SEARCH · PubMed Health

Results for “ADAR”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources

PubMed2024

Global quantification of off-target activity by base editors.

Base editors are engineered deaminases combined with CRISPR components. These engineered deaminases are designed to target specific sites within DNA or RNA to make a precise change in the molecule. In therapeutics, they hold promise for correcting mutations associated with genetic diseases. However, a key challenge is minimizing unintended edits at off-target sites, which could lead to harmful mutations. Researchers are actively addressing this concern through a variety of optimization efforts that aim to improve the precision of base editors and minimize off-target activity. Here, we examine the various types of off-target activity, and the methods used to evaluate them. Current methods for finding off-target activity focus on identifying similar sequences in the genome or in the transcriptome, assuming the guide RNA misdirects the editor. The main method presented here, that was originally developed to quantify editing levels mediated by the ADAR enzyme, takes a different approach, investigating the inherent activity of base editors themselves, which might lead to off-target edits beyond sequence similarity. The editing index tool quantifies global off-target editing, eliminates the need to detect individual off-target sites, and allows for assessment of the global load of mutations.

Gene Editing↗

PubMed2005

Retention and repression: fates of hyperedited RNAs in the nucleus.

Double-stranded RNA (dsRNA) is often formed in the nuclei of mammalian cells, but in this compartment it does not induce the effects characteristic of cytoplasmic dsRNA. Rather, recent work has suggested that nuclear dsRNA is a target for the ADAR class of enzymes, which deaminate adenosines to inosines. Further, there are a number of distinct fates of such edited RNA, including nuclear retention and perhaps also gene silencing.

Active Transport, Cell Nucleus↗

PubMed2005

Vigilins bind to promiscuously A-to-I-edited RNAs and are involved in the formation of heterochromatin.

The fate of double-stranded RNA (dsRNA) in the cell depends on both its length and location . The expression of dsRNA in the nucleus leads to several distinct consequences. First, the promiscuous deamination of adenosines to inosines by dsRNA-specific adenosine deaminase (ADAR) can lead to the nuclear retention of edited transcripts . Second, dsRNAs might induce heterochromatic gene silencing through an RNAi-related mechanism . Is RNA editing also connected to heterochromatin? We report that members of the conserved Vigilin class of proteins have a high affinity for inosine-containing RNAs. In agreement with other work , we find that these proteins localize to heterochromatin and that mutation or depletion of the Drosophila Vigilin, DDP1, leads to altered nuclear morphology and defects in heterochromatin and chromosome segregation. Furthermore, nuclear Vigilin is found in complexes containing not only the editing enzyme ADAR1 but also RNA helicase A and Ku86/70. In the presence of RNA, the Vigilin complex recruits the DNA-PKcs enzyme, which appears to phosphorylate a discrete set of targets, some or all of which are known to participate in chromatin silencing. These results are consistent with a mechanistic link between components of the DNA-repair machinery and RNA-mediated gene silencing.

Animals↗

PubMed2006

Computational analyses show A-to-G mutations correlate with nascent mRNA hairpins at somatic hypermutation hotspots.

Activation-induced cytidine deaminase (AID) initiates Phase I somatic hypermutation (SHM) of antibody genes by deaminating deoxy-cytosine to deoxy-uracil (C-to-U). These lesions trigger Phase II, a poorly understood process of error-prone repair targeting A-T pairs by DNA polymerase eta (Pol eta). Since Pol eta is also a reverse transcriptase, Phase II could involve copying off RNA as well as DNA templates. We explore this idea further since in an RNA-based pathway it is conceivable that adenosine-to-inosine (A-to-I) RNA editing causes A-to-G transitions since I like G pairs with C. Adenosine deaminases (ADARs) are known to preferentially edit A nucleotides that are preceded by an A or U (W) in double-stranded RNA substrates. On this assumption and using a theoretical bioinformatics approach we show that a significant and specific correlation (P<0.002) exists between the frequency of WA-to-WG mutations and the number of mRNA hairpins that could potentially form at the mutation site. This implies roles for both RNA editing and reverse transcription during SHM in vivo and suggests definitive genetic experiments targeting the appropriate ADAR1 isoform (gammaINF-ADAR1) and/or Ig pre-mRNA templates.

Computational Biology↗

PubMed2026

Whole-exome characterization of host genetic variation in HIV-associated genes across the high-prevalence Mizo population, Northeast India.

