RNA Editing Enzyme Adenosine Deaminase Research Articles

Metformin switches cell death modes to soothe the apical periodontitis via ZBP1.

Apical periodontitis (AP) is a disease caused by pathogenic microorganisms and featured with the degradation of periapical hard tissue. Our recent research showed the crucial role of Z-DNA binding protein 1 (ZBP1)-mediated necroptosis and apoptosis in the pathogenesis of AP. However, the specific regulatory mechanisms of ZBP1 in AP are not fully elucidated. It was found that metformin has a regulatory role in cell necroptosis and apoptosis. But whether and how metformin regulates necroptosis and apoptosis through the ZBP1 in the context of AP remains unknown. This study provided evidence that lipopolysaccharide (LPS) promotes the synthesis of left-handed Z-nucleic acids (Z-NA), which in turn activates ZBP1. Knockout of Zbp1 by CRISPR/Cas9 technology significantly reduced LPS-induced necroptosis and apoptosis invitro. By using Zbp1-knockout mice, periapical bone destruction was alleviated. Moreover, type I interferon induced the expression of interferon-stimulated genes (ISGs), which serve as a major source of Z-NA. In addition, the RNA-editing enzyme Adenosine Deaminase RNA specific 1 (ADAR1) prevented the accumulation of endogenous Z-NA. Meanwhile, metformin suppressed the ZBP1-mediated necroptosis by inhibiting the expression of ZBP1 and the accumulation of ISGs. Metformin also promoted mitochondrial apoptosis, which is critical for the elimination of intracellular bacterial infection. The enhanced apoptosis further promoted the healing of infected apical bone tissues. In summary, these results demonstrated that the recognition of Z-NA by ZBP1 plays an important role in AP pathogenesis. Metformin suppressed ZBP1-mediated necroptosis and promoted apoptosis, thereby contributing to the soothing of inflammation and bone healing in AP.

Generation of a new Adar1p150-/- mouse demonstrates isoform-specific roles in embryonic development and adult homeostasis.

The RNA editing enzyme Adenosine deaminase acting on RNA 1 (ADAR1) is an essential regulator of innate immune activation by both cellular and viral dsRNA. Adenosine-to-Inosine (A-to-I) editing by ADAR1 modifies the sequence and structure of endogenous dsRNA and masks it from the cytoplasmic dsRNA sensor melanoma differentiation-associated protein 5 (MDA5), preventing innate immune activation. Loss of function mutations in ADAR are associated with rare autoinflammatory disorders including Aicardi-Goutieres Syndrome (AGS), defined by a constitutive systemic upregulation of type I interferon (IFN). The murine Adar gene encodes two protein isoforms with distinct functions: ADAR1p110 is constitutively expressed and localizes to the nucleus, whereas ADAR1p150 is primarily cytoplasmic and is inducible by IFN. Recent studies have demonstrated the critical requirement for ADAR1p150 to suppress innate immune activation by self dsRNAs, however, detailed in vivo characterization of the role of ADAR1p150 during development and in adult mice is lacking. We developed a new ADAR1p150-specific knockout mouse mutant based on a single nucleotide deletion that resulted in the loss of the ADARp150 protein without affecting ADAR1p110 expression. The Adar1p150-/- died embryonically at E11.5-E12.5 due to cell death in the fetal liver accompanied by an activated IFN response. Somatic loss of ADAR1p150 in adults was lethal and caused rapid hematopoietic failure, demonstrating an ongoing requirement for ADAR1p150 in vivo. The generation and characterization of this mouse model demonstrates the essential role of ADAR1p150 in vivo and provides a new tool for dissecting the functional differences between ADAR1 isoforms and their physiological contributions.

Immunosuppressive lncRNA LINC00624 promotes tumor progression and therapy resistance through ADAR1 stabilization

BackgroundDespite the success of HER2-targeted therapy in achieving prolonged survival in approximately 50% of treated individuals, treatment resistance is still an important challenge for HER2+ breast cancer (BC) patients. The...

