Simultaneous profiling of the RNA targets of two RNA-binding proteins using TRIBE-STAMP.

RNA-binding proteins (RBPs) are central players in RNA homeostasis and the control of gene expression. The identification of RBP targets, interactions, and the regulatory networks they control is crucial for understanding their cellular functions. Traditional methods for identifying RBP targets across the transcriptome have been insightful but are limited by their focus on a single RBP at a time and their general inability to identify individual RNA molecules that are bound by RBPs of interest. Recently, we overcame these limitations by developing TRIBE-STAMP, a method which enables concurrent identification of the RNA targets of two RBPs of interest with single-molecule resolution. TRIBE-STAMP works by tagging desired RBPs with either the ADAR or APOBEC1 RNA editing enzymes and expressing them in cells, followed by RNA-seq. Subsequent computational identification of A-to-I and C-to-U editing events enables the simultaneous identification of the ADAR- and APOBEC1-fused RBP target RNAs, respectively. Here, we present a detailed protocol for TRIBE-STAMP, including considerations for fusion protein expression in cells and step-by-step computational analysis of sequencing data. TRIBE-STAMP is a simple and highly versatile approach for single-molecule identification of the targets of RBPs which enables unprecedented insights into the biological interplay between RBP pairs in cells.

ADAR1 Promotes Myogenic Proliferation and Differentiation of Goat Skeletal Muscle Satellite Cells.

As one of the most important economic traits for domestic animal husbandry, skeletal muscle is regulated by an intricate molecular network. Adenosine deaminase acting on RNA (ADAR1) involves various physiological processes and diseases, such as innate immunity and the development of lung adenocarcinoma, breast cancer, gastric cancer, etc. However, its role in skeletal muscle growth requires further clarification. Here, we explored the functions of ADAR1 in the myogenic process of goat skeletal muscle satellite cells (MuSCs). The ADAR1 transcripts were noticeably enriched in goat visceral tissues compared to skeletal muscle. Additionally, its levels in slow oxidative muscles like the psoas major and minor muscles were higher than in the fast oxidative glycolytic and fast glycolytic muscles. Among the two common isoforms from ADAR1, p110 is more abundant than p150. Moreover, overexpressing ADAR1 enhanced the proliferation and myogenic differentiation of MuSCs. The mRNA-seq performed on MuSCs' knockdown of ADAR1 obtained 146 differentially expressed genes (DEGs), 87 upregulated and 59 downregulated. These DEGs were concentrated in muscle development and process pathways, such as the MAPK and cAMP signaling pathways. Furthermore, many DEGs as the key nodes defined by protein-protein interaction networks (PPI), including STAT3, MYH3/8, TGFß2, and ACTN4, were closely related to the myogenic process. Finally, RNA immunoprecipitation combined with qPCR (RIP-qPCR) showed that ADAR1 binds to PAX7 and MyoD mRNA. This study indicates that ADAR1 promotes the myogenic development of goat MuSCs, which provides a useful scientific reference for further exploring the ADAR1-related regulatory networks underlying mammal skeletal muscle growth.

Targeting exosomal double-stranded RNA-TLR3 signaling pathway attenuates morphine tolerance and hyperalgesia.

Long-term morphine use leads to tolerance and hyperalgesia in patients with chronic pain, with neuroinflammation playing a key role, but its underlying mechanisms remain elusive. This study determines that repeated intrathecal morphine injections increase double-stranded RNA (dsRNA) production in spinal neurons, due to downregulated adenosine deaminase RNA specific 1 (ADAR1) expression. Lentivirus-induced ADAR1 elevation decreases the high levels of intracellular dsRNA and attenuates morphine tolerance and hyperalgesia. dsRNA is released into cerebrospinal fluid via exosomes (Exos) after repeated morphine injections and is taken up by microglia for TLR3-TRIF-IL-6 signaling activation. Blocking Exos release with GW4869 or inhibition of TLR3 signaling mitigates neuroinflammation, preventing the development of morphine tolerance and hyperalgesia. Intrathecal injection of TLR3 inhibitor alone shows analgesic effects in neuropathic pain, and co-administration with morphine amplifies the analgesic efficacy of morphine. These findings demonstrate that targeting dsRNA-TLR3 signaling to mitigate neuroinflammation could be a promising treatment for morphine tolerance.