BACKGROUND: The Mizoram state of Northeast India has one of the highest HIV prevalence rates in Asia, yet the host genetic factors influencing HIV susceptibility in this Tibeto-Burman population remain uncharacterised. METHODS: We performed whole-exome sequencing using Illumina NovaSeq 6000, mean coverage 100X on 76 HIV-negative Mizo individuals. Variants were called using GATK HaplotypeCaller v4.3 against GRCh38p14, annotated with ANNOVAR, and filtered using hard-quality thresholds (QD ≥ 2, SOR ≤ 3, MQ ≥ 40, DP ≥ 10, GQ ≥ 20). The allele frequencies were compared against gnomAD v2.1.1 population databases. Hardy-Weinberg equilibrium was assessed using the Wigginton exact test with Bonferroni correction. RESULTS: Post-quality filtering resulted in 12,011 sample-variants across 2,821 unique positions from 36 HIV-associated loci (33 protein-coding genes, 2 chemokine ligands, and 3 lncRNA targets). Of these, 784 observations (51 unique positions) were high-impact nonsynonymous or loss-of-function variants. ADAR rs2229857 (p.K384R, NM_015840) was the most frequently observed variant (Mizo carrier frequency = 0.895; 95% CI: 0.806-0.946). CXCR1 rs16858808 (p.R335C) showed the greatest population enrichment (Mizo carrier frequency = 0.197; 95% CI: 0.123-0.300; 7.65-fold carrier-frequency enrichment versus gnomAD South Asian; CADD = 15.60). Sixteen of 20 tested variants deviated from Hardy-Weinberg equilibrium after Bonferroni correction (p < 0.0025), predominantly showing excess homozygosity consistent with the endogamous Mizo population. The protective variant CCR5-Δ32 was absent in all the 76 individuals tested. CONCLUSION: This first whole-exome characterization of HIV host genes in the Mizo population identifies CXCR1 rs16858808 as the most population-enriched functional variant and reveals a pervasive endogamy signature. These findings provide a population-specific genetic framework for future HIV susceptibility studies and ART pharmacogenomics research.

Humans↗

PubMed2004

Altered RNA editing of serotonin 5-HT2C receptor induced by interferon: implications for depression associated with cytokine therapy.

Members of the ADAR (adenosine deaminases acting on RNA) gene family are involved in one type of RNA editing that converts adenosine residues to inosine. The A-to-I editing of serotonin receptor subtype 2C (5-HT(2C)R) mRNA leads to replacement of three amino acid residues located within the intracellular loop II domain, resulting in dramatic alterations in G-protein coupling functions of the receptor. It has been speculated that RNA editing may play a role in several pharmacological and behavioral processes where the serotonergic plasticity is mediated through 5-HT(2C)R. Interferon-alpha (IFN-alpha) often causes severe depression in patients treated for chronic viral hepatitis and certain malignancies. In this study, we examined the effects of IFN-alpha on RNA editing in human glioblastoma cell lines, which express 5-HT(2C)R mRNAs. ADAR1 expression and the pattern of the 5-HT(2C)R mRNA editing rapidly changed in response to IFN-alpha, leading to the dominant expression of the 5-HT(2C)R-VSI isoform predicted to have reduced G-protein coupling functions. Our results support the hypothesis that 5-HT(2C)R mRNA editing has causative relevance in the pathophysiology of depression associated with cytokine therapy.

Adenosine Deaminase↗

PubMedDate not supplied

Candidate editases for GluR channels in single neurons of rat hippocampus and cerebellum.

RNA editing by site selective adenosine deamination changes codons in several nuclear transcripts in the mammalian brain and affects critical properties of the encoded proteins, as exemplified by the calcium permeability of AMPA receptor channels. The recently cloned RNA dependent adenosine deaminases ADAR1, ADAR2 and ADAR3 form a small family of sequence-related candidate editases which are expressed in brain and other tissues at distinct levels and patterns. We have employed single-cell polymerase chain reaction of hippocampal CA1 and CA3 pyramidal neurons and cerebellar Purkinje and Bergmann glial cells in an attempt to evaluate the expression of these enzymes at a cellular level. We found ADAR2 expressed in all cells analyzed; approximately 50% of the cells co-expressed ADAR1 or ADAR3. The differential ADAR expression revealed by our study might underlie the distinct editing efficiencies and selectivities in different GluR subunit transcripts.

