Temperature-dependent RNA editing in octopus extensively recodes the neural proteome.

In poikilotherms, temperature changes challenge the integration of physiological function. Within the complex nervous systems of the behaviorally sophisticated coleoid cephalopods, these problems are substantial. RNA editing by adenosine deamination is a well-positioned mechanism for environmental acclimation. We report that the neural proteome of Octopus bimaculoides undergoes massive reconfigurations via RNA editing following a temperature challenge. Over 13,000 codons are affected, and many alter proteins that are vital for neural processes. For two highly temperature-sensitive examples, recoding tunes protein function. For synaptotagmin, a key component of Ca2+-dependent neurotransmitter release, crystal structures and supporting experiments show that editing alters Ca2+ binding. For kinesin-1, a motor protein driving axonal transport, editing regulates transport velocity down microtubules. Seasonal sampling of wild-caught specimens indicates that temperature-dependent editing occurs in the field as well. These data show that A-to-I editing tunes neurophysiological function in response to temperature in octopus and most likely other coleoids.

Trade-off between Transcriptome Plasticity and Genome Evolution in Cephalopods.

RNA editing, a post-transcriptional process, allows the diversification of proteomes beyond the genomic blueprint; however it is infrequently used among animals for this purpose. Recent reports suggesting increased levels of RNA editing in squids thus raise the question of the nature and effects of these events. We here show that RNA editing is particularly common in behaviorally sophisticated coleoid cephalopods, with tens of thousands of evolutionarily conserved sites. Editing is enriched in the nervous system, affecting molecules pertinent for excitability and neuronal morphology. The genomic sequence flanking editing sites is highly conserved, suggesting that the process confers a selective advantage. Due to the large number of sites, the surrounding conservation greatly reduces the number of mutations and genomic polymorphisms in protein-coding regions. This trade-off between genome evolution and transcriptome plasticity highlights the importance of RNA recoding as a strategy for diversifying proteins, particularly those associated with neural function. PAPERCLIP.

In search of critical dsRNA targets of ADAR1.

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.

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.

The New RNA-Editing Era - Ethical Considerations.

The characteristics of RNA editing, including the lower risk compared with genome editing, may loosen the ethical barriers that are currently imposed on genetic engineering, thus opening new possibilities for research, therapy, and human enhancement. We should start considering the future ethical and social implications of this new and promising technology.

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.

A-to-I RNA editing - immune protector and transcriptome diversifier.

Modifications of RNA affect its function and stability. RNA editing is unique among these modifications because it not only alters the cellular fate of RNA molecules but also alters their sequence relative to the genome. The most common type of RNA editing is A-to-I editing by double-stranded RNA-specific adenosine deaminase (ADAR) enzymes. Recent transcriptomic studies have identified a number of 'recoding' sites at which A-to-I editing results in non-synonymous substitutions in protein-coding sequences. Many of these recoding sites are conserved within (but not usually across) lineages, are under positive selection and have functional and evolutionary importance. However, systematic mapping of the editome across the animal kingdom has revealed that most A-to-I editing sites are located within mobile elements in non-coding parts of the genome. Editing of these non-coding sites is thought to have a critical role in protecting against activation of innate immunity by self-transcripts. Both recoding and non-coding events have implications for genome evolution and, when deregulated, may lead to disease. Finally, ADARs are now being adapted for RNA engineering purposes.

Deep transcriptome profiling reveals limited conservation of A-to-I RNA editing in Xenopus.

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.

Systematic identification of A-to-I RNA editing in zebrafish development and adult organs.

A-to-I RNA editing is a common post transcriptional mechanism, mediated by the Adenosine deaminase that acts on RNA (ADAR) enzymes, that increases transcript and protein diversity. The study of RNA editing is limited by the absence of editing maps for most model organisms, hindering the understanding of its impact on various physiological conditions. Here, we mapped the vertebrate developmental landscape of A-to-I RNA editing, and generated the first comprehensive atlas of editing sites in zebrafish. Tens of thousands unique editing events and 149 coding sites were identified with high-accuracy. Some of these edited sites are conserved between zebrafish and humans. Sequence analysis of RNA over seven developmental stages revealed high levels of editing activity in early stages of embryogenesis, when embryos rely on maternal mRNAs and proteins. In contrast to the other organisms studied so far, the highest levels of editing were detected in the zebrafish ovary and testes. This resource can serve as the basis for understanding of the role of editing during zebrafish development and maturity.