Activation of PKR by a short-hairpin RNA.

Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54's potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation.

Revealing the hidden RBP-RNA interactions with RNA modification enzyme-based strategies.

RNA-binding proteins (RBPs) are powerful and versatile regulators in living creatures, playing fundamental roles in organismal development, metabolism, and various diseases by the regulation of gene expression at multiple levels. The requirements of deep research on RBP function have promoted the rapid development of RBP-RNA interplay detection methods. Recently, the detection method of fusing RNA modification enzymes (RME) with RBP of interest has become a hot topic. Here, we reviewed RNA modification enzymes in adenosine deaminases that act on RNA (ADAR), terminal nucleotidyl transferase (TENT), and activation-induced cytosine deaminase/ApoB mRNA editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family, regarding the biological function, biochemical activity, and substrate specificity originated from enzyme selves, their domains and partner proteins. In addition, we discussed the RME activity screening system, and the RME mutations with engineered enzyme activity. Furthermore, we provided a systematic overview of the basic principles, advantages, disadvantages, and applications of the RME-based and cross-linking and immunopurification (CLIP)-based RBP target profiling strategies, including targets of RNA-binding proteins identified by editing (TRIBE), RNA tagging, surveying targets by APOBEC-mediated profiling (STAMP), CLIP-seq, and their derivative technology. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Processing > RNA Editing and Modification.

Electroacupuncture Relieves Neuropathic Pain via Adenosine 3 Receptor Activation in the Spinal Cord Dorsal Horn of Mice.

Neuropathic pain (NPP) is a devastating and unbearable painful condition. As prevailing treatment strategies have failed to mitigate its complications, there remains a demand for effective therapies. Electroacupuncture (EA) has proved a potent remedial strategy in NPP management in humans and mammals. However, past studies have investigated the underlying mechanism of the analgesic effects of EA on NPP, focusing primarily on adenosine receptors in peripheral tissues. Herein, we elucidate the role of the adenosine (Adora-3) signaling pathway in mediating pain relief through EA in the central nervous system, which is obscure in the literature and needs exploration. Specific pathogen-free (SPF) male adult mice (C57BL/6 J) were utilized to investigate the effect of EA on adenosine metabolism (CD73, ADA) and its receptor activation (Adora-3), as potential mechanisms to mitigate NPP in the central nervous system. NPP was induced via spared nerve injury (SNI). EA treatment was administered seven times post-SNI surgery, and lumber (L4-L6) spinal cord was collected to determine the molecular expression of mRNA and protein levels. In the spinal cord of mice, following EA application, the expression results revealed that EA upregulated (p < 0.05) Adora-3 and CD73 by inhibiting ADA expression. In addition, EA triggered the release of adenosine (ADO), which modulated the nociceptive responses and enhanced neuronal activation. Meanwhile, the interplay between ADO levels and EA-induced antinociception, using an Adora-3 agonist and antagonist, showed that the Adora-3 agonist IB-MECA significantly increased (p < 0.05) nociceptive thresholds and expression levels. In contrast, the antagonist MRS1523 exacerbated neuropathic pain. Furthermore, an upregulated effect of EA on Adora-3 expression was inferred when the Adora-3 antagonist was administered, and the EA treatment increased the fluorescent intensity of Adora-3 in the spinal cord. Taken together, EA effectively modulates NPP by regulating the Adora-3 signaling pathway under induced pain conditions. These findings enhance our understanding of NPP management and offer potential avenues for innovative therapeutic interventions.

Engineering TadA ortholog-derived cytosine base editor without motif preference and adenosine activity limitation.

The engineered TadA variants used in cytosine base editors (CBEs) present distinctive advantages, including a smaller size and fewer off-target effects compared to cytosine base editors that rely on natural deaminases. However, the current TadA variants demonstrate a preference for base editing in DNA with specific motif sequences and possess dual deaminase activity, acting on both cytosine and adenosine in adjacent positions, limiting their application scope. To address these issues, we employ TadA orthologs screening and multi sequence alignment (MSA)-guided protein engineering techniques to create a highly effective cytosine base editor (aTdCBE) without motif and adenosine deaminase activity limitations. Notably, the delivery of aTdCBE to a humanized mouse model of Duchenne muscular dystrophy (DMD) mice achieves robust exon 55 skipping and restoration of dystrophin expression. Our advancement in engineering TadA ortholog for cytosine editing enriches the base editing toolkits for gene-editing therapy and other potential applications.