Rescue of autosomal dominant hearing loss by in vivo delivery of mini dCas13X-derived RNA base editor

Programmable RNA editing tools enable the reversible correction of mutant transcripts, reducing the potential risk associated with permanent genetic changes associated with the use of DNA editing tools. However, the potential of these RNA tools to treat disease remains unknown. Here, we evaluated RNA correction therapy with Cas13-based RNA base editors in the myosin VI p.C442Y heterozygous mutation (Myo6C442Y/+) mouse model that recapitulated the phenotypes of human dominant-inherited deafness. We first screened several variants of Cas13-based RNA base editors and guide RNAs (gRNAs) targeting Myo6C442Y in cultured cells and found that mini dCas13X.1-based adenosine base editor (mxABE), composed of truncated Cas13X.1 and the RNA editing enzyme adenosine deaminase acting on RNA 2 deaminase domain variant (ADAR2ddE488Q), exhibited both high efficiency of A > G conversion and low frequency of off-target edits. Single adeno-associated virus (AAV)-mediated delivery of mxABE in the cochlea corrected the mutated Myo6C442Y to Myo6WT allele in homozygous Myo6C442Y/C442Y mice and resulted in increased Myo6WT allele in the injected cochlea of Myo6C442Y/+ mice. The treatment rescued auditory function, including auditory brainstem response and distortion product otoacoustic emission up to 3 months after AAV-mxABE-Myo6 injection in Myo6C442Y/+ mice. We also observed increased survival rate of hair cells and decreased degeneration of hair bundle morphology in the treated compared to untreated control ears. These findings provide a proof-of-concept study for RNA editing tools as a therapeutic treatment for various semidominant forms of hearing loss and other diseases.

ADAR1 prevents autoinflammation by suppressing spontaneous ZBP1 activation.

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)1. In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi-Goutières syndrome (AGS)2. In mice, complete loss of ADAR1 activity is embryonically lethal3-6, and mutations similar to those found in patients with AGS cause autoinflammation7-12. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR3,7-10,13,14. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient inthe expression of both ADAR and MAVS. The Z-nucleic-acid-binding Zα domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Zα domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function.

CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo.

RNA base editing represents a promising alternative to genome editing. Recent approaches harness the endogenous RNA-editing enzyme adenosine deaminase acting on RNA (ADAR) to circumvent problems caused by ectopic expression of engineered editing enzymes, but suffer from sequence restriction, lack of efficiency and bystander editing. Here we present in silico-optimized CLUSTER guide RNAs that bind their target messenger RNAs in a multivalent fashion, achieve editing with high precision and efficiency and enable targeting of sequences that were not accessible using previous gRNA designs. CLUSTER gRNAs can be genetically encoded and delivered using viruses, and are active in a wide range of cell lines. In cell culture, CLUSTER gRNAs achieve on-target editing of endogenous transcripts with yields of up to 45% without bystander editing. In vivo, CLUSTER gRNAs delivered to mouse liver by hydrodynamic tail vein injection edited reporter constructs at rates of up to 10%. The CLUSTER approach opens avenues for drug development in the field of RNA base editing.

Adenosine-to-inosine Alu RNA editing controls the stability of the pro-inflammatory long noncoding RNA NEAT1 in atherosclerotic cardiovascular disease

Long non-coding RNAs (lncRNAs) have emerged as critical regulators in human disease including atherosclerosis. However, the mechanisms involved in the post-transcriptional regulation of the expression of disease-associated lncRNAs are not fully understood. Gene expression studies revealed that Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) lncRNA expression was increased by >2-fold in peripheral blood mononuclear cells (PBMCs) derived from patients with coronary artery disease (CAD) or in carotid artery atherosclerotic plaques. We observed a linear association between NEAT1 lncRNA expression and prevalence of CAD which was independent of age, sex, cardiovascular traditional risk factors and renal function. NEAT1 expression was induced by TNF-α, while silencing of NEAT1 profoundly attenuated the TNF-α-induced vascular endothelial cell pro-inflammatory response as defined by the expression of CXCL8, CCL2, VCAM1 and ICAM1. Overexpression of the RNA editing enzyme adenosine deaminase acting on RNA-1 (ADAR1), but not of its editing-deficient mutant, upregulated NEAT1 levels. Conversely, silencing of ADAR1 suppressed the basal levels and the TNF-α-induced increase of NEAT1. NEAT1 lncRNA expression was strongly associated with ADAR1 in CAD and peripheral arterial vascular disease. RNA editing mapping studies revealed the presence of several inosines in close proximity to AU-rich elements within the AluSx3+/AluJo‐ double-stranded RNA complex. Silencing of the stabilizing RNA-binding protein AUF1 reduced NEAT1 levels while silencing of ADAR1 profoundly affected the binding capacity of AUF1 to NEAT1. Together, our findings propose a mechanism by which ADAR1-catalyzed A-to-I RNA editing controls NEAT1 lncRNA stability in ASCVD.

