The Role of ADAR1 in Biology and Disease
Adenosine deaminases acting on RNA (ADAR) proteins catalyze the post-transcriptional modification of adenosine to inosine (A-to-I) on double-stranded RNA (dsRNA) molecules. The ADAR proteins edit A to I non-selectively in extended perfect double-stranded RNA duplexes, an activity that is of ancient origin and highly conserved. ADAR1 functions as a major RNA editor, resulting in the differential regulation of RNA processing by other RNA-binding proteins (reviewed in 1, 2, 3). In humans, ADAR1 and ADAR2 have deaminating activity while ADAR3 lacks catalytic activity. ADAR1 has two isoforms, ADAR1p110 and ADAR1p150. The larger p150 isoform contains a nuclear export sequence, enabling its expression in the cytoplasm in addition to the nucleus, whereas the ADAR1p110 isoform and ADAR2 lack this domain and are localized mainly within the nucleus.
Notably, ADAR1 regulates innate immunity by suppressing pattern recognition receptor mechanisms. During viral infection, long stretches of exogenous dsRNA are recognized by melanoma differentiation-associated protein 5 (MDA5), protein kinase R (PKR), and 2’-5’ Oligoadenylate Synthetase (OAS), initiating three pathways leading to apoptosis to prevent viral spread. The MDA5 pathway leads to induction of interferon (IFN) genes critical in fighting viral infections. In the absence of viral infection, endogenous sequences of dsRNA are modified by ADAR1, preventing their recognition by these innate immune sensors. Although essential in cellular discrimination of exogenous or endogenous dsRNA, modifications of RNA by ADAR1 can directly lead to amino acid substitutions, which can lead to disease.
ADAR1 also contributes to alterations in the processing and function of microRNAs (miRNAs) both positively and negatively. A-to-I editing of primary miRNA transcripts (pri-miRNA) and pre-miRNA inhibits both DROSHA and DICER-mediated processing, which suppresses the downstream silencing effects of miRNA. On the other hand, ADAR1 can directly dimerize with DICER to promote cleavage of pre-miRNA, independently of its deaminase activity, resulting in increased amounts of miRNA. Such divergent effects on miRNA remain to be fully understood, particularly how it relates to disease. Some evidence suggests an association between ADAR1 miRNA editing and certain cancers.
Finally, ADAR1 and ADAR2 play an important role in the post-transcriptional expansion of protein diversity, as they can introduce stops codons, alternative splice sites, and missense mutations in endogenous pre-mRNAs. The A-to-I editing of pre-mRNAs, which is read as G by the translation machinery, sometimes results in a different amino acid, so that the amino acid sequence of a protein differs from the gene-coded sequence. These changes depend on the formation of a loop forming a double strand in the pre-mRNA, with the edit occurring in the exon. Some of these divergent proteins play a role during development and are regulated by the tissue-specific and temporal expression of the editor.
Other changes, observed mostly in the nervous system (neurotransmitters, ion channels, G protein-coupled receptors), expand protein functionality. A well-known example is the Glutamate receptor, in which a change of CAG to CGG (glutamine Q to arginine R) by ADAR2 results in a dramatic increase in calcium permeability. Serotonin receptor 5-HT2C contains 3 potential modification sites in its messenger RNA. A Q/R site is the most highly edited with up to 99% of transcripts modified, resulting in changes in intracellular signaling that affect appetite and mood.
ADAR1 in disease
Specific genetic mutations of ADAR1 leading to impaired protein function have been associated with several IFN-mediated diseases, including Aicardi-Goutieres syndrome, dyschromatosis symmetrica hereditaria, bilateral striatal necrosis, and spastic paraplegia. Autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) have also been linked to ADAR1, with overexpression of ADAR1p150 observed in RA patients and high levels of A-to-I editing observed in SLE patients.
Importantly, ADAR1 overactivity and loss of function have both been linked to various cancers and offer new therapeutic opportunities. With the emergence of checkpoint blockade immunotherapy, researchers continue to search for mechanisms of enhancing treatment efficacy, especially against solid tumors. ADAR1 itself may function as an immune checkpoint. Studies have now identified that inactivating ADAR1 in tumor cells renders them highly susceptible to immunotherapy, a process linked to unedited dsRNA and activation of pattern recognition receptors inducing IFN pathways.
