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At least 19 recordsLinked to original sources
PubMed2005

Evolutionarily conserved human targets of adenosine to inosine RNA editing.

A-to-I RNA editing by ADARs is a post-transcriptional mechanism for expanding the proteomic repertoire. Genetic recoding by editing was so far observed for only a few mammalian RNAs that are predominantly expressed in nervous tissues. However, as these editing targets fail to explain the broad and severe phenotypes of ADAR1 knockout mice, additional targets for editing by ADARs were always expected. Using comparative genomics and expressed sequence analysis, we identified and experimentally verified four additional candidate human substrates for ADAR-mediated editing: FLNA, BLCAP, CYFIP2 and IGFBP7. Additionally, editing of three of these substrates was verified in the mouse while two of them were validated in chicken. Interestingly, none of these substrates encodes a receptor protein but two of them are strongly expressed in the CNS and seem important for proper nervous system function. The editing pattern observed suggests that some of the affected proteins might have altered physiological properties leaving the possibility that they can be related to the phenotypes of ADAR1 knockout mice.

Adenosine↗
PubMed2005

Is abundant A-to-I RNA editing primate-specific?

A-to-I RNA editing is common in all eukaryotes, and is associated with various neurological functions. Recently, A-to-I editing was found to occur frequently in the human transcriptome. In this article, we show that the frequency of A-to-I editing in humans is at least an order of magnitude higher than in the mouse, rat, chicken or fly genomes. The extraordinary frequency of RNA editing in human is explained by the dominance of the primate-specific Alu element in the human transcriptome, which increases the number of double-stranded RNA substrates.

3' Untranslated Regions↗
PubMed2025

Obstacles in quantifying A-to-I RNA editing by Sanger sequencing.

Adenosine-to-Inosine (A-to-I) RNA editing is the most prevalent type of RNA editing, in which adenosine within a completely or largely double-stranded RNA (dsRNA) is converted to inosine by deamination. RNA editing was shown to be involved in many neurological diseases and cancer; therefore, detection of A-to-I RNA editing and quantitation of editing levels are necessary for both basic and clinical biomedical research. While high-throughput sequencing (HTS) is widely used for global detection of editing events, Sanger sequencing is the method of choice for precise characterization of editing site clusters (hyper-editing) and for comparing levels of editing at a particular site under different environmental conditions, developmental stages, genetic backgrounds, or disease states. To detect A-to-I editing events and quantify them using Sanger sequencing, RNA samples are reverse transcribed, cDNA is amplified using gene-specific primers, and then sequenced. The chromatogram outputs are then compared to the genomic DNA sequence. As editing occurs in the context of dsRNA, the reverse transcription step is performed at a temperature as high as 65 °C, using thermostable reverse transcriptase to open double-stranded structures. However, this measure alone is insufficient for transcripts possessing long stems comprised of hundreds of nucleotide pairs. Consequently, the editing levels detected by Sanger sequencing are significantly lower than those obtained by HTS, and the amplification yield is low. We suggest that the reverse transcription is biased towards unedited transcripts, and the severity of the bias is dependent on the transcript's secondary structure. Here, we show how this bias can be significantly reduced to allow reliable detection of editing levels and sufficient product yield.

RNA Editing↗
PubMed2026

A-to-I RNA editing remodels 5'-UTR initiation codons to tune translational output.

A-to-I RNA editing is a prevalent post-transcriptional modification in higher eukaryotes that converts adenosine to inosine within RNA molecules. Because inosine is interpreted as guanosine during translation, editing can alter codon identity and potentially influence translation initiation signals. Here, we examined whether A-to-I editing within the 5' untranslated region (5'-UTR) can remodel upstream initiation codons and thereby tune downstream translation. Using luciferase-based reporter systems, we show that AUA-to-AUI editing generates an initiation-competent inosine-containing codon, whereas AUG-to-IUG editing markedly attenuates initiation and can relieve uORF-mediated repression. Quantitative in vitro and cellular assays establish the initiation hierarchy AUA&#x2009;<&#x2009;AUI&#x2009;<&#x2009;AUG, with IUG exhibiting strongly reduced initiation efficiency. Importantly, AUI-mediated upstream initiation did not behave like a canonical AUG-initiated uORF in the tested contexts; its effect on downstream ORF translation was modest and context-dependent. Transcriptome-wide bioinformatic analysis identified endogenous human transcripts whose 5'-UTRs harbor editing sites compatible with initiation-codon gain or attenuation. Reporter validation using native 5'-UTR sequences supports the possibility that editing-dependent initiation-codon remodeling can tune translational output in living cells, particularly through AUG-to-IUG-mediated derepression. Together, these findings establish a reporter-based framework in which A-to-I editing can remodel 5'-UTR initiation codons, while highlighting the need for endogenous protein-level and native-locus validation to determine physiological relevance.

