A-to-I editing of coding and non-coding RNAs by ADARs.

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. This A-to-I editing occurs not only in protein-coding regions of mRNAs, but also frequently in non-coding regions that contain inverted Alu repeats. Editing of coding sequences can result in the expression of functionally altered proteins that are not encoded in the genome, whereas the significance of Alu editing remains largely unknown. Certain microRNA (miRNA) precursors are also edited, leading to reduced expression or altered function of mature miRNAs. Conversely, recent studies indicate that ADAR1 forms a complex with Dicer to promote miRNA processing, revealing a new function of ADAR1 in the regulation of RNA interference.

ADAR1 forms a complex with Dicer to promote microRNA processing and RNA-induced gene silencing.

Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosine residues to inosine specifically in double-stranded RNAs. In this study, we investigated the interaction of the RNA editing mechanism with the RNA interference (RNAi) machinery and found that ADAR1 forms a complex with Dicer through direct protein-protein interaction. Most importantly, ADAR1 increases the maximum rate (Vmax) of pre-microRNA (miRNA) cleavage by Dicer and facilitates loading of miRNA onto RNA-induced silencing complexes, identifying a new role of ADAR1 in miRNA processing and RNAi mechanisms. ADAR1 differentiates its functions in RNA editing and RNAi by the formation of either ADAR1/ADAR1 homodimer or Dicer/ADAR1 heterodimer complexes, respectively. As expected, the expression of miRNAs is globally inhibited in ADAR1(-/-) mouse embryos, which, in turn, alters the expression of their target genes and might contribute to their embryonic lethal phenotype.

Functions and regulation of RNA editing by ADAR deaminases.

One type of RNA editing converts adenosines to inosines (A-->I editing) in double-stranded RNA (dsRNA) substrates. A-->I RNA editing is mediated by adenosine deaminase acting on RNA (ADAR) enzymes. A-->I RNA editing of protein-coding sequences of a limited number of mammalian genes results in recoding and subsequent alterations of their functions. However, A-->I RNA editing most frequently targets repetitive RNA sequences located within introns and 5' and 3' untranslated regions (UTRs). Although the biological significance of noncoding RNA editing remains largely unknown, several possibilities, including its role in the control of endogenous short interfering RNAs (esiRNAs), have been proposed. Furthermore, recent studies have revealed that the biogenesis and functions of certain microRNAs (miRNAs) are regulated by the editing of their precursors. Here, I review the recent findings that indicate new functions for A-->I editing in the regulation of noncoding RNAs and for interactions between RNA editing and RNA interference mechanisms.

Dynamic landscape and regulation of RNA editing in mammals.

Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.

Processing of Alu small RNAs by DICER/ADAR1 complexes and their RNAi targets.

In addition to adenosine-to-inosine RNA editing activities, ADAR1 has been shown to have various RNA editing-independent activities including modulation of RNAi efficacy. We previously reported that ADAR1 forms a heterodimer complex with DICER and facilitates processing of pre-miRNAs to mature miRNAs. In addition to miRNA synthesis, DICER is involved in processing of long dsRNAs into small RNAs (endo-siRNAs). Generation of retrotransposon-derived endo-siRNAs by DICER and their functions in regulation of transcripts in mouse oocytes has been previously reported. However, the synthesis and functions of endo-siRNAs in somatic cells remain largely unknown. Here, we report that ADAR1 together with DICER generates endogenous small RNAs, Alu endo-siRNAs by cleaving long double-stranded regions of inverted Alu repeats. We identified AGO2-loaded Alu endo-siRNAs, which are highly expressed in commonly used cell lines. These Alu endo-siRNAs carrying both sense and antisense Alu sequences seem to target a set of genes containing a single Alu sequence, either antisense or sense, respectively, within their 3'UTR. In silico screening identified potential RNA silencing target genes for these Alu endo-siRNAs. We present results of a proof-of-concept experiment, in which sense Alu endo-siRNAs derived from AluSz and AluJr family elements target CUB Domain Containing Protein 1 mRNAs containing an antisense copy of AluJb in their 3'UTRs and consequently induce apoptosis in HeLa cells. Our results clearly indicate that Alu endo-siRNAs are functional also in somatic cells.

