A mammalian reporter system for fast and quantitative detection of intracellular A-to-I RNA editing levels.
ABSTRACT: An important molecular mechanism to create protein diversity from a limited set of genes is A-to-I RNA editing. RNA editing converts single adenosines into inosines in pre-mRNA. These single base conversions can have a wide variety of consequences. Editing can lead to codon changes and, consequently, altered protein function. Moreover, editing can alter splice sites and influences miRNA biogenesis and target recognition. The two enzymes responsible for editing in mammals are adenosine deaminase acting on RNA (ADAR) 1 and 2. However, it is currently largely unknown how the activity of these enzymes is regulated in vivo. Editing activity does not always correlate with ADAR expression levels, suggesting posttranscriptional or posttranslational mechanisms for controlling activity. To investigate how editing is regulated in mammalian cells, we have developed a straightforward quantitative reporter system to detect editing levels. By employing luciferase activity as a readout, we could easily detect different levels of editing in a cellular context. In addition, increased levels of ADAR2 correlated with increased levels of luciferase activity. This reporter system therefore sets the stage for the effective screening of cDNA libraries or small molecules for strong modulators of intracellular editing to ultimately elucidate how A-to-I editing is regulated in vivo.
Project description:ADAR RNA editing enzymes are high-affinity dsRNA-binding proteins that deaminate adenosines to inosines in pre-mRNA hairpins and also exert editing-independent effects. We generated a Drosophila AdarE374A mutant strain encoding a catalytically inactive Adar with CRISPR/Cas9. We demonstrate that Adar adenosine deamination activity is necessary for normal locomotion and prevents age-dependent neurodegeneration. The catalytically inactive protein, when expressed at a higher than physiological level, can rescue neurodegeneration in Adar mutants, suggesting also editing-independent effects. Furthermore, loss of Adar RNA editing activity leads to innate immune induction, indicating that Drosophila Adar, despite being the homolog of mammalian ADAR2, also has functions similar to mammalian ADAR1. The innate immune induction in fly Adar mutants is suppressed by silencing of Dicer-2, which has a RNA helicase domain similar to MDA5 that senses unedited dsRNAs in mammalian Adar1 mutants. Our work demonstrates that the single Adar enzyme in Drosophila unexpectedly has dual functions.
Project description:The conversion of adenosine to inosine within RNA transcripts is regulated by a family of double-stranded RNA-specific adenosine deaminases referred to as adenosine deaminases that act on RNA (ADARs). Little is known regarding the developmental expression of ADAR family members or the mechanisms responsible for the specific patterns of editing observed for ADAR substrates. We have examined the spatiotemporal expression patterns for ADAR1 and ADAR2 in mouse forebrain. ADAR1 and ADAR2 are broadly distributed in most regions of the mouse forebrain by P0, including the cerebral cortex, hippocampus, and diencephalon. High expression levels were maintained into adulthood. Colocalization studies demonstrated ADAR1 and ADAR2 expression in neurons but not astrocytes. Editing for specific ADAR mRNA targets precedes high expression of ADAR proteins, suggesting that region-specific differences in editing patterns may not be mediated solely by ADAR expression levels.
Project description:Members of the double-stranded RNA- (dsRNA) specific adenosine deaminase gene family convert adenosine residues into inosines in dsRNA and are involved in A-to-I RNA editing of transcripts of glutamate receptor (GluR) subunits and serotonin receptor subtype 2C (5-HT(2C)R). We have isolated hADAR3, the third member of this class of human enzyme and investigated its editing site selectivity using in vitro RNA editing assay systems. As originally reported for rat ADAR3 or RED2, purified ADAR3 proteins could not edit GluR-B RNA at the "Q/R" site, the "R/G" site, and the intronic "hot spot" site. In addition, ADAR3 did not edit any of five sites discovered recently within the intracellular loop II region of 5-HT(2C)R RNAs, confirming its total lack of editing activity for currently known substrate RNAs. Filter-binding analyses revealed that ADAR3 is capable of binding not only to dsRNA but also to single-stranded RNA (ssRNA). Deletion mutagenesis identified a region rich in arginine residues located in the N-terminus that is responsible for binding of ADAR3 to ssRNA. The presence of this ssRNA-binding domain as well as its expression in restricted brain regions and postmitotic neurons make ADAR3 distinct from the other two ADAR gene family members, editing competent ADAR1 and ADAR2. ADAR3 inhibited in vitro the activities of RNA editing enzymes of the ADAR gene family, raising the possibility of a regulatory role in RNA editing.