There are several distinct mechanisms by which ADAR1 can affect oncogenesis. Firstly, ADAR1 RNA editing of specific transcripts has been associated with certain types of cancers and their pathogenicity. In particular, editing of AZIN1, DHFR, FAK, GLI1, MYC, and RHOQ mRNA has been linked to increased oncogenesis or increased treatment resistance. Secondly, editing of tumor suppressor miRNAs, particularly let-7 miRNAs, leads to their decreased biogenesis, thus promoting Chronic Myeloid Leukemia self-renewal. On the other hand, ADAR1 activity on other miRNAs exerts a tumor suppressive effect. For example, ADAR1 decreases levels of miR-378 and miR-455, which are inhibitors of tumor suppressors. This regulation is associated with limiting melanoma progression and growth. Lastly, ADAR1 editing can restrict alternative splicing in cancer, as observed with CCDC15 exon 9, a tumorigenic splice variant that is reduced by the activity of ADAR1.
ADAR-based Editors
Scientists have also been taking advantage of the RNA-editing capability of ADAR to advance novel gene therapies to the clinic. In some instances, the editor is added to immune stem cells together with a guide RNA and a mutant Cas9 protein that only nicks the DNA (Cas9n), which is then base-edited during repair. Prime editors have been shown to cause fewer off-target modifications compared to CRISPR/Cas9 editing, and recently reached the clinic (4). The significance of using single-base gene editing for gene therapy should not be overlooked: about 50% of the 75,000 gene variants known to be pathogenic correspond to a point mutation that may be corrected using single-base editing. Adenine base editors (ABE) have been optimized to improve their activity while decreasing off-target modifications (for example ABE7.10, ABEmax, and ABE8e).
In other instances, scientists aim directly at the mRNA of mutant proteins, with the idea that ADAR editors can selectively repair the sequence of mutant proteins during protein production. For example, the 3-letter codon AAG (lysine) is edited as IAG, which is read as GAG by the ribosome and translated into glutamate. Thus, using the A-to-I ADAR editor can change a lysine to a glutamate in a protein of interest (5). Optimized ADAR proteins can be delivered using viral vectors, if the protein is engineered to be small enough to accommodate the limited cargo capacity of AAVs. Alternatively, endogenous ADAR can be redirected using naked antisense oligonucleotides. This strategy avoids the delivery issues associated with having to administer exogenous enzymes to patients using viral vectors, clearing a faster path to the clinic (6). Since many patients have pre-existing antibodies against AAVs or develop antibodies soon after the first treatment, bypassing the need for viral vectors could prove very advantageous. As proof-of-concept, the SERPINA1/AAT mRNA editor designed to treat Alpha-1 antitrypsin deficiency (AATD) has become the first ADAR-based editor to reach clinical trial and is now being tested in patients. AATD is caused by a mutation in gene SERPINA1, encoding Alpha-1 antitrypsin. Many patients have a mutation that replaces a glutamate with lysine, which can be directly reversed by ADAR editing. RNA editing, it is hoped, will be safer compared to DNA-based gene therapy, as it does not manipulate the genome.
Novel luciferase reporter cell lines for studying ADAR1
Based on a similar reporter system as described by K. Fritzell, et al. (7), BPS Bioscience has developed three luciferase-based reporter cell lines for studying ADAR1. Several ADAR reporter constructs were designed and evaluated for their relative response to ADAR1 overexpression. They all feature an ADAR1 hairpin target with a stop codon (UAG) which is susceptible to ADAR1-mediated editing to a tryptophan (UUG), located upstream of a Firefly luciferase reporter. ADAR1 activity, therefore, directly correlates with luciferase activity. The best reporter was selected based on optimization and development experiments that have been described in our poster presented at AACR Annual Meeting 2024 entitled: Development of a genetically validated, cell-based reporter assay for ADAR1 editing activity.
| Cell Line | HEK293 | ADAR1 Responsive Luciferase Reporter | ADAR1 Activity Luciferase Reporter | ADAR1 Activity TWO-Luciferase Reporter |
|---|---|---|---|---|
| Cat # | N/A | 82239 | 82239 | 82240 |
| Endogenous ADAR1 Expression | Low | Low | Low | Low |
| Exogenous ADAR1 Expression | No | No | Yes | Yes |
| ADAR1-sensitive firefly luciferase | No | Yes | Yes | Yes |
| Constitutive renilla luciferase | No | No | No | Yes |
| Readout reagent | N/A | ONE-Step™ (#60690) | ONE-Step™ (#60690) | TWO-Step (#60683) |
The ADAR1 Responsive Luciferase Reporter HEK293 Cell Line is designed to monitor the activity of an exogenous ADAR protein introduced by transfection or transduction. The cells express the optimal ADAR1 reporter construct but display very low levels of endogenous ADAR1 and luciferase activity is minimal. Forced ADAR1 expression replaces the stop codon with tryptophan and allows luciferase expression and activity. These cells must be transfected to express ADAR1 for luciferase to be induced. They can be used to transfect ADAR mutants and variants for structure-function studies, or when designing new ADAR1 variants for RNA editing.
