Search Results (96)

RNA editing is a critical post-transcriptional modification that contributes to transcriptomic and proteomic diversity. The most common A-to-I (recognized as G) RNA editing enzymes are adenosine deaminases acting on RNA 1 and 2 (ADAR1 and ADAR2, respectively), which mediate alterations across all regions of mRNA molecules. However, a systematic cross-tissue view of RNA editing and its molecular correlates is still lacking. Here, we developed a rapid method for ADAR editing assessment based on 24 frequently edited positions in coding regions, which enables faster estimation of RNA editing levels than previous methods. We applied this metric to assess RNA editing in normal and cancerous lung and colorectal tissues. We analyzed RNA and whole exome sequencing profiles of experimental 172 colorectal and 144 lung cancer samples, and literature 646 colorectal and 1037 lung cancer samples. We also examined two types of control tissues: tumor-matched normal tissues (51 colorectal and 108 lung samples) and healthy tissues (6 colorectal and 7 lung samples). Overall ADAR-mediated RNA editing levels were ~2.9- and ~4.7-fold higher in healthy controls than in colorectal and lung cancers, respectively. In addition to their well-known association with immune cells, we identified positive correlations of ADAR editing with 740 molecular pathways including those responsible for extracellular matrix organization, RAS-MAPK axis and G2/M phase cell cycle arrest, and negative—with 139 pathways responsible for DNA repair, apoptosis, expression of transposable elements, and other factors. Full article

RNA editing is a way to diversify, regulate expression, and expand the cell transcriptome. The most common RNA editing is the reversible conversion of adenosine (A) to inosine (I) driven by double-stranded RNA-binding adenosine deaminases (ADARs). As inosine is recognized as guanosine (G) during translation, the RNA editing may result in non-synonymous codon changes. For this reason, ADARs have gained attention as promising enzymes to rewrite mRNA. Many efforts were undertaken to engineer a precise, effective, and controllable ADAR-based system to target certain Adenines on RNA to repair pathological mutations. This review summarizes the advances in ADAR-mediated RNA editing, evolving from systems using antisense oligonucleotides as guide RNA to recruit endogenous or overexpressed ADARs, through more complex setups additionally expressing other RNA-binding proteins, to rational designs harnessing ADARs to convert other nucleotides and amplify the low initial signal. Increasing the specificity and yield of RNA editing, expanding the number of targetable sites, and reducing off-target and bystander activity remain key challenges for these technologies. Improving delivery efficiency across a broad range of cell types, as well as optimizing delivery routes in in vivo studies are also critical to harness them as advantageous tools for both research and therapy. Full article

Adenosine deaminase acting on RNA 1 (ADAR1) is an essential RNA-editing enzyme responsible for the hydrolytic deamination of adenosine to inosine (A-to-I) in double-stranded RNA. This editing mechanism plays a critical role in gene regulation, particularly in neural and immune contexts. Dysregulation of ADAR1 activity has been implicated in neurological disorders, cancer progression, and immune dysfunction, making ADAR1 an emerging therapeutic target. However, progress in therapeutic development has been hindered by the lack of structural insight into the full-length protein and how its dynamic behavior influences RNA-editing specificity and protein–protein interactions. In this study, we present computational models of the full-length ADAR1p150 isoform generated by homology modeling and further analyzed using molecular dynamics (MD) simulations and principal component analysis (PCA). Our analyses reveal that the dsRBD3 and CDD remain structurally stable, crucial for protein binding and catalytic function, whereas ZBDs and dsRBD1/2 exhibit extensive flexibility, particularly in inter-domain loops, facilitating RNA recognition indicative of conformational selection and fly-casting mechanisms. Free-energy landscape mapping identifies multiple low-energy conformations, highlighting conserved domain cores and flexible loop arrangements. Together, these findings underscore the importance of ADAR1’s dynamic architecture in regulating its function. By linking static structural information with dynamic behavior, the full-length models and dynamic insights presented here provide a valuable framework for future studies of ADAR1 complex formation, editing specificity, and therapeutic targeting. Full article

