Double-stranded RNA-specific adenosine deaminase is an enzyme that in humans is encoded by the ADARgene. Adenosine deaminases acting on RNA are enzymes responsible for binding to double stranded RNA and converting adenosine to inosine by deamination. ADAR protein is a RNA-binding protein, which functions in RNA-editing through post-transcriptional modification of mRNA transcripts by changing the nucleotide content of the RNA. The conversion from A to I in the RNA disrupt the normal A:U pairing which makes the RNA unstable. Inosine is structurally similar to that of guanine which leads to I to cytosine binding. Inosine typically mimicks guanosine during translation. Codon changes can arise from editing which may lead to changes in the coding sequences for proteins and their functions. Most editing sites are found in noncoding regions of RNA such as untranslated regions, Alu elements, and long interspersed nuclear element. Mutations in this gene have been associated with dyschromatosis symmetrica hereditaria, as well as Aicardi–Goutières syndrome. Alternate transcriptional splice variants, encoding different isoforms, have been characterized. ADAR also impacts the transcriptome in editing-independent ways, likely by interfering with other RNA-binding proteins.
Discovery
Adenosine deaminase acting on RNA and its gene were first discovered accidentally in 1987 as a result of research by Brenda Bass and Harold Weintraub. These researchers were using antisense RNA inhibition to determine which genes play a key role in the development of Xenopus laevis embryos. Previous research on Xenopus oocytes had been successful. However, when Bass and Weintraub applied identical protocols to Xenopus embryos, they were unable to determine the embryo’s developmental genes. In an attempt to understand why the method was unsuccessful, they began comparing duplex RNA in both oocytes and embryos. This led them to discover that a developmentally regulated activity denatures RNA:RNA hybrids in embryos. In 1988, Richard Wagner et al. further studied the activity occurring on Xenopus embryos. They determined that a protein was responsible for the unwinding of RNA due to the absence of activity after proteinase treatment. It was also shown that this protein is specific for double stranded RNA, or dsRNA, and does not require ATP. Additionally, it became evident that the protein’s activity on dsRNA modifies it beyond a point of rehybridization, but does not fully denature it. Finally, the researchers determined that this unwinding is due to the deamination of adenosine residues to inosine. This modification results in mismatched base-pairing between inosine and uridine, leading to the destabilization and unwinding of dsRNA.
Function and origin
ADARs acting on RNA is one of the most common forms of RNA editing, and has both selective and non-selective activity. ADAR is able to both modify and regulate the output of gene product, as inosine is interpreted by the cell to be guanosine. ADAR has also been determined to change the functionality of small RNA molecules. Recently, ADARs have also been discovered as a splicing regulator with their editing capability or RNA binding function. It is believed that ADAR evolved from ADAT, a critical protein present in all eukaryotes, early in the metazoan period through the addition of a dsRNA binding domain. This likely occurred in the lineage which leads to the crown Metazoa when a duplicate ADAT gene was coupled to a gene encoding at least one double stranded RNA binding. The ADAR family of genes has been largely conserved over the history of its existence. This, along with its presence in the majority of modern phyla, indicates that RNA editing is an essential regulatory gene for metazoan organisms. ADAR has not been discovered in a variety of non-metazoan eukaryotes, such as plants, fungi and choanoflagellates.
Types
In mammals, there are three types of ADARs, 1, 2 and 3. ADAR1 and ADAR2 are found in many tissues in the body while ADAR3 is only found in the brain. ADAR1 and ADAR2 are known to be catalytically active while ADAR3 is thought to be inactive. ADAR1 has two known isoforms known as ADAR1p150 and ADAR1p110. ADAR1p110 is usually only found in the nucleus while ADAR1p150 shuffles between the nucleus to the cytoplasm and is mostly present in the cytoplasm. Although ADAR1 and ADAR2 share many common functional domains as well as commonality in terms of expression pattern, structure of protein and requirements of substrates having double stranded RNA structures, they differ in their editing activity.
Catalytic activity
Biochemical reaction
ADARs catalyze the reaction from A to I by hydrolytic deamination. It does this by the use of an activated water molecule for a nucelophilic attack. It is done by the addition of water to carbon 6 and removal of ammonia with a hydrated intermediate.
Active site
In humans, the enzyme's active site has 2-3 amino-terminal dsRNA binding domains and one carboxy terminal catalytic deaminase domain. In the dsRBD domain there is a conserved α-β-β-β-α configuration present. ADAR1 contains two areas for binding Z-DNA known as Zα and Zβ. ADAR2 and ADAR3 have an arginine rich single stranded RNA binding domain. A crystal structure of ADAR2 has been solved. In the enzyme active site, there is a glutamic acid residue that hydrogen bonds to a water. There is a histidine and two cysteine restudies that coordinates a zinc ion. The zinc activates the water molecule for the nucelophilic hydrolytic deamination. Within the catalytic core there is an inositol hexakisphosphate, which stabilizes arginine and lysine residues.