RNA Editing by ADAR Adenosine Deaminases in the Cell Models of CAG Repeat Expansion Diseases: Significant Effect of Differentiation from Stem Cells into Brain Organoids in the Absence of Substantial Influence of CAG Repeats on the Level of Editing.

Expansion of CAG repeats in certain genes is a known cause of several neurodegenerative diseases, but exact mechanism behind this is not yet fully understood. It is believed that the double-stranded RNA regions formed by CAG repeats could be harmful to the cell. This study aimed to test the hypothesis that these RNA regions might potentially interfere with ADAR RNA editing enzymes, leading to the reduced A-to-I editing of RNA and activation of the interferon response. We studied induced pluripotent stem cells (iPSCs) derived from the patients with Huntington's disease or ataxia type 17, as well as midbrain organoids developed from these cells. A targeted panel for next-generation sequencing was used to assess editing in the specific RNA regions. Differentiation of iPSCs into brain organoids led to increase in the ADAR2 gene expression and decrease in the expression of protein inhibitors of RNA editing. As a result, there was increase in the editing of specific ADAR2 substrates, which allowed identification of differential substrates of ADAR isoforms. However, comparison of the pathology and control groups did not show differences in the editing levels among the iPSCs. Additionally, brain organoids with 42-46 CAG repeats did not exhibit global changes. On the other hand, brain organoids with the highest number of CAG repeats in the huntingtin gene (76) showed significant decrease in the level of RNA editing of specific transcripts, potentially involving ADAR1. Notably, editing of the long non-coding RNA PWAR5 was nearly absent in this sample. It could be stated in conclusion that in most cultures with repeat expansion, the hypothesized effect on RNA editing was not confirmed.

An orthology-based methodology as a complementary approach to retrieve evolutionarily conserved A-to-I RNA editing sites.

Adar-mediated adenosine-to-inosine (A-to-I) mRNA editing is a conserved mechanism that exerts diverse regulatory functions during the development, evolution, and adaptation of metazoans. The accurate detection of RNA editing sites helps us understand their biological significance. In this work, with an improved genome assembly of honeybee (Apis mellifera), we used a new orthology-based methodology to complement the traditional pipeline of (de novo) RNA editing detection. Compared to the outcome of traditional pipeline, we retrieved many novel editing sites in CDS that are deeply conserved between honeybee and other distantly related insects. The newly retrieved sites were missed by the traditional de novo identification due to the stringent criteria for controlling false-positive rate. Caste-specific editing sites are identified, including an Ile>Met auto-recoding site in Adar. This recoding was even conserved between honeybee and bumblebee, suggesting its putative regulatory role in shaping the phenotypic plasticity of eusocial Hymenoptera. In summary, we proposed a complementary approach to the traditional pipeline and retrieved several previously unnoticed CDS editing sites. From both technical and biological aspects, our works facilitate future researches on finding the functional editing sites and advance our understanding on the connection between RNA editing and the great phenotypic diversity of organisms.

Role of thoracoscopy in diagnosis of tubercular pleural effusion in low ADA setting- A prospective, observational study.

BACKGROUND: Tubercular Pleural effusion (TBPE) is one of most common extrapulmonary tuberculosis. It can be difficult to diagnose due to low sensitivity of pleural fluid smear, culture and CBNAAT. Diagnosis of TBPE is then dependent on the level of pleural fluid Adenosine Deaminase (ADA). Thoracoscopic pleural biopsy gives definite diagnosis specially in Low Pleural fluid ADA setting. AIMS AND OBJECTIVE: This study was planned to find out the prevalence of tubercular etiology in patients of exudative pleural effusion with low ADA (ADA <40 IU/L). MATERIAL AND METHODS: A Prospective, observational study was carried out in a tertiary teaching institute in north India. Total 142 patients of pleural effusion with low ADA were enrolled. All patients underwent rigid thoracoscopy for confirmation of their diagnosis. RESULTS: Out of 142 patients, male were 78 (55%) and female were 64 (45%). Mean age of patients were 57.4 years. Tuberculosis was diagnosed as a cause of effusion in 22 (15.5%) out of 142 patients. Majority of TBPE patients had pleural thickening as thoracoscopic finding. Mean ADA level in TBPE was 27.36 ± 11.6 as compared to 18.55 ± 9.02 in non tubercular pleural effusion patients and this difference was significant statistically (P- 0.002). CONCLUSION: The diagnosis of patients having exudative, low ADA pleural effusion can be very easily confirmed by thoracoscopy guided pleural biopsy which has a very high diagnostic yield.