Background: ADAR1 (ADAR), ADAR2 (ADARB1), and ADAR3 (ADARB2) are deaminase adenosine RNA-specific enzymes that play a significant role in RNA metabolism. ADAR1 (ADAR) and ADAR2 (ADARB1) catalyze A-to-I editing and ADAR3 (ADARB2) plays a regulatory role. The role of these three genes still remains unknown in head and neck cancers (HNSCC). The aim of this study is to reveal the role of deaminase adenosine RNA-specific enzymes in pathomechanisms of HNSCC and to investigate their potential utility as diagnostic and/or prognostic biomarkers. Methods: The quantitative PCR analysis was conducted using RNA isolated from 22 pairs of matched tumor and adjacent normal tissues, 76 formalin-fixed paraffin-embedded (FFPE) tumor samples, and a panel of HNSCC cell lines (DOK, SCC-25, SCC-40, FaDu, and CAL-27). In parallel, transcriptomic and clinical data from the Cancer Genome Atlas HNSCC cohort were analyzed. Patients were stratified into high- and low-expression groups, and statistical assessments included overall survival and progression-free interval analyses, evaluation of gene expression in relation to clinicopathological parameters, correlation with other genes, and functional pathway exploration using gene set enrichment analysis. Results: ADARB2 was significantly downregulated in HNSCC tumor tissues compared to adjacent normal mucosa (p = 0.044), with discriminatory potential to distinguish malignant from non-malignant tissues (AUC = 0.692, p = 0.029). TCGA data confirmed ADAR (p < 0.0001) and ADARB1 (p < 0.0001) upregulation in tumors, while ADARB2 was markedly reduced (p = 0.04). Patients with high ADARB2 expression showed significantly longer overall survival (pa = 0.0121; pb = 0.0098), with a trend toward improved progression-free survival (pb = 0.0681). Subsite analysis revealed high ADAR expression correlated with poor OS in pharyngeal tumors (p < 0.05), whereas high ADARB2 expression was linked to improved DFS (pa = 0.0023, pb = 0.0047). GSEA indicated that low ADARB2 expression was enriched in oncogenic pathways, including Wnt/β-catenin (p = 0.006), MYC targets (p = 0.009), and TGF-β1 (p = 0.009). Conclusions: ADARB2 expression was significantly reduced in HNSCC tumor tissues compared to normal mucosa and demonstrated strong discriminatory power for distinguishing malignant from non-malignant samples. High ADARB2 expression was associated with markedly improved overall survival, whereas low expression correlated with enrichment of oncogenic pathways, including Wnt/β-catenin, Notch, and Hedgehog, consistent with a poorer clinical prognosis. These findings highlight ADARB2 as a promising diagnostic biomarker and independent prognostic factor in HNSCC. Full article

This paper summarizes the results of multi-year studies performed by our research team, focusing on an analysis of protein recoding mediated by messenger RNA editing by ADAR adenosine deaminases. Searching for ADAR-mediated protein recoding was performed in the central nervous system of the model organisms, fruit fly and mouse, as well as in the human proteomic datasets. The proteogenomic approach has made it possible to identify dozens of editing events in the proteome, thus validating the results of transcriptomic studies. The observed recoding events in animals, ranging from insects to mammals, mainly affect the cytoskeletal components and proteins involved in synaptic transmission. In humans, recoding changes are most often observed in the central nervous system or tumor tissues. Over 15 million editing sites have been identified in humans; only a few thousand of those can potentially yield amino acid substitutions. Using a proteogenomic approach, dozens of protein recoding sites are identified, demonstrating their origin in ADAR RNA editing. Moreover, this revealed that the level of recoding at specific sites is not directly related to the abundance of ADAR enzymes per se or their target proteins. The recoding processes probably have differential regulation of interactions at the mRNA level that is yet to be clarified. Full article

Long non-coding RNAs (lncRNAs) are transcripts over 200 nucleotides long that do not encode proteins. Although many lncRNAs remain uncharacterized, they are known to play diverse regulatory roles in gene expression. A group of lncRNAs called natural antisense transcripts can form double-stranded structures with their sense partners due to sequence complementarity. These duplexes can become substrates for A-to-I RNA editing, an epitranscriptomic modification mediated by ADAR enzymes. RNA editing is known to influence transcript splicing, affect the resulting gene expression product or alter RNA stability, all of which can impact cancer cell biology. Here, we show a novel natural antisense transcript, UGGT1-AS1, that we have identified and characterized in terms of its cellular localization and sense partner interactions. Furthermore, we demonstrate that UGGT1-AS1 affects cell proliferation and regulates the stability of the UGGT1 sense transcript. Finally, using publicly available RNA sequencing data, we identify A-to-I RNA editing events in the protein-coding gene UGGT1 and further confirm them by RT-PCR and Sanger sequencing in MCF7 cell lines. We hypothesize that UGGT1-AS1 may act as a triggering factor for the A-to-I RNA editing process in its sense partner. Our findings highlight the regulatory role of UGGT1-AS1 and suggest its involvement in RNA editing and cancer biology. Full article