Dimerization
It has been found in mammals that the conversion from A to I requires homodimerization of ADAR1 and ADAR2, but not ADAR3. In vivo studies have not yet been conclusive if RNA binding is required for dimerization. A study with ADAR1 and 2 mutants which were not able to bind to dsRNA were still able to dimerize, showing they may bind based on protein-protein interactions
Model organisms
Model organisms have been used in the study of ADAR function. A conditional knockout mouse line, called Adartm1aWtsi was generated as part of the International Knockout Mouse Consortium program — a high-throughput mutagenesis project to generate and distribute animal models of disease to interested scientists Male and female animals underwent a standardized phenotypic screen to determine the effects of deletion. Twenty five tests were carried out on mutant mice and two significant abnormalities were observed. Few homozygous mutant embryos were identified during gestation, and none survived until weaning. The remaining tests were carried out on heterozygous mutant adult mice and no abnormalities were observed in these animals.
Role in disease
Aicardi–Goutières syndrome and Bilateral Striatal Necrosis/Dystonia
ADAR1 is one of multiple genes which can contribute to Aicardi–Goutières syndrome when mutated. This is a genetic inflammatory disease primarily affecting the skin and the brain. The inflammation is caused by incorrect activation of interferon inducible genes such as those activated to fight off viral infections. Mutation and loss of function of ADAR1 prevents destabilization of double stranded RNA and the body mistakes this for viral RNA resulting in an autoimmune response. The phenotype in Adar knock-out mice is rescued by the p150 form of ADAR1 containing the Zα domain that binds specifically to the left-handed double-stranded conformation found in Z-DNA and Z-RNA, but not by the p110 isoform that lacks this domain. In humans, the P193A mutation in the Zα domain is causal for Aicardi–Goutières syndrome and for the more severe phenotype found in Bilateral Striatal Necrosis/Dystonia. The findings establish a biological role for the left-handed Z-DNA conformation.
Research has shown that ADAR1 can be both beneficial and a hindrance in a cells ability to fight off HIV infection. Expression levels of the ADAR1 protein have shown to be elevated during HIV infection and it has been suggested that it is responsible for A to G mutations in the HIV genome, inhibiting replication. The authors of this study also suggest that mutation of the HIV genome by ADAR1 might in some cases lead to beneficial viral mutations which could contribute to drug resistance.
Hepatocellular carcinoma
Studies of samples from patients with hepatocellular carcinoma have shown that ADAR1 is frequently upregulated and ADAR2 is frequently downregulated in the disease. It has been suggested that this is responsible for the disrupted A to I editing pattern seen in HCC and that ADAR1 acts as an oncogene in this context whilst ADAR2 has tumor suppressor activities. The imbalance of ADAR expression could change the frequency of A to I transitions in the protein coding region of genes, resulting in mutated proteins which drive the disease. The dysregulation of ADAR1 and ADAR2 could be used as a possible poor prognostic marker.
Melanoma
In contrast to hepatocellular carcinoma, several research studies have indicated that loss of ADAR1 contributes to melanoma growth and metastasis. It is known that ADAR can act on microRNA and affect its biogenesis, stability and/or its binding target. It has been suggested that ADAR1 is downregulated by cAMP- response element binding protein, limiting its ability to act on miRNA. One such example is miR-455-5p which is edited by ADAR1. When ADAR is downregulated by CREB the unedited miR-455-5p downregulates a tumor suppressor protein called CPEB1, contributing to melanoma progression in an in vivo model.
A Gly1007Arg mutation in ADAR1, as well as other truncated versions, have been implicated as a cause in some cases of DSH1. This is a disease characterized by hyperpigmentation in the hands and feet and can occur in Japanese and Chinese families.
Viral activity
Antiviral
ADAR1 is an interferon -inducible protein, so it would make sense that it would assist with a cell’s immune pathway. This seems to be true for the HCV replicon, Lymphocytic choriomeningitis LCMV, and polyomavirus
Proviral
ADAR1 is known to be proviral in other circumstances. ADAR1’s A to I editing has been found in many viruses including measles virus, influenza virus, lymphocytic choriomeningitis virus, polyomavirus, hepatitis delta virus, and hepatitis C virus. Although ADAR1 has been seen in other viruses, it has only been studied extensively in a few; one of those is measles virus. Research done on MV has shown that ADAR1 enhances viral replication. This is done through two different mechanisms: RNA editing and inhibition of dsRNA-activated protein kinase. Specifically, viruses are thought to use ADAR1 as a positive replication factor by selectively suppressing dsRNA-dependent and antiviral pathways.