Adaptive Proteome Diversification by Nonsynonymous A-to-I RNA Editing in Coleoid Cephalopods.

RNA editing by the ADAR enzymes converts selected adenosines into inosines, biological mimics for guanosines. By doing so, it alters protein-coding sequences, resulting in novel protein products that diversify the proteome beyond its genomic blueprint. Recoding is exceptionally abundant in the neural tissues of coleoid cephalopods (octopuses, squids, and cuttlefishes), with an over-representation of nonsynonymous edits suggesting positive selection. However, the extent to which proteome diversification by recoding provides an adaptive advantage is not known. It was recently suggested that the role of evolutionarily conserved edits is to compensate for harmful genomic substitutions, and that there is no added value in having an editable codon as compared with a restoration of the preferred genomic allele. Here, we show that this hypothesis fails to explain the evolutionary dynamics of recoding sites in coleoids. Instead, our results indicate that a large fraction of the shared, strongly recoded, sites in coleoids have been selected for proteome diversification, meaning that the fitness of an editable A is higher than an uneditable A or a genomically encoded G.

Genome-wide quantification of ADAR adenosine-to-inosine RNA editing activity.

Adenosine-to-inosine (A-to-I) RNA editing by the adenosine deaminase that acts on RNA (ADAR) enzymes is a common RNA modification, preventing false activation of the innate immune system by endogenous double-stranded RNAs. Methods for quantification of ADAR activity are sought after, due to an increasing interest in the role of ADARs in cancer and autoimmune disorders, as well as attempts to harness the ADAR enzymes for RNA engineering. Here, we present the Alu editing index (AEI), a robust and simple-to-use computational tool devised for this purpose. We describe its properties and demonstrate its superiority to current quantification methods of ADAR activity. The AEI is used to map global editing across a large dataset of healthy human samples and identify putative regulators of ADAR, as well as previously unknown factors affecting the observed Alu editing levels. These should be taken into account in future comparative studies of ADAR activity. The AEI tool is available at https://github.com/a2iEditing/RNAEditingIndexer.

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.

Spatially regulated editing of genetic information within a neuron.

In eukaryotic cells, with the exception of the specialized genomes of mitochondria and plastids, all genetic information is sequestered within the nucleus. This arrangement imposes constraints on how the information can be tailored for different cellular regions, particularly in cells with complex morphologies like neurons. Although messenger RNAs (mRNAs), and the proteins that they encode, can be differentially sorted between cellular regions, the information itself does not change. RNA editing by adenosine deamination can alter the genome's blueprint by recoding mRNAs; however, this process too is thought to be restricted to the nucleus. In this work, we show that ADAR2 (adenosine deaminase that acts on RNA), an RNA editing enzyme, is expressed outside of the nucleus in squid neurons. Furthermore, purified axoplasm exhibits adenosine-to-inosine activity and can specifically edit adenosines in a known substrate. Finally, a transcriptome-wide analysis of RNA editing reveals that tens of thousands of editing sites (>70% of all sites) are edited more extensively in the squid giant axon than in its cell bodies. These results indicate that within a neuron RNA editing can recode genetic information in a region-specific manner.

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.

Human cancer tissues exhibit reduced A-to-I editing of miRNAs coupled with elevated editing of their targets.