Immune-related IncRNA LINC00944 responds to variations in ADAR1 levels and it is associated with breast cancer prognosis

AimsBreast cancer is one of the leading causes of woman deaths worldwide, being a major public health problem. It has been reported that the expression of the RNA-editing enzyme Adenosine Deaminase Acting on RNAs 1 (ADAR1) is upregulated in breast cancer, predicting poor prognosis in patients. A few reports in literature examine ADAR1 and long non-coding RNAs (lncRNAs) interplay in cancer and suggest key roles in cancer-related pathways. This study aimed to investigate whether ADAR1 could alter the expression levels of lncRNAs and explore how those changes are related to breast cancer biology. Main methodsADAR1 overexpression and knockdown studies were performed in breast cancer cell lines to analyze the effects over lncRNAs expression. Guilt-by-Association correlation analysis of the TCGA-BRCA cohort was performed to predict the function of the lncRNA LINC00944. Key findingsHere, we show that LINC00944 is responsive to ADAR1 up- and downregulation in breast cancer cells. We found that LINC00944 expression has a strong relationship with immune signaling pathways. Further assessment of the TCGA-BRCA cohort showed that LINC00944 expression was positively correlated to tumor-infiltrating T lymphocytes and pro-apoptotic markers. Moreover, we found that LINC00944 expression was correlated to the age at diagnosis, tumor size, and estrogen and progesterone receptor expression. Finally, we show that low expression of LINC00944 is correlated to poor prognosis in breast cancer patients. SignificanceOur study provides further evidence of the effect of ADAR1 over lncRNA expression levels, and on the participation of LINC00944 in breast cancer, suggesting to further investigate its potential role as prognostic biomarker.

The p150 Isoform of ADAR1 Blocks Sustained RLR signaling and Apoptosis during Influenza Virus Infection.

Signaling through retinoic acid inducible gene I (RIG-I) like receptors (RLRs) is tightly regulated, with activation occurring upon sensing of viral nucleic acids, and suppression mediated by negative regulators. Under homeostatic conditions aberrant activation of melanoma differentiation-associated protein-5 (MDA5) is prevented through editing of endogenous dsRNA by RNA editing enzyme Adenosine Deaminase Acting on RNA (ADAR1). In addition, ADAR1 is postulated to play pro-viral and antiviral roles during viral infections that are dependent or independent of RNA editing activity. Here, we investigated the importance of ADAR1 isoforms in modulating influenza A virus (IAV) replication and revealed the opposing roles for ADAR1 isoforms, with the nuclear p110 isoform restricting versus the cytoplasmic p150 isoform promoting IAV replication. Importantly, we demonstrate that p150 is critical for preventing sustained RIG-I signaling, as p150 deficient cells showed increased IFN-β expression and apoptosis during IAV infection, independent of RNA editing activity. Taken together, the p150 isoform of ADAR1 is important for preventing sustained RIG-I induced IFN-β expression and apoptosis during viral infection.

ADAR2 mislocalization and widespread RNA editing aberrations in C9orf72-mediated ALS/FTD.

