RNA Pol II-dependent transcription efficiency fine-tunes A-to-I editing levels.

A-to-I RNA editing is a widespread epitranscriptomic phenomenon leading to the conversion of adenosines to inosines, which are primarily interpreted as guanosines by cellular machines. Consequently, A-to-I editing can alter splicing or lead to recoding of transcripts. As misregulation of editing can cause a variety of human diseases, A-to-I editing requires tight regulation of the extent of deamination, particularly in protein-coding regions. The bulk of A-to-I editing occurs cotranscriptionally. Thus, we studied A-to-I editing regulation in the context of transcription and pre-mRNA processing. We show that stimulation of transcription impacts editing levels. Activation of the transcription factor MYC leads to an up-regulation of A-to-I editing, particularly in transcripts that are suppressed upon MYC activation. Moreover, low pre-mRNA synthesis rates and low pre-mRNA expression levels support high levels of editing. We also show that editing levels greatly differ between nascent pre-mRNA and mRNA in a cellular system, as well as in mouse tissues. Editing levels can increase or decrease from pre-mRNA to mRNA and can vary across editing targets and across tissues, showing that pre-mRNA processing is an important layer of editing regulation. Several lines of evidence suggest that the differences emerge during pre-mRNA splicing. Moreover, actinomycin D treatment of primary neuronal cells and editing level analysis suggests that regulation of editing levels also depends on transcription.

The ADAR1 editome reveals drivers of editing-specificity for ADAR1-isoforms.

Adenosine deaminase acting on RNA ADAR1 promotes A-to-I conversion in double-stranded and structured RNAs. ADAR1 has two isoforms transcribed from different promoters: cytoplasmic ADAR1p150 is interferon-inducible while ADAR1p110 is constitutively expressed and primarily localized in the nucleus. Mutations in ADAR1 cause Aicardi - Goutières syndrome (AGS), a severe autoinflammatory disease associated with aberrant IFN production. In mice, deletion of ADAR1 or the p150 isoform leads to embryonic lethality driven by overexpression of interferon-stimulated genes. This phenotype is rescued by deletion of the cytoplasmic dsRNA-sensor MDA5 indicating that the p150 isoform is indispensable and cannot be rescued by ADAR1p110. Nevertheless, editing sites uniquely targeted by ADAR1p150 remain elusive. Here, by transfection of ADAR1 isoforms into ADAR-less mouse cells we detect isoform-specific editing patterns. Using mutated ADAR variants, we test how intracellular localization and the presence of a Z-DNA binding domain-α affect editing preferences. These data show that ZBDα only minimally contributes to p150 editing-specificity while isoform-specific editing is primarily directed by the intracellular localization of ADAR1 isoforms. Our study is complemented by RIP-seq on human cells ectopically expressing tagged-ADAR1 isoforms. Both datasets reveal enrichment of intronic editing and binding by ADAR1p110 while ADAR1p150 preferentially binds and edits 3'UTRs.

Filamin A pre-mRNA editing modulates vascularization and tumor growth.

Adenosine to inosine (A to I) editing is mediated by adenosine deaminases acting on RNA (ADAR) enzymes. Inosines are interpreted as guanosines by the translational machinery. Consequently, A to I editing in mRNAs can lead to their recoding and the formation of proteins not encoded in the genome. Filamin A is an actin-crosslinking protein. A to I editing in the filamin pre-mRNA leads to the exchange of a glutamine to an arginine in a highly interactive domain of the protein. However, the consequences of this editing event are still poorly understood. Here we show, using transgenic mice expressing either constitutively edited or constitutively uneditable filamin A that filamin A editing critically controls angiogenesis in tumors but also in a mouse ischemia model. Hyper-editing reduces angiogenesis, while hypoediting leads to increased angiogenesis, possibly by altering vascular endothelial growth factor receptor 2 (VEGFR2) turnover. Further, FLNA editing of the tumor itself seemingly affects its metastatic potential by changing its interaction with the extracellular matrix. We therefore identify filamin A editing as a critical component for angiogenesis, tumor growth, and metastasis formation.

How RNA editing keeps an I on physiology.

