts adenosine residues to inosine (A-to-I RNA editing) in double-stranded RNA (dsRNA). Three ADAR gene family members (ADAR1-3) have been identified in vertebrates. A-to-I editing of protein-coding mRNAs results in synthesis of proteins not directly encoded by the genome. This protein-recoding type RNA editing occurs to only a limited number of ion channel and neurotransmitter gene transcripts, and represents a miniscule fraction of total edited RNAs. The biological significance of the bulk of A-to-I RNA editing and the majority of ADAR functions largely remain to be established. During the past 22 years, this grant has enabled us to clone ADAR1, the first identified member of the ADAR gene family. This in turn has led to the identification and cloning of ADAR2 and ADAR3. We determined substrate specificity and enzymatic characteristics of ADARs and demonstrated a requirement of RNA-binding independent homodimerization for enzymatic activity of ADAR1 and ADAR2. Furthermore, we have created an ADAR1-/- mutation in mice that causes widespread apoptosis and consequent embryonic lethal phenotype. Finally, we demonstrated that both ADAR1 and ADAR2 edit specific adenosine residues of certain miRNA precursor dsRNAs (pri-miRNAs). A-to-I editing of pri-miRNAs inhibits expression and function of mature miRNAs, revealing the presence of an antagonistic interaction between RNA editing and RNAi pathways. During the current grant support period, we discovered a new and A-to-I RNA editing-independent function of ADAR1 in the RNAi mechanism. ADAR1 forms an RNA-binding independent complex with Dicer via its second dsRNA binding domain (dsRBD2) and the DEAD-box RNA helicase and DUP283 domains of Dicer. ADAR1 in the Dicer complex facilitates the pre-miRNA dicing reaction and promotes RISC loading of miRNA, revealing the presence of a completely different type of interaction, stimulative interaction, of the RNA editing and the RNAi machineries. Furthermore, we found that ADAR1 partitioned between its two functional roles (RNAi and RNA editing) by forming either Dicer/ADAR1 heterodimers or ADAR1 homodimer complexes through its dsRBD2 and dsRBD3 domains, respectively. In this application, we will focus our research efforts on this newly discovered stimulative interaction between RNA editing and RNAi. Specifically, we will determine: 1) the mechanism that regulates the switch of ADAR1 function from A-to-I editor to RNAi silencer. 2) the stress-responsive miRNAs generated by the Dicer/ADAR1 complex and their target genes. 3) the importance of ADAR1 RNAi function during development by phenotypic analysis of new mutant mouse lines defective specifically in either formation of the Dicer/ADAR1 complex or A-to-I RNA editing, respectively.

Public Health Relevance

MiRNAs play critical roles in many biological processes, such as tissue differentiation, cell proliferation, embryonic development, and apoptosis. For this reason, the mutation of miRNAs, the dysfunction of miRNA biogenesis, and the dysregulation of miRNAs result in various diseases including cancers, cardiovascular disease, and diabetes. Dicer is absolutely required for processing of miRNAs, and thus, it is important to better understand the mechanism that controls activities of Dicer. Our recent findings indicate that ADAR1 interacts with Dicer and promotes its function in miRNA processing and RNAi mechanisms. Therefore, our proposal to expand our previous studies and define the function of ADAR1 in the control of Dicer activities is highly relevant to many human diseases.

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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Bender, Michael T
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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
Sakurai, Masayuki; Shiromoto, Yusuke; Ota, Hiromitsu et al. (2017) ADAR1 controls apoptosis of stressed cells by inhibiting Staufen1-mediated mRNA decay. Nat Struct Mol Biol 24:534-543
Tan, Meng How; Li, Qin; Shanmugam, Raghuvaran et al. (2017) Dynamic landscape and regulation of RNA editing in mammals. Nature 550:249-254
Song, Chunzi; Sakurai, Masayuki; Shiromoto, Yusuke et al. (2016) Functions of the RNA Editing Enzyme ADAR1 and Their Relevance to Human Diseases. Genes (Basel) 7:
Miyake, Kotaro; Ohta, Toshio; Nakayama, Hisako et al. (2016) CAPS1 RNA Editing Promotes Dense Core Vesicle Exocytosis. Cell Rep 17:2004-2014
Nishikura, Kazuko (2016) A-to-I editing of coding and non-coding RNAs by ADARs. Nat Rev Mol Cell Biol 17:83-96
Gumireddy, Kiranmai; Li, Anping; Kossenkov, Andrew V et al. (2016) The mRNA-edited form of GABRA3 suppresses GABRA3-mediated Akt activation and breast cancer metastasis. Nat Commun 7:10715
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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