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Adenosine deaminases acting on RNA (ADARs) are enzymes that catalyze the hydrolytic deamination of adenosine to inosine. The editing feature of ADARs has garnered much attention as a therapeutic tool to repurpose ADARs to correct disease-causing mutations at the mRNA level in a technique called site-directed RNA editing (SDRE). Administering a short guide RNA oligonucleotide that hybridizes to a mutant sequence forms the requisite dsRNA substrate, directing ADARs to edit the desired adenosine. However, much is still unknown about ADARs' selectivity and sequence-specific effects on editing. Atomic-resolution structures can help provide additional insight to ADARs' selectivity and lead to novel guide RNA designs. Indeed, recent structures of ADAR domains have expanded our understanding on RNA binding and the base-flipping catalytic mechanism. These efforts have enabled the rational design of improved ADAR guide strands and advanced the therapeutic potential of the SDRE approach. While no full-length structure of any ADAR is known, this review presents an exposition of the structural basis for function of the different ADAR domains, focusing on human ADAR2. Key insights are extrapolated to human ADAR1, which is of substantial interest because of its widespread expression in most human tissues.

BackgroundRNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants (SNVs) in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme that mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung adenocarcinoma cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, the use of short-read RNA-seq has limited the community's ability to interrogate both forms of RNA variation simultaneously.ResultsWe employ long-read sequencing technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We develop a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generate nanopore data with high sequence accuracy from H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We apply our workflow to identify key inosine isoform associations to help clarify the prominence of ADAR in tumorigenesis.ConclusionsUltimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.

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. [ABSTRACT FROM AUTHOR]

Simple Summary: MicroRNAs (miRNAs) play a significant role as epigenetic regulators in cancer. IsomiRs are miRNA molecules that undergo small modifications during miRNA processing, which can affect their stability and their interaction with mRNA targets. While some isomiRs are linked to specific cancers, many, including those in the lung, remain understudied. To address this, small RNA sequencing data from lung adenocarcinoma (LUAD) and adult non-malignant lung (ANL) samples were analyzed to quantify isomiR expression. This analysis identified 16 A-to-I edited isomiRs, 213 5′ isomiRs, 128 3′ adenylated isomiRs, and 100 3′ uridylated isomiRs. A-to-I editing rates correlated with the expression of editing enzymes ADAR and ADARB1, both deregulated in LUAD. LUAD samples had lower A-to-I editing and 3′ adenylation rates compared to ANL. Machine learning models based on isomiR data effectively distinguished ANL from stage I/II LUAD samples, suggesting that isomiRs hold potential as cancer biomarkers. 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. [ABSTRACT FROM AUTHOR]

RNA-binding proteins (RBPs) play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the adenosine deaminase acting on RNA family of RBPs and the sole adenosine-to-inosine RNA-editing enzyme in Caenorhabditis elegans , can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RBPs that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogeneous nuclear ribonucleoprotein family of RBPs. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing–dependent and RNA editing–independent mechanisms in regulating embryogenesis. [ABSTRACT FROM AUTHOR]

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Measures of life outlook in older adults have been investigated in connection to pain, as both pain management and outlook are important factors of successful aging. We hypothesized that higher pain is associated with lower optimism among community-dwelling older adults. We utilized data from the UC San Diego Successful Aging Evaluation (SAGE), a prospective longitudinal cohort study initiated in 2010, to evaluate the relationship between pain and optimism in 378 community-dwelling adults aged ≥50 years. We used the revised Life Orientation Test (LOT-R) to measure optimism and three pain subscales–PROMIS Pain Interference, PROMIS Pain Intensity, and MOS 36-Item Short-Form Health Survey (SF-36)–as pain measures. Regression analyses reveal negative relationships between pain and optimism for all three pain scales, with regression coefficients of −0.277 (p <.0001), −0.246 (p <.0001), and 0.269 (p <.0001) respectively. This indicates value in considering physical and psychological elements in future intervention research to promote healthy aging. [ABSTRACT FROM AUTHOR]

Underrepresented minorities (URMs) are disproportionately affected with aging-related conditions and have inadequate representation in gerontology and geriatrics professions. The Mentorship for Advancing Undergraduate Research on Aging (MADURA) Program aims to increase inclusion of URMs by improving undergraduate retention and success, increasing rates of graduate/medical school applications, and increasing entry into aging research/clinical employment. MADURA provides cohorts with faculty and peer mentorship, research skills training, paid research lab experiences and professional development opportunities. About 87% of the 2023 MADURA cohort intends to take 1+ year after receiving a Bachelor's degree, to prepare for graduate education. Planned activities include gaining work experience, preparing for standardized tests, and obtaining formal training to strengthen graduate/medical school applications. In addition to immediate graduate program acceptances, other student outcomes should be assessed. Longitudinal research on the effectiveness of various post-graduation pathways could assist Mentorship programs in supporting their graduates' longer term educational and career goal attainment. [ABSTRACT FROM AUTHOR]

The National Institute of Health R25 Research Education Program was evaluated in the second year of implementation. Twelve mentors and 20 underrepresented minority students (URMs) scholars from partnerships and collaborations among five colleges and universities were added to the program to provide a more diverse research experience. Findings reveal that 100% of research mentors agree that the approachableness and accessibility of the program coordinator were beneficial in achieving mentorship goals and objectives. In addition, 85% of the students strongly agreed that the presentation of their research findings and the weekly reflection on goals, identification of accomplishments, and obstacles through the individual development plan were very effective. Of the 23 successfully tracked students for 2 years, six URMs (26.09%) obtained a bachelor's degree and were admitted into a graduate program; two were directly admitted to a PhD program in biomedical sciences. [ABSTRACT FROM AUTHOR]