The hexanucleotide repeat expansion GGGGCC (G4C2)n in the C9orf72 gene is the most common genetic abnormality associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent findings suggest that dysfunction of nuclear-cytoplasmic trafficking could affect the transport of RNA binding proteins in C9orf72 ALS/FTD. Here, we provide evidence that the RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) is mislocalized in C9orf72 repeat expansion mediated ALS/FTD. ADAR2 is responsible for adenosine (A) to inosine (I) editing of double-stranded RNA, and its function has been shown to be essential for survival. Here we show the mislocalization of ADAR2 in human induced pluripotent stem cell-derived motor neurons (hiPSC-MNs) from C9orf72 patients, in mice expressing (G4C2)149, and in C9orf72 ALS/FTD patient postmortem tissue. As a consequence of this mislocalization we observe alterations in RNA editing in our model systems and across multiple brain regions. Analysis of editing at 408,580 known RNA editing sites indicates that there are vast RNA A to I editing aberrations in C9orf72-mediated ALS/FTD. These RNA editing aberrations are found in many cellular pathways, such as the ALS pathway and the crucial EIF2 signaling pathway. Our findings suggest that the mislocalization of ADAR2 in C9orf72 mediated ALS/FTD is responsible for the alteration of RNA processing events that may impact vast cellular functions, including the integrated stress response (ISR) and protein translation.

Genetic code restoration by artificial RNA editing of Ochre stop codon with ADAR1 deaminase

Site directed mutagenesis is a very effective approach to recode genetic information. Proper linking of the catalytic domain of the RNA editing enzyme adenosine deaminase acting on RNA (ADAR) to an antisense guide RNA can convert specific adenosines (As) to inosines (Is), with the latter recognized as guanosines (Gs) during the translation process. Efforts have been made to engineer the deaminase domain of ADAR1 and the MS2 system to target specific A residues to restore G→A mutations. The target consisted of an ochre (TAA) stop codon, generated from the TGG codon encoding amino acid 58 (Trp) of enhanced green fluorescent protein (EGFP). This system had the ability to convert the stop codon (TAA) to a readable codon (TGG), thereby restoring fluorescence in a cellular system, as shown by JuLi fluorescence and LSM confocal microscopy. The specificity of the editing was confirmed by polymerase chain reaction-restriction fragment length polymorphism, as the restored EGFP mRNA could be cleaved into fragments of 160 and 100 base pairs. Direct sequencing analysis with both sense and antisense primers showed that the restoration rate was higher for the 5' than for the 3'A. This system may be very useful for treating genetic diseases that result from G→A point mutations. Successful artificial editing of RNA in vivo can accelerate research in this field, and pioneer genetic code restoration therapy, including stop codon read-through therapy, for various genetic diseases.

Pahse 2 clinical trial with perampanel for sporadic ayotrophic lateral sclerosis

Background: Perampanel, a selective non-competitive AMPA receptor antagonist, has been reported to prevent the progression of the model mouse for sporadic amyotrophic lateral sclerosis (ALS) (AR2 mice), in which an RNA editing enzyme adenosine deaminase acting on RNA2 (ADAR2) is conditionally knocked out in the motor neurons. Because of therapeutic potency of perampanel for sporadic ALS, we planned to perform a clinical trial with perampanel for sporadic ALS.

Role of RPL39 in Metaplastic Breast Cancer.

Background: Metaplastic breast cancer is one of the most therapeutically challenging forms of breast cancer because of its highly heterogeneous and chemoresistant nature. We have previously demonstrated that ribosomal protein L39 (RPL39) and its gain-of-function mutation A14V have oncogenic activity in triple-negative breast cancer and this activity may be mediated through inducible nitric oxide synthase (iNOS). The function of RPL39 and A14V in other breast cancer subtypes is currently unknown. The objective of this study was to determine the role and mechanism of action of RPL39 in metaplastic breast cancer. Methods: Both competitive allele-specific and droplet digital polymerase chain reaction were used to determine the RPL39 A14V mutation rate in metaplastic breast cancer patient samples. The impact of RPL39 and iNOS expression on patient overall survival was estimated using the Kaplan-Meier method. Co-immunoprecipitation and immunoblot analyses were used for mechanistic evaluation of RPL39. Results: The RPL39 A14V mutation rate was 97.5% (39/40 tumor samples). High RPL39 (hazard ratio = 0.71, 95% confidence interval = 0.55 to 0.91, P = .006) and iNOS expression (P = .003) were associated with reduced patient overall survival. iNOS inhibition with the pan-NOS inhibitor NG-methyl-L-arginine acetate decreased in vitro proliferation and migration, in vivo tumor growth in both BCM-4664 and BCM-3807 patient-derived xenograft models (P = .04 and P = .02, respectively), and in vitro and in vivo chemoresistance. Mechanistically, RPL39 mediated its cancer-promoting actions through iNOS signaling, which was driven by the RNA editing enzyme adenosine deaminase acting on RNA 1. Conclusion: NOS inhibitors and RNA editing modulators may offer novel treatment options for metaplastic breast cancer.