Adenosine deaminases acting on RNAs convert adenosines (A) to inosines (I) in structured or double-stranded RNAs. In mammals, this process is widespread. In the human transcriptome, more than a million different sites have been identified that undergo an ADAR-mediated A-to-I exchange Inosines have an altered base pairing potential due to the missing amino group when compared to the original adenosine. Consequently, inosines prefer to base pair with cytosines but can also base pair with uracil or adenine. This altered base pairing potential not only affects protein decoding at the ribosome but also influences the folding of RNAs and the proteins that can associate with it. Consequently, an A to I exchange can also affect RNA processing and turnover (Nishikura K. Annu Rev Biochem 79: 321-349, 2010; Brümmer A, Yang Y, Chan TW, Xiao X. Nat Commun 8: 1255, 2017). All of these events will interfere with gene expression and therefore, can also affect cellular and organismic physiology. As double-stranded RNAs are a hallmark of viral pathogens RNA-editing not only affects RNA-processing, coding, and gene expression but also controls the antiviral response to double-stranded RNAs. Most interestingly, recent advances in our understanding of ADAR enzymes reveal multiple layers of regulation by which ADARs can control antiviral programs. In this review, we focus on the recoding of mRNAs where the altered translation products lead to physiological changes. We also address recent advances in our understanding of the multiple layers of antiviral responses and innate immune modulations mediated by ADAR1.

A-to-I RNA editing of Filamin A regulates cellular adhesion, migration and mechanical properties.

A-to-I RNA editing by ADARs is an abundant epitranscriptomic RNA-modification in metazoa. In mammals, Flna pre-mRNA harbours a single conserved A-to-I RNA editing site that introduces a Q-to-R amino acid change in Ig repeat 22 of the encoded protein. Previously, we showed that FLNA editing regulates smooth muscle contraction in the cardiovascular system and affects cardiac health. The present study investigates how ADAR2-mediated A-to-I RNA editing of Flna affects actin crosslinking, cell mechanics, cellular adhesion and cell migration. Cellular assays and AFM measurements demonstrate that the edited version of FLNA increases cellular stiffness and adhesion but impairs cell migration in both, mouse fibroblasts and human tumour cells. In vitro, edited FLNA leads to increased actin crosslinking, forming actin gels of higher stress resistance. Our study shows that Flna RNA editing is a novel regulator of cytoskeletal organisation, affecting the mechanical property and mechanotransduction of cells.

Site-directed RNA editing: recent advances and open challenges.

RNA editing by cytosine and adenosine deaminases changes the identity of the edited bases. While cytosines are converted to uracils, adenines are converted to inosines. If coding regions of mRNAs are affected, the coding potential of the RNA can be changed, depending on the codon affected. The recoding potential of nucleotide deaminases has recently gained attention for their ability to correct genetic mutations by either reverting the mutation itself or by manipulating processing steps such as RNA splicing. In contrast to CRISPR-based DNA-editing approaches, RNA editing events are transient in nature, therefore reducing the risk of long-lasting inadvertent side-effects. Moreover, some RNA-based therapeutics are already FDA approved and their use in targeting multiple cells or organs to restore genetic function has already been shown. In this review, we provide an overview on the current status and technical differences of site-directed RNA-editing approaches. We also discuss advantages and challenges of individual approaches.

An I for an A: Dynamic Regulation of Adenosine Deamination-Mediated RNA Editing.

RNA-editing by adenosine deaminases acting on RNA (ADARs) converts adenosines to inosines in structured RNAs. Inosines are read as guanosines by most cellular machineries. A to I editing has two major functions: first, marking endogenous RNAs as "self", therefore helping the innate immune system to distinguish repeat- and endogenous retrovirus-derived RNAs from invading pathogenic RNAs; and second, recoding the information of the coding RNAs, leading to the translation of proteins that differ from their genomically encoded versions. It is obvious that these two important biological functions of ADARs will differ during development, in different tissues, upon altered physiological conditions or after exposure to pathogens. Indeed, different levels of ADAR-mediated editing have been observed in different tissues, as a response to altered physiology or upon pathogen exposure. In this review, we describe the dynamics of A to I editing and summarize the known and likely mechanisms that will lead to global but also substrate-specific regulation of A to I editing.