A new function for the RNA-editing enzyme ADAR1.

ADAR1 catalyzes the deamination of adenosine to inosine in double-stranded RNA. This RNA-editing enzyme is now shown to be involved in hematopoiesis, where it acts to suppress interferon signaling and to block premature apoptosis.

Elucidating the inosinome: global approaches to adenosine-to-inosine RNA editing.

Catalysed by members of the adenosine deaminase acting on RNA (ADAR) family of enzymes, adenosine-to-inosine (A-to-I) editing converts adenosines in RNA molecules to inosines, which are functionally equivalent to guanosines. Recently, global approaches to studying this widely conserved phenomenon have emerged. The use of bioinformatics, high-throughput sequencing and other approaches has increased the number of known editing sites by several orders of magnitude, and we now have a greater understanding of the control and the biological significance of editing. This Progress article reviews some of these recent global studies and their results.

A-to-I editing of protein coding and noncoding RNAs.

Adenosine deaminase acting on RNA (ADAR) catalyzes the hydrolytic deamination of adenosine to inosine in double-stranded RNA (dsRNA) substrates. Inosine pairs preferentially with cytidine, as opposed to uridine; therefore, ADAR editing alters the sequence and base pairing properties of both protein-coding and non-coding RNA. Editing can directly alter the sequence of protein-coding transcripts and modify splicing, or affect a variety of non-coding targets, including microRNA, small interfering RNA, viral transcripts, and repeat elements such as Alu and LINE. Such editing has a wide range of physiological effects, including modification of targets in the brain and in disease states.

ADAR1 protein induces adenosine-targeted DNA mutations in senescent Bcl6 gene-deficient cells.

Somatic mutations accumulate in senescent cells. Bcl6, which functions as a transcriptional repressor, has been identified as a potent inhibitor of cell senescence, but a role of Bcl6 in the accumulation of somatic mutations has remained unclear. Ig class-switch recombination simultaneously induces somatic mutations in an IgM class-switch (Ig-Sµ) region of IgG B cells. Surprisingly, mutations were detected in the Ig-Sµ region of Bcl6-deficient IgM B cells without class-switch recombination, and these mutations were mainly generated by conversion of adenosine to guanosine, suggesting a novel DNA mutator in the B cells. The ADAR1 (adenosine deaminase acting on RNA1) gene was overexpressed in Bcl6-deficient cells, and its promoter analysis revealed that ADAR1 is a molecular target of Bcl6. Exogenous ADAR1 induced adenosine-targeted DNA mutations in IgM B cells from ADAR1-transgenic mice and in wild-type mouse embryonic fibroblasts (MEFs). These mutations accumulated in senescent MEFs accompanied with endogenous ADAR1 expression, and the frequency in senescent Bcl6-deficient MEFs was higher than senescent wild-type MEFs. Thus, Bcl6 protects senescent cells from accumulation of adenosine-targeted DNA mutations induced by ADAR1.

Enhancement of alcohol drinking in mice depends on alterations in RNA editing of serotonin 2C receptors.

