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 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

BACKGROUND: Adenosine-to-inosine RNA editing is a co-transcriptional/post-transcriptional modification of double-stranded RNA, catalysed by one of two active adenosine deaminases acting on RNA (ADARs), ADAR1 and ADAR2. ADARB1 encodes the enzyme ADAR2 that is highly expressed in the brain and essential to modulate the function of glutamate and serotonin receptors. Impaired ADAR2 editing causes early onset progressive epilepsy and premature death in mice. In humans, ADAR2 dysfunction has been very recently linked to a neurodevelopmental disorder with microcephaly and epilepsy in four unrelated subjects. METHODS: We studied three children from two consanguineous families with severe developmental and epileptic encephalopathy (DEE) through detailed physical and instrumental examinations. Exome sequencing (ES) was used to identify ADARB1 mutations as the underlying genetic cause and in vitro assays with transiently transfected cells were performed to ascertain the impact on ADAR2 enzymatic activity and splicing. RESULTS: All patients showed global developmental delay, intractable early infantile-onset seizures, microcephaly, severe-to-profound intellectual disability, axial hypotonia and progressive appendicular spasticity. ES revealed the novel missense c.1889G>A, p.(Arg630Gln) and deletion c.1245_1247+1 del, p.(Leu415PhefsTer14) variants in ADARB1 (NM_015833.4). The p.(Leu415PhefsTer14) variant leads to incorrect splicing resulting in frameshift with a premature stop codon and loss of enzyme function. In vitro RNA editing assays showed that the p.(Arg630Gln) variant resulted in a severe impairment of ADAR2 enzymatic activity. CONCLUSION: In conclusion, these data support the pathogenic role of biallelic ADARB1 variants as the cause of a distinctive form of DEE, reinforcing the importance of RNA editing in brain function and development.

• Keywords
• DNA, epilepsy, missense, mutation, nervous system diseases, sequence analysis,
• MeSH
• Adenosine Deaminase genetics   metabolism   MeSH
• Alleles MeSH
• Child MeSH
• RNA, Double-Stranded metabolism   MeSH
• RNA Editing MeSH
• Epilepsy enzymology   genetics   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mutation MeSH
• Brain Diseases enzymology   genetics   metabolism   MeSH
• Neurodevelopmental Disorders enzymology   genetics   MeSH
• Consanguinity MeSH
• Child, Preschool MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Pedigree MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ADARB1 protein, human MeSH Browser
• Adenosine Deaminase MeSH
• RNA, Double-Stranded MeSH
• RNA-Binding Proteins MeSH

The RNA editing enzyme ADAR2 is essential for the recoding of brain transcripts. Impaired ADAR2 editing leads to early-onset epilepsy and premature death in a mouse model. Here, we report bi-allelic variants in ADARB1, the gene encoding ADAR2, in four unrelated individuals with microcephaly, intellectual disability, and epilepsy. In one individual, a homozygous variant in one of the double-stranded RNA-binding domains (dsRBDs) was identified. In the others, variants were situated in or around the deaminase domain. To evaluate the effects of these variants on ADAR2 enzymatic activity, we performed in vitro assays with recombinant proteins in HEK293T cells and ex vivo assays with fibroblasts derived from one of the individuals. We demonstrate that these ADAR2 variants lead to reduced editing activity on a known ADAR2 substrate. We also demonstrate that one variant leads to changes in splicing of ADARB1 transcript isoforms. These findings reinforce the importance of RNA editing in brain development and introduce ADARB1 as a genetic etiology in individuals with intellectual disability, microcephaly, and epilepsy.

• Keywords
• ADAR2, RNA editing, epilepsy, intellectual disability, microcephaly, migrating focal seizures,
• MeSH
• Adenosine Deaminase genetics   MeSH
• Alleles MeSH
• Alternative Splicing genetics   MeSH
• Child MeSH
• Genetic Predisposition to Disease genetics   MeSH
• Genetic Variation genetics   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Intellectual Disability genetics   MeSH
• Microcephaly genetics   MeSH
• Child, Preschool MeSH
• RNA-Binding Proteins genetics   MeSH
• RNA Splicing genetics   MeSH
• Seizures genetics   MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Male MeSH
• Child, Preschool MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• ADARB1 protein, human MeSH Browser
• Adenosine Deaminase MeSH
• RNA-Binding Proteins MeSH

ADAR RNA editing enzymes are high-affinity dsRNA-binding proteins that deaminate adenosines to inosines in pre-mRNA hairpins and also exert editing-independent effects. We generated a Drosophila AdarE374A mutant strain encoding a catalytically inactive Adar with CRISPR/Cas9. We demonstrate that Adar adenosine deamination activity is necessary for normal locomotion and prevents age-dependent neurodegeneration. The catalytically inactive protein, when expressed at a higher than physiological level, can rescue neurodegeneration in Adar mutants, suggesting also editing-independent effects. Furthermore, loss of Adar RNA editing activity leads to innate immune induction, indicating that Drosophila Adar, despite being the homolog of mammalian ADAR2, also has functions similar to mammalian ADAR1. The innate immune induction in fly Adar mutants is suppressed by silencing of Dicer-2, which has a RNA helicase domain similar to MDA5 that senses unedited dsRNAs in mammalian Adar1 mutants. Our work demonstrates that the single Adar enzyme in Drosophila unexpectedly has dual functions.

• MeSH
• Adenosine Deaminase chemistry   genetics   MeSH
• Adenosine Monophosphate metabolism   MeSH
• Point Mutation genetics   MeSH
• Nerve Degeneration pathology   MeSH
• Drosophila melanogaster genetics   immunology   MeSH
• RNA Editing genetics   MeSH
• Catalysis MeSH
• Locomotion MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Brain metabolism   MeSH
• Immunity, Innate genetics   MeSH
• Protein Domains MeSH
• Drosophila Proteins chemistry   genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Ribonuclease III metabolism   MeSH
• RNA Helicases metabolism   MeSH
• Aging pathology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Adar protein, Drosophila MeSH Browser
• Adenosine Deaminase MeSH
• Adenosine Monophosphate MeSH
• DCR-2 protein, Drosophila MeSH Browser
• RNA, Messenger MeSH
• Drosophila Proteins MeSH
• Ribonuclease III MeSH
• RNA Helicases MeSH