Transportin-1: A Nuclear Import Receptor with Moonlighting Functions.

Transportin-1 (Trn1), also known as karyopherin-ß2 (Kapß2), is probably the best-characterized nuclear import receptor of the karyopherin-ß family after Importin-ß, but certain aspects of its functions in cells are still puzzling or are just recently emerging. Since the initial identification of Trn1 as the nuclear import receptor of hnRNP A1 ∼25 years ago, several molecular and structural studies have unveiled and refined our understanding of Trn1-mediated nuclear import. In particular, the understanding at a molecular level of the NLS recognition by Trn1 made a decisive step forward with the identification of a new class of NLSs called PY-NLSs, which constitute the best-characterized substrates of Trn1. Besides PY-NLSs, many Trn1 cargoes harbour NLSs that do not resemble the archetypical PY-NLS, which complicates the global understanding of cargo recognition by Trn1. Although PY-NLS recognition is well established and supported by several structures, the recognition of non-PY-NLSs by Trn1 is far less understood, but recent reports have started to shed light on the recognition of this type of NLSs. Aside from its principal and long-established activity as a nuclear import receptor, Trn1 was shown more recently to moonlight outside nuclear import. Trn1 has for instance been caught in participating in virus uncoating, ciliary transport and in modulating the phase separation properties of aggregation-prone proteins. Here, we focus on the structural and functional aspects of Trn1-mediated nuclear import, as well as on the moonlighting activities of Trn1.

ADAR-deficiency perturbs the global splicing landscape in mouse tissues.

Adenosine-to-inosine RNA editing and pre-mRNA splicing largely occur cotranscriptionally and influence each other. Here, we use mice deficient in either one of the two editing enzymes ADAR (ADAR1) or ADARB1 (ADAR2) to determine the transcriptome-wide impact of RNA editing on splicing across different tissues. We find that ADAR has a 100× higher impact on splicing than ADARB1, although both enzymes target a similar number of substrates with a large common overlap. Consistently, differentially spliced regions frequently harbor ADAR editing sites. Moreover, catalytically dead ADAR also impacts splicing, demonstrating that RNA binding of ADAR affects splicing. In contrast, ADARB1 editing sites are found enriched 5' of differentially spliced regions. Several of these ADARB1-mediated editing events change splice consensus sequences, therefore strongly influencing splicing of some mRNAs. A significant overlap between differentially edited and differentially spliced sites suggests evolutionary selection toward splicing being regulated by editing in a tissue-specific manner.

An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype.

The RNA-editing protein ADAR is essential for early development in the mouse. Genetic evidence suggests that A to I editing marks endogenous RNAs as 'self'. Today, different Adar knockout alleles have been generated that show a common phenotype of apoptosis, liver disintegration, elevated immune response and lethality at E12.5. All the Adar knockout alleles can be rescued by a concomitant deletion of the innate immunity genes Mavs or Ifih1 (MDA5), albeit to different extents. This suggests multiple functions of ADAR. We analyze AdarΔ7-9 mice that show a unique growth defect phenotype when rescued by Mavs. We show that AdarΔ7-9 can form a truncated, unstable, editing deficient protein that is mislocalized. Histological and hematologic analysis of these mice indicate multiple tissue- and hematopoietic defects. Gene expression profiling shows dysregulation of Rps3a1 and Rps3a3 in rescued AdarΔ7-9. Consistently, a distortion in 40S and 60S ribosome ratios is observed in liver cells. This dysregulation is also seen in AdarΔ2-13; Mavs-/- but not in AdarE861A/E861A; Ifih1-/- mice, suggesting editing-independent functions of ADAR in regulating expression levels of Rps3a1 and Rps3a3. In conclusion, our study demonstrates the importance of ADAR in post-natal development which cannot be compensated by ADARB1.

The Editor's I on Disease Development.

Adenosine-to-inosine (A-to-I) editing of RNA leads to deamination of adenosine to inosine. Inosine is interpreted as guanosine by the cellular machinery, thus altering the coding, folding, splicing, or transport of transcripts. A-to-I editing is tightly regulated. Altered editing has severe consequences for human health and can cause interferonopathies, neurological disorders, and cardiovascular disease, as well as impacting on cancer progression. ADAR1-mediated RNA editing plays an important role in antiviral immunity and is essential for distinguishing between endogenous and viral RNA, thereby preventing autoimmune disorders. Interestingly, A-to-I editing can be used not only to correct genomic mutations at the RNA level but also to modulate tumor antigenicity with large therapeutic potential. We highlight recent developments in the field, focusing on cancer and other human diseases.