RNA Editing↗
PubMed2006

A-to-I RNA editing and human disease.

The post-transcriptional modification of mammalian transcripts by A-to-I RNA editing has been recognized as an important mechanism for the generation of molecular diversity and also regulates protein function through recoding of genomic information. As the molecular players of editing are characterized and an increasing number of genes become identified that are subject to A-to-I modification, the potential impact of editing on the etiology or progression of human diseases is realized. Here we review the recent knowledge on where disturbances in A-to-I RNA editing have been correlated with human disease phenotypes.

Adenosine↗
PubMed2026

EndoV does not measurably affect TadA-dependent A-to-I RNA editing in Escherichia coli under exponential-growth conditions in rich medium.

Adenosine-to-inosine (A-to-I) mRNA editing changes the genetic information post-transcriptionally and was only recently reported to occur in bacteria. Here, we examined whether endonuclease V (EndoV; encoded by nfi) cleaves inosine-containing RNAs in vivo and thereby influences the abundance and fate of A-to-I-edited mRNAs in Escherichia coli. We generated an nfi loss-of-function mutant carrying a premature stop codon and performed RNA sequencing alongside the isogenic wild-type strain. We observed that global and site-specific editing occurrence or levels in both mRNAs and tRNAs were indistinguishable between strains. Moreover, overexpression of EndoV did not affect the number of edited sites, motif enrichment, or editing levels compared with a control strain overexpressing mCherry. Our findings suggest that, in contrast to human EndoV, bacterial EndoV does not regulate the steady-state pool of edited mRNAs in E. coli under nutrient-rich, exponential-growth conditions in vivo.IMPORTANCEAdenosine-to-inosine (A-to-I) mRNA editing is an emerging regulatory layer in bacteria, but the factors that act on edited transcripts are largely unknown. Endonuclease V (EndoV) was a prime candidate because it cleaves inosine-containing nucleic acids and can act on inosine-containing RNA in vitro. By combining loss-of-function and overexpression of EndoV with genome-wide RNA editing measurements, we show that EndoV does not measurably influence TadA-dependent A-to-I mRNA editing in Escherichia coli under standard laboratory conditions. This negative result is important because it rules out a natural effector candidate and redirects attention to other bacterial pathways that may process edited RNAs. Our work, therefore, sharpens mechanistic models for bacterial RNA editing and helps focus future searches for its regulators and physiological roles.

Escherichia coli↗
PubMed2024

Aptazyme-directed A-to-I RNA editing.

As a promising therapeutic approach, the RNA editing process can correct pathogenic mutations and is reversible and tunable, without permanently altering the genome. RNA editing mediated by human ADAR proteins offers unique advantages, including high specificity and low immunogenicity. Compared to CRISPR-based gene editing techniques, RNA editing events are temporary, which can reduce the risk of long-term unintended side effects, making off-target edits less concerning than DNA-targeting methods. Moreover, ADAR-based RNA editing tools are less likely to elicit immune reactions because ADAR proteins are of human origin, and their small size makes them relatively easy to incorporate into gene therapy vectors, such as adeno-associated virus vectors (AAVs), which have limited space. Despite the promise of RNA editing as a therapeutic approach, precise temporal and spatial control of RNA editing is still lacking. Therefore, we have developed a small molecule-inducible RNA editing strategy by incorporating aptazymes into the guide RNA of the BoxB-&#x3bb;N-ADAR system. This chapter provides detailed protocols for targeted RNA editing by ADAR deaminases using aptazyme-based guide RNAs controlled by exogenous small molecules, marking the earliest use of aptazymes to regulate RNA editing strategies. Once small molecules are added or removed, aptazymes trigger self-cleavage to release the guide RNA, thus achieving small molecule-controlled RNA editing. To satisfy different RNA editing applications, we have realized the conditional activation and deactivation of A-to-I RNA editing of target mRNA using switch aptazymes. We provide step-by-step protocols for constructing guide RNA plasmids for regulatory purposes and conducting small molecule-induced RNA regulatory editing experiments in cells.

Animals↗
PubMed2004

Widespread A-to-I RNA editing of Alu-containing mRNAs in the human transcriptome.

