Written by Emily Locke
While at newspaper publishers the editor-in-chief still adjusts texts before they are published, there are also certain master regulators in the cell that can induce changes to the mRNA before it is translated into a protein. An important player in this so-called RNA-editing is the protein ADAR1 (adenosine deaminase acting on RNA). ADAR1 is an RNA-specific adenosine deaminase that catalyzes the conversion of adenosine nucleoside building blocks to inosine [1]. This enzyme is found in all vertebrate tissue types and plays a critical role in promoting protein evolution [2]. In addition, it appears to play an important function in the innate immune response, which is why mutations in the adar1 gene are associated with a variety of diseases [3]. Thus, the protein could be a promising target for various therapies and is therefore being studied in numerous research fields.
These topics await you:
1) From A to I: How ADAR1 edits RNA
2) ADAR1 as a Master Regulator of the Innate Immune Response
3) ADAR1: A Promising Target in Research
4) ADAR1 Dual Luciferase Reporter HEK293 Cell Line
Subscribe to the free Biomol newsletter and never miss a blog article again!
From A to I: How ADAR1 edits RNA
ADAR enzymes can alter the nucleotide sequence of double-stranded RNA (dsRNA) post-transcriptionally. To do this, they bind to the dsRNA and convert the nucleoside adenosine to an inosine by deamination. Inosine is a rare nucleoside in RNA that consists of the purine base hypoxanthine and D-ribose. The biochemical reaction catalyzed by ADAR1 proceeds as follows: An activated water molecule reacts with the amino group on the 6th carbon atom of an adenosine in a nucleophilic substitution reaction [4]. This results in a hydrated intermediate, which, however, decomposes after a short time because the amino group of the adenine exits as an ammonium ion (Fig. 1).
The resulting inosine destabilizes the RNA because it does not bind to the complementary uracil in the same way as adenosine. Due to its structural similarity to guanine, a bond to cytosine forms instead, which can lead to codon changes. These in turn lead to changes in the coding sequence of proteins as well as to alternative transcriptional splicing variants, and thus ultimately to other protein functions [1].
Figure 1: Deamination of adenosine to inosine by ADAR1. ADAR1 catalyzes the attack of a water molecule on the C6 amino group of an adenosine, creating a hydrated intermediate. After a short time, the nucleoside inosine is formed by the exit of an ammonium ion [5].
The editing of RNA by ADAR1 leads to a considerable increase in protein diversity, which is vital for organisms. Many proteins can only be generated in this way, including, for example, the glutamate receptor GRIA2, which is essential for neuronal communication [6]. However, more and more research results indicate that ADAR1, in addition to increasing transcriptional diversity, also plays a crucial role in the innate immune response and in the development of autoimmune diseases.
ADAR1 as a Master Regulator of the Innate Immune Response
Our immune system is specialized to recognize specific molecular patterns that indicate the presence of pathogenic organisms. These so-called PAMPs (pathogen-associated molecular patterns) are detected by corresponding receptors, the pattern recognition receptors (PRRs), whereupon the innate immune response is initiated. Possible PAMPs include foreign double-stranded RNA in the cytoplasm, as this is often produced as an intermediate in viral replication. Since dsRNA is rather uncommon in eukaryotic cells, our immune system immediately raises the alarm upon detecting such RNA and signals the invasion of a pathogen [3].
But in fact, there are also endogenous RNAs that can form double-stranded structures. These include, for example, RNAs transcribed from retrotransposons or RNAs from the mitochondrial matrix [3]. These RNAs would therefore also have to be sensed by the PRRs and would thus lead to autoimmunity. However, this does not normally occur - but why? How can the cell distinguish between foreign and endogenous dsRNA?
One process that appears to play a role in differentiation is RNA editing. Base exchange from A to I catalyzed by ADAR1 can destabilize dsRNA as Watson-Crick AU base pairs are replaced by so-called IU wobble pairs [7]. This unstable base pairing causes cellular dsRNA to revert to a single-stranded structure in which it does not stimulate an immune response. However, as soon as the amount of dsRNA in the cytoplasm exceeds a certain threshold, for example during a viral infection, it is detected by PRRs and appropriate defense mechanisms are initiated.
A growing body of research indicates that destabilization of dsRNA plays a critical role in maintaining self-tolerance to such RNA and preventing autoimmunity [3]. For example, a mutated adar1 gene may contribute to the development of Aicardi-Goutières syndrome. This is a rare inherited disease in which inflammatory leukodystrophy occurs [8]. It is characterized by elevated levels of IFN-α in cerebrospinal fluid. This is due in part to loss of ADAR1 protein function, which prevents dsRNA from being destabilized. The resulting accumulation of dsRNA stimulates IFN-α production, although no (viral) infection is present, and thus leads to an autoimmune response [9].
ADAR1: A Promising Target in Research
ADAR1 is an exciting protein for scientists, especially because of its diverse involvement in the development of various diseases. The protein's function is also being studied in depth in cancer research, as RNA editing catalyzed by ADAR1 can lead to dangerous, cancer-promoting amino acid exchanges [10]. Moreover, ADAR1 is frequently overexpressed in breast, lung, liver, and esophageal cancers, where it promotes cancer cell proliferation. Thus, the enzyme represents a promising therapeutic target for novel approaches to treat cancer.