An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation.

Adar null mutant mouse embryos die with aberrant double-stranded RNA (dsRNA)-driven interferon induction, and Adar Mavs double mutants, in which interferon induction is prevented, die soon after birth. Protein kinase R (Pkr) is aberrantly activated in Adar Mavs mouse pup intestines before death, intestinal crypt cells die, and intestinal villi are lost. Adar Mavs Eifak2 (Pkr) triple mutant mice rescue all defects and have long-term survival. Adenosine deaminase acting on RNA 1 (ADAR1) and PKR co-immunoprecipitate from cells, suggesting PKR inhibition by direct interaction. AlphaFold studies on an inhibitory PKR dsRNA binding domain (dsRBD)-kinase domain interaction before dsRNA binding and on an inhibitory ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA provide a testable model of the inhibition. Wild-type or editing-inactive human ADAR1 expressed in A549 cells inhibits activation of endogenous PKR. ADAR1 dsRNA binding is required for, but is not sufficient for, PKR inhibition. Mutating the ADAR1 dsRBD3-PKR contact prevents co-immunoprecipitation, ADAR1 inhibition of PKR activity, and co-localization of ADAR1 and PKR in cells.

ADAR1 Is Essential for Smooth Muscle Homeostasis and Vascular Integrity.

Vascular smooth muscle cells (VSMCs) play a critical role in maintaining vascular integrity. VSMC dysfunction leads to numerous vascular diseases. Adenosine deaminases acting on RNA 1 (ADAR1), an RNA editing enzyme, has shown both RNA editing and non-editing functions. Global deletion of ADAR1 causes embryonic lethality, but the phenotype of homozygous ADAR1 deletion specifically in SMCs (ADAR1sm-/-) remains to be determined. By crossing ADAR1fl/fl mice with Myh11-CreERT2 mice followed by Tamoxifen induction, we found that ADAR1sm-/- leads to lethality in adult mice 14 days after the induction. Gross examination revealed extensive hemorrhage and detrimental vascular damage in different organs. Histological analyses revealed destruction of artery structural integrity with detachment of elastin laminae from VSMCs in ADAR1sm-/- aortas. Furthermore, ADAR1sm-/- resulted in severe VSMC apoptosis and mitochondrial dysfunction. RNA sequencing analyses of ADAR1sm-/- aorta segments demonstrated profound transcriptional alteration of genes impacting vascular health including a decrease in fibrillin-1 expression. More importantly, ADAR1sm-/- disrupts the elastin and fibrillin-1 interaction, a molecular event essential for artery structure. Our results indicate that ADAR1 plays a critical role in maintaining SMC survival and vascular stability and resilience.

An improved SNAP-ADAR tool enables efficient RNA base editing to interfere with post-translational protein modification.

RNA base editing relies on the introduction of adenosine-to-inosine changes into target RNAs in a highly programmable manner in order to repair disease-causing mutations. Here, we propose that RNA base editing could be broadly applied to perturb protein function by removal of regulatory phosphorylation and acetylation sites. We demonstrate the feasibility on more than 70 sites in various signaling proteins and identify key determinants for high editing efficiency and potent down-stream effects. For the JAK/STAT pathway, we demonstrate both, negative and positive regulation. To achieve high editing efficiency over a broad codon scope, we applied an improved version of the SNAP-ADAR tool. The transient nature of RNA base editing enables the comparably fast (hours to days), dose-dependent (thus partial) and reversible manipulation of regulatory sites, which is a key advantage over DNA (base) editing approaches. In summary, PTM interference might become a valuable field of application of RNA base editing.

Bioinformatics for Inosine: Tools and Approaches to Trace This Elusive RNA Modification.