Serotonin 2C receptors (5-HT(2C)R) are G-protein-coupled receptors with various actions, including involvement in drug addiction. 5-HT2CR undergoes mRNA editing, converting genomically encoded adenosine residues to inosines via adenosine deaminases acting on RNA (ADARs). Here we show that enhanced alcohol drinking behaviour in mice is associated with the degree of 5-HT(2C)R mRNA editing in the nucleus accumbens and dorsal raphe nuceus, brain regions important for reward and addiction. Following chronic alcohol vapour exposure, voluntary alcohol intake increased in C57BL/6J mice, but remained unchanged in C3H/HeJ and DBA/2J mice. 5-HT(2C)R mRNA editing frequency in both regions increased significantly in C57BL/6J mice, as did expressions of 5-HT(2C)R, ADAR1 and ADAR2, but not in other strains. Moreover, mice that exclusively express the unedited isoform (INI) of 5-HT(2C)R mRNA on a C57BL/6J background did not exhibit increased alcohol intake compared with wild-type mice. Our results indicate that alterations in 5-HT(2C)R mRNA editing underlie alcohol preference in mice.

Modulation of microRNA expression and function by ADARs.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by preventing the translation of specific messenger RNAs. Adenosine deaminases acting on RNAs (ADARs) catalyze adenosine-to-inosine (A-to-I) RNA editing, the conversion of adenosines into inosines, in double-stranded RNAs. Because inosine preferentially base pairs with cytidine, this conversion is equivalent to an adenosine to guanosine change. Over the past seven years, an increasing number of edited adenosines have been identified in miRNAs. Editing of miRNAs affects their biogenesis, causes their degradation or alters the set of messenger RNAs that they regulate. Recently, ADARs have been shown to also affect the miRNA phenomenon by sequestering miRNAs or by editing the messenger RNAs they regulate. This article reviews the recent attempts to identify miRNA editing sites and elucidate the effects of ADARs on miRNA expression and function.

Antagonistic and stimulative roles of ADAR1 in RNA silencing.

Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosine residues to inosine specifically in double-stranded RNAs (dsRNA). This A-to-I RNA editing pathway and the RNA interference (RNAi) pathway seem to interact antagonistically by competing for their common dsRNA substrates. For instance, A-to-I editing of certain microRNA (miRNA) precursors by ADAR1 and ADAR2 inhibits their processing to mature miRNAs. Recent studies unexpectedly revealed the presence of a completely different type of interaction between the RNA editing mechanism and the RNAi machinery. ADAR1 forms a complex via direct protein-protein interaction with Dicer, an RNase III gene family member involved in the RNAi mechanism. ADAR1 in the Dicer complex promotes pre-miRNA cleavage by Dicer and facilitates loading of miRNA onto RNA-induced silencing complexes, giving rise to an unsuspected stimulative function of ADAR1 on miRNA processing and RNAi mechanisms. ADAR1 differentiates its functions in RNA editing and RNAi by formation of either ADAR1-ADAR1 homodimer or Dicer-ADAR1 heterodimer complexes. Expression of miRNAs is globally inhibited in ADAR1-null mouse embryos, which, in turn, alters expression of their target genes and may contribute to their embryonic lethal phenotype.

The role of RNA editing enzyme ADAR1 in human disease.

Adenosine deaminase acting on RNA (ADAR) catalyzes the posttranscriptional conversion of adenosine to inosine in double-stranded RNA (dsRNA), which can lead to the creation of missense mutations in coding sequences. Recent studies show that editing-dependent functions of ADAR1 protect dsRNA from dsRNA-sensing molecules and inhibit innate immunity and the interferon-mediated response. Deficiency in these ADAR1 functions underlie the pathogenesis of autoinflammatory diseases such as the type I interferonopathies Aicardi-Goutieres syndrome and dyschromatosis symmetrica hereditaria. ADAR1-mediated editing of endogenous coding and noncoding RNA as well as ADAR1 editing-independent interactions with DICER can also have oncogenic or tumor suppressive effects that affect tumor proliferation, invasion, and response to immunotherapy. The combination of proviral and antiviral roles played by ADAR1 in repressing the interferon response and editing viral RNAs alters viral morphogenesis and cell susceptibility to infection. This review analyzes the structure and function of ADAR1 with a focus on its position in human disease pathways and the mechanisms of its disease-associated effects. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

ADAR1 downregulation by autophagy drives senescence independently of RNA editing by enhancing p16INK4a levels.