RNA editing by adenosine deamination generates RNA and protein diversity through the posttranscriptional modification of single nucleotides in RNA sequences. Few mammalian A-to-I edited genes have been identified despite evidence that many more should exist. Here we identify intramolecular pairs of Alu elements as a major target for editing in the human transcriptome. An experimental demonstration in 43 genes was extended by a broader computational analysis of more than 100,000 human mRNAs. We find that 1,445 human mRNAs (1.4%) are subject to RNA editing at more than 14,500 sites, and our data further suggest that the vast majority of pre-mRNAs (greater than 85%) are targeted in introns by the editing machinery. The editing levels of Alu-containing mRNAs correlate with distance and homology between inverted repeats and vary in different tissues. Alu-mediated RNA duplexes targeted by RNA editing are formed intramolecularly, whereas editing due to intermolecular base-pairing appears to be negligible. We present evidence that these editing events can lead to the posttranscriptional creation or elimination of splice signals affecting alternatively spliced Alu-derived exons. The analysis suggests that modification of repetitive elements is a predominant activity for RNA editing with significant implications for cellular gene expression.

5' Untranslated Regions↗
PubMed2000

Comparative analysis of the DRADA A-to-I RNA editing gene from mammals, pufferfish and zebrafish.

The DRADA gene in mammals encodes an A-to-I RNA editase, an adenosine deaminase that acts on pre-mRNAs to produce site specific inosines. DRADA has been shown to deaminate specific adenosine residues in a subset of glutamate and serotonin receptors, and this editing results in proteins of altered sequences and functional properties. DRADA thus plays a role in creating protein diversity. To study the evolutionary significance of this gene, we have characterized the genomic structure of DRADA from Fugu rubripes, and compared the protein sequences of DRADA from mammals, pufferfish and zebrafish. The DRADA gene from Fugu is three-fold compacted with respect to the human gene, and contains a novel intron within the large second coding exon. DRADA cDNAs were isolated from zebrafish and a second pufferfish, Tetraodon fluviatilis. Comparisons among fish, and between fish and mammals, of the protein sequences show that the catalytic domains are highly conserved for each gene, while the RNA binding domains vary within a single protein in their levels of conservation. Conservation within the Z DNA binding domain has also been assessed. Different levels of conservation among domains of different functional roles may reflect differences in editase substrate specificity and/or substrate sequence conservation.

Adenosine Deaminase↗
PubMed2005

Modulation of ADAR1 editing activity by Z-RNA in vitro.

RNA editing by A-to-I modification has been recognized as an important molecular mechanism for generating RNA and protein diversity. In mammals, it is mediated by a family of adenosine deaminases that act on RNAs (ADARs). The large version of the editing enzyme ADAR1 (ADAR1-L), expressed from an interferon-responsible promoter, has a Z-DNA/Z-RNA binding domain at its N-terminus. We have tested the in vitro ability of the enzyme to act on a 50 bp segment of dsRNA with or without a Z-RNA forming nucleotide sequence. A-to-I editing efficiency is markedly enhanced in presence of the sequence favoring Z-RNA. In addition, an alteration in the pattern of modification along the RNA duplex becomes evident as reaction times decrease. These results suggest that the local conformation of dsRNA molecules might be an important feature for target selectivity by ADAR1 and other proteins with Z-RNA binding domains.

Adenosine Deaminase↗
PubMed2022

ADAR regulates APOL1 via A-to-I RNA editing by inhibition of MDA5 activation in a paradoxical biological circuit.

APOL1 risk variants are associated with increased risk of kidney disease in patients of African ancestry, but not all individuals with the APOL1 high-risk genotype develop kidney disease. As APOL1 gene expression correlates closely with the degree of kidney cell injury in both cell and animal models, the mechanisms regulating APOL1 expression may be critical determinants of risk allele penetrance. The APOL1 messenger RNA includes Alu elements at the 3' untranslated region that can form a double-stranded RNA structure (Alu-dsRNA) susceptible to posttranscriptional adenosine deaminase acting on RNA (ADAR)-mediated adenosine-to-inosine (A-to-I) editing, potentially impacting gene expression. We studied the effects of ADAR expression and A-to-I editing on APOL1 levels in podocytes, human kidney tissue, and a transgenic APOL1 mouse model. In interferon-&#x3b3; (IFN-&#x3b3;)-stimulated human podocytes, ADAR down-regulates APOL1 by preventing melanoma differentiation-associated protein 5 (MDA5) recognition of dsRNA and the subsequent type I interferon (IFN-I) response. Knockdown experiments showed that recognition of APOL1 messenger RNA itself is an important contributor to the MDA5-driven IFN-I response. Mathematical modeling suggests that the IFN-ADAR-APOL1 network functions as an incoherent feed-forward loop, a biological circuit capable of generating fast, transient responses to stimuli. Glomeruli from human kidney biopsies exhibited widespread editing of APOL1 Alu-dsRNA, while the transgenic mouse model closely replicated the edited sites in humans. APOL1 expression in mice was inversely correlated with Adar1 expression under IFN-&#x3b3; stimuli, supporting the idea that ADAR regulates APOL1 levels in&#xa0;vivo. ADAR-mediated A-to-I editing is an important regulator of APOL1 expression that could impact both penetrance and severity of APOL1-associated kidney disease.