We examined the relationship between subjective and objective sleep outcomes and loneliness in older women at risk for Alzheimer's disease (AD). Our sample consisted of 39 participants (aged 65+) with mild cognitive deficits who completed the UCLA Loneliness Scale, the Pittsburgh Sleep Quality Index (PSQI), and an at home sleep test, to determine presence of obstructive sleep apnea. Based on sleep quality scores, individuals categorized as "poor sleepers" had significantly higher loneliness scores than "good sleepers." However, total loneliness scores did not significantly differ between groups with or without sleep apnea. We found that higher loneliness was significantly associated to lower habitual sleep efficiency and sleep duration and was also influenced by use of sleep medication. Our findings suggest that increased loneliness relates to worse subjective sleep quality, but not to sleep apnea. These findings suggest that combined interventions targeting loneliness and sleep quality may be important for older women. [ABSTRACT FROM AUTHOR]

This article offers a general approach to plan and implement an educational program by illustrating the evaluation process and data from the AGE-ADAR Scholars Program. A well-designed program evaluation will also include a plan for stakeholder dissemination. We adapted a framework from the Centers for Disease Control and Prevention which includes hard and soft outcomes. Hard outcomes include improved academic metrics, including test scores and graduation rates. Soft outcomes include changes in students' attitudes, particularly those related to reduced ageism as well as readiness to pursue graduate study. While our hard and soft outcomes suggest that the ADAR program is effective in increasing interest in rural health disparities and aging research, we are hopeful that our impact will continue to enrich the lives of our students and the communities in which they live and work. [ABSTRACT FROM AUTHOR]

Background: The Cultivating Health and Aging Researchers by Integrating Science, Medicine, and Aging (CHARISMA) program at the University of Chicago (UC), is an NIA-funded longitudinal clinical research training program for undergraduate students from groups underrepresented in the sciences and medicine. CHARISMA students participate in an aging-focused: 1) realistic research career experience, 2) didactic curriculum, and 3) multi-tiered mentorship program. This manuscript describes CHARISMA and early quantitative data demonstrating its success. Methods: Students apply for and are accepted into CHARISMA in year-long intervals, with programming lasting from June to May. Short-term outcomes are measured using student surveys, including an adapted 23-question version of the critical research appraisal inventory (CRAI), the Geriatrics Attitude Scale for Primary Care Residents, and questions rating the overall program, mentor, importance of aging research, and career interests. Results: Twenty-two students have completed CHARISMA. After completing CHARISMA, student aggregate CRAI scores increased (6.8 to 7.5, p = 0.04). Additionally, the substantial majority of students completing CHARISMA were definitely, very or somewhat Additionally, 87% (19/22), 73% (16/22), and 82% (18/22) of students were definitely, very, or somewhat interested in pursuing a career in medicine that serves older adults, pursuing a career in clinical research, or pursuing a career focused on aging-related research. Conclusion: Early data demonstrates that CHARISMA increases undergraduate student knowledge of and interest in aging-related clinical research. [ABSTRACT FROM AUTHOR]

The purpose of this study was to examine the effects of far-infrared (FIR) heat on quality of life (QOL) in older adults. Participants were assigned to either a convective heat group (CON) or a convective and FIR group. Participants received six, 30-min heat sessions over the course of three weeks. Pre- and post-assessments included physical measures such as range of motion, gait speed, Timed Up and Go, and hand grip strength. Standardized questionnaires were used to determine pain severity and its interference with daily life, and the impact pain had on overall QOL. Pain severity was significantly reduced (from 3.31 to 2.5, p <.05) in the FIR group from pre-to-post, and pain interference was significantly reduced (from 1.26 to 0.43, p <.05) in the CON group from pre-to-post testing. Findings suggest that heat therapy was successful in reducing pain over time. [ABSTRACT FROM AUTHOR]

Abstract 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.

Abstract Background RNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants (SNVs) in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme that mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung adenocarcinoma cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, the use of short-read RNA-seq has limited the community’s ability to interrogate both forms of RNA variation simultaneously. Results We employ long-read sequencing technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We develop a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generate nanopore data with high sequence accuracy from H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We apply our workflow to identify key inosine isoform associations to help clarify the prominence of ADAR in tumorigenesis. Conclusions Ultimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.

Abstract Background Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder affecting women of reproductive ages. Our previous study has implicated a possible link between RNA editing and PCOS, yet the actual role of RNA editing, its association with clinical features, and the underlying mechanisms remain unclear. Methods Ten RNA-Seq datasets containing 269 samples of multiple tissue types, including granulosa cells, T helper cells, placenta, oocyte, endometrial stromal cells, endometrium, and adipose tissues, were retrieved from public databases. Peripheral blood samples were collected from twelve PCOS and ten controls and subjected to RNA-Seq. Transcriptome-wide RNA-Seq data analysis was conducted to identify differential RNA editing (DRE) between PCOS and controls. The functional significance of DRE was evaluated by luciferase reporter assays and overexpression in human HEK293T cells. Dehydroepiandrosterone and lipopolysaccharide were used to stimulate human KGN granulosa cells to evaluate gene expression. Results RNA editing dysregulations across multiple tissues were found to be associated with PCOS in public datasets. Peripheral blood transcriptome analysis revealed 798 DRE events associated with PCOS. Through weighted gene co-expression network analysis, our results revealed a set of hub DRE events in PCOS blood. A DRE event in the eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2:chr2:37,100,559) was associated with PCOS clinical features such as luteinizing hormone (LH) and the ratio of LH over follicle-stimulating hormone. Luciferase assays, overexpression, and knockout of RNA editing enzyme adenosine deaminase RNA specific (ADAR) showed that the ADAR-mediated editing cis-regulated EIF2AK2 expression. EIAF2AK2 showed a higher expression after dehydroepiandrosterone and lipopolysaccharide stimulation, triggering changes in the downstrean MAPK pathway. Conclusions Our study presented the first evidence of cross-tissue RNA editing dysregulation in PCOS and its clinical associations. The dysregulation of RNA editing mediated by ADAR and the disrupted target EIF2AK2 may contribute to PCOS development via the MPAK pathway, underlining such epigenetic mechanisms in the disease.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study presents the design of a controller for a two-degree-of-freedom (2-DOF) helicopter based on sequential invariant manifolds with exponential convergence. The system is decomposed into two subsystems for pitch and yaw angles, and exponentially stable manifolds are constructed for each subsystem. The control law is found based on sequential manifolds and the Analytical Design of Aggregated Regulators (ADAR) method. The controller is designed to increase the system's stability against disturbances while ensuring stability over a finite period of time. The response time of the system can be evaluated in advance through the parameters of the designed manifold. The robustness of the control law for external disturbances was proven using the Lyapunov function in the design process. Finally, the effectiveness of the proposed controller based on the synergetic control theory is demonstrated by numerical simulation results and a comparison with the backstepping controller. [ABSTRACT FROM AUTHOR]

Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification mediated by the adenosine deaminases acting on RNA (ADAR) family of enzymes, expanding the transcriptome by altering selected nucleotides A to I in RNA molecules. Recently, A-to-I editing has been explored for correcting disease-causing mutations in RNA using therapeutic guide oligonucleotides to direct ADAR editing at specific sites. Humans have two active ADARs whose preferences and specificities are not well understood. To investigate their substrate specificity, we introduced hADAR1 and hADAR2, respectively, into Schizosaccharomyces pombe (S. pombe), which lacks endogenous ADARs, and evaluated their editing activities in vivo. Using transcriptome sequencing of S. pombe cultured at optimal growth temperature (30 °C), we identified 483 A-to-I high-confident editing sites for hADAR1 and 404 for hADAR2, compared with the non-editing wild-type control strain. However, these sites were mostly divergent between hADAR1 and hADAR2-expressing strains, sharing 33 common sites that are less than 9% for each strain. Their differential specificity for substrates was attributed to their differential preference for neighboring sequences of editing sites. We found that at the -3-position relative to the editing site, hADAR1 exhibits a tendency toward T, whereas hADAR2 leans toward A. Additionally, when varying the growth temperature for hADAR1- and hADAR2-expressing strains, we observed increased editing sites for them at both 20 and 35 °C, compared with them growing at 30 °C. However, we did not observe a significant shift in hADAR1 and hADAR2's preference for neighboring sequences across three temperatures. The vast changes in RNA editing sites at lower and higher temperatures were also observed for hADAR2 previously in budding yeast, which was likely due to the influence of RNA folding at these different temperatures, among many other factors. We noticed examples of longer lengths of dsRNA around the editing sites that induced editing at 20 or 35 °C but were absent at the other two temperature conditions. We found genes' functions can be greatly affected by editing of their transcripts, for which over 50% of RNA editing sites for both hADAR1 and hADAR2 in S. pombe were in coding sequences (CDS), with more than 60% of them resulting in amino acid changes in protein products. This study revealed the extensive differences in substrate selectivity between the two active human ADARS, i.e., ADAR1 and ADAR2, and provided novel insight when utilizing the two different enzymes for in vivo treatment of human genetic diseases using the RNA editing approach. [ABSTRACT FROM AUTHOR]

Cancer is a complex and heterogeneous disease, in which a number of genetic and epigenetic changes occur in tumor onset and progression. Recent studies indicate that changes at the RNA level are also involved in tumorigenesis, such as adenosine-to-inosine (A-to-I) RNA editing. Here, we systematically investigate transcriptome-wide A-to-I editing events in a large number of samples from Non-Hodgkin lymphomas (NHLs). Using a computational pipeline that determines significant differences in editing level between NHL and normal samples at known A-to-I editing sites, we identify a number of differentially edited editing sites between NHL subtypes and normal samples. Most of the differentially edited sites are located in non-coding regions, and many such sites show a strong correlation between gene expression level and editing efficiency, indicating that RNA editing might have direct consequences for the cancer cell's aberrant gene regulation status in these cases. Moreover, we establish a strong link between RNA editing and NHL by demonstrating that NHL and normal samples and even NHL subtypes can be distinguished based on genome-wide RNA editing profiles alone. Our study establishes a strong link between RNA editing, cancer and aberrant gene regulation in NHL. [ABSTRACT FROM AUTHOR]

Background: Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder affecting women of reproductive ages. Our previous study has implicated a possible link between RNA editing and PCOS, yet the actual role of RNA editing, its association with clinical features, and the underlying mechanisms remain unclear. Methods: Ten RNA-Seq datasets containing 269 samples of multiple tissue types, including granulosa cells, T helper cells, placenta, oocyte, endometrial stromal cells, endometrium, and adipose tissues, were retrieved from public databases. Peripheral blood samples were collected from twelve PCOS and ten controls and subjected to RNA-Seq. Transcriptome-wide RNA-Seq data analysis was conducted to identify differential RNA editing (DRE) between PCOS and controls. The functional significance of DRE was evaluated by luciferase reporter assays and overexpression in human HEK293T cells. Dehydroepiandrosterone and lipopolysaccharide were used to stimulate human KGN granulosa cells to evaluate gene expression. Results: RNA editing dysregulations across multiple tissues were found to be associated with PCOS in public datasets. Peripheral blood transcriptome analysis revealed 798 DRE events associated with PCOS. Through weighted gene co-expression network analysis, our results revealed a set of hub DRE events in PCOS blood. A DRE event in the eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2:chr2:37,100,559) was associated with PCOS clinical features such as luteinizing hormone (LH) and the ratio of LH over follicle-stimulating hormone. Luciferase assays, overexpression, and knockout of RNA editing enzyme adenosine deaminase RNA specific (ADAR) showed that the ADAR-mediated editing cis-regulated EIF2AK2 expression. EIAF2AK2 showed a higher expression after dehydroepiandrosterone and lipopolysaccharide stimulation, triggering changes in the downstrean MAPK pathway. Conclusions: Our study presented the first evidence of cross-tissue RNA editing dysregulation in PCOS and its clinical associations. The dysregulation of RNA editing mediated by ADAR and the disrupted target EIF2AK2 may contribute to PCOS development via the MPAK pathway, underlining such epigenetic mechanisms in the disease. [ABSTRACT FROM AUTHOR]