Mesothelioma is a rare and aggressive cancer linked to asbestos exposure and characterized by rapid metastasis and poor prognosis. Inhibition of adenosine deaminase acting on dsRNA 2 (ADAR2) RNA binding but not ADAR2 editing has shown antitumor effects in mesothelioma. Natural compounds from the Traditional Chinese Medicine (TCM) database were docked to the RNA-binding interface of ADAR2’s second dsRNA binding domain (dsRBD2), and their drug-likeness and predicted safety were assessed. Eight ligands (ZINC000085597263, ZINC000085633079, ZINC000014649947, ZINC000034512861, ZINC000070454124, ZINC000085594944, ZINC000085633008, and ZINC000095909822) showed high binding affinity to dsRBD2 from molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations. Protein–ligand interactions were analyzed to identify key residues contributing to these binding affinities. Molecular dynamics (MD) simulations of dsRBD–ligand–RNA complexes revealed that four compounds (ZINC000085597263, ZINC000085633079, ZINC000014649947, and ZINC000034512861) had negative binding affinities to dsRBD2 in the presence of the RNA substrate GluR-2. Key residues, including Val164, Met165, Lys209, and Lys212, were crucial for ligand binding, even with RNA present, suggesting these compounds could inhibit dsRBD2’s RNA-binding function. The predicted biological activities of these compounds indicate potential anticancer properties, particularly for the treatment of mesothelioma. These compounds are structurally similar to known anti-mesothelioma agents or anticancer drugs, highlighting their therapeutic potential. Current mesothelioma treatments are limited. Optimization of these compounds, alone or in combination with current therapeutics, has potential for mesothelioma treatment. Additionally, five high-quality full-length ADAR2 models were developed. These models provide insights into ADAR2 function, mutation impacts, and potential areas for protein engineering to enhance stability, RNA-binding specificity, or protein interactions, particularly concerning dimerization or complex formation with other proteins and RNAs. Full article

The double-stranded RNA editing enzyme ADAR1 connects two forms of genetic programming, one based on codons and the other on flipons. ADAR1 recodes codons in pre-mRNA by deaminating adenosine to form inosine, which is translated as guanosine. ADAR1 also plays essential roles in the immune defense against viruses and cancers by recognizing left-handed Z-DNA and Z-RNA (collectively called ZNA). Here, we review various aspects of ADAR1 biology, starting with codons and progressing to flipons. ADAR1 has two major isoforms, with the p110 protein lacking the p150 Zα domain that binds ZNAs with high affinity. The p150 isoform is induced by interferon and targets ALU inverted repeats, a class of endogenous retroelement that promotes their transcription and retrotransposition by incorporating Z-flipons that encode ZNAs and G-flipons that form G-quadruplexes (GQ). Both p150 and p110 include the Zβ domain that is related to Zα but does not bind ZNAs. Here we report strong evidence that Zβ binds the GQ that are formed co-transcriptionally by ALU repeats and within R-loops. By binding GQ, ADAR1 suppresses ALU-mediated alternative splicing, generates most of the reported nonsynonymous edits and promotes R-loop resolution. The recognition of the various alternative nucleic acid conformations by ADAR1 connects genetic programming by flipons with the encoding of information by codons. The findings suggest that incorporating G-flipons into editmers might improve the therapeutic editing efficacy of ADAR1. Full article

SNAP-tag and Halo-tag have been employed to achieve targeted RNA editing by directing the deaminase domain of human ADAR to specific sites in the transcriptome. This targeting is facilitated by short guide RNAs (gRNAs) complementary to the target transcript, which are chemically modified with benzylguanine or chloroalkane moieties to enable covalent binding to the respective self-labeling enzymes. However, broad application of this approach has been limited by challenges such as low scalability, the requirement for specialized chemical expertise and equipment, and labor-intensive protocols. In this study, we introduce streamlined, efficient protocols for the synthesis and purification of these linkers, suitable for SNAP-tag and Halo-tag applications, without the need for advanced chemical equipment. Our methods enable linker coupling in a kit-like manner and support the high-yield production of modified gRNAs. We demonstrate that the newly synthesized linkers and gRNA designs perform similarly to previously published constructs with regard to RNA editing efficiency. Moreover, large-scale production of modified gRNAs facilitates their use in studies involving cellular uptake and in vivo experiments. Full article

In the field of RNA therapy, innovative approaches based on adenosine deaminases acting on RNA (ADAR)-mediated site-directed RNA editing (SDRE) have been established, providing an exciting opportunity for RNA therapeutics. ADAR1 and ADAR2 enzymes are accountable for the predominant form of RNA editing in humans, which involves the hydrolytic deamination of adenosine (A) to inosine (I). This inosine is subsequently interpreted as guanosine (G) by the translational and splicing machinery because of their structural similarity. Intriguingly, the novel SDRE system leverages this recoding ability of ADAR proteins to correct the pathogenic G to A nucleotide mutations through a short, engineered guide RNA (gRNA). Thus, ADAR-mediated SDRE is emerging as a powerful tool to manipulate the genetic information at the RNA level and correct disease-causing mutations without causing damage to the genome. Further it is emerging as a new instrument for personalized medicine, since treatments can be tailored to the unique genetic mutations present in an individual patient. In this short review, we aimed to described the main approached bases on ADARs activity, highlighting their advantages and disadvantages. Full article