Adenosine Deaminase↗

PubMed2000

A-to-I pre-mRNA editing in Drosophila is primarily involved in adult nervous system function and integrity.

Specific A-to-I RNA editing, like that seen in mammals, has been reported for several Drosophila ion channel genes. Drosophila possesses a candidate editing enzyme, dADAR. Here, we describe dADAR deletion mutants that lack ADAR activity in extracts. Correspondingly, all known Drosophila site-specific RNA editing (25 sites in three ion channel transcripts) is abolished. Adults lacking dADAR are morphologically wild-type but exhibit extreme behavioral deficits including temperature-sensitive paralysis, locomotor uncoordination, and tremors which increase in severity with age. Neurodegeneration accompanies the increase in phenotypic severity. Surprisingly, dADAR mutants are not short-lived. Thus, A-to-I editing of pre-mRNAs in Drosophila acts predominantly through nervous system targets to affect adult nervous system function, integrity, and behavior.

Adenosine Deaminase↗

PubMed2002

Transcript mutations of the alpha regulatory subunit of protein kinase A and up-regulation of the RNA-editing gene transcript in lupus T lymphocytes.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disorder characterised by diverse dysfunctions of immune effector cells, including proliferation and cytotoxicity. In T cells from patients with SLE, activity of type 1 protein kinase A isozymes is greatly reduced because of decreased expression of the alpha and beta regulatory subunits (RI alpha and RI beta). We aimed to identify a molecular mechanism or mechanisms for this isozyme deficiency by assessing occurrence of mutations in transcripts of the RI alpha subunit in patients with SLE. METHODS: We cloned and sequenced cDNA of RI alpha and corresponding genomic DNA of the coding region to detect sequence changes from eight patients with SLE and six healthy controls. Because transcript editing is regulated by adenosine deaminases that act on RNA (ADAR), we quantified expression of ADAR1 transcripts in SLE and control T cells by competitive PCR. FINDINGS: Sequence analyses of cDNA showed heterogeneous transcript mutations, including deletions, transitions, and transversions. We identified 1.22 x 10(-3)/bp transcript mutations in SLE T cells-a frequency 7.5 times higher than that in control T cells. By contrast, we identified no genomic mutations. Two hotspots were identified in the RI alpha subunit transcripts from SLE T cells, one located adjacent to a pseudosubstrate site of the RI alpha subunit and the other a component of the cAMP binding A domain. ADAR1 mRNA content was 3.5 times higher in SLE cells than in control T cells (p=0.001). INTERPRETATION: An RNA-editing enzyme could be converting adenosine to inosine within double-stranded regions of RNA, resulting in transcript mutations. This process could be one mechanism resulting in mutations in the RI alpha subunit of type 1 protein kinase A.

Adenosine Deaminase↗

PubMed2000

Sequence, genomic organization and functional expression of the murine tRNA-specific adenosine deaminase ADAT1.

We have recently identified the first mammalian tRNA-specific adenosine deaminase human ADAT1, a member of the ADAR family of RNA editing enzymes. This protein is responsible for the first step of the unique A(37) to m(1)I(37) modification in eukaryotic tRNA(Ala). Here, we present the genomic structure of murine ADAT1 and the functional expression of mADAT1 cDNA. In mouse, as well as in human, ADAT1 is expressed from a single copy gene. The coding region of the mADAT1 gene is spread over nine exons, covering approximately 30kb of genomic DNA and encodes a protein of 499 amino acids. Overall, mADAT1 shares 81% nucleotide homology and 87.5% protein homology with the human ortholog. The recombinant mouse protein is active specifically and with a high efficiency on human tRNA(Ala) in vitro. Its genomic organization is compared to the structures of the sequence-related, pre-mRNA specific adenosine deaminases ADAR1 and ADAR2.

Adenosine Deaminase↗

PubMed2001

RNA editing by base deamination: more enzymes, more targets, new mysteries.

The posttranscriptional modification of messenger RNA precursors (pre-mRNAs) by base deamination can profoundly alter the physiological function of the encoded proteins. The recent identification of tRNA-specific adenosine deaminases (ADATs) has led to the suggestion that these enzymes, as well as the cytidine and adenosine deaminases acting on pre-mRNAs (CDARs and ADARs), belong to a superfamily of RNA-dependent deaminases. This superfamily might have evolved from an ancient cytidine deaminase. This article reviews the reactions catalysed by these enzymes and discusses their evolutionary relationships.