The dynamic process of Drosophila spermatogenesis involves asymmetric division, mitosis, and meiosis, which ultimately results in the production of mature spermatozoa. Disorders of spermatogenesis can lead to infertility in males. ADAR (adenosine deaminase acting on RNA) mutations in Drosophila cause male infertility, yet the causative factors remain unclear. In this study, immunofluorescence staining was employed to visualize endogenous ADAR proteins and assess protein levels via fluorescence-intensity analysis. In addition, the early differentiation disorders and homeostatic alterations during early spermatogenesis in the testes were examined through quantification of transit-amplifying region length, counting the number of GSCs (germline stem cells), and fertility experiments. Our findings suggest that deletion of ADAR causes testicular tip transit-amplifying cells to accumulate and become infertile in older male Drosophila. By overexpressing ADAR in early germline cells, male infertility can be partially rescued. Transcriptome analysis showed that ADAR maintained early spermatogenesis homeostasis through the bone-morphogenetic-protein (BMP) signaling pathway. Taken together, these findings have the potential to help explore the role of ADAR in early spermatogenesis. [ABSTRACT FROM AUTHOR]

BACKGROUND: RNA-Seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme which mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung ADC cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, short read RNA-Seq has limited the community's ability to interrogate both forms of RNA variation simultaneously. RESULTS: We employed long-read technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We have developed a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generated nanopore data with high sequence accuracy of H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We applied our workflow to identify key inosine-isoform associations to help clarify the prominence of ADAR in tumorigenesis. CONCLUSIONS: Ultimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.

RNA therapeutics have had a tremendous impact on medicine, recently exemplified by the rapid development and deployment of mRNA vaccines to combat the COVID-19 pandemic. In addition, RNA-targeting drugs have been developed for diseases with significant unmet medical needs through selective mRNA knockdown or modulation of pre-mRNA splicing. Recently, RNA editing, particularly antisense RNA-guided adenosine deaminase acting on RNA (ADAR)-based programmable A-to-I editing, has emerged as a powerful tool to manipulate RNA to enable correction of disease-causing mutations and modulate gene expression and protein function. Beyond correcting pathogenic mutations, the technology is particularly well suited for therapeutic applications that require a transient pharmacodynamic effect, such as the treatment of acute pain, obesity, viral infection, and inflammation, where it would be undesirable to introduce permanent alterations to the genome. Furthermore, transient modulation of protein function, such as altering the active sites of enzymes or the interface of protein-protein interactions, opens the door to therapeutic avenues ranging from regenerative medicine to oncology. These emerging RNA-editing-based toolsets are poised to broadly impact biotechnology and therapeutic applications. Here, we review the emerging field of therapeutic RNA editing, highlight recent laboratory advancements, and discuss the key challenges on the path to clinical development.

This review aims to highlight the structures of ADAR proteins that have been crucial in the discernment of their functions and are relevant to future therapeutic development. ADAR proteins can correct or diversify genetic information, underscoring their pivotal contribution to protein diversity and the sophistication of neuronal networks. ADAR proteins have numerous functions in RNA editing independent roles and through the mechanisms of A-I RNA editing that continue to be revealed. Provided is a detailed examination of the ADAR family members—ADAR1, ADAR2, and ADAR3—each characterized by distinct isoforms that offer both structural diversity and functional variability, significantly affecting RNA editing mechanisms and exhibiting tissue-specific regulatory patterns, highlighting their shared features, such as double-stranded RNA binding domains (dsRBD) and a catalytic deaminase domain (CDD). Moreover, it explores ADARs’ extensive roles in immunity, RNA interference, and disease modulation, demonstrating their ambivalent nature in both the advancement and inhibition of diseases. Through this comprehensive analysis, the review seeks to underline the potential of targeting ADAR proteins in therapeutic strategies, urging continued investigation into their biological mechanisms and health implications.

ABSTRACTAlthough A-to-I RNA editing leads to similar effects to A-to-G DNA mutation, nonsynonymous RNA editing (recoding) is believed to confer its adaptiveness by ‘epigenetically’ regulating proteomic diversity in a temporospatial manner, avoiding the pleiotropic effect of genomic mutations. Recent discoveries on the evolutionary trajectory of Ser>Gly auto-editing site in insect Adar gene demonstrated a selective advantage to having an editable codon compared to uneditable ones. However, apart from pure observations, quantitative approaches for justifying the adaptiveness of individual RNA editing sites are still lacking. We performed a comparative genomic analysis on 113 Diptera species, focusing on the Adar Ser>Gly auto-recoding site in Drosophila. We only found one species having a derived Gly at the corresponding site, and this occurrence was significantly lower than genome-wide random expectation. This suggests that the Adar Ser>Gly site is unlikely to be genomically replaced with G during evolution, and thus indicating the advantage of editable status over hardwired genomic alleles. Similar trends were observed for the conserved Ile>Met recoding in gene Syt1. In the light of evolution, we established a comparative genomic approach for quantitatively justifying the adaptiveness of individual editing sites. Priority should be given to such adaptive editing sites in future functional studies.