Humans↗
PubMed2000

Comparative analysis of the RED1 and RED2 A-to-I RNA editing genes from mammals, pufferfish and zebrafish.

One type of RNA editing involves the deamination of adenosine (A) residues to inosines (I) at specific sites in specific pre-mRNAs. These inosines are subsequently read as guanosines by the ribosome, with potentially significant consequences for protein sequence. In mammals, two such A-to-I RNA editases are RED1, which edits some serotonin and glutamate receptors, and RED2, with unidentified substrates. To study the evolutionary conservation among these editases, we have isolated homologous genes from the Japanese pufferfish, Fugu rubripes. Fugu has two genes homologous to Red1 that are similar in size and organization and that show a fivefold compaction relative to the human gene; they differ, however, in their base compositional features. The Fugu gene for RED2 is unusually large, spanning more than 50kb; within the largest intron, there is evidence for a novel gene on the opposite strand. Because of these unusual features, the partial genomic structure was determined for the mouse RED2 gene. A partial cDNA for RED1 was also isolated from zebrafish. Comparisons between fish and between fish and mammals of the protein sequences show that the catalytic domains are highly conserved for each gene, while the RNA-binding domains vary within a single protein in their levels of conservation. Different levels of conservation among domains of different functional roles may reflect differences in editase substrate specificity and/or substrate sequence conservation.

Adenosine Deaminase↗
PubMed2024

Bioinformatic approaches for accurate assessment of A-to-I editing in complete transcriptomes.

A-to-I RNA editing is an RNA modification that alters the RNA sequence relative to the its genomic blueprint. It is catalyzed by double-stranded RNA-specific adenosine deaminase (ADAR) enzymes, and contributes to the complexity and diversification of the proteome. Advancement in the study of A-to-I RNA editing has been facilitated by computational approaches for accurate mapping and quantification of A-to-I RNA editing based on sequencing data. In this chapter we review some of the main computational approaches currently used, describe potential hurdles, challenges and pitfalls, and discuss possible ways to mitigate them.

RNA Editing↗
PubMed2019

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 &#x223c;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.

Adenosine Deaminase↗
PubMed2005

Letter from the editor: Adenosine-to-inosine RNA editing in Alu repeats in the human genome.

Adenosine-to-inosine (A-to-I) RNA editing increases the complexity of the human transcriptome and is essential for maintenance of normal life in mammals. Most A-to-I substitutions occur within repetitive elements in the genome, mainly in Alu repeats. The phenomenon of A-to-I editing is far less abundant in mice, rats, chickens and flies than in humans, which correlates with the relative under-representation of Alu repeats in these non-primate genomes. Here, we review the recent results of bioinformatic and laboratory approaches that have estimated the extent of the editing phenomenon. We discuss the possible biological relevance of the editing pathway, its possible interaction with other cellular pathways that respond to double-stranded RNA and its possible contribution to the accelerated evolution of primates.

Adenosine↗
PubMed2006

RNA editing level in the mouse is determined by the genomic repeat repertoire.

A-to-I RNA editing is the conversion of adenosine to inosine in double-stranded cellular and viral RNAs. Recently, abundant editing of human transcripts affecting thousands of genes has been reported. Most editing sites are confined to the primate-specific Alu repeats. Notably, the editing level in mouse was shown to be much lower. In order to find the reason for this dramatic difference, here we identify editing sites within mouse repeats and analyze the sequence properties required for RNA editing. Our results show that the overall rate of RNA editing is determined by specific properties of different repeat families such as abundance, length, and divergence. We show that the striking difference in editing levels between human and mouse is mostly due to the higher divergence of the different mouse repeats.

Animals↗