Project description:ADAR enzymes convert adenosines to inosines within double-stranded RNAs, including microRNA (miRNA) precursors, with important consequences on miRNA retargeting and expression. ADAR2 activity is impaired in glioblastoma and its rescue has anti-tumoral effects. However, how ADAR2 activity may impact the miRNome and the progression of glioblastoma is not known.By integrating deep-sequencing and array approaches with bioinformatics analyses and molecular studies, we show that ADAR2 is essential to edit a small number of mature miRNAs and to significantly modulate the expression of about 90 miRNAs in glioblastoma cells. Specifically, the rescue of ADAR2 activity in cancer cells recovers the edited miRNA population lost in glioblastoma cell lines and tissues, and rebalances expression of onco-miRNAs and tumor suppressor miRNAs to the levels observed in normal human brain. We report that the major effect of ADAR2 is to reduce the expression of a large number of miRNAs, most of which act as onco-miRNAs. ADAR2 can edit miR-222/221 and miR-21 precursors and decrease the expression of the corresponding mature onco-miRNAs in vivo and in vitro, with important effects on cell proliferation and migration.Our findings disclose an additional layer of complexity in miRNome regulation and provide information to better understand the impact of ADAR2 editing enzyme in glioblastoma. We propose that ADAR2 is a key factor for maintaining edited-miRNA population and balancing the expression of several essential miRNAs involved in cancer.
Project description:Adenosine deaminases that act on RNA (ADARs) deaminate adenosines in dsRNA to produce inosines. ADARs are essential in mammals and are particularly important in the nervous system. Altered levels of adenosine-to-inosine (A-to-I) editing are observed in several diseases. The extent to which an adenosine is edited depends on sequence context. Human ADAR2 (hADAR2) has 5' and 3' neighbor preferences, but which amino acids mediate these preferences, and by what mechanism, is unknown. We performed a screen in yeast to identify mutations in the hADAR2 catalytic domain that allow editing of an adenosine within a disfavored triplet. Binding affinity, catalytic rate, base flipping, and preferences were monitored to understand the effects of the mutations on ADAR reactivity. Our data provide information on the amino acids that affect preferences and point to a conserved loop as being of key importance. Unexpectedly, our data suggest that hADAR2's preferences derive from differential base flipping rather than from direct recognition of neighboring bases. Our studies set the stage for understanding the basis of altered editing levels in disease and for developing therapeutic reagents.
Project description: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.
Project description: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.
Project description:The adenosine deaminases acting on RNA (ADARs) comprise a family of RNA editing enzymes that selectively modify single codons within RNA primary transcripts with often profound impact on protein function. Little is known about the mechanisms that regulate nuclear RNA editing activity. Editing levels show cell-type specific and developmental modulation that does not strictly coincide with observed expression levels of ADARs. Here, we provide evidence for a molecular mechanism that might control nuclear import of specific ADARs and, in turn, nuclear RNA editing. We identify an in vivo ADAR3 interaction partner, importin alpha 1 (KPNA2) that specifically recognizes an arginine-rich ADAR3 sequence motif and show that it acts as a functional nuclear localization sequence. Furthermore, whereas KPNA2, but not KPNA1 or KNPA3, recognizes the ADAR3 NLS, we observe the converse binding specificity with ADAR2. Interestingly, alternative splicing of ADAR2 pre-mRNA introduces an ADAR3-like NLS that alters the interaction profile with the importins. Thus, in vivo RNA editing might be regulated, in part, through controlled subcellular localization of ADARs, which in turn is governed by the coordinated local expression of importin alpha proteins and ADAR protein variants.
Project description:Background: Adenosine deaminases that act on RNA (ADARs) bind to double-stranded and structured RNAs and deaminate adenosines to inosines. This A to I editing is widespread and required for normal life and development. Besides mRNAs and repetitive elements, ADARs can target miRNA precursors. Editing of miRNA precursors can affect processing efficiency and alter target specificity. Interestingly, ADARs can also influence miRNA abundance independent of RNA-editing. In mouse embryos where editing levels are low, ADAR2 was found to be the major ADAR protein that affects miRNA abundance. Here we extend our analysis to adult mouse brains where high editing levels are observed. Results: Using Illumina deep sequencing we compare the abundances of mature miRNAs and editing events within them, between wild-type and ADAR2 knockout mice in the adult mouse brain. Reproducible changes in abundance of specific miRNAs are observed in ADAR2 deficient mice. Most of these quantitative changes seem unrelated to A to I editing events. However, many A to G transitions in cDNAs prepared from mature miRNA sequences, reflecting A to I editing events in the RNA, are observed with frequencies reaching up to 80%. About half of these editing events are primarily caused by ADAR2 while a few miRNAs show increased editing in the absence of ADAR2, suggesting preferential editing by ADAR1. Moreover, novel, previously unknown editing events were identified in several miRNAs. In general 64% of all editing events are located within the seed region of mature miRNAs. In one of these cases retargeting of the edited miRNA could be verified in reporter assays. Also, altered processing efficiency upon editing near a processing site could be experimentally verified. Conclusions: ADAR2 can significantly influence the abundance of certain miRNAs in the brain. Only in a few cases changes in miRNA abundance can be explained by miRNA editing. Thus, ADAR2 binding to miRNA precursors, without editing them, may influence their processing and thereby abundance. ADAR1 and ADAR2 have both overlapping and distinct specificities for editing of miRNA editing sites. Over 60% of editing occurs in the seed region possibly changing target specificities for many edited miRNAs. Examination of the effect of ADAR2 on mature miRNA abundance and sequence in adult mouse brain.
Project description: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.