86,940 research outputs found
ADAR-1 editing and DAg accumulation.
No full textA) Schematic representation of the ADAR-1 locus targeted by the different sgRNAs for KO generation (Upper) and the ADAR-1 isoforms, p110 and p150, including the functional domains for each isoform and the localization of the sgRNAs targeted regions (Lower). Western blot analysis of WT, ADAR-1 KO or overexpressing cells lines. Protein extracts from ADAR-1 overexpressing (p110 or p150), ADAR-1 KO (p110 and p150 or p150 only) and control Huh7.5 and HEK293T cell lines were analyzed by western blot to detect ADAR-1 expression and β-actin B) Western blot analysis of the human (Upper), rodent (Lower) DAg forms in ADAR-1 overexpressing (p110 or p150), ADAR-1 KO (p110 and p150 or p150 only) and control Huh7.5 (Left panels) and HEK293T (Right panels) cell lines. Cells were transfected with an empty pcDNA3.1 plasmid or with pcDNA3.1 plasmids encoding dimers of the HDV or RDeV genomes and collected 9 days post-transfection (d.p.t.) for HDV transfected Huh7.5 cells, 6 d.p.t for HDV transfected HEK293T cells, 6 d.p.t. for RDeV transfected Huh7.5 and HEK293T cells. Protein extracts were analyzed by western blot for DAg and β-actin expression. (TIF)</p
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Phylogenetic and structural analysis of Hydra ADAR
No full textAdenosine deaminases acting on RNAs (ADARs) perform adenosine-to-inosine (A-to-I) RNA editing for essential biological functions. While studies of editing sites in diverse animals have revealed unique biological roles of ADAR editing including temperature adaptation and reproductive maturation, rigorous biochemical and structural studies of these ADARs are lacking. Here, we present a phylogenetic sequence analysis and AlphaFold computational structure prediction to reveal that medusozoan ADAR2s contain five dsRNA binding domains (dsRBDs) with several RNA binding residues in the dsRBDs and deaminase domain conserved. Additionally, we identified evolutionary divergence between the medusozoan (e.g. Hydra) and anthozoan cnidarian subphyla. The anthozoan ADAR deaminase domains more closely resemble human ADARs with longer 5' RNA binding loops, glutamate base-flipping residues, and a conserved TWDG dimerization motif. Conversely, medusozoan ADAR deaminase domains have short 5' binding loops, glutamine flipping residues, and non-conserved helix dimerization motif. We also report the direct detection of A-to-I RNA editing by an ADAR ortholog from the freshwater cnidarian Hydra vulgaris (hyADAR). We solved the crystal structure of the monomeric deaminase domain of hyADAR (hyADARd) to 2.0 Å resolution, showing conserved active site architecture and the presence of a buried inositol hexakisphosphate known to be required for ADAR activity. In addition, these data demonstrate that medusozoans have evolved novel ADAR structural features, however the physiological consequence of this remains unknown. In addition, these results provide a framework for biochemically and structurally characterizing ADARs from evolutionarily distant organisms to understand the diverse roles of ADAR editing amongst metazoans
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The Impact of Sequence Context and Sugar Modifications to Human ADAR Function
No full textAdenosine Deaminases Acting on RNA (ADARs) are responsible for the deamination of adenosine (A) to inosine in double-stranded RNAs (dsRNA). Inosine (I) is read as guanosine (G) by molecular machinery so ADARs effectively catalyze an A to G point mutation. This RNA editing ability has designated the ADAR family as a target for therapeutic development in a method called ADAR-mediated site directed RNA editing (SDRE). This modality involves introducing guide oligonucleotides which specifically hybridize to a disease-causing mutation in the target RNA and recruiting ADAR to edit that specific mutation. Thus, ADAR-mediated RNA editing has strong potential to treat genetic disorders at the RNA level and mitigate or eliminate symptoms. However, ADARs do not edit every adenosine in dsRNA equally; details such as sequence context, RNA secondary structures, and chemical modifications factor into how well ADAR can edit a particular A. The broad goal of the works in this dissertation is to gain insight into the structures and modifications that can be included in the substrate RNA which can facilitate specific efficient editing at a desired site. Chapter 1 provides a general introduction of methods used to treat genetic disorders and how ADARs have been adopted into this field of therapeutics. A brief background of the work done to investigate the structure and function of ADARs is included to describe the potential and limitations associated with ADAR-mediated SDRE.
