The ADARB1 gene encodes the adenosine deaminase acting on RNA 2 (ADAR2) RNA editing enzyme, which edits the GRIA2 transcript Q/R editing site with almost 100% efficiency in the nervous system. The edited GRIA2 R transcript encodes the GLUA2 R subunit isoform of tetrameric α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which is essential to prevent seizures associated with aberrantly elevated AMPA receptor cation permeability. Rare biallelic variants in ADARB1 cause severe infant and childhood seizures and developmental delays in seven cases we previously described. Here, we report two new homozygous ADARB1 variants and study ADAR2 variant editing activities at the GRIA2 Q/R site and other editing sites in cell cultures. One new variant in the second double-stranded RNA binding domain (dsRBD II) retains up to 60% editing activity, whereas another, in the deaminase domain, eliminates RNA editing activity. Reduced GRIA2 Q/R site editing increases AMPA receptor permeability by upregulating the expression of the GLUA2 Q isoform and reducing overall GLUA2 subunit levels, resulting in AMPA receptors that lack GLUA2 and are calcium-permeable. Because failure to edit the GRIA2 Q/R site leads to failure of intron 11 splicing, we also examined the effects of ADAR2 variants on the splicing of a mouse Gria2-based reporter and concluded that ADAR2 variants affect splicing only through their effects on RNA editing activity. To expand the number of variants in ADARB1, some variants reported in ClinVar have also been analyzed by in silico methods to predict which are likely to be most deleterious and associated with seizures in patients.

• Keywords
• ADAR2, ADARB1, RNA editing, seizures,
• Publication type
• Journal Article MeSH

The RNA editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) is essential for correct functioning of innate immune responses. The ADAR1p110 isoform is mainly nuclear and ADAR1p150, which is interferon (IFN) inducible, is predominately cytoplasmic. Using three different methods - co-immunoprecipitation (co-IP) of endogenous ADAR1, Strep-tag co-IP and BioID with individual ADAR1 isoforms - a comprehensive interactome was generated during both homeostasis and the IFN response. Both known and novel interactors as well as editing regulators were identified. Nuclear proteins were detected as stable interactors with both ADAR1 isoforms. In contrast, BioID identified distinct protein networks for each ADAR1 isoform, with nuclear components observed with ADAR1p110 and components of cytoplasmic cellular condensates with ADAR1p150. RNase A digestion distinguished between distal and proximal interactors, as did a double-stranded RNA (dsRNA)-binding mutant of ADAR1 which demonstrated the importance of dsRNA binding for ADAR1 interactions. IFN treatment did not affect the core ADAR1 interactomes but resulted in novel interactions, the majority of which are proximal interactions retained after RNase A treatment. Short treatment with high molecular weight poly(I:C) during the IFN response resulted in dsRNA-binding-dependent changes in the proximal protein network of ADAR1p110 and association of the ADAR1p150 proximal protein network with some components of antiviral stress granules.

• MeSH
• Adenosine Deaminase * metabolism   genetics   MeSH
• Cell Nucleus * metabolism   MeSH
• Cytoplasm * metabolism   MeSH
• RNA, Double-Stranded metabolism   genetics   MeSH
• RNA Editing MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Interferons metabolism   genetics   MeSH
• Humans MeSH
• Protein Interaction Maps MeSH
• Poly I-C pharmacology   MeSH
• Protein Isoforms * metabolism   genetics   MeSH
• RNA-Binding Proteins * metabolism   genetics   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• ADAR protein, human MeSH Browser
• Adenosine Deaminase * MeSH
• RNA, Double-Stranded MeSH
• Interferons MeSH
• Poly I-C MeSH
• Protein Isoforms * MeSH
• RNA-Binding Proteins * MeSH

Adenosine deaminase acting on RNA 1 (ADAR1) is the principal enzyme for the adenosine-to-inosine RNA editing that prevents the aberrant activation of cytosolic nucleic acid sensors by endogenous double stranded RNAs and the activation of interferon-stimulated genes. In mice, the conditional neural crest deletion of Adar1 reduces the survival of melanocytes and alters the differentiation of Schwann cells that fail to myelinate nerve fibers in the peripheral nervous system. These myelination defects are partially rescued upon the concomitant removal of the Mda5 antiviral dsRNA sensor in vitro, suggesting implication of the Mda5/Mavs pathway and downstream effectors in the genesis of Adar1 mutant phenotypes. By analyzing RNA-Seq data from the sciatic nerves of mouse pups after conditional neural crest deletion of Adar1 (Adar1cKO), we here identified the transcription factors deregulated in Adar1cKO mutants compared to the controls. Through Adar1;Mavs and Adar1cKO;Egr1 double-mutant mouse rescue analyses, we then highlighted that the aberrant activation of the Mavs adapter protein and overexpression of the early growth response 1 (EGR1) transcription factor contribute to the Adar1 deletion associated defects in Schwann cell development in vivo. In silico and in vitro gene regulation studies additionally suggested that EGR1 might mediate this inhibitory effect through the aberrant regulation of EGR2-regulated myelin genes. We thus demonstrate the role of the Mda5/Mavs pathway, but also that of the Schwann cell transcription factors in Adar1-associated peripheral myelination defects.