Cellular senescence plays a causal role in ageing and, in mice, depletion of p16INK4a-expressing senescent cells delays ageing-associated disorders1,2. Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes that are also implicated as important regulators of human ageing, and ADAR inactivation causes age-associated pathologies such as neurodegeneration in model organisms3,4. However, the role, if any, of ADARs in cellular senescence is unknown. Here we show that ADAR1 is post-transcriptionally downregulated by autophagic degradation to promote senescence through p16INK4a upregulation. The ADAR1 downregulation is sufficient to drive senescence in both in vitro and in vivo models. Senescence induced by ADAR1 downregulation is p16INK4a-dependent and independent of its RNA-editing function. Mechanistically, ADAR1 promotes SIRT1 expression by affecting its RNA stability through HuR, an RNA-binding protein that increases the half-life and steady-state levels of its target mRNAs. SIRT1 in turn antagonizes translation of mRNA encoding p16INK4a. Hence, downregulation of ADAR1 and SIRT1 mediates p16INK4a upregulation by enhancing its mRNA translation. Finally, Adar1 is downregulated during ageing of mouse tissues such as brain, ovary and intestine, and Adar1 expression correlates with Sirt1 expression in these tissues in mice. Together, our study reveals an RNA-editing-independent role for ADAR1 in the regulation of senescence by post-transcriptionally controlling p16INK4a expression.

Editing of Epstein-Barr virus-encoded BART6 microRNAs controls their dicer targeting and consequently affects viral latency.

Certain primary transcripts of miRNA (pri-microRNAs) undergo RNA editing that converts adenosine to inosine. The Epstein-Barr virus (EBV) genome encodes multiple microRNA genes of its own. Here we report that primary transcripts of ebv-miR-BART6 (pri-miR-BART6) are edited in latently EBV-infected cells. Editing of wild-type pri-miR-BART6 RNAs dramatically reduced loading of miR-BART6-5p RNAs onto the microRNA-induced silencing complex. Editing of a mutation-containing pri-miR-BART6 found in Daudi Burkitt lymphoma and nasopharyngeal carcinoma C666-1 cell lines suppressed processing of miR-BART6 RNAs. Most importantly, miR-BART6-5p RNAs silence Dicer through multiple target sites located in the 3'-UTR of Dicer mRNA. The significance of miR-BART6 was further investigated in cells in various stages of latency. We found that miR-BART6-5p RNAs suppress the EBNA2 viral oncogene required for transition from immunologically less responsive type I and type II latency to the more immunoreactive type III latency as well as Zta and Rta viral proteins essential for lytic replication, revealing the regulatory function of miR-BART6 in EBV infection and latency. Mutation and A-to-I editing appear to be adaptive mechanisms that antagonize miR-BART6 activities.

Modulation of microRNA processing and expression through RNA editing by ADAR deaminases.

Adenosine deaminases acting on RNA (ADARs) are involved in editing of adenosine residues to inosine in double-stranded RNA (dsRNA). Although this editing recodes and alters functions of several mammalian genes, its most common targets are noncoding repeat sequences, indicating the involvement of this editing system in currently unknown functions other than recoding of protein sequences. Here we show that specific adenosine residues of certain microRNA (miRNA) precursors are edited by ADAR1 and ADAR2. Editing of pri-miR-142, the precursor of miRNA-142, expressed in hematopoietic tissues, resulted in suppression of its processing by Drosha. The edited pri-miR-142 was degraded by Tudor-SN, a component of RISC and also a ribonuclease specific to inosine-containing dsRNAs. Consequently, mature miRNA-142 expression levels increased substantially in ADAR1 null or ADAR2 null mice. Our results demonstrate a new function of RNA editing in the control of miRNA biogenesis.

TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing.

MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the post-transcriptional level. Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex. These pre-miRNAs are cleaved by the RNase III Dicer to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein), which contains three double-stranded, RNA-binding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer-TRBP with Argonaute 2 (Ago2), the catalytic engine of RISC. The physical association of Dicer-TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer-TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer-TRBP complex not only in miRNA processing but also as a platform for RISC assembly.

Regulation of innate immune responses by DAI (DLM-1/ZBP1) and other DNA-sensing molecules.

DNA, whether it is microbe-derived or host-derived, evokes immune responses when exposed to the cytosol of a cell. We previously reported that DNA-dependent activator of IFN regulatory factors (DAI), also referred to as DLM-1/ZBP1, functions as a DNA sensor that activates the innate immune system. In the present study, we examined the regulation of the complex DNA-sensing system by DAI and other molecules. We first show that DAI directly interacts with DNA in vitro and that it requires three DNA-binding domains for full activation in vivo. We also show that the artificially induced dimerization of DAI results in the DNA-independent activation of type I IFN genes, thereby providing a better understanding for the molecular basis of DAI activation. Furthermore, we provide evidence for the presence of additional DNA sensors, either positively or negatively regulating cytosolic DNA-mediated innate immune responses. These results in toto provide insights into the mechanism of DAI activation and reveal the complex regulatory mechanisms underlying DNA-mediated protective and pathologic immune responses.

ADAR1 RNA editing enzyme regulates R-loop formation and genome stability at telomeres in cancer cells.

ADAR1 is involved in adenosine-to-inosine RNA editing. The cytoplasmic ADAR1p150 edits 3'UTR double-stranded RNAs and thereby suppresses induction of interferons. Loss of this ADAR1p150 function underlies the embryonic lethality of Adar1 null mice, pathogenesis of the severe autoimmune disease Aicardi-Goutières syndrome, and the resistance developed in cancers to immune checkpoint blockade. In contrast, the biological functions of the nuclear-localized ADAR1p110 remain largely unknown. Here, we report that ADAR1p110 regulates R-loop formation and genome stability at telomeres in cancer cells carrying non-canonical variants of telomeric repeats. ADAR1p110 edits the A-C mismatches within RNA:DNA hybrids formed between canonical and non-canonical variant repeats. Editing of A-C mismatches to I:C matched pairs facilitates resolution of telomeric R-loops by RNase H2. This ADAR1p110-dependent control of telomeric R-loops is required for continued proliferation of telomerase-reactivated cancer cells, revealing the pro-oncogenic nature of ADAR1p110 and identifying ADAR1 as a promising therapeutic target of telomerase positive cancers.

Frequency and fate of microRNA editing in human brain.

Primary transcripts of certain microRNA (miRNA) genes (pri-miRNAs) are subject to RNA editing that converts adenosine to inosine (A-->I RNA editing). However, the frequency of the pri-miRNA editing and the fate of edited pri-miRNAs remain largely to be determined. Examination of already known pri-miRNA editing sites indicated that adenosine residues of the UAG triplet sequence might be edited more frequently. In the present study, therefore, we conducted a large-scale survey of human pri-miRNAs containing the UAG triplet sequence. By direct sequencing of RT-PCR products corresponding to pri-miRNAs, we examined 209 pri-miRNAs and identified 43 UAG and also 43 non-UAG editing sites in 47 pri-miRNAs, which were highly edited in human brain. In vitro miRNA processing assay using recombinant Drosha-DGCR8 and Dicer-TRBP (the human immuno deficiency virus transactivating response RNA-binding protein) complexes revealed that a majority of pri-miRNA editing is likely to interfere with the miRNA processing steps. In addition, four new edited miRNAs with altered seed sequences were identified by targeted cloning and sequencing of the miRNAs that would be processed from edited pri-miRNAs. Our studies predict that approximately 16% of human pri-miRNAs are subject to A-->I editing and, thus, miRNA editing could have a large impact on the miRNA-mediated gene silencing.