A-to-I RNA editing is an important post-transcriptional modification, known to be altered in tumors. It targets dozens of sites within miRNAs, some of which impact miRNA biogenesis and function, as well as many miRNA recognition sites. However, the full extent of the effect of editing on regulation by miRNAs and its behavior in human cancers is still unknown. Here we systematically characterized miRNA editing in 10 593 human samples across 32 cancer types and normal controls. We find that the majority of previously reported sites show little to no evidence for editing in this dataset, compile a list of 58 reliable miRNA editing sites, and study them across normal and cancer samples. Edited miRNA versions tend to suppress expression of known oncogenes, and, consistently, we observe a clear global tendency for hypo-editing in tumors, in strike contrast to the behavior for mRNA editing, allowing an accurate classification of normal/tumor samples based on their miRNA editing profile. In many cancers this profile correlates with patients' survival. Finally, thousands of miRNA binding sites are differentially edited in cancer. Our study thus establishes the important effect of RNA editing on miRNA-regulation in the tumor cell, with prospects for diagnostic and prognostic applications.

ADAR2/miR-589-3p axis controls glioblastoma cell migration/invasion.

Recent studies have reported the emerging role of microRNAs (miRNAs) in human cancers. We systematically characterized miRNA expression and editing in the human brain, which displays the highest number of A-to-I RNA editing sites among human tissues, and in de novo glioblastoma brain cancer. We identified 299 miRNAs altered in their expression and 24 miRNAs differently edited in human brain compared to glioblastoma tissues. We focused on the editing site within the miR-589-3p seed. MiR-589-3p is a unique miRNA almost fully edited (∼100%) in normal brain and with a consistent editing decrease in glioblastoma. The edited version of miR-589-3p inhibits glioblastoma cell proliferation, migration and invasion, while the unedited version boosts cell proliferation and motility/invasion, thus being a potential cancer-promoting factor. We demonstrated that the editing of this miRNA is mediated by ADAR2, and retargets miR-589-3p from the tumor-suppressor PCDH9 to ADAM12, which codes for the metalloproteinase 12 promoting glioblastoma invasion. Overall, our study dissects the role of a unique brain-specific editing site within miR-589-3p, with important anticancer features, and highlights the importance of RNA editing as an essential player not only for diversifying the genomic message but also for correcting not-tolerable/critical genomic coding sites.

Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior.

The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior.

A-to-I RNA Editing in the Earliest-Diverging Eumetazoan Phyla.

The highly conserved ADAR enzymes, found in all multicellular metazoans, catalyze the editing of mRNA transcripts by the deamination of adenosines to inosines. This type of editing has two general outcomes: site specific editing, which frequently leads to recoding, and clustered editing, which is usually found in transcribed genomic repeats. Here, for the first time, we looked for both editing of isolated sites and clustered, non-specific sites in a basal metazoan, the coral Acropora millepora during spawning event, in order to reveal its editing pattern. We found that the coral editome resembles the mammalian one: it contains more than 500,000 sites, virtually all of which are clustered in non-coding regions that are enriched for predicted dsRNA structures. RNA editing levels were increased during spawning and increased further still in newly released gametes. This may suggest that editing plays a role in introducing variability in coral gametes.

Reduced levels of protein recoding by A-to-I RNA editing in Alzheimer's disease.

Adenosine to inosine (A-to-I) RNA editing, catalyzed by the ADAR enzyme family, acts on dsRNA structures within pre-mRNA molecules. Editing of the coding part of the mRNA may lead to recoding, amino acid substitution in the resulting protein, possibly modifying its biochemical and biophysical properties. Altered RNA editing patterns have been observed in various neurological pathologies. Here, we present a comprehensive study of recoding by RNA editing in Alzheimer's disease (AD), the most common cause of irreversible dementia. We have used a targeted resequencing approach supplemented by a microfluidic-based high-throughput PCR coupled with next-generation sequencing to accurately quantify A-to-I RNA editing levels in a preselected set of target sites, mostly located within the coding sequence of synaptic genes. Overall, editing levels decreased in AD patients' brain tissues, mainly in the hippocampus and to a lesser degree in the temporal and frontal lobes. Differential RNA editing levels were observed in 35 target sites within 22 genes. These results may shed light on a possible association between the neurodegenerative processes typical for AD and deficient RNA editing.