Adenosine deaminase acting on RNA 1 (ADAR1) is the principal enzyme for the adenosine-to-inosine RNA editing that prevents the aberrant activation of cytosolic nucleic acid sensors by endogenous double stranded RNAs and the activation of interferon-stimulated genes. In mice, the conditional neural crest deletion of Adar1 reduces the survival of melanocytes and alters the differentiation of Schwann cells that fail to myelinate nerve fibers in the peripheral nervous system. These myelination defects are partially rescued upon the concomitant removal of the Mda5 antiviral dsRNA sensor in vitro, suggesting implication of the Mda5/Mavs pathway and downstream effectors in the genesis of Adar1 mutant phenotypes. By analyzing RNA-Seq data from the sciatic nerves of mouse pups after conditional neural crest deletion of Adar1 (Adar1cKO), we here identified the transcription factors deregulated in Adar1cKO mutants compared to the controls. Through Adar1;Mavs and Adar1cKO;Egr1 double-mutant mouse rescue analyses, we then highlighted that the aberrant activation of the Mavs adapter protein and overexpression of the early growth response 1 (EGR1) transcription factor contribute to the Adar1 deletion associated defects in Schwann cell development in vivo. In silico and in vitro gene regulation studies additionally suggested that EGR1 might mediate this inhibitory effect through the aberrant regulation of EGR2-regulated myelin genes. We thus demonstrate the role of the Mda5/Mavs pathway, but also that of the Schwann cell transcription factors in Adar1-associated peripheral myelination defects. Full article

Adenosine Deaminases Acting on RNA (ADARs) are members of a family of RNA editing enzymes that catalyze the conversion of adenosine into inosine in double-stranded RNA (dsRNA). ADARs’ selective activity on dsRNA presents the ability to correct mutations at the transcriptome level using guiding oligonucleotides. However, this approach is limited by ADARs’ preference for specific sequence contexts to achieve efficient editing. Substrates with a guanosine adjacent to the target adenosine in the 5′ direction (5′-GA) are edited less efficiently compared to substrates with any other canonical nucleotides at this position. Previous studies showed that a G/purine mismatch at this position results in more efficient editing than a canonical G/C pair. Herein, we investigate a series of modified oligonucleotides containing purine or size-expanded nucleoside analogs on guide strands opposite the 5′-G (−1 position). The results demonstrate that modified adenosine and inosine analogs enhance editing at 5′-GA sites. Additionally, the inclusion of a size-expanded cytidine analog at this position improves editing over a control guide bearing cytidine. High-resolution crystal structures of ADAR:/RNA substrate complexes reveal the manner by which both inosine and size-expanded cytidine are capable of activating editing at 5′-GA sites. Further modification of these altered guide sequences for metabolic stability in human cells demonstrates that the incorporation of specific purine analogs at the −1 position significantly improves editing at 5′-GA sites. Full article

Background: MicroRNAs (miRNAs) modulate the expression of oncogenes and tumor suppressor genes, functioning as significant epigenetic regulators in cancer. IsomiRs are miRNA molecules that have undergone small modifications during miRNA processing. These modifications can alter an isomiR’s binding stability with mRNA targets, and certain isomiRs have been implicated in the development of specific cancers. Still, the isomiRomes of many tissues, including the lung, have not been characterized; Methods: In this study, we analyzed small RNA sequencing data for three cohorts of lung adenocarcinoma (LUAD) and adult non-malignant lung (ANL) samples. Results: We quantified isomiR expression and found 16 A-to-I edited isomiRs expressed in multiple cohorts, as well as 213 5′ isomiRs, 128 3′ adenylated isomiRs, and 100 3′ uridylated isomiRs. Rates of A-to-I editing at editing hotspots correlated with mRNA expression of the editing enzymes ADAR and ADARB1, which were both observed to be deregulated in LUAD. LUAD samples displayed lower overall rates of A-to-I editing and 3′ adenylation than ANL samples. Support vector machines and random forest models were trained on one cohort to distinguish ANL and stage I/II LUAD samples using reads per million (RPM) and frequency data for different types of isomiRs. Models trained on A-to-I editing rates at editing hotspots displayed high accuracy when tested on the other two cohorts and compared favorably to classifiers trained on miRNA expression alone; Conclusions: We have identified isomiRs in the human lung and found that their expression differs between non-malignant and tumor tissues, suggesting they hold potential as cancer biomarkers. Full article

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. Full article

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. Full article