ABSTRACTAdar-mediated adenosine-to-inosine (A-to-I) RNA editing mainly occurs in nucleus and diversifies the transcriptome in a flexible manner. It has been a challenging task to identify beneficial editing sites from the sea of total editing events. The functional Ser>Gly auto-recoding site in insect Adar gene has uneditable Ser codons in ancestral nodes, indicating the selective advantage to having an editable status. Here, we extended this case study to more metazoan species, and also looked for all Drosophila recoding events with potential uneditable synonymous codons. Interestingly, in D. melanogaster, the abundant nonsynonymous editing is enriched in the codons that have uneditable counterparts, but the Adar Ser>Gly case suggests that the editable orthologous codons in other species are not necessarily edited. The use of editable versus ancestral uneditable codon is a smart way to infer the selective advantage of RNA editing, and priority might be given to these editing sites for functional studies due to the feasibility to construct an uneditable allele. Our study proposes an idea to narrow down the candidates of beneficial recoding sites. Meanwhile, we stress that the matched transcriptomes are needed to verify the conservation of editing events during evolution.

Head grooming in Drosophila consists of repeated sweeps of the legs across the head, comprising regular cycles. We used the GAL4-UAS system to study the effects of overexpressing shibirets1 and of Adar knockdown via RNA interference, on the period of head-grooming cycles in Drosophila. Overexpressing shibirets1 interferes with synaptic vesicle recycling and thus with cell communication, while Adar knockdown reduces RNA editing of neuronal transcripts for a large number of genes. All transgenic flies and their controls were tested at 22° to avoid temperature effects; in wild type, cycle frequency varied with temperature with a Q10 of 1.3. Two experiments were performed with transgenic shibirets1: (1) each fly was heat-shocked for 10 min at 30° immediately before testing at 22° and (2) flies were not heat shocked. In both experiments, cycle period was increased when shibirets1 was overexpressed in all neurons, but was not increased when shibirets1 was overexpressed in motoneurons alone. We hypothesize that grooming cycles in flies overexpressing shibirets1 are lengthened because of synaptic impairment in neural circuits that control head-grooming cycles. In flies with constitutive, pan-neuronal Adar knockdown, cycle period was more variable within individuals, but mean cycle period was not significantly altered. We conclude that RNA editing is essential for the maintenance of within-individual stereotypy of head-grooming cycles. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 has accumulated many mutations since its emergence in late 2019. Nucleotide substitutions leading to amino acid replacements constitute the primary material for natural selection. Insertions, deletions, and substitutions appear to be critical for coronavirus's macro- and microevolution. Understanding the molecular mechanisms of mutations in the mutational hotspots (positions, loci with recurrent mutations, and nucleotide context) is important for disentangling roles of mutagenesis and selection. In the SARS-CoV-2 genome, deletions and insertions are frequently associated with repetitive sequences, whereas C>U substitutions are often surrounded by nucleotides resembling the APOBEC mutable motifs. We describe various approaches to mutation spectra analyses, including the context features of RNAs that are likely to be involved in the generation of recurrent mutations. We also discuss the interplay between mutations and natural selection as a complex evolutionary trend. The substantial variability and complexity of pipelines for the reconstruction of mutations and the huge number of genomic sequences are major problems for the analyses of mutations in the SARS-CoV-2 genome. As a solution, we advocate for the development of a centralized database of predicted mutations, which needs to be updated on a regular basis. [ABSTRACT FROM AUTHOR]

The dynamic interaction of RNA-binding proteins (RBPs) with their target RNAs contributes to the diversity of ribonucleoprotein (RNP) complexes that are involved in a myriad of biological processes. Identifying the RNP components at high resolution and defining their interactions are key to understanding their regulation and function. Expressing fusions between an RBP of interest and an RNA editing enzyme can result in nucleobase changes in target RNAs, representing a recent addition to experimental approaches for profiling RBP/RNA interactions. Here, we have used the MS2 protein/RNA interaction to test four RNA editing proteins for their suitability to detect target RNAs of RBPs in planta. We have established a transient test system for fast and simple quantification of editing events and identified the hyperactive version of the catalytic domain of an adenosine deaminase (hADARcd) as the most suitable editing enzyme. Examining fusions between homologs of polypyrimidine tract binding proteins (PTBs) from Arabidopsis thaliana and hADARcd allowed determining target RNAs with high sensitivity and specificity. Moreover, almost complete editing of a splicing intermediate provided insight into the order of splicing reactions and PTB dependency of this particular splicing event. Addition of sequences for nuclear localisation of the fusion protein increased the editing efficiency, highlighting this approach’s potential to identify RBP targets in a compartment-specific manner. Our studies have established the editing-based analysis of interactions between RBPs and their RNA targets in a fast and straightforward assay, offering a new system to study the intricate composition and functions of plant RNPs in vivo.Key message: We have established a fast and straightforward editing-based assay to analyse RBP/RNA interactions, demonstrating PTB binding to an intronic sequence adjacent to a cassette exon as prerequisite for its inclusion. [ABSTRACT FROM AUTHOR]