Inosine is a nucleotide resulting from the deamination of adenosine in RNA. This chemical modification process, known as RNA editing, is typically mediated by a family of double-stranded RNA binding proteins named Adenosine Deaminase Acting on dsRNA (ADAR). While the presence of ADAR orthologs has been traced throughout the evolution of metazoans, the existence and extension of RNA editing have been characterized in a more limited number of animals so far. Undoubtedly, ADAR-mediated RNA editing plays a vital role in physiology, organismal development and disease, making the understanding of the evolutionary conservation of this phenomenon pivotal to a deep characterization of relevant biological processes. However, the lack of direct high-throughput methods to reveal RNA modifications at single nucleotide resolution limited an extended investigation of RNA editing. Nowadays, these methods have been developed, and appropriate bioinformatic pipelines are required to fully exploit this data, which can complement existing approaches to detect ADAR editing. Here, we review the current literature on the "bioinformatics for inosine" subject and we discuss future research avenues in the field.

Rational design of base, sugar and backbone modifications improves ADAR-mediated RNA editing.

AIMers are short, chemically modified oligonucleotides that induce A-to-I RNA editing through interaction with endogenous adenosine deaminases acting on RNA (ADAR) enzymes. Here, we describe the development of new AIMer designs with base, sugar and backbone modifications that improve RNA editing efficiency over our previous design. AIMers incorporating a novel pattern of backbone and 2' sugar modifications support enhanced editing efficiency across multiple sequences. Further efficiency gains were achieved through incorporation of an N-3-uridine (N3U), in place of cytidine (C), in the 'orphan base' position opposite the edit site. Molecular modeling suggests that N3U might enhance ADAR catalytic activity by stabilizing the AIMer-ADAR interaction and potentially reducing the energy required to flip the target base into the active site. Supporting this hypothesis, AIMers containing N3U consistently enhanced RNA editing over those containing C across multiple target sequences and multiple nearest neighbor sequence combinations. AIMers combining N3U and the novel pattern of 2' sugar chemistry and backbone modifications improved RNA editing both in vitro and in vivo. We provide detailed N3U synthesis methods and, for the first time, explore the impact of N3U and its analogs on ADAR-mediated RNA editing efficiency and targetable sequence space.

ADARp150 counteracts whole genome duplication.

Impaired control of the G1/S checkpoint allows initiation of DNA replication under non-permissive conditions. Unscheduled S-phase entry is associated with DNA replication stress, demanding for other checkpoints or cellular pathways to maintain proliferation. Here, we uncovered a requirement for ADARp150 to sustain proliferation of G1/S-checkpoint-defective cells under growth-restricting conditions. Besides its well-established mRNA editing function in inversely oriented short interspersed nuclear elements (SINEs), we found ADARp150 to exert a critical function in mitosis. ADARp150 depletion resulted in tetraploidization, impeding cell proliferation in mitogen-deprived conditions. Mechanistically we show that ADAR1 depletion induced aberrant expression of Cyclin B3, which was causative for mitotic failure and whole-genome duplication. Finally, we find that also in vivo ADAR1-depletion-provoked tetraploidization hampers tumor outgrowth.

Factors influencing accelerated aging in patients with type 2 diabetes mellitus and coronary heart disease.

Objective: To investigate the factors influencing accelerated aging in patients with type 2 diabetes mellitus (T2DM) and coronary heart disease (CHD). Methods: A total of 216 patients diagnosed with T2DM and CHD between August 2019 and August 2023 at Xuzhou Central Hospital were selected. Patients were divided into an aging group and a non-aging group, based on the positive or negative values of phenotypic age acceleration (PhenoAgeAccel). Logistic regression analysis was conducted. Variables that had a univariate analysis P< 0.05 were included in the multivariate analysis to identify factors influencing aging in patients with T2DM and CHD, and the area under the curve of the model was reported. Results: This study included 216 patients, with 89 in the accelerated aging group, and 127 in the non-accelerated aging group. The average age of patients was 70.40 (95% CI: 69.10-71.69) years, with 137 males (63.4%). Compared with the non-accelerated aging group, patients in the accelerated aging group were older, with a higher proportion of males, and a higher prevalence of hypertension, stable angina pectoris, and unstable angina pectoris. Multivariate Logistic regression analysis indicated that the absolute value of neutrophils (NEUT#), urea (UREA), adenosine deaminase (ADA), and the triglyceride-glucose index (TyG) were risk factors for accelerated aging, while cholinesterase (CHE) was a protective factor. For each unit increase in NEUT#, UREA, ADA, and TyG, the risk of aging increased by 64%, 48%, 10%, and 789%, respectively. The overall area under the receiver operating characteristic (ROC) curve of the model in the training set was 0.894, with a 95% confidence interval (CI) of 0.851-0.938. Conclusion: NEUT#, CHE, UREA, ADA, and TyG are predictors of accelerated aging in patients with T2DM and CHD, with the model showing favorable overall predictive performance.