Figure 1: Illustration of the mechanism of action of ADAR1 Responsive Luciferase Reporter HEK293 Cell Line.
Figure 2. The ADAR1 Responsive Reporter Cell Line responds to ADAR1 but not to ADAR2 expression. Cells were plated at 30,000 cells/well in a 96-well plate. The day after, they were transfected with increasing amounts of either ADAR1 (p150 or p110 isoform) or ADAR2-encoding plasmids and incubated for 24 hours. Luciferase activity was measured using ONE-Step™ Luciferase Assay System (#60690). Results are expressed as fold induction of luminescence signal compared to mock-transfected controls.
The ADAR1 Activity Luciferase Reporter HEK293 Cell Line contains the optimal ADAR1 reporter construct and was engineered to express ADAR1, therefore basal luciferase activity is high. These cells are ideal for the screening and profiling of compounds that inhibit ADAR1 activity.
Figure 3: Illustration of the mechanism of action of ADAR1 Activity Luciferase Reporter HEK293 Cell Line.
The ADAR1 Activity TWO-Luciferase Reporter HEK293 Cell Line was engineered to constitutively express Renilla luciferase in addition to ADAR1 Firefly luciferase reporter and ADAR1 protein. Renilla luciferase activity serves as a proxy for cell viability, allowing the user to assess compound toxicity in parallel with its effect on ADAR1 activity.
Figure 4: Illustration of the mechanism of action of ADAR1 Activity TWO-Luciferase Reporter HEK293 Cell Line.
Figure 5: Firefly luciferase and Renilla luciferase signals correlate with cell numbers in ADAR1 Activity TWO-Luciferase Reporter Cell Line. Cells were plated at increasing densities in a 96-well plate. Firefly and Renilla luciferase activities were measured using the TWO-Step Luciferase Assay System (BPS Bioscience #60683).
Conclusion
We have developed sensitive and robust cell-based assays for measuring ADAR1 activity. Our three cell lines provide well-validated resources to study different aspects of ADAR1 function in a cellular context.
- ADAR1 Responsive Luciferase Reporter HEK293 Cell Line is designed to compare the effect of ADAR1 modifications and mutations on ADAR1 activity, for example when studying structure/function relationships, or when designing ADAR1 variants for editing. For a simple readout, use with ONE-Step™ Luciferase Assay system.
- ADAR1 Activity Luciferase Reporter HEK293 Cell Line stably expresses ADAR1 and is suitable to assess the efficacy of ADAR1 modulators such as small molecule inhibitors. This assay is amenable to high-throughput screening. Use with ONE-Step™ Luciferase Assay system.
- ADAR1 Activity TWO-Luciferase Reporter HEK293 Cell Line allows the multiplexing of efficacy and toxicity measurements for ADAR1-directed compounds such as small molecule inhibitors. Use with the TWO-Step Luciferase Assay system.
Illustrations were created with Biorender.com.
References
1. B. Song, et al. 2022. Wiley Interdiscip Rev RNA. 13(10):e1665. Pubmed
2. L. Xu and M. Ohman. 2018. Genes (Basel). 10(1):12. Pubmed
3. M. Lamers, et al. 2019. Front Immunol. 10:1763. Pubmed
4. A. Mullard. 2024. Nat Rev Drug Discov. 10.1038/d41573-024-00070-y. Pubmed
5. LT. Stafforst and M.F. Schneider. 2012. Angew Chem Int Ed Engl. 51: 11166. Pubmed
6. T. Merkle, et al. 2019. Nature Biotechnology. 37: 133. Pubmed
7. K. Fritzell, et al. 2019. Nucleic Acids Res. 47(4):e22. Pubmed
│Protein Products
│Biochemical Assay Kits
│Cell Lines
- ADAR1 Responsive Luciferase Reporter HEK293 Cell Line
- ADAR1 Activity Luciferase Reporter HEK293 Cell Line
- ADAR1 Activity TWO-Luciferase Reporter HEK293 Cell Line