Adenosine Deaminase↗

PubMed2000

dADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing.

We have identified a homolog of the ADAR (adenosine deaminases that act on RNA) class of RNA editases from Drosophila, dADAR. The dADAR locus has been localized to the 2B6-7 region of the X chromosome and the complete genomic sequence organization is reported here. dADAR is most homologous to the mammalian RNA editing enzyme ADAR2, the enzyme that specifically edits the Q/R site in the pre-mRNA encoding the glutamate receptor subunit GluR-B. Partially purified dADAR expressed in Pichia pastoris has robust nonspecific A-to-I deaminase activity on synthetic dsRNA substrates. Transcripts of the dADAR locus originate from two regulated promoters. In addition, alternative splicing generates at least four major dADAR isoforms that differ at their amino-termini as well as altering the spacing between their dsRNA binding motifs. dADAR is expressed in the developing nervous system, making it a candidate for the editase that acts on para voltage-gated Na+ channel transcripts in the central nervous system. Surprisingly, dADAR itself undergoes developmentally regulated RNA editing that changes a conserved residue in the catalytic domain. Taken together, these findings show that both transcription and processing of dADAR transcripts are under strict developmental control and suggest that the process of RNA editing in Drosophila is dynamically regulated.

Adenosine Deaminase↗

PubMed2000

Analysis of the RNA-editing reaction of ADAR2 with structural and fluorescent analogues of the GluR-B R/G editing site.

ADARs are adenosine deaminases responsible for RNA editing reactions that occur in eukaryotic pre-mRNAs, including the pre-mRNAs of glutamate and serotonin receptors. Here we describe the generation and analysis of synthetic ADAR2 substrates that differ in structure around an RNA editing site. We find that five base pairs of duplex secondary structure 5' to the editing site increase the single turnover rate constant for deamination 17-39-fold when compared to substrates lacking this structure. ADAR2 deaminates an adenosine in the sequence context of a natural editing site >90-fold more rapidly and to a higher yield than an adjacent adenosine in the same RNA structure. This reactivity is minimally dependent on the base pairing partner of the edited nucleotide; adenosine at the editing site in the naturally occurring A.C mismatch is deaminated to approximately the same extent and only 4 times faster than adenosine in an A.U base pair at this site. A steady-state rate analysis at a saturating concentration of the most rapidly processed substrate indicates that product formation is linear with time through at least three turnovers with a slope of 13 +/- 1.5 nM.min(-1) at 30 nM ADAR2 for a k(ss) = 0.43 +/- 0.05 min(-1). In addition, ADAR2 induces a 3.3-fold enhancement in fluorescence intensity and a 14 nm blue shift in the emission maximum of a duplex substrate with 2-aminopurine located at the editing site, consistent with a mechanism whereby ADAR2 flips the reactive nucleotide out of the double helix prior to deamination.

2-Aminopurine↗

PubMed1978

Raman spectra of heme a, cytochrome oxidase-ligand complexes, and alkaline denatured oxidase.

We report 441.6 nm excitation resonance Raman spectra of oxidized and reduced monomeric heme a-imidazole, cytochrome oxidase-exogenous ligand complexes in various redox states, and alkaline denatured oxidase. These data show that, in reduced oxidase, the cytochrome a3 Raman spectrum has bands at 215, 364, 1230, and 1670 cm-1 not observed in the cytochrome a spectrum. The appearance of these bands in the reduced cytochrome a3 spectrum is due to interactions between the heme a of cytochrome a3 and its protein environment and not to intrinsic properties of heme a. These interactions are pH sensitive and strongly influence the vibrational spectra of both heme a groups. We assign the 1670-cm-1 band to the heme a formyl substituent and propose that the intensity of the 1670 cm-1 is high for reduced cytochrome a3 because the C==O lies in the porphyrin plane and is very weak for oxidized and reduced cytochrome a, oxidized cytochrome a3, and oxidized and reduced heme a-imidazole because the C==O lies out of the plane. We suggest that movement of the C==O in and out of the plane explains the ligand induced spectral shift in the optical absorption spectrum of reduced cytochrome a3. Finally, we confirm the observation of Adar & Yonetani (private communication) that, under laser illumination, resting oxidase is photoreactive.