RNA editing regulates host immune response and T cell homeostasis in SARS-CoV-2 infection.

Adenosine to inosine (A-to-I) RNA editing by ADAR1 has been implicated in maintaining self-tolerance, preventing autoimmunity, and mediating antiviral immunity. Foreign viral double-stranded RNA triggers rapid interferon response and activates ADAR1 in the host immune system. Emerging data points to a role of ADAR1 A-to-I editing in the inflammatory response associated with severe COVID-19 disease. We identify A-to-I editing events within human whole transcriptome data from SARS-CoV-2 infected individuals, non-infected individuals, and individuals with other viral illnesses from nasopharyngeal swabs. High levels of RNA editing in host cells are associated with low SARS-CoV-2 viral load (p = 9.27 E-06), suggesting an inhibitory effect of ADAR1 on viral infection. Additionally, we find differentially expressed genes associated with RNA-modifications and interferon response. Single cell RNA-sequencing analysis of SARS-CoV-2 infected nasopharyngeal swabs reveals that cytotoxic CD8 T cells upregulate ADAR1 in COVID-19 positive samples (p = 0.0269). We further reveal ADAR1 expression increases with CD4 and CD8 T cell activation, and knockdown of ADAR1 leads to apoptosis and aberrant IL-2 secretion. Together, our data suggests A-to-I RNA editing is required to maintain healthy homeostasis of activated T cells to combat SARS-CoV-2 infection.

Adaptive evolution of A-to-I auto-editing site in Adar of eusocial insects.

BACKGROUND: Adenosine-to-inosine (A-to-I) RNA editing is a co-/post-transcriptional modification introducing A-to-G variations in RNAs. There is extensive discussion on whether the flexibility of RNA editing exerts a proteomic diversification role, or it just acts like hardwired mutations to correct the genomic allele. Eusocial insects evolved the ability to generate phenotypically differentiated individuals with the same genome, indicating the involvement of epigenetic/transcriptomic regulation. METHODS: We obtained the genomes of 104 Hymenoptera insects and the transcriptomes of representative species. Comparative genomic analysis was performed to parse the evolutionary trajectory of a regulatory Ile > Met auto-recoding site in Adar gene. RESULTS: At genome level, the pre-editing Ile codon is conserved across a node containing all eusocial hymenopterans. At RNA level, the editing events are confirmed in representative species and shows considerable condition-specificity. Compared to random expectation, the editable Ile codon avoids genomic substitutions to Met or to uneditable Ile codons, but does not avoid mutations to other unrelated amino acids. CONCLUSIONS: The flexibility of Adar auto-recoding site in Hymenoptera is selectively maintained, supporting the flexible RNA editing hypothesis. We proposed a new angle to view the adaptation of RNA editing, providing another layer to explain the great phenotypical plasticity of eusocial insects.

Programmed RNA editing with an evolved bacterial adenosine deaminase.

Programmed RNA editing presents an attractive therapeutic strategy for genetic disease. In this study, we developed bacterial deaminase-enabled recoding of RNA (DECOR), which employs an evolved Escherichia coli transfer RNA adenosine deaminase, TadA8e, to deposit adenosine-to-inosine editing to CRISPR-specified sites in the human transcriptome. DECOR functions in a variety of cell types, including human lung fibroblasts, and delivers on-target activity similar to ADAR-overexpressing RNA-editing platforms with 88% lower off-target effects. High-fidelity DECOR further reduces off-target effects to basal level. We demonstrate the clinical potential of DECOR by targeting Van der Woude syndrome-causing interferon regulatory factor 6 (IRF6) insufficiency. DECOR-mediated RNA editing removes a pathogenic upstream open reading frame (uORF) from the 5' untranslated region of IRF6 and rescues primary ORF expression from 12.3% to 36.5%, relative to healthy transcripts. DECOR expands the current portfolio of effector proteins and opens new territory in programmed RNA editing.