A high resolution A-to-I editing map in the mouse identifies editing events controlled by pre-mRNA splicing.

Pre-mRNA-splicing and adenosine to inosine (A-to-I) RNA-editing occur mostly cotranscriptionally. During A-to-I editing, a genomically encoded adenosine is deaminated to inosine by adenosine deaminases acting on RNA (ADARs). Editing-competent stems are frequently formed between exons and introns. Consistently, studies using reporter assays have shown that splicing efficiency can affect editing levels. Here, we use Nascent-seq and identify ∼90,000 novel A-to-I editing events in the mouse brain transcriptome. Most novel sites are located in intronic regions. Unlike previously assumed, we show that both ADAR (ADAR1) and ADARB1 (ADAR2) can edit repeat elements and regular transcripts to the same extent. We find that inhibition of splicing primarily increases editing levels at hundreds of sites, suggesting that reduced splicing efficiency extends the exposure of intronic and exonic sequences to ADAR enzymes. Lack of splicing factors NOVA1 or NOVA2 changes global editing levels, demonstrating that alternative splicing factors can modulate RNA editing. Finally, we show that intron retention rates correlate with editing levels across different brain tissues. We therefore demonstrate that splicing efficiency is a major factor controlling tissue-specific differences in editing levels.

Dynamic Interactions Between the Genome and an Endogenous Retrovirus: Tirant in Drosophila simulans Wild-Type Strains.

All genomes contain repeated sequences that are known as transposable elements (TEs). Among these are endogenous retroviruses (ERVs), which are sequences similar to retroviruses and are transmitted across generations from parent to progeny. These sequences are controlled in genomes through epigenetic mechanisms. At the center of the epigenetic control of TEs are small interfering RNAs of the piRNA class, which trigger heterochromatinization of TE sequences. The tirant ERV of Drosophila simulans displays intra-specific variability in copy numbers, insertion sites, and transcription levels, providing us with a well-suited model to study the dynamic relationship between a TE family and the host genome through epigenetic mechanisms. We show that tirant transcript amounts and piRNA amounts are positively correlated in ovaries in normal conditions, unlike what was previously described following divergent crosses. In addition, we describe tirant insertion polymorphism in the genomes of three D. simulans wild-type strains, which reveals a limited number of insertions that may be associated with gene transcript level changes through heterochromatin spreading and have phenotypic impacts. Taken together, our results participate in the understanding of the equilibrium between the host genome and its TEs.

Inosine induces context-dependent recoding and translational stalling.

RNA modifications are present in all classes of RNAs. They control the fate of mRNAs by affecting their processing, translation, or stability. Inosine is a particularly widespread modification in metazoan mRNA arising from deamination of adenosine catalyzed by the RNA-targeting adenosine deaminases ADAR1 or ADAR2. Inosine is commonly thought to be interpreted as guanosine by cellular machines and during translation. Here, we systematically test ribosomal decoding using mass spectrometry. We show that while inosine is primarily interpreted as guanosine it can also be decoded as adenosine, and rarely even as uracil. Decoding of inosine as adenosine and uracil is context-dependent. In addition, mass spectrometry analysis indicates that inosine causes ribosome stalling especially when multiple inosines are present in the codon. Indeed, ribosome profiling data from human tissues confirm inosine-dependent ribosome stalling in vivo. To our knowledge this is the first study where decoding of inosine is tested in a comprehensive and unbiased way. Thus, our study shows novel, unanticipated functions for inosines in mRNAs, further expanding coding potential and affecting translational efficiency.

RNA editing of Filamin A pre-mRNA regulates vascular contraction and diastolic blood pressure.