In Chapter 2, a systematic evolution of ligands by exponential enrichment (SELEX) approach is used to filter a large library of RNA substrates for the highest affinity binding substrates to determine how secondary structures in the RNA duplex can impact binding to ADAR and the subsequent editing with the discovered structures.
Chapters 3 and 4 investigate the effect of 2’-sugar modifications on ADAR editing activity. Chapter 3 details my contribution to a fellow lab member’s project investigating the effect of 2’-Fluoro (2’-F) modifications to RNA residues within a particular binding domain on the substrate RNA. My work here provides insight into the effects a 2’-F has on ADAR-RNA interactions that led to increased editing rates using a 2’-F. Chapter 4 establishes the baseline for the use of copper-click handles on the 2’-sugar to be used in substrate RNAs so we can investigate the effects of propargyl groups and a variety of triazole products on RNA editing
Fmrp-Adar interaction in zebrafish.
No full text<p><b>A.</b> Phylogenetic tree of zebrafish and human Adar proteins. Sequences are labeled with gene names, chromosomal locations, and accession numbers. To standardize and simplify the nomenclature, we named the genes Adar1-3, as indicated on the right side of each clade. Similarity values of each Adar member appear on top of each clade. <b>B.</b> Sequence conservation and motif distribution of Adar proteins in zebrafish and humans. Protein domains: adenosine deaminase domain (deaminase, white), double-stranded RNA binding motif (dsRBM, black) and zDNA binding domain (z_alpha, light grey). <b>C-R.</b> <i>In situ</i> hybridization showing lateral (<b>C</b>, <b>E</b>, <b>F</b>, <b>H</b>, <b>I</b>, <b>K</b>, <b>L</b>, <b>N</b>, <b>O</b>, <b>Q</b>) and dorsal (<b>D</b>, <b>G</b>, <b>J</b>, <b>M</b>, <b>P</b>, <b>R</b>) views of the spatial expression pattern of all four <i>adar</i> genes in 2 dpf <b>(C-D, F-G, I-J, L-M)</b> and 6 dpf <b>(E, H, K, N)</b> WT larvae. Expression is detected primarily in the nervous system. <b>O-R.</b> Selected regions (black frames in <b>L</b> and <b>M</b>) show <i>adar2b</i> (<b>O-P)</b> and <i>adar3</i> (<b>Q-R)</b> expression in the spinal cord of 2 dpf WT embryo. <b>S.</b> HEK-293T cells were transiently transfected with the zebrafish proteins Adar2a and Fmrp fused to EGFP and MYC, respectively (EGFP-Adar2a and MYC-Fmrp). Co-immunoprecipitation was used to detect Adar2a and Fmrp interaction. Actin was used as a negative control. The cell lysate was immunoprecipitated with anti-actin, anti-MYC, or anti-EGFP. Proteins were purified from the complexes and separated by SDS-PAGE. <b>T.</b> Western blot shows the protein content following the transfection prior to the immunoprecipitation. The proteins were detected with specific antibodies against MYC, EGFP, and actin. <b>U.</b> Computational sequence homology predicted the number of RNA recognition elements (RREs) in the CDS of <i>adar</i> genes that are recognized by Fmrp. <b>V.</b> RNA immunoprecipitation (RIP) assays show that Fmrp binds <i>adar1</i>. PCR amplification of <i>adar1</i> on RNA extracted from a RIP experiment conducted with anti-Actin and anti-MYC antibodies, and on total RNA extracted from HEK293T cells. <b>W.</b> RT-PCR assays showed that the mRNA expression levels of all four <i>adar</i> genes increased in 6 dpf <i>fmr1</i>-/- larvae (grey bars) when compared with WT larvae (white bars). Values are represented as means ± SEM. *<i>p</i><0.05, **<i>p</i><0.005, two-way <i>t</i>-test assuming unequal variances. <b>X.</b> Adar2 protein expression was analyzed by Western blot with specific antibodies against Adar2 and actin as a loading control. Elevated Adar2 protein levels of approximately 30% are present in <i>fmr1</i>-/- brains.</p
Genome Research / ADAR-deficiency perturbs the global splicing landscape in mouse tissues
No full textAdenosine-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
Global Transcriptome Analysis of RNA Abundance Regulation by ADAR in Lung Adenocarcinoma
No full textDespite tremendous advances in targeted therapies against lung adenocarcinoma, the majority of patients do not benefit from personalized treatments. A deeper understanding of potential therapeutic targets is crucial to increase the survival of patients. One promising target, ADAR, is amplified in 13% of lung adenocarcinomas and in-vitro studies have demonstrated the potential of its therapeutic inhibition to inhibit tumor growth. ADAR edits millions of adenosines to inosines within the transcriptome, and while previous studies of ADAR in cancer have solely focused on protein-coding edits, >99% of edits occur in non-protein coding regions. Here, we develop a pipeline to discover the regulatory potential of RNA editing sites across the entire transcriptome and apply it to lung adenocarcinoma tumors from The Cancer Genome Atlas. This method predicts that 1413 genes contain regulatory edits, predominantly in non-coding regions. Genes with the largest numbers of regulatory edits are enriched in both apoptotic and innate immune pathways, providing a link between these known functions of ADAR and its role in cancer. We further show that despite a positive association between ADAR RNA expression and apoptotic and immune pathways, ADAR copy number is negatively associated with apoptosis and several immune cell types' signatures
Variations on the Author
No full text“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Editing activity in ADAR-expressing yeast strains is strongest for mallard duck ADAR1.