While in most cases increased ADAR1 expression promotes cancer development and progression, there are also a few cancers caused by low activity of the protein. For example, studies have shown that loss of ADAR1 protein can lead to melanoma development and metastasis: ADAR enzymes can affect the biogenesis and stability of microRNAs (miRNAs). As melanoma develops, ADAR1 is often downregulated by the protein CREB (cAMP-response element binding protein), preventing it from affecting the activity of miRNAs. This includes miR-455-5p, which in its non-edited state inhibits the tumor suppressor protein CPEB1 and thus contributes to melanoma development [11].
In addition to the examples mentioned here, the protein ADAR1 regulates numerous other biochemical processes in the cell in a variety of ways. Researchers in various fields such as immunology, oncology and virology are particularly interested in the importance of the enzyme in immune defense and its role in the development of various diseases. Do you also deal with ADAR1 in your research? Our manufacturer BPS Bioscience offers three high-quality products to support you in your analysis of this exciting protein!
ADAR1 Dual Luciferase Reporter HEK293 Cell Line
The ADAR1 Dual Luciferase Reporter HEK293 cell line can be used to study the RNA editing activity of ADAR1. The cell line stably expresses ADAR1 under a CMV promoter and also contains an ADAR editing reporter construct with genes for two different luciferases. The reporter gene, which is constitutively expressed, encodes the Firefly luciferase. This is followed by a gene for the ADAR substrate GluA2 and the gene for Renilla luciferase.
The GluA2 sequence has been modified to contain the amber codon UAG. When this stop codon is converted by ADAR1, it becomes UIG by the exchange of adenosine to inosine, which is read as tryptophan (UGG) by the translational machinery of the cell. This change allows translation to continue to the end of the reporter mRNA, which also expresses Renilla luciferase (Fig. 2, top). However, if ADAR1 enzyme activity is inhibited, translation terminates at the amber codon and Renilla luciferase expression does not occur (Fig. 2, bottom). This cell line is therefore used to screen for potential ADAR1 inhibitors. By comparing the expression of Firefly and Renilla luciferase, the inhibition of ADAR1 activity can be measured.
Figure 2: The ADAR1 editing reporter construct of the ADAR1 dual luciferase reporter HEK293 cell line. Top: In the presence of the ADAR1 enzyme, the amber codon UAG is converted to UIG, continuing translation to the end of the reporter mRNA and expressing Renilla luciferase. Bottom: When ADAR1 is down-regulated or inactivated by inhibitors, there is no change in the amber codon, so translation terminates here and Renilla luciferase is not expressed.
ADAR1, FLAG-Tag
In addition to the HEK293 cell line, BPS Bioscience also offers the recombinant protein ADAR1, which can be used in immunotherapy studies, for example. The 137 kDa heavy protein is expressed in a HEK293 cell expression system and has a FLAG tag at the N-terminus. A FLAG tag is a protein tag used in protein purification and characterization of recombinant proteins. It represents one of the most specific tags and can be used in many different assays where the tag is to be recognized and bound by antibodies. In order to proteolytically cleave the FLAG tag after purification, a protease cleavage site can also be inserted between the recombinant protein and the FLAG tag [12].
ADAR2 (ADARB1), FLAG-Tag
Although so far, we have mainly referred to ADAR1, the ADAR gene family consists of three different types of ADAR enzymes: ADAR or ADAR1, ADARB1 or ADAR2, and ADARB2 or ADAR3 [1]. While ADAR3 is found only in the brain and has a slightly different mechanism, ADAR1 and ADAR2 share the same functional domains. Both are found in many tissues in the body and share similar expression patterns as well as protein structures. ADAR2 also uses dsRNA as a substrate, but differs from ADAR1 in its editing activity [13]. The 78 kDa heavy recombinant protein was purified by affinity chromatography and also has an N-terminal FLAG tag. It can be used in human cells, for example, in the field of RNA epigenetics.
Sources
[1] Savva, Y.A., Rieder, L.E., Reenan, R.A. The ADAR protein family. Genome Biol. 13, 252 (2012).
[2] https://en.wikipedia.org/wiki/ADAR, 14.03.2023
[3] Lamers, M., van den Hoogen, B., Haagmans, B. ADAR1: “Editor-in-Chief” of Cytoplasmic Innate Immunity. Front Immunol. 10 (2019).
[4] Samuel, CE. Adenosine deaminases acting on RNA (ADARs) and A-to-I editing. Springer. ISBN 978-3-642-22800-1 (2012).
[5] https://commons.wikimedia.org/wiki/File:ADAR_reaction.jpg, 15.03.2023
[6] https://www.uniprot.org/uniprotkb/P55265/entry, 14.03.2023
[7] Samuel, CE. ADARs: viruses and innate immunity. Curr Top Microbiol Immunol. 353:163-95 (2012).
[8] https://elaev.de/leukodystrophien/arten/aicardi-goutieres-syndrom/, 14.03.2023
[9] Liddicoat, B. J. et al. RNA editing by ADAR1 prevents MDA5 sensing of endogenous dsRNA as nonself. Science. 349:6252:1115-1120 (2015).
[10] Gallo A, Galardi S. A-to-I RNA editing and cancer: from pathology to basic science. RNA Biol. 5(3):135-9 (2008).
[11] Shoshan, E., Mobley, A., Braeuer, R. et al. Reduced adenosine-to-inosine miR-455-5p editing promotes melanoma growth and metastasis. Nat Cell Biol. 17, 311–321 (2015).
[12] https://en.wikipedia.org/wiki/FLAG-tag, 14.03.2023
[13] Higuchi, M., Maas, S., Single, F. et al. Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature. 406, 78–81 (2000).
Preview image: https://commons.wikimedia.org/wiki/File:ADAR_Protein_3.png