The AMPA receptor antagonist perampanel robustly rescues amyotrophic lateral sclerosis (ALS) pathology in sporadic ALS model mice.

Both TDP-43 pathology and failure of RNA editing of AMPA receptor subunit GluA2, are etiology-linked molecular abnormalities that concomitantly occur in the motor neurons of the majority of patients with amyotrophic lateral sclerosis (ALS). AR2 mice, in which an RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) is conditionally knocked out in the motor neurons, exhibit a progressive ALS phenotype with TDP-43 pathology in the motor neurons through a Ca2+-permeable AMPA receptor-mediated mechanism. Therefore, amelioration of the increased Ca2+ influx by AMPA receptor antagonists may be a potential ALS therapy. Here, we showed that orally administered perampanel, a selective, non-competitive AMPA receptor antagonist significantly prevented the progression of the ALS phenotype and normalized the TDP-43 pathology-associated death of motor neurons in the AR2 mice. Given that perampanel is an approved anti-epileptic drug, perampanel is a potential candidate ALS drug worthy of a clinical trial.

A bimodular nuclear localization signal assembled via an extended double-stranded RNA-binding domain acts as an RNA-sensing signal for transportin 1.

The human RNA-editing enzyme adenosine deaminase acting on RNA (ADAR1) carries a unique nuclear localization signal (NLS) that overlaps one of its double-stranded RNA-binding domains (dsRBDs). This dsRBD-NLS is recognized by the nuclear import receptor transportin 1 (Trn1; also called karyopherin-β2) in an RNA-sensitive manner. Most Trn1 cargos bear a well-characterized proline-tyrosine-NLS, which is missing from the dsRBD-NLS. Here, we report the structure of the dsRBD-NLS, which reveals an unusual dsRBD fold extended by an additional N-terminal α-helix that brings the N- and C-terminal flanking regions in close proximity. We demonstrate experimentally that the atypical ADAR1-NLS is bimodular and is formed by the combination of the two flexible fragments flanking the folded domain. The intervening dsRBD acts only as an RNA-sensing scaffold, allowing the two NLS modules to be properly positioned for interacting with Trn1. We also provide a structural model showing how Trn1 can recognize the dsRBD-NLS and how dsRNA binding can interfere with Trn1 binding.

Rescue of amyotrophic lateral sclerosis phenotype in a mouse model by intravenous AAV9‐ADAR2 delivery to motor neurons

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease, and the lack of effective therapy results in inevitable death within a few years of onset. Failure of GluA2 RNA editing resulting from downregulation of the RNA-editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) occurs in the majority of ALS cases and causes the death of motor neurons via a Ca2+-permeable AMPA receptor-mediated mechanism. Here, we explored the possibility of gene therapy for ALS by upregulating ADAR2 in mouse motor neurons using an adeno-associated virus serotype 9 (AAV9) vector that provides gene delivery to a wide array of central neurons after peripheral administration. A single intravenous injection of AAV9-ADAR2 in conditional ADAR2 knockout mice (AR2), which comprise a mechanistic mouse model of sporadic ALS, caused expression of exogenous ADAR2 in the central neurons and effectively prevented progressive motor dysfunction. Notably, AAV9-ADAR2 rescued the motor neurons of AR2 mice from death by normalizing TDP-43 expression. This AAV9-mediated ADAR2 gene delivery may therefore enable the development of a gene therapy for ALS.