No full text(A) Res-scanner detection of editing sites (Methods) for five yeast strains expressing different active ADAR enzymes results in varying amounts of sites, spanning two to three orders of magnitude. Note that due to the large numbers for mdADAR1 (113,672 sites, in a 12Mbp genome) and hbADAR2 (10,443), the results for the other strains are invisible in the main graph, and thus these results are plotted again in the inset. The (three rightmost) control strains show no excess of A-to-G mismatches, attesting for no editing activity as expected. (B) Distribution of the neighboring nucleotides to the predicted editing sites reveals the familiar editing motif (mostly a depletion of G upstream) for all strains expressing active ADARs, but not for the control ones. (C) Thermodynamic stability of the predicted secondary structures surrounding the detected sites (Methods). Lower (more negative) ΔG indicates a more stable structure. Note that no structure was found for 113, 379, 939, 2526 and 32197 sites, for the five organisms, respectively. Their ΔG was set to zero. (D) Genome-wide editing index (Methods) for the five active ADAR strains and three control ones. The dashed line represents the base-line noise level. (E-F) ADAR expression levels (TPM) (E) and protein levels (F) for the different strains do not account for the wide differences in the number of sites detected. Logarithmically growing cells carrying the indicated plasmids were grown in SC-URA+Raf (ADAR expression is off). 2% Gal was then added for 4 hours, to induce the ADAR genes expression. Cell lysates from the SC-URA+Raf and SC-URA+Gal (t-0 and t-4 respectively) were separated by SDS-PAGE and immunoblotted with anti-FLAG (α-FLAG. Anti-3-phosphoglycerate kinase 1 (α-PGK1) was used as a loading control. Relative intensity was measured as pixel density for each sample with consideration of the loading control. Protein levels were quantitated relative to the loading control in three independent experiments. Whiskers represent the minimum and maximum values measured from three independent experiments. Stars above the boxplots indicate a statistically significant difference between the means of two samples (ns: p > 0.05; *: p < = 0.05; **: p < = 0.01; ***: p < = 0.001; ****: p < = 0.0001).</p
[Newspaper Clipping: Author Claims Evidence of Second JFK Assassin #1]
No full textNewspaper article titled "Author Claims Evidence of Second JFK Assassin." The article states that author Richard J. Whalen concluded "that there is circumstantial evidence to support the theory of a second assassin in the shooting of President John F. Kennedy.
ADARs and the balance game between virus infection and innate immune cell response
No full textAll viruses that have dsRNA structures at any stages of their life cycle may potentially undergo RNA editing events mediated by ADAR enzymes. Indeed, an increasing number of studies that describe A-to-I sequence changes in viral genomes and/or transcripts, consistent with ADAR deaminase activity, has been reported so far. These modifications can appear as either hyperediting or specific RNA editing events in viral dsRNAs. It is now well established that ADAR enzymes can affect viral life cycles in an editing-dependent and -independent manner, with ADARs acting as pro- or antiviral factors. Despite the discovery of editing events in viral RNAs dates back to thirty years ago, the biological consequences of A-to-I changes during viral infection is still far to be completely elucidated. In this review, past and recent studies on the importance of ADAR enzymes on several viruses will be examined
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