• Keywords
• ADAR1, EGR1, MAVS, Schwann cells, differentiation, neural crest,
• MeSH
• Adenosine Deaminase * genetics   metabolism   MeSH
• Cell Differentiation * genetics   MeSH
• Neural Crest * metabolism   MeSH
• Interferon-Induced Helicase, IFIH1 genetics   metabolism   MeSH
• Myelin Sheath metabolism   MeSH
• Mice, Knockout * MeSH
• Mice MeSH
• Schwann Cells * metabolism   pathology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ADAR1 protein, mouse MeSH Browser
• Adenosine Deaminase * MeSH
• Ifih1 protein, mouse MeSH Browser
• Interferon-Induced Helicase, IFIH1 MeSH

The adenosine deaminase acting on RNA (ADAR) enzymes that catalyze the conversion of adenosine to inosine in double-stranded (ds)RNA are evolutionarily conserved and are essential for many biological functions including nervous system function, hematopoiesis, and innate immunity. Initially it was assumed that the wide-ranging biological roles of ADARs are due to inosine in mRNA being read as guanosine by the translational machinery, allowing incomplete RNA editing in a target codon to generate two different proteins from the same primary transcript. In humans, there are approximately seventy-six positions that undergo site-specific editing in tissues at greater than 20% efficiency that result in recoding. Many of these transcripts are expressed in the central nervous system (CNS) and edited by ADAR2. Exploiting mouse genetic models revealed that transgenic mice lacking the gene encoding Adar2 die within 3 weeks of birth. Therefore, the role of ADAR2 in generating protein diversity in the nervous system is clear, but why is ADAR RNA editing activity essential in other biological processes, particularly editing mainly involving ADAR1? ADAR1 edits human transcripts having embedded Alu element inverted repeats (AluIRs), but the link from this activity to innate immunity activation was elusive. Mice lacking the gene encoding Adar1 are embryonically lethal, and a major breakthrough was the discovery that the role of Adar1 in innate immunity is due to its ability to edit such repetitive element inverted repeats which have the ability to form dsRNA in transcripts. The presence of inosine prevents activation of the dsRNA sensor melanoma differentiation-associated protein 5 (Mda5). Thus, inosine helps the cell discriminate self from non-self RNA, acting like a barcode on mRNA. As innate immunity is key to many different biological processes, the basis for this widespread biological role of the ADAR1 enzyme became evident.Our group has been studying ADARs from the outset of research on these enzymes. In this Account, we give a historical perspective, moving from the initial purification of ADAR1 and ADAR2 and cloning of their encoding genes up to the current research focus in the field and what questions still remain to be addressed. We discuss the characterizations of the proteins, their localizations, posttranslational modifications, and dimerization, and how all of these affect their biological activities. Another aspect we explore is the use of mouse and Drosophila genetic models to study ADAR functions and how these were crucial in determining the biological functions of the ADAR proteins. Finally, we describe the severe consequences of rare mutations found in the human genes encoding ADAR1 and ADAR2.

• MeSH
• Adenosine Deaminase * genetics   metabolism   MeSH
• RNA, Double-Stranded * genetics   MeSH
• Inosine genetics   metabolism   MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• Mice MeSH
• Immunity, Innate MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ADAR1 protein, mouse MeSH Browser
• Adenosine Deaminase * MeSH
• RNA, Double-Stranded * MeSH
• Inosine MeSH
• RNA, Messenger MeSH

The adenosine deaminase acting on RNA (ADAR) enzymes are essential for neuronal function and innate immune control. ADAR1 RNA editing prevents aberrant activation of antiviral dsRNA sensors through editing of long, double-stranded RNAs (dsRNAs). In this review, we focus on the ADAR2 proteins involved in the efficient, highly site-specific RNA editing to recode open reading frames first discovered in the GRIA2 transcript encoding the key GLUA2 subunit of AMPA receptors; ADAR1 proteins also edit many of these sites. We summarize the history of ADAR2 protein research and give an up-to-date review of ADAR2 structural studies, human ADARBI (ADAR2) mutants causing severe infant seizures, and mouse disease models. Structural studies on ADARs and their RNA substrates facilitate current efforts to develop ADAR RNA editing gene therapy to edit disease-causing single nucleotide polymorphisms (SNPs). Artificial ADAR guide RNAs are being developed to retarget ADAR RNA editing to new target transcripts in order to correct SNP mutations in them at the RNA level. Site-specific RNA editing has been expanded to recode hundreds of sites in CNS transcripts in Drosophila and cephalopods. In Drosophila and C. elegans, ADAR RNA editing also suppresses responses to self dsRNA.

• Keywords
• ADAR, ADARB1, dsRNA, neurons, recoding RNA editing,
• MeSH
• Adenosine Deaminase * metabolism   MeSH
• Receptors, AMPA genetics   metabolism   MeSH
• Antiviral Agents MeSH
• Caenorhabditis elegans genetics   MeSH
• Drosophila genetics   MeSH
• RNA, Double-Stranded genetics   MeSH
• Genetic Therapy MeSH
• Humans MeSH
• Mice MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• ADARB1 protein, human MeSH Browser
• Adenosine Deaminase * MeSH
• Receptors, AMPA MeSH
• Antiviral Agents MeSH
• RNA, Double-Stranded MeSH
• RNA-Binding Proteins MeSH

Eukaryotic mRNAs are modified by several chemical marks which have significant impacts on mRNA biology, gene expression, and cellular metabolism as well as on the survival and development of the whole organism. The most abundant and well-studied mRNA base modifications are m6A and ADAR RNA editing. Recent studies have also identified additional mRNA marks such as m6Am, m5C, m1A and Ψ and studied their roles. Each type of modification is deposited by a specific writer, many types of modification are recognized and interpreted by several different readers and some types of modifications can be removed by eraser enzymes. Several works have addressed the functional relationships between some of the modifications. In this review we provide an overview on the current status of research on the different types of mRNA modifications and about the crosstalk between different marks and its functional consequences.

• Keywords
• ADAR, Inosine, epitranscriptome, m1A, m5C, m6A, m6Am, pseudouridine,
• MeSH
• Epigenesis, Genetic * MeSH
• Epigenomics methods   MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• RNA Processing, Post-Transcriptional * MeSH
• Transcriptome * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• RNA, Messenger MeSH