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.

• Klíčová slova
• ADAR2, ADARB1, RNA editing, seizures,
• Publikační typ
• časopisecké články 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
• adenosindeaminasa * genetika   metabolismus   MeSH
• dvouvláknová RNA * genetika   MeSH
• inosin genetika   metabolismus   MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• myši MeSH
• přirozená imunita MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ADAR1 protein, mouse MeSH Prohlížeč
• adenosindeaminasa * MeSH
• dvouvláknová RNA * MeSH
• inosin MeSH
• messenger RNA 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.

• Klíčová slova
• ADAR, ADARB1, dsRNA, neurons, recoding RNA editing,
• MeSH
• adenosindeaminasa * metabolismus   MeSH
• AMPA receptory genetika   metabolismus   MeSH
• antivirové látky MeSH
• Caenorhabditis elegans genetika   MeSH
• Drosophila genetika   MeSH
• dvouvláknová RNA genetika   MeSH
• genetická terapie MeSH
• lidé MeSH
• myši MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• ADARB1 protein, human MeSH Prohlížeč
• adenosindeaminasa * MeSH
• AMPA receptory MeSH
• antivirové látky MeSH
• dvouvláknová RNA MeSH
• proteiny vázající RNA 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.

• Klíčová slova
• DNA, epilepsy, missense, mutation, nervous system diseases, sequence analysis,
• MeSH
• adenosindeaminasa genetika   metabolismus   MeSH
• alely MeSH
• dítě MeSH
• dvouvláknová RNA metabolismus   MeSH
• editace RNA MeSH
• epilepsie enzymologie   genetika   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mutace MeSH
• nemoci mozku enzymologie   genetika   metabolismus   MeSH
• neurovývojové poruchy enzymologie   genetika   MeSH
• pokrevní příbuzenství MeSH
• předškolní dítě MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• rodokmen MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ADARB1 protein, human MeSH Prohlížeč
• adenosindeaminasa MeSH
• dvouvláknová RNA MeSH
• proteiny vázající RNA MeSH