Cytochromes↗

PubMed2001

Structure of the DLM-1-Z-DNA complex reveals a conserved family of Z-DNA-binding proteins.

The first crystal structure of a protein, the Z alpha high affinity binding domain of the RNA editing enzyme ADAR1, bound to left-handed Z-DNA was recently described. The essential set of residues determined from this structure to be critical for Z-DNA recognition was used to search the database for other proteins with the potential for Z-DNA binding. We found that the tumor-associated protein DLM-1 contains a domain with remarkable sequence similarities to Z alpha(ADAR). Here we report the crystal structure of this DLM-1 domain bound to left-handed Z-DNA at 1.85 A resolution. Comparison of Z-DNA binding by DLM-1 and ADAR1 reveals a common structure-specific recognition core within the binding domain. However, the domains differ in certain residues peripheral to the protein-DNA interface. These structures reveal a general mechanism of Z-DNA recognition, suggesting the existence of a family of winged-helix proteins sharing a common Z-DNA binding motif.

Adenosine Deaminase↗

PubMed2004

Control of human potassium channel inactivation by editing of a small mRNA hairpin.

Genomic recoding by A-->I RNA editing plays an important role in diversifying the proteins involved in electrical excitability. Here, we describe editing of an intronless potassium channel gene. A small region of human K(V)1.1 mRNA sequence directs efficient modification of one adenosine by human adenosine deaminase acting on RNA 2 (hADAR2). Mutational analysis shows that this region adopts a hairpin structure. Electrophysiological characterization reveals that the editing event (I/V) profoundly affects channel inactivation conferred by accessory beta subunits. Drosophila melanogaster Shaker channels, mimicking this editing event through mutation, exhibit a similar effect. In addition, we demonstrate that mRNAs for the paralogous D. melanogaster Shab potassium channel are edited at the same position by fly ADAR-a clear example of convergent evolution driven by adenosine deamination. These results suggest an ancient and key regulatory role for this residue in K(V) channels.

Amino Acid Sequence↗

PubMed2006

RNA editing and alternative splicing: the importance of co-transcriptional coordination.

The carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (pol II) is essential for several co-transcriptional pre-messenger RNA processing events, including capping, 3'-end processing and splicing. We investigated the role of the CTD of RNA pol II in the coordination of A to I editing and splicing of the ADAR2 (ADAR: adenosine deaminases that act on RNA) pre-mRNA. The auto-editing of Adar2 intron 4 by the ADAR2 adenosine deaminase is tightly coupled to splicing, as the modification of the dinucleotide AA to AI creates a new 3' splice site. Unlike other introns, the CTD is not required for efficient splicing of intron 4 at either the normal 3' splice site or the alternative site created by editing. However, the CTD is required for efficient co-transcriptional auto-editing of ADAR2 intron 4. Our results implicate the CTD in site-selective RNA editing by ADAR2 and in coordination of editing with alternative splicing.

Adenosine Deaminase↗

PubMed2003

Widespread inosine-containing mRNA in lymphocytes regulated by ADAR1 in response to inflammation.

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional modification of pre-mRNA catalysed by an RNA-specific adenosine deaminase (ADAR). A-to-I RNA editing has been previously reported in the pre-mRNAs of brain glutamate and serotonin receptors and in lung tissue during inflammation. Here we report that systemic inflammation markedly induces inosine-containing mRNA to approximately 5% of adenosine in total mRNA. Induction was the result of up-regulation of A-to-I RNA editing as both dsRNA editing activity and ADAR1 expression were increased in the spleen, thymus and peripheral lymphocytes from endotoxin-treated mice. Up-regulation of ADAR1 was confirmed in vitro in T lymphocytes and macrophages stimulated with a variety of inflammatory mediators including tumour necrosis factor-alpha and interferon-gamma. A late induction of RNA editing was detected in concanavalin A-activated splenocytes stimulated with interleukin-2 in vitro. Taken together, these data suggest that a large number of inosine-containing mRNAs are produced during acute inflammation via up-regulation of ADAR1-mediated RNA editing. These events may affect the inflammatory and immune response through modulation of protein production.

Acute Disease↗