Recent studies have underscored the pivotal role of adenosine-to-inosine RNA editing by ADAR in suppressing innate immune interferon responses triggered by cellular double-stranded RNA (dsRNA). Transcriptome-wide analyses of ADAR editing patterns, as well as a study of the evolutionary dynamics of dsRNAs, suggest that the vast majority of ADAR targets are not immunogenic, or at least do not necessarily lead to an interferon response, even in their unedited form. Identifying the specific ADAR editing targets crucial for its immune protection regulatory function is an open unmet challenge. The role of immunogenic dsRNAs and their editing in inflammatory and autoimmune diseases and cancer is increasingly appreciated. Comprehensive understanding of dsRNA immunogenicity may promote our understanding of these diseases and open doors to therapeutic avenues. Recent studies have underscored the pivotal role of adenosine-to-inosine RNA editing, catalyzed by ADAR1, in suppressing innate immune interferon responses triggered by cellular double-stranded RNA (dsRNA). However, the specific ADAR1 editing targets crucial for this regulatory function remain elusive. We review analyses of transcriptome-wide ADAR1 editing patterns and their evolutionary dynamics, which offer valuable insights into this unresolved query. The growing appreciation of the significance of immunogenic dsRNAs and their editing in inflammatory and autoimmune diseases and cancer calls for a more comprehensive understanding of dsRNA immunogenicity, which may promote our understanding of these diseases and open doors to therapeutic avenues. [ABSTRACT FROM AUTHOR]

The conversion of Adenosine (A) to Inosine (I), by Adenosine Deaminases Acting on RNA or ADARs, is an essential post-transcriptional modification that contributes to proteome diversity and regulation in metazoans including humans. In addition to its transcriptome-regulating role, ADARs also play a major part in immune response to viral infection, where an interferon response activates interferon-stimulated genes, such as ADARp150, in turn dynamically regulating host-virus interactions. A previous report has shown that infection from reoviruses, despite strong activation of ADARp150, does not influence the editing of some of the major known editing targets, while likely editing others, suggesting a potentially nuanced editing pattern that may depend on different factors. However, the results were based on a handful of selected editing sites and did not cover the entire transcriptome. Thus, to determine whether and how reovirus infection specifically affects host ADAR editing patterns, we analyzed a publicly available deep-sequenced RNA-seq dataset, from murine fibroblasts infected with wild-type and mutant reovirus strains that allowed us to examine changes in editing patterns on a transcriptome-wide scale. To the best of our knowledge, this is the first transcriptome-wide report on host editing changes after reovirus infection. Our results demonstrate that reovirus infection induces unique nuanced editing changes in the host, including introducing sites uniquely edited in infected samples. Genes with edited sites are overrepresented in pathways related to immune regulation, cellular signaling, metabolism, and growth. Moreover, a shift in editing targets has also been observed, where the same genes are edited in infection and control conditions but at different sites, or where the editing rate is increased for some and decreased for other differential targets, supporting the hypothesis of dynamic and condition-specific editing by ADARs.

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.

The ongoing arms race between viruses and their hosts is constantly evolving. One of the ways in which cells defend themselves against invading viruses is by using restriction factors (RFs), which are cell-intrinsic antiviral mechanisms that block viral replication and transcription. Recent research has identified a specific group of RFs that belong to the cellular epigenetic machinery and are able to restrict the gene expression of certain viruses. These RFs can be referred to as epigenetic restriction factors or eRFs. In this review, eRFs have been classified into two categories. The first category includes eRFs that target viral chromatin. So far, the identified eRFs in this category include the PML-NBs, the KRAB/KAP1 complex, IFI16, and the HUSH complex. The second category includes eRFs that target viral RNA or, more specifically, the viral epitranscriptome. These epitranscriptomic eRFs have been further classified into two types: those that edit RNA bases—adenosine deaminase acting on RNA (ADAR) and pseudouridine synthases (PUS), and those that covalently modify viral RNA—the N6-methyladenosine (m6A) writers, readers, and erasers. We delve into the molecular machinery of eRFs, their role in limiting various viruses, and the mechanisms by which viruses have evolved to counteract them. We also examine the crosstalk between different eRFs, including the common effectors that connect them. Finally, we explore the potential for new discoveries in the realm of epigenetic networks that restrict viral gene expression, as well as the future research directions in this area. [ABSTRACT FROM AUTHOR]

The ability to sense and respond to infection is essential for life. Viral infection produces double-stranded RNAs (dsRNAs) that are sensed by proteins that recognize the structure of dsRNA. This structure-based recognition of viral dsRNA allows dsRNA sensors to recognize infection by many viruses, but it comes at a cost—the dsRNA sensors cannot always distinguish between "self" and "nonself" dsRNAs. "Self" RNAs often contain dsRNA regions, and not surprisingly, mechanisms have evolved to prevent aberrant activation of dsRNA sensors by "self" RNA. Here, we review current knowledge about the life of endogenous dsRNAs in mammals—the biosynthesis and processing of dsRNAs, the proteins they encounter, and their ultimate degradation. We highlight mechanisms that evolved to prevent aberrant dsRNA sensor activation and the importance of competition in the regulation of dsRNA sensors and other dsRNA-binding proteins. Recognition of foreign and endogenous dsRNAs is complicated by the sequence-independent binding of dsRNA-binding proteins. Cottrell, Andrews, and Bass review the synthesis, processing, binding, and degradation of dsRNAs. They highlight the mechanisms that prevent dsRNA sensors from binding to endogenous dsRNAs and activating innate immunity pathways. [ABSTRACT FROM AUTHOR]

ADAR (Adenosine Deaminases Acting on RNA) proteins are a group of enzymes that play a vital role in RNA editing by converting adenosine to inosine in RNAs. This process is a frequent post-transcriptional event observed in metazoan transcripts. Recent studies indicate widespread dysregulation of ADAR-mediated RNA editing across many immune-related diseases, such as human cancer. We comprehensively review ADARs' function as pattern recognizers and their capability to contribute to mediating immune-related pathways. We also highlight the potential role of site-specific RNA editing in maintaining homeostasis and its relationship to various diseases, such as human cancers. More importantly, we summarize the latest cutting-edge computational approaches and data resources for predicting and analyzing RNA editing sites. Lastly, we cover the recent advancement in site-directed ADAR editing tool development. This review presents an up-to-date overview of ADAR-mediated RNA editing, how site-specific RNA editing could potentially impact disease pathology, and how they could be harnessed for therapeutic applications. [ABSTRACT FROM AUTHOR]

In this paper, the authors present a new method to synthesize an adaptive sliding controller for Twin Rotor MIMO System (TRMS) based on Synergetic Control Theory (SCT). This system represents a prototype of a helicopter with two degrees of freedom and is widely used in automatic control laboratories. The complexity of the control problem is due to the nonlinear cross-coupling between the main and tail rotors. Uncertainty in system parameters further increases the complexity of the control problem. In Synergetic Control Theory, manifolds are designed for each channel. The control law is found based on sequential manifolds and the Analytical Design of Aggregated Regulators (ADAR) method. The adaptive law when the parameters are uncertain is given based on the analysis of system stability thanks to the Lyapunov function of the first manifold. Finally, the effectiveness of the proposed controller is demonstrated by numerical simulation results and comparison with conventional Sliding Mode Control (SMC). [ABSTRACT FROM AUTHOR]

Adenosine deaminases acting on RNA (ADARs) are endogenous enzymes catalyzing the deamination of adenosines to inosines, which are then read as guanosines during translation. This ability to recode makes ADAR an attractive therapeutic tool to edit genetic mutations and reprogram genetic information at the mRNA level. Using the endogenous ADARs and guiding them to a selected target has promising therapeutic potential. Indeed, different studies have reported several site-directed RNA-editing approaches for making targeted base changes in RNA molecules. The basic strategy has been to use guide RNAs (gRNAs) that hybridize and form a double-stranded RNA (dsRNA) structure with the desired RNA target because of ADAR activity in regions of dsRNA formation. Here we report on a novel pipeline for identifying disease-causing variants as candidates for RNA editing, using a yeast-based screening system to select efficient gRNAs for editing of nonsense mutations, and test them in a human cell line reporter system. We have used this pipeline to modify the sequence of transcripts carrying nonsense mutations that cause inherited retinal diseases in the FAM161A, KIZ, TRPM1, and USH2A genes. Our approach can serve as a basis for gene therapy intervention in knockin mouse models and ultimately in human patients.

Abstract Background Xenopus has served as a valuable model system for biomedical research over the past decades. Notably, ADAR was first detected in frog oocytes and embryos as an activity that unwinds RNA duplexes. However, the scope of A-to-I RNA editing by the ADAR enzymes in Xenopus remains underexplored. Results Here, we identify millions of editing events in Xenopus with high accuracy and systematically map the editome across developmental stages, adult organs, and species. We report diverse spatiotemporal patterns of editing with deamination activity highest in early embryogenesis before zygotic genome activation and in the ovary. Strikingly, editing events are poorly conserved across different Xenopus species. Even sites that are detected in both X. laevis and X. tropicalis show largely divergent editing levels or developmental profiles. In protein-coding regions, only a small subset of sites that are found mostly in the brain are well conserved between frogs and mammals. Conclusions Collectively, our work provides fresh insights into ADAR activity in vertebrates and suggest that species-specific editing may play a role in each animal’s unique physiology or environmental adaptation.

To date, few studies have investigated whether the RNA-editing enzymes adenosine deaminases acting on RNA (ADARs) influence RNA functioning in lung adenocarcinoma (LUAD). To investigate the role of ADAR in lung cancer, we leveraged the advantages of The Cancer Genome Atlas (TCGA) database, from which we obtained transcriptome data and clinical information from 539 patients with LUAD. First, we compared ARAR expression levels in LUAD tissues with those in normal lung tissues using paired and unpaired analyses. Next, we evaluated the influence of ADARs on multiple prognostic indicators, including overall survival at 1, 3 and 5 years, as well as disease-specific survival and progression-free interval, in patients with LUAD. We also used Kaplan-Meier survival curves to estimate overall survival and Cox regression analysis to assess covariates associated with prognosis. A nomogram was constructed to validate the impact of the ADARs and clinicopathological factors on patient survival probabilities. The volcano plot and heat map revealed the differentially expressed genes associated with ADARs in LUAD. Finally, we examined ADAR expression versus immune cell infiltration in LUAD using Spearman's analysis. Using the Gene Expression Profiling Interactive Analysis (GEPIA2) database, we identified the top 100 genes most significantly correlated with ADAR expression, constructed a protein-protein interaction network and performed a Gene Ontology/Kyoto Encyclopedia of Genes and Genomes analysis on these genes. Our results demonstrate that ADARs are overexpressed in LUAD and correlated with poor patient prognosis. ADARs markedly increase the infiltration of T central memory, T helper 2 and T helper cells, while reducing the infiltration of immature dendritic, dendritic and mast cells. Most immune response markers, including T cells, tumor-associated macrophages, T cell exhaustion, mast cells, macrophages, monocytes and dendritic cells, are closely correlated with ADAR expression in LUAD.

Interferon (IFN)-β is the first-line disease management choice in multiple sclerosis (MS) with profound effects; however, in up to 50% of patients, clinical response does not occur. Ascertaining the responding state, need a long-term clinical follow-up, and this may lead to delay in use of other effective medications. IFN-induced cascade and its regulation is considered to play a major role in MS. Adenosine deaminase, RNA-specific (ADAR) dysregulation is important to IFN signaling pathway as an activity suppressor. Hence, we investigated the expression of ADAR and its single nucleotide variants of rs2229857 association with response to IFN-β in relapsing-remitting MS patients. mRNA levels and genotyping of rs2229857 in 167 MS patients were investigated via SYBR Green real-time (RT)-quantitative polymerase chain reaction and high-resolution melting RT PCR, respectively. The allele-A in rs2229857 and higher expression of ADAR were associated with poor response to IFN-β. Two response groups were significantly different in terms of annualized relapse rate, first symptoms, first extended disability status scale (EDSS), current EDSS, and the MS severity score. According to this study's findings, assessment of transcript levels and also variants in ADAR may be useful in identifying patients' response to IFN-β before starting treatment. Further investigations are needed to determine the potency of ADAR to be a predictive biomarker in drug responsiveness.

Abstract Background Conversion or editing of adenosine (A) into inosine (I) catalyzed by specialized cellular enzymes represents one of the most common post-transcriptional RNA modifications with emerging connection to disease. A-to-I conversions can happen at specific sites and lead to increase in proteome diversity and changes in RNA stability, splicing, and regulation. Such sites can be detected as adenine-to-guanine sequence changes by next-generation RNA sequencing which resulted in millions reported sites from multiple genome-wide surveys. Nonetheless, the lack of extensive independent validation in such endeavors, which is critical considering the relatively high error rate of next-generation sequencing, leads to lingering questions about the validity of the current compendiums of the editing sites and conclusions based on them. Results Strikingly, we found that the current analytical methods suffer from very high false positive rates and that a significant fraction of sites in the public databases cannot be validated. In this work, we present potential solutions to these problems and provide a comprehensive and extensively validated list of A-to-I editing sites in a human cancer cell line. Our findings demonstrate that most of true A-to-I editing sites in a human cancer cell line are located in the non-coding transcripts, the so-called RNA 'dark matter'. On the other hand, many ADAR editing events occurring in exons of human protein-coding mRNAs, including those that can recode the transcriptome, represent false positives and need to be interpreted with caution. Nonetheless, yet undiscovered authentic ADAR sites that increase the diversity of human proteome exist and warrant further identification. Conclusions Accurate identification of human ADAR sites remains a challenging problem, particularly for the sites in exons of protein-coding mRNAs. As a result, genome-wide surveys of ADAR editome must still be accompanied by extensive Sanger validation efforts. However, given the vast number of unknown human ADAR sites, there is a need for further developments of the analytical techniques, potentially those that are based on deep learning solutions, in order to provide a quick and reliable identification of the editome in any sample.

Background: Xenopus has served as a valuable model system for biomedical research over the past decades. Notably, ADAR was first detected in frog oocytes and embryos as an activity that unwinds RNA duplexes. However, the scope of A-to-I RNA editing by the ADAR enzymes in Xenopus remains underexplored. Results: Here, we identify millions of editing events in Xenopus with high accuracy and systematically map the editome across developmental stages, adult organs, and species. We report diverse spatiotemporal patterns of editing with deamination activity highest in early embryogenesis before zygotic genome activation and in the ovary. Strikingly, editing events are poorly conserved across different Xenopus species. Even sites that are detected in both X. laevis and X. tropicalis show largely divergent editing levels or developmental profiles. In protein-coding regions, only a small subset of sites that are found mostly in the brain are well conserved between frogs and mammals. Conclusions: Collectively, our work provides fresh insights into ADAR activity in vertebrates and suggest that species-specific editing may play a role in each animal's unique physiology or environmental adaptation. [ABSTRACT FROM AUTHOR]

Simple Summary: Recent evidence from whole-exome and whole-genome sequencing approaches revealed a complex genomic landscape for many cancers. In addition to somatic mutations and alternative splicing changes, genetic information can also be altered by RNA editing, which enables alterations of genome information in a very dynamic and flexible way. Influenced by both external factors and microenvironmental signals, RNA editing deeply contributes to cancer morphological plasticity. Coding and noncoding RNA molecules play their roles in ensuring cell function and tissue homeostasis in an ordered and systematic fashion. RNA chemical modifications can occur both at bases and ribose sugar, and, similarly to DNA and histone modifications, can be written, erased, and recognized by the corresponding enzymes, thus modulating RNA activities and fine-tuning gene expression programs. RNA editing is one of the most prevalent and abundant forms of post-transcriptional RNA modification in normal physiological processes. By altering the sequences of mRNAs, it makes them different from the corresponding genomic template. Hence, edited mRNAs can produce protein isoforms that are functionally different from the corresponding genome-encoded variants. Abnormalities in regulatory enzymes and changes in RNA-modification patterns are closely associated with the occurrence and development of various human diseases, including cancer. To date, the roles played by RNA modifications in cancer are gathering increasing interest. In this review, we focus on the role of RNA editing in cancer transformation and provide a new perspective on its impact on tumorigenesis, by regulating cell proliferation, differentiation, invasion, migration, stemness, metabolism, and drug resistance. [ABSTRACT FROM AUTHOR]

Coleoid cephalopods (octopus, squid and cuttlefish) have unusually complex nervous systems. The coleoid nervous system is also the only one currently known to recode the majority of expressed proteins through A-to-I RNA editing. The deamination of adenosine by adenosine deaminase acting on RNA (ADAR) enzymes produces inosine, which is interpreted as guanosine during translation. If this occurs in an open reading frame, which is the case for tens of thousands of editing sites in coleoids, it can recode the encoded protein. Here, we describe recent findings aimed at deciphering the mechanisms underlying high-level recoding and its adaptive potential. We describe the complement of ADAR enzymes in cephalopods, including a recently discovered novel domain in sqADAR1. We further summarize current evidence supporting an adaptive role of high-level RNA recoding in coleoids, and review recent studies showing that a large proportion of recoding sites is temperature-sensitive. Despite these new findings, the mechanisms governing the high level of RNA recoding in coleoid cephalopods remain poorly understood. Recent advances using genome editing in squid may provide useful tools to further study A-to-I RNA editing in these animals. [ABSTRACT FROM AUTHOR]