ADAR1 ablation decreases bone mass by impairing osteoblast function in mice

Bone mass is controlled through a delicate balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We show here that RNA editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) is critical for proper control of bone mass. Postnatal conditional knockout of Adar1 (the gene encoding ADAR1) resulted in a severe osteopenic phenotype. Ablation of the Adar1 gene significantly suppressed osteoblast differentiation without affecting osteoclast differentiation in bone. In vitro deletion of the Adar1 gene decreased expression of osteoblast-specific osteocalcin and bone sialoprotein genes, alkaline phosphatase activity, and mineralization, suggesting a direct intrinsic role of ADAR1 in osteoblasts. ADAR1 regulates osteoblast differentiation by, at least in part, modulation of osterix expression, which is essential for bone formation. Further, ablation of the Adar1 gene decreased the proliferation and survival of bone marrow stromal cells and inhibited the differentiation of mesenchymal stem cells towards osteoblast lineage. Finally, shRNA knockdown of the Adar1 gene in MC-4 pre-osteoblasts reduced cyclin D1 and cyclin A1 expression and cell growth. Our results identify ADAR1 as a new key regulator of bone mass and suggest that ADAR1 functions in this process mainly through modulation of the intrinsic properties of osteoblasts (i.e., proliferation, survival and differentiation).

Deletion of the RNA‐editing enzyme ADAR1 causes regression of established chronic myelogenous leukemia in mice

Patients with chronic myelogenous leukemia (CML) respond well to tyrosine kinase inhibitors (TKIs) of the Bcr-Abl oncoprotein. However, intolerance and resistance to these agents remains a challenge, and TKIs are unable to eradicate rare leukemia-initiating cells. Leukemia treatment would benefit from a better understanding of molecular signals that are necessary for the survival of leukemia-initiating cells but dispensable for normal hematopoietic stem cells. Leukemia-initiating cells in CML can arise from myeloid progenitor cells, a population that we have reported in normal hematopoiesis to depend on the RNA-editing enzyme adenosine deaminase acting on RNA-1 (ADAR1). We now report that Bcr-Abl transformed leukemic cells were ADAR1-dependent in a conditional ADAR1 knockout mouse model. ADAR1 deletion reversed leukocytosis and splenomegaly, and preferentially depleted primitive Lin-Sca+Kit+ (LSK) leukemic cells but not LSK cells lacking the leukemic oncoprotein. ADAR1 deletion ultimately normalized the peripheral white blood count, eliminating leukemic cells as assessed by PCR. These results uncover a novel requirement for ADAR1 in myeloid leukemic cells and indicate that ADAR1 may comprise a new molecular target for CML-directed therapeutics.

ADAR1 isoform involvement in embryonic lethality

Letters to the editor recently published in PNAS (1, 2) challenged and defended a claim made on a recent article (3). Matthaei et al. (1) questioned whether the animal model used by Ward et al. (3) was adequate to support their claim that the P150 isoform of the RNA editing enzyme adenosine deaminase 1 (ADAR1) is required for embryogenesis. We believe the question remains open and present data indicating that a more complete assessment of ADAR1 isoform protein expression in the model of Ward et al. (3) is necessary to draw firm conclusions from it.

Reply to Matthaei et al.: The p150 isoform of the RNA editing enzyme Adenosine Deaminase 1 is required for embryogenesis

Matthaei et al. (1) raise the possibility that specific targeting of the p150 isoform of the RNA editing enzyme Adenosine Deaminase 1 (ADAR1) may have resulted in the generation of a hypomorph of the p110 isoform, thereby generating a functional double deletion similar to previous Adar1 gene disruptions (2, 3). On this basis, they question our conclusion that the p150 isoform of ADAR1 is required for embryogenesis. Specifically, Matthaei et al. call into question the results reported in figure 1E of our article (4), which supported the conclusion that expression of the exon 1B-containing transcript …