Epitranscriptomic events such as adenosine-to-inosine (A-to-I) RNA editing by ADAR can recode mRNAs to translate novel proteins. Editing of the mRNA that encodes actin crosslinking protein Filamin A (FLNA) mediates a Q-to-R transition in the interactive C-terminal region. While FLNA editing is conserved among vertebrates, its physiological function remains unclear. Here, we show that cardiovascular tissues in humans and mice show massive editing and that FLNA RNA is the most prominent substrate. Patient-derived RNA-Seq data demonstrate a significant drop in FLNA editing associated with cardiovascular diseases. Using mice with only impaired FLNA editing, we observed increased vascular contraction and diastolic hypertension accompanied by increased myosin light chain phosphorylation, arterial remodeling, and left ventricular wall thickening, which eventually causes cardiac remodeling and reduced systolic output. These results demonstrate a causal relationship between RNA editing and the development of cardiovascular disease indicating that a single epitranscriptomic RNA modification can maintain cardiovascular health.

Organ-wide profiling in mouse reveals high editing levels of Filamin B mRNA in the musculoskeletal system.

Adenosine to inosine RNA editing in protein-coding messenger RNAs (mRNAs) potentially leads to changes in the amino acid composition of the encoded proteins. The mRNAs encoding the ubiquitously expressed actin-crosslinking proteins Filamin A and Filamin B undergo RNA editing leading to a highly conserved glutamine to arginine exchange at the identical position in either protein. Here, by targeted amplicon sequencing we analysed the RNA editing of Filamin B across several mouse tissues during post-natal development. We find highest filamin B editing levels in skeletal muscles, cartilage and bones, tissues where Filamin B function seems most important. Through the analysis of Filamin B editing in mice deficient in either ADAR1 or 2, we identified ADAR2 as the enzyme responsible for Filamin B RNA editing. We show that in neuronal tissues Filamin B editing drops in spliced transcripts indicating regulated maturation of edited transcripts. We show further that the variability of Filamin B editing across several organs correlates with its mRNA expression.

Positioning Europe for the EPITRANSCRIPTOMICS challenge.

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.

Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells.

Telomeres cap the ends of eukaryotic chromosomes, protecting them from degradation and erroneous recombination events which may lead to genome instability. Telomeres are transcribed giving rise to telomeric repeat-containing RNAs, called TERRA. The TERRA long noncoding RNAs have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. While TERRA RNAs are predominantly nuclear and localize at telomeres, little is known about the dynamics and function of TERRA molecules expressed from individual telomeres. Herein, we developed an assay to image endogenous TERRA molecules expressed from a single telomere in living human cancer cells. We show that single-telomere TERRA can be detected as TERRA RNA single particles which freely diffuse within the nucleus. Furthermore, TERRA molecules aggregate forming TERRA clusters. Three-dimensional size distribution and single particle tracking analyses revealed distinct sizes and dynamics for TERRA RNA single particles and clusters. Simultaneous time lapse confocal imaging of TERRA particles and telomeres showed that TERRA clusters transiently co-localize with telomeres. Finally, we used chemically modified antisense oligonucleotides to deplete TERRA molecules expressed from a single telomere. Single-telomere TERRA depletion resulted in increased DNA damage at telomeres and elsewhere in the genome. These results suggest that single-telomere TERRA transcripts participate in the maintenance of genomic integrity in human cancer cells.

The Other Face of an Editor: ADAR1 Functions in Editing-Independent Ways.

The RNA editing enzyme ADAR1 seemingly has more functions besides RNA editing. Mouse models lacking ADAR1 and sensors of foreign RNA show that RNA editing by ADAR1 plays a crucial role in the innate immune response. Still, RNA editing alone cannot explain all observed phenotypes. Thus, additional roles for ADAR1 must exist. Binding of ADAR1 to RNA is independent of its RNA editing function. Thus, ADAR1 may compete with other RNA-binding proteins. A very recent manuscript elaborates on this and reports competition of ADAR1 with STAUFEN1, thereby modulating RNA-degradation. ADAR1 is also known to recruit proteins such as DROSHA to nascent transcripts. Still, many open questions remain. For instance, the biological role of the Z-DNA binding domains in ADAR1 is not defined. Moreover, the impact of ADAR1 on the RNA-folding landscape is unclear. In sum, moonlighting functions of ADAR1 may be manifold and have a great impact on the transcriptome.