Identification of exceptionally potent adenosine deaminases RNA editors from high body temperature organisms.

The most abundant form of RNA editing in metazoa is the deamination of adenosines into inosines (A-to-I), catalyzed by ADAR enzymes. Inosines are read as guanosines by the translation machinery, and thus A-to-I may lead to protein recoding. The ability of ADARs to recode at the mRNA level makes them attractive therapeutic tools. Several approaches for Site-Directed RNA Editing (SDRE) are currently under development. A major challenge in this field is achieving high on-target editing efficiency, and thus it is of much interest to identify highly potent ADARs. To address this, we used the baker yeast Saccharomyces cerevisiae as an editing-naïve system. We exogenously expressed a range of heterologous ADARs and identified the hummingbird and primarily mallard-duck ADARs, which evolved at 40-42°C, as two exceptionally potent editors. ADARs bind to double-stranded RNA structures (dsRNAs), which in turn are temperature sensitive. Our results indicate that species evolved to live with higher core body temperatures have developed ADAR enzymes that target weaker dsRNA structures and would therefore be more effective than other ADARs. Further studies may use this approach to isolate additional ADARs with an editing profile of choice to meet specific requirements, thus broadening the applicability of SDRE.

Global quantification exposes abundant low-level off-target activity by base editors.

Base editors are dedicated engineered deaminases that enable directed conversion of specific bases in the genome or transcriptome in a precise and efficient manner, and hold promise for correcting pathogenic mutations. A major concern limiting application of this powerful approach is the issue of off-target edits. Several recent studies have shown substantial off-target RNA activity induced by base editors and demonstrated that off-target mutations may be suppressed by improved deaminases versions or optimized guide RNAs. Here, we describe a new class of off-target events that are invisible to the established methods for detection of genomic variations and were thus far overlooked. We show that nonspecific, seemingly stochastic, off-target events affect a large number of sites throughout the genome or the transcriptome, and account for the majority of off-target activity. We develop and employ a different, complementary approach that is sensitive to the stochastic off-target activity and use it to quantify the abundant off-target RNA mutations due to current, optimized deaminase editors. We provide a computational tool to quantify global off-target activity, which can be used to optimize future base editors. Engineered base editors enable directed manipulation of the genome or transcriptome at single-base resolution. We believe that implementation of this computational approach would facilitate design of more specific base editors.

Increased A-to-I RNA editing in atherosclerosis and cardiomyopathies.

Adenosine-to-inosine RNA editing is essential to prevent undesired immune activation. This diverse process alters the genetic content of the RNA and may recode proteins, change splice sites and miRNA targets, and mimic genomic mutations. Recent studies have associated or implicated aberrant editing with pathological conditions, including cancer, autoimmune diseases, and neurological and psychiatric conditions. RNA editing patterns in cardiovascular tissues have not been investigated systematically so far, and little is known about its potential role in cardiac diseases. Some hints suggest robust editing in this system, including the fact that ADARB1 (ADAR2), the main coding-sequence editor, is most highly expressed in these tissues. Here we characterized RNA editing in the heart and arteries and examined a contributory role to the development of atherosclerosis and two structural heart diseases -Ischemic and Dilated Cardiomyopathies. Analyzing hundreds of RNA-seq samples taken from the heart and arteries of cardiac patients and controls, we find that global editing, alongside inflammatory gene expression, is increased in patients with atherosclerosis, cardiomyopathies, and heart failure. We describe a single recoding editing site and suggest it as a target for focused research. This recoding editing site in the IGFBP7 gene is one of the only evolutionary conserved sites between mammals, and we found it exhibits consistently increased levels of editing in these patients. Our findings reveal that RNA editing is abundant in arteries and is elevated in several key cardiovascular conditions. They thus provide a roadmap for basic and translational research of RNA as a mediator of atherosclerosis and non-genetic cardiomyopathies.

REDIportal: millions of novel A-to-I RNA editing events from thousands of RNAseq experiments.

RNA editing is a relevant epitranscriptome phenomenon able to increase the transcriptome and proteome diversity of eukaryotic organisms. ADAR mediated RNA editing is widespread in humans in which millions of A-to-I changes modify thousands of primary transcripts. RNA editing has pivotal roles in the regulation of gene expression or modulation of the innate immune response or functioning of several neurotransmitter receptors. Massive transcriptome sequencing has fostered the research in this field. Nonetheless, different aspects of the RNA editing biology are still unknown and need to be elucidated. To support the study of A-to-I RNA editing we have updated our REDIportal catalogue raising its content to about 16 millions of events detected in 9642 human RNAseq samples from the GTEx project by using a dedicated pipeline based on the HPC version of the REDItools software. REDIportal now allows searches at sample level, provides overviews of RNA editing profiles per each RNAseq experiment, implements a Gene View module to look at individual events in their genic context and hosts the CLAIRE database. Starting from this novel version, REDIportal will start collecting non-human RNA editing changes for comparative genomics investigations. The database is freely available at http://srv00.recas.ba.infn.it/atlas/index.html.

The cell line A-to-I RNA editing catalogue.

Adenosine-to-inosine (A-to-I) RNA editing is a common post transcriptional modification. It has a critical role in protecting against false activation of innate immunity by endogenous double stranded RNAs and has been associated with various regulatory processes and diseases such as autoimmune and cardiovascular diseases as well as cancer. In addition, the endogenous A-to-I editing machinery has been recently harnessed for RNA engineering. The study of RNA editing in humans relies heavily on the usage of cell lines as an important and commonly-used research tool. In particular, manipulations of the editing enzymes and their targets are often developed using cell line platforms. However, RNA editing in cell lines behaves very differently than in normal and diseased tissues, and most cell lines exhibit low editing levels, requiring over-expression of the enzymes. Here, we explore the A-to-I RNA editing landscape across over 1000 human cell lines types and show that for almost every editing target of interest a suitable cell line that mimics normal tissue condition may be found. We provide CLAIRE, a searchable catalogue of RNA editing levels across cell lines available at http://srv00.recas.ba.infn.it/atlas/claire.html, to facilitate rational choice of appropriate cell lines for future work on A-to-I RNA editing.

Decreased A-to-I RNA editing as a source of keratinocytes' dsRNA in psoriasis.

Recognition of dsRNA molecules activates the MDA5-MAVS pathway and plays a critical role in stimulating type-I interferon responses in psoriasis. However, the source of the dsRNA accumulation in psoriatic keratinocytes remains largely unknown. A-to-I RNA editing is a common co- or post-transcriptional modification that diversifies adenosine in dsRNA, and leads to unwinding of dsRNA structures. Thus, impaired RNA editing activity can result in an increased load of endogenous dsRNAs. Here we provide a transcriptome-wide analysis of RNA editing across dozens of psoriasis patients, and we demonstrate a global editing reduction in psoriatic lesions. In addition to the global alteration, we also detect editing changes in functional recoding sites located in the IGFBP7, COPA, and FLNA genes. Accretion of dsRNA activates autoimmune responses, and therefore the results presented here, linking for the first time an autoimmune disease to reduction in global editing level, are relevant to a wide range of autoimmune diseases.

Altered RNA Editing in Atopic Dermatitis Highlights the Role of Double-Stranded RNA for Immune Surveillance.

Atopic dermatitis (AD) is associated with dysregulated type 1 IFN‒mediated responses, in parallel with the dominant type 2 inflammation. However, the pathophysiology of this dysregulation is largely unknown. Adenosine-to-inosine RNA editing plays a critical role in immune regulation by preventing double-stranded RNA recognition by MDA5 and IFN activation. We studied global adenosine-to-inosine editing in AD to elucidate the role played by altered editing in the pathophysiology of this disease. Analysis of three RNA-sequencing datasets of AD skin samples revealed reduced levels of adenosine-to-inosine RNA editing in AD. This reduction was seen globally throughout Alu repeats as well as in coding genes and in specific pre-mRNA loci expected to create long double-stranded RNA, the main substrate of MDA5 leading to type I IFN activation. Consistently, IFN signature genes were upregulated. In contrast, global editing was not altered in systemic lupus erythematosus and systemic sclerosis, despite IFN activation. Our results indicate that altered editing leading to impairment of the innate immune response may be involved in the pathogenesis of AD. Possibly, it may be relevant for additional autoimmune and inflammatory diseases.

Landscape of adenosine-to-inosine RNA recoding across human tissues.

RNA editing by adenosine deaminases changes the information encoded in the mRNA from its genomic blueprint. Editing of protein-coding sequences can introduce novel, functionally distinct, protein isoforms and diversify the proteome. The functional importance of a few recoding sites has been appreciated for decades. However, systematic methods to uncover these sites perform poorly, and the full repertoire of recoding in human and other mammals is unknown. Here we present a new detection approach, and analyze 9125 GTEx RNA-seq samples, to produce a highly-accurate atlas of 1517 editing sites within the coding region and their editing levels across human tissues. Single-cell RNA-seq data shows protein recoding contributes to the variability across cell subpopulations. Most highly edited sites are evolutionary conserved in non-primate mammals, attesting for adaptation. This comprehensive set can facilitate understanding of the role of recoding in human physiology and diseases.

Increased RNA editing in maternal immune activation model of neurodevelopmental disease.

The etiology of major neurodevelopmental disorders such as schizophrenia and autism is unclear, with evidence supporting a combination of genetic factors and environmental insults, including viral infection during pregnancy. Here we utilized a mouse model of maternal immune activation (MIA) with the viral mimic PolyI:C infection during early gestation. We investigated the transcriptional changes in the brains of mouse fetuses following MIA during the prenatal period, and evaluated the behavioral and biochemical changes in the adult brain. The results reveal an increase in RNA editing levels and dysregulation in brain development-related gene pathways in the fetal brains of MIA mice. These MIA-induced brain editing changes are not observed in adulthood, although MIA-induced behavioral deficits are observed. Taken together, our findings suggest that MIA induces transient dysregulation of RNA editing at a critical time in brain development.

The impact of individual comorbidities on non-relapse mortality following allogeneic hematopoietic stem cell transplantation.

Comorbidity burden is a well-established risk factor for non-relapse mortality (NRM) following allogeneic stem cell transplantation (allo-SCT). We evaluated whether individual comorbidities could better characterize NRM risk. Furthermore, given differing toxicity profiles of conditioning agents, we hypothesized that the hazard of comorbidities is exerted in a regimen-specific manner. This retrospective study included 875 adults treated with an allo-SCT. Six conditioning regimens were considered. Across the entire cohort and within each regimen, the hazard ratio (HR) for NRM associated with individual comorbidities was assessed using multivariable Cox regressions. In the overall population, renal dysfunction, hypoalbuminemia, and severe hepatic disease were associated with the highest risk of NRM (HR 2.1, HR 1.9, HR 1.7, respectively). The risk associated with specific comorbidities was modified by the conditioning regimen and was not correlated with intensity. In patients conditioned with fludarabine/busulfan (Flu/Bu4), NRM risk was increased with cardiac disease (HR 5.54). Severe pulmonary disease and a pre-existing infection were associated with increased NRM risk in patients receiving fludarabine/melphalan (HR 4.9) and fludarabine/treosulfan (HR 3.6), respectively. Comorbidities may exert effects unique to particular conditioning regimens, suggesting that regimen selection should be driven